Lung cancer differential marker

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage application filed under 35 USC .sctn.371 of PCT/JP2011/070635, filed Sep. 9, 2011, which claims the benefit of Japanese Patent Application No. 2010-209932, filed Sep. 17, 2010, both of which are incorporated by reference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is 119244_00053_Revised_Sequence_Listing. The size of the text file is 206 KB, and the text file was created on Jan. 26, 2016.

TECHNICAL FIELD

The present invention relates to a lung cancer differential marker glycoprotein having a glycan or a fragment thereof, and a method for determining the presence or absence of lung cancer or the histological type of lung cancer using the same.

BACKGROUND ART

Lung cancer is a typical example of intractable cancers and is the first and second leading causes of cancer deaths in Japanese men and women, respectively, in 2009. The types of lung cancer are broadly classified into small cell cancer (small cell lung cancer) accounting for approximately 10% and non-small cell cancer accounting for approximately 90%. The types of non-small cell cancer are further classified into adenocarcinoma (lung adenocarcinoma) (60%), squamous cell carcinoma (lung squamous cell carcinoma) (25%), and large-cell cancer (large-cell lung cancer) (5%).

The small cell lung cancer is a very high-grade cancer and therefore has a strong tendency to metastasize even in an early stage. As seen in previous cases, it is likely that the small cell lung cancer has already metastasized systemically when found. Thus, non-surgical therapy is commonly selected for this cancer even if its metastasis to lymph nodes or other tissues is not confirmed. Since this cancer is highly sensitive to chemotherapy or radiation, chemotherapy is central to the non-surgical therapy.

By contrast, the non-small cell lung cancer, which constitutes a large portion of lung cancer cases, is low sensitive to chemotherapy or radiation. For its treatment, it is important to find the cancer relatively early and remove the lesion by surgical therapy.

Tests for lung cancer can be broadly classified, depending on the purposes of the tests, into three: (1) lung cancer assessment to test the probability of lung cancer; (2) definite diagnosis of lung cancer to confirm that this probable lung cancer is definitely lung cancer; and (3) determination of the stage of lung cancer progression to test the histological type and stage of progression of the definitely diagnosed lung cancer.

These tests generally adopt a method which involves detecting an abnormal shadow in the lung field by chest X-ray examination or CT scan and subsequently finally determining a cancer type and comprehensively determining the stage of progression by bronchoscopy or by the pathological diagnosis of biopsy samples obtained using biopsy or the like. However, cases with small cell cancer coexisting with non-small cell cancer or borderline cancers might be given different diagnostic outcomes among pathologists. Accurate definite diagnosis has not yet been established for lung cancer.

In recent years, tumor markers have been used in cancer prognosis or the like. The tumor markers refer to substances produced by cancer cells or substances produced by cells in response to cancer cells. The amounts of the tumor markers contained in serum reflect the amount or histological type of tumor. The tumor markers can therefore serve as an index for, for example, determining the presence or absence of cancer and as such, can be used in diagnostic aids, the prediction of a histological type or the stage of progression, the determination of therapeutic effects, the prediction of recurrence, prognosis, etc. Currently, some tumor markers, such as CEA, CYFRA, NSE, ProGRP, SCC, and SLX, are also known for lung cancer (Non Patent Literatures 1 to 3). All of these tumor markers, however, are based on the difference in protein expression level, i.e., increase or decrease in protein expression, in blood or tissue between healthy persons and lung cancer patients. These tumor markers are usually expressed even in normal cells and are thus low specific for lung cancer. Hence, the obtained results present the problem of low reliability or detection sensitivity. In addition, lung cancer markers useful in determining the histological type of detected lung cancer have not yet been found.

CITATION LIST

Non Patent Literature

Non Patent Literature 1: Molina R, et al., (2005) Anticancer Res 25: 1773-1778. Non Patent Literature 2: Mizuguchi S, et al., (2007) Ann Thorac Surg 83: 216-221. Non Patent Literature 3: Holdenrieder S, et al., (2008) Clin Cancer Res 14: 7813-7821.

SUMMARY OF INVENTION

Technical Problem

An object of the present invention is to develop a lung cancer differential marker with which lung cancer can be diagnosed conveniently and highly sensitively without depending only on increase or decrease in protein expression level between cancer patients and healthy persons. More specifically, an object of the present invention is to develop a lung cancer-specific differential marker glycoprotein and a fragment thereof, which serve as an indication for suffering lung cancer.

Another object of the present invention is to develop a lung cancer differential marker capable of determining the histological type of lung cancer.

A further object of the present invention is to develop a glycan probe for differential diagnosis of lung cancer, with which the presence or absence of lung cancer and further, its histological type can be determined by histological staining.

Solution to Problem

The compositional and structural diversities of glycans on proteins secreted from cells are controlled by the balanced expression of hundreds of glycan-related genes and vary depending on the degrees of cell differentiation and cancer progression. Glycoproteins whose glycan structures are altered can be used as disease condition index markers including tumor markers. In recent years, such glycan-related tumor markers based on proteomics have been searched for actively. In the pipeline of the marker search, first, candidate molecules are identified by large-scale analysis at phase 1. Subsequently, the candidate molecules are tested by quantitative analysis at phase 2 to narrow down the candidates. Then, a validation test is conducted at phase 3.

In order to attain the objects, the present inventors have searched for lung cancer differential markers using glycoproteomics based on the marker search pipeline. As a result, the present inventors have successfully identified, from among serum glycoproteins, novel glycoprotein or glycopeptide groups having lung cancer-specific structures detected in a lung cancer cell culture supernatant. The present inventors have also revealed that the presence or absence of lung cancer and the histological type of lung cancer can be determined using these glycoprotein or glycopeptide groups. The present invention is based on these findings and provides the followings:

(1) A lung cancer differential marker glycoprotein listed in Table 1 or 2, being glycosylated with a glycan at the asparagine residue(s) at the glycosylation site(s) shown in Table 1 or 2:

TABLE-US-00001 TABLE 1 Protein Small cell cancer Adenocarcinoma Glycosylation SEQ # Protein name gi(ID) AAL ConA AAL ConA site Peptide sequence NO. 1 acid alpha-glucosidase gi|119393891, .largecircle. X X X 390 QVVENMTR 1 gi|119393893 470 GVFITNETGQPLIGK 2 882 GAYTQVIFLARNNTIVNELVR 3 2 biotinidase gi|4557373 X X .largecircle. X 119 DVQIIVFPEDGIHGFNFTR 4 150 FNDTEVLQR 5 349 SHLIIAQVAKNPVGLIGAENATGETDPSHSK 6 349 NPVGLIGAENATGETDPSHSKFLK 7 3 cathepsin D gi|4503143 X X .largecircle. X 263 YYKGSLSYLNVTR 8 4 cathepsin L1 gi|22202619, .largecircle. X X X 221 YNPKYSVANDTGFVDIPKQEK 9 gi|4503155 221 YSVANDTGFVDIPK 10 5 cathepsin L2 gi|23110960 .largecircle. X X X 221 YRPENSVANDTGFTVVAPGKEK 11 292 NLDHGVLVVGYGFEGANSNNSK 12 6 cell adhesion molecule 4 gi|21686977 .largecircle. X X X 67 QTLFFNGTR 13 7 deoxyribonuclease II, gi|4503349 .largecircle. X X X 86 SNTSQLAFLLYNDQPPQPSK 14 lysosomal 8 fibronectin 1 gi|47132557 (isoform 1), X X .largecircle. X 430 GGNSNGALCHFPFLYNNHNYTDCTSEGR 15 gi|47132551 (isoform 2), 528 DQCIVDDITYNVNDTFHK 16 gi|16933542 (isoform 3), 542 RHEEGHMLNCTCFGQGR 17 gi|47132555 (isoform 4), 542 HEEGHMLNCTCFGQGR 18 gi|47132553 (isoform 5), 1007 ESKPLTAQQTTKLDAPTNLQFVNETDSTVLVR 19 gi|47132549 (isoform 6), 1007 LDAPTNLQFVNETDSTVLVR 20 gi|47132547 (isoform 7) 1291 WTPLNSSTIIGYR 21 9 galectin-3-binding gi|122937327 .largecircle. X X X 44 ADVGGEAAGTSINHSQA- VLQR 22 protein-like 61 QGNASDVVLR 23 307 FFDVNGSAFLPR 24 10 insulin-like growth gi|119964726 .largecircle. X X X 112 SLLEFNTTVSCDQQGTNHR 25 factor 2 receptor 435 MSVINFECNKTAGNDGK 26 582 TNITLVCKPGDLESAPVLR 27 2085 GYPCGGNKTASSVIELTCTK 28 11 insulin-like growth factor gi|62243248 (isoform a), X X .largecircle. X 116 GLCVNASAVSR 29 binding protein 3 gi|62243068 (isoform b) 205 YKVDYESQSTDTQNFSSESKR 30 12 insulin-like growth factor gi|56090548 X .largecircle. X X 166 DGPCEFAPVVVVPPRSVHNVTGAQVGLSCE- VR 31 binding protein-like 1 166 SVHNVTGAQVGLSCEVR 32 13 integral membrane protein 1 gi|22749415 X .largecircle. X X 548 TILVDNNTWNNTHISR 33 14 L1 cell adhesion molecule gi|4557707 (isoform 1), X X .largecircle. .largecircle. 433 ILTADNQTYMAVQGSTAYLLCK 34 gi|13435353 (isoform 2) 671 WYSLGKVPGNQTSTTLK 35 777 VQWRPQGTRGPWQEQIVSDPFLVVSNTSTFVPYEIK 36 979 THNLTDLSPHLR 37 15 lysosomal acid phosphatase 2 gi|4557010 .largecircle. X X X 133 FNPNISWQPIPVHTVPITEDR 38 167 YEQLQNETRQTPEYQNESSR 39 177 QTPEYQNESSR 40 16 melanoma cell adhesion gi|71274107 .largecircle. X X X 56 CGLSQSQGNLSHVDWFSVHK 41 molecule 418 CVASVPSIPGLNR 42 17 melanoma-associated antigen gi|134244281 (isoform 1), .largecircle. .largecircle. X X 38 WCATSDPEQHKCGNMSEAFR 43 p97 gi|16163666 (isoform 2) 515 DCDVLTAVSEFFNASCVPVNNPK 44 18 neogenin homolog 1 gi|4505375 X .largecircle. X X 73 GSSVILNCSAYSEPSPK 45 210 VIKLPSGMLVISNATEGDGGLYR 46 470 TPASDPHGDNLTYSVFYTK 47 19 neural cell adhesion gi|94420689 (isoform 1), .largecircle. X X X 347 TSTRNISSEEK 48 molecule 1 gi|115529482 (isoform 2), 449 DGQLLPSSNYSNIK 49 gi|115529478 (isoform 3) 478 IYNTPSASYLEVTPDSENDFGNYNCTAVNR 50 20 neuronal pentraxin 11 gi|28195384 .largecircle. X X X 148 ANVSNAGLPGDFR 51 189 VAELEDEKSLLHNETSAHR 52 21 neuronal pentraxin receptor gi|17402888 X .largecircle. X X 42 ALPGGADNASVASGAAASPGPQR 53 22 ribonuclease T2 gi|5231228 X X .largecircle. X 106 AYWPDVIHSFPNR 54 212 QDQQLQNCTEPGEQPSPK 55 23 secretogranin III gi|19557645 .largecircle. .largecircle. X X 68 KTYPPENKPGQSNYSFVDNLNL- LK 56 346 NKLEKNATDNISK 57 24 sel-1 suppressor of gi|19923669 X .largecircle. X X 608 EASIVGENETYPR 58 lin-12-like 25 sparc/osteonectin, cwcv and gi|7662036 .largecircle. .largecircle. X X 225 LRDWFQLLHENSKQNGSASSVA- GPASGLDK 59 kazal-like domains 225 QNGSASSVAGPASGLDK 60 proteoglycan (testican) 2 26 Thy-1 cell surface antigen gi|19923362 .largecircle. .largecircle. X X 42 LDCRHENTSSSPIQYEFS- LTR 61 27 tubulointerstitial gi|11545918 X X .largecircle. X 78 GRADDCALPYLGAICYC- DLFCNR 62 nephritis antigen-like 1 161 AINQGNYGWQAGNHSAFWGMTLDEGIR 63 28 v-kit Hardy-Zuckerman 4 gi|4557695 (isoform 1), .largecircle. .largecircle. X X 130 SLYGKEDNDTLVR 64 feline sarcoma viral gi|148005039 (isoform 2) 367 TFTDKWEDYPKSENESNIR 65 oncogene homolog 463 CSASVLPVDVQTLNSSGPPFGK 66 486 LVVQSSIDSSAFKHNGTVECK 67 29 laminin alpha 5 gi|21264602 X X .largecircle. .largecircle. 95 LVGGPVAGGDPNQTIR 68 921 LNLTSPDLFWLVFR 69 1330 VWQGHANASFCPHGYGCR 70 1529 TIPPDCLLCQPQTFGCHPLVGCEECNCSGPGIQELT DPTCDTDSGQCK 71 2019 CEICAPGFYGNALLPGNCTR 72 2196 GINASSMAWAR 73 2209 LHRLNASIADLQSQLR 74 2303 TLSELMSQTGHLGLANASAPSGEQLLR 75 2423 DNATLQATLHAAR 76 2501 LVEAAEAHAQQLGQLALNLSSIILDVNQDRLTQR 77 2568 QGLVDRAQQLLANSTALEEAMLQEQQR 78 2707 GVHNASLALSASIGR 79 3107 LNTTGVSAGCTADLLVGR 80 3287 VFDLQQNLGSVNVSTGCAPALQAQTPGLGPR 81 30 laminin, beta 1 gi|4504951 X X .largecircle. .largecircle. 1041 KCVCNYLGTVQEHCNGSDCQCDK- 82 1279 LSDTTSQSNSTAK 83 1487 QSAEDILLKTNATK 84 1643 AIKQADEDIQGTQNLLTSIESETAASEETLFNASQR 85 31 phospholipid transfer gi|5453914 (isoform a), X X .largecircle. .largecircle. 64 GKEGHFYYNISEVK 86 protein gi|33356541 (isoform b) 143 MKVSNVSCQASVSR 87 143 VSNVSCQASVSR 88 245 GAFFPLTERNWSLPNR 89 398 FRIYSNHSALESLALIPLQAPLK 90 193 GAFFPLTERNWSLPNR 91

TABLE-US-00002 TABLE 2 Protein Small cell cancer Adenocarcinoma Glycosylation SEQ # Protein name gi(ID) AAL ConA AAL ConA site Peptide sequence NO 1 activated leukocyte cell gi|68163411 X .largecircle. X X 167 KLGDCISEDSYPDGNITWYR 92 adhesion molecule 265 NAIKEGDNITLK 93 265 EGDNITLK 94 361 NATVVWMKDNIR 95 480 YYSKIIISPEENVTLTCTAENQLER 96 2 alpha 2 type V collagen gi|89363017 X X X .largecircle. 1400 EASQNITYICK 97 preproprotein 3 amiloride binding gi|73486661 X X .largecircle. .largecircle. 538 LENITNPWSPR 98 protein 1 4 aspartate beta-hydroxylase gi|14589866 (isoform a) X X X .largecircle. 452 LVQLFPNDTSLKNDLGVGYLLIGDNDNAKK 99 5 aspartate beta-hydroxylase gi|14589864 (isoform b) .largecircle. .largecircle. X X 64 DFRYNLSEVLQGK 100 6 beta-1,3-galactosyl-O- gi|148277029, X X .largecircle. X 58 HLELAGENPSSD- INCTK 101 glycosyl-glycoprotein gi|148277031, 95 WTPDDYINMTSDCSSFIK 102 beta-1,6-N-acetyl- gi|148277033, glucosaminyltransferase gi|148277035, gi|21614523 7 bone morphogenetic gi|4502421 X X .largecircle. .largecircle. 142 AATSRP- ERVWPDGVIPFVIGGNFTGSQR 103 protein 1 363 ISVTPGEKIILNFTSLDLYR 104 599 LNGSITSPGWPK 105 8 calsyntenin 2 gi|11545861 .largecircle. X X X 98 IHGQELPFEAVVLNKTSGEGR 106 374 NLTDQFTITMWMK 107 716 QECLELNHSELHQR 108 729 HLDATNSTAGYSIYGVGSMSR 109 9 carboxypeptidase D gi|22202611 X .largecircle. X X 172 LLNTTDVYLLPSLNPDGFERAR 110 522 RFANEYPNITR 111 978 HIWSLEISNKPNVSEPEEPKIR 112 1070 GKDLDTDFTNNASQPETK 113 10 CD47 antigen gi|4502673 (isoform 1), X .largecircle. X X 73 GRDIYTFDGALNK 114 gi|38683836 (isoform 2), 73 DIYTFDGALNKSTVPTDFSSAK 115 gi|68223315 (isoform 3) 111 MDKSDAVSHTGNYTCEVTELTR 116 11 CD63 antigen gi|4502679 (isoform A), X .largecircle. X X 130 QQMENYPKNNHTASILDR 117 gi|91199546 (isoform B) 130 NNHTASILDR 118 172 NRVPDSCCINVTVGCGINFNEK 119 12 CD97 antigen gi|17978491 (isoform 1), X X .largecircle. X 108 TFKNESENTCQDVDECQQNPR 120 gi|17978489 (isoform 2), 453 RLSAVNSIFLSHNNTK 121 gi|68508955 (isoform 3) 360 RLSAVNSIFLSHNNTK 122 108 TFKNESENTCQDVDECQQNPR 123 404 RLSAVNSIFLSHNNTK 124 13 complement factor I gi|119392081 X X .largecircle. X 103 FLNNGTCTAEGK 125 177 FKLSDLSINSTECLHVHCR 126 177 LSDLSINSTECLHVHCR 127 464 SIPACVPWSPYLFQPNDTCIVSGWGR 128 494 LISNCSKFYGNR 129 14 cystatin F gi|20302139 X X .largecircle. .largecircle. 84 YSVEKFNNCTNDMFLFK 130 84 FNNCTNDMFLFKESR 131 137 LDDCDFQTNHTLK 132 15 desmocollin 2 gi|13435364 X X X .largecircle. 546 SLDREAETIKNGIYNITVLASDQGGR 133 629 AINDTAAR 134 16 epithelial V-like gi|21536337 X X X .largecircle. 39 VLEAVNGTDAR 135 antigen 1 118 LQFDDNGTYTCQVK 136 17 FAT tumor suppressor 1 gi|66346693 X X .largecircle. .largecircle. 333 AIGGIDWDSHPFGYNLTLQAK 1- 37 998 QVYNLTVR 138 1551 IVVNVSDTNDHAPWFTASSYK 139 3716 QLLHKINSSVTDIEEIIGVR 140 18 fibrinogen-like 2 gi|5730075 X X .largecircle. .largecircle. 263 LDGSTNFTR 141 336 LHVGNYNGTAGDALR 142 19 Fraser syndrome 1 gi|108773804 X X .largecircle. .largecircle. 1107 IHTPSLHVNGSLILPIGSIKP- LDFSLLNVQDQEGR 143 1503 IVYNITLPLHPNQGIIEHR 144 1776 ISGSEVEELSEVSNFTMEDINNKK 145 2562 YTSYNVSEK 146 2667 VIINDTEDEPTLEFDKK 147 20 growth differentiation gi|4758936 X X X .largecircle. 70 LRANQSWEDSNTDL- VPAPAVR 148 factor 15 70 ANQSWEDSNTDLVPAPAVRILTPEVR 149 21 immunoglobulin gi|148664190 (isoform 1), .largecircle. X X X 101 FQLLNFSSSELK 150 superfamily, member 4D gi|148664211 (isoform 2) 113 VSLTNVSISDEGR 151 isoform 1 22 integrin, alpha 1 gi|31657142 X X X .largecircle. 418 NTTFNVESTK 152 883 DSCESNHNITCK 153 1113 SENASLVLSSSNQK 154 23 intercellular adhesion gi|4557878 X X .largecircle. X 202 TELDLRPQGLELFENTSAPYQLQTFVLPA- TPPQLVSPR 155 molecule 1 267 LNPTVTYGNDSFSAK 156 24 interleukin 6 receptor gi|4504673 X X X .largecircle. 93 SVQLHDSGNYSCYR 157 25 latent transforming growth gi|18497288 X X .largecircle. X 89 DSCQQGSNMTLIGENGHSTDTLTGSGFR 15- 8 factor beta binding 349 RLNSTHCQDINECAMPGVCR 159 protein 3 845 DRSHCEDIDECDFPAACIGGDCINTNGSYR 160 26 mucin 16 gi|83367077 X X .largecircle. X 12586 NTSVGLLYSGCR 161 13193 KFNITESVLQGLLKPLFK 162 14363 NIEDALNQLFRNSSIK 163 14417 NGTQLQNFTLDR 164 27 netrin 4 gi|93204871 X X .largecircle. X 56 KLWADTTCGQNATELYCFYSENTDLTC- RQPK 165 163 YFATNCSATFGLEDDVVKK 166 28 neuronal cell adhesion gi|81158226 X .largecircle. X X 223 FNHTQTIQQK 167 molecule isoform A 245, 251 VISVDELNDTIAANLSDTEFYGAK 168 276 ERPPTFLTPEGNASNKEELR 169 314 EDGMLPKNR 170 507 GSALHEDIYVLHENGTLEIPVAQKDSTGTYTCVAR 171 858 VNVVNSTLAEVHWDPVPLK 172 29 olfactomedin related ER gi|17136143 (isoform 1), .largecircle. .largecircle. X X 85 QLLEKVQNMSQSIEVLDR 173 localized protein gi|5453547 (isoform 2) 85 VQNMSQSIEVLDRR 174 270 SMVDFMNTDNFTSHR 175 376 LDPVSLQTLQTWNTSYPKR 176 85 QLLEKVQNMSQSIEVLDR 177 30 osteoprotegerin gi|148743793 X X .largecircle. .largecircle. 98 ELQYVKQ- ECNR 178 152 CPDGFFSNETSSKAPCR 179 178 GNATHDNICSGNSESTQK 180 289 HIGHANLTFEQLR 181 31 palmitoyl-protein gi|4506031 X X X .largecircle. 212 GINESYKK 182 thioesterase 1 (ceroid- 232 FLNDSIVDPVDSEWFGFYR 183 lipofuscinosis, neuronal 1, infantile) 32 peptidylprolyl isomerase gi|4758950 X X X .largecircle. 148 HYGPGWVSMANAGKDTNGSQFFITTVK - 184 B 33 plasminogen activator, gi|4505861 X X .largecircle. .largecircle. 152 GTWSTAESGAECTNW- NSSALAQKPYSGR 185 tissue type I 219 AGKYSSEFCSTPACSEGNSDCYFGNGSAYR 186 219 YSSEFCSTPACSEGNSDCYFGNGSAYR 187 483 CTSQHLLNRTVTDNMLCAGDTR 188 34 prion protein gi|122056623, X X X .largecircle. 197 QHTVTTTTKGENFTETDVK- 189 gi|122056625, 197 GENFTETDVK 190 gi|122056628, gi|34335270, gi|4506113, 35 prostaglandin H2 D- gi|32171249 X X X .largecircle. 51 WFSAGLASNSSWLR 191 isomerase 78 SVVAPATDGGLNLTSTFLR 192 36 protein tyrosine gi|109633041 (isoform 1), .largecircle. X X X 721 KVEVEPLNSTAVHVYWK 193 phosphatase, receptor gi|109633039 (isoform 2), 966 DINSQQELQNITTDTRFTLTGLKPDTTYDIK 194 type, F 721 KVEVEPLNSTAVHVYWK 195 957 DINSQQELQNITTDTRFTLTGLKPDTTYDIK 196 37 protein tyrosine gi|110735404 (isoform 1), X X .largecircle. X 410 QLTLQWEPLGYNVTR 197 phosphatase, receptor gi|110735406 (isoform 2), type, U gi|l10735402 (isoform 3) 38 seizure related 6 gi|6912612 (isoform 1), .largecircle. .largecircle. X X 177 LLANSSMLGEGQVLR 198 homolog (mouse)-like 2 gi|42491358 (isoform 2) 303 IVSPEPGGAVGPNLTCR 199 247 LLANSSMLGEGQVLR 200 373 IVSPEPGGAVGPNLTCR 201 39 seizure related 6 gi|32261332 .largecircle. X X X 328 SVNLSDGELLSIR 202 homolog (mouse)-like 40 seizure related 6 gi|148839280 (isoform 1), X .largecircle. X X 399, 422 HLTCLNATQPFWDSKEPVCIAACGGVIRNATTGR 203 homolog gi|148839346 (isoform 2) 436, 440 IVSPGFPGNYSNNLTCHWLLEAPEGQR 204 41 serine carboxypeptidase gi|83641874, .largecircle. .largecircle. X X 34- 6 QAIHVGNQTFNDGTIVEK 205 vitellogenic-like gi|83641876 42 solute carrier family 39 gi|55741750 X X .largecircle. X 191, 198 LHHHLDHNNTHHFHNDSITPSER 206 (zinc transporter), 218 GEPSNEPSTETNKTQEQSDVKLPK 207 member 10 339 KDLNEDDHHHECLNVTQLLK 208 43 tenascin C (hexabrachion) gi|4504549 X X .largecircle. X 38 QSGVNATLPEENQPVVFNHVYNIK- 209 327 CINGTCYCEEGFTGEDCGKPTCPHACHTQGR 210 788 QTGLAPGQEYEISLHIVKNNTRGPGLK 211 1018 LNYSLPTGQWVGVQLPR 212 1034 NTTSYVLRGLEPGQEYNVLLTAEK 213 1079 VKASTEQAPELENLTVTEVGWDGLR 214 1093 LNWTAADQAYEHFIIQVQEANKVEAAR 215 1485 LLETVEYNISGAER 216 44 tissue factor pathway gi|5454114 X X .largecircle. X 145 YFYNNQTK 217 inhibitor 45 transforming growth gi|63025222 X X .largecircle. .largecircle. 82 LRLASPPSQGEVPPGPLPE- AVLALYNSTR 218 factor, beta 1 46 tumor-associated calcium gi|4505059, X X .largecircle. X 111 QCNGTSTCWCVNTAGVR 219 signal transducer 1 gi|4505057 168 HRPTAGAFNHSDLDAELR 220

47 UDP-GIcNAc:betaGal beta- gi|9845238 X X .largecircle. X 89 LSNISHLNYCEPDLR 221 1,3-N-acetyl- glucosaminyltransferase 2 173 ESWGQESNAGNQTVVR 222 48 von Willebrand factor A gi|38348304 X X .largecircle. .largecircle. 147 NASVPQILIIVTDGK 223 domain containing 2

(2) The lung cancer differential marker glycoprotein according to (1), wherein the sugar chain is at least one glycan selected from the group consisting of a fucosylated glycan, a high mannose-type glycan, a hybrid-type glycan, a biantennary complex-type glycan, chitin, polylactosamine, and a .beta.1,3-galactose epitope.

(3) The lung cancer differential marker glycoprotein according to (2), wherein the glycoprotein is for differential diagnosis of small cell lung cancer or lung adenocarcinoma.

(4) The lung cancer differential marker glycoprotein according to (3), wherein the glycoprotein is for differential diagnosis of small cell lung cancer and is at least one glycoprotein selected from the group consisting of neural cell adhesion molecule (NCAM1), secretogranin III, and insulin-like growth factor-binding protein-L1 (IGFBP-L1).

(5) The lung cancer differential marker glycoprotein according to (3), wherein the glycoprotein is for differential diagnosis of lung adenocarcinoma and is fibronectin 1.

(6) A fragment of a lung cancer differential marker glycoprotein according to any of (1) to (5), comprising at least one asparagine residue at a glycosylation site shown in Table 1 or 2 being glycosylated with a glycan.

(7) A method for determining lung cancer, comprising detecting at least one lung cancer differential marker glycoprotein shown in Table 1 or 2 being glycosylated with a glycan at the asparagine residue(s) at the glycosylation site(s) shown in Table 1 or 2, and/or at least one fragment thereof, the fragment comprising at least one asparagine residue at the glycosylation site shown in Table 1 or 2 being glycosylated with a glycan, from a sample obtained from a test subject, wherein the detection of the glycoprotein or fragment determines that the test subject suffers lung cancer.

(8) The method according to (7), wherein the lung cancer differential marker glycoprotein and/or the fragment thereof are detected using at least one glycan probe binding to the glycan.

(9) The method according to (8), wherein the glycan probe binds to a fucosylated glycan, a high mannose-type glycan, a hybrid-type glycan, a biantennary complex-type glycan, chitin, polylactosamine, or a .beta.1,3-galactose epitope.

(10) The method according to (8) or (9), wherein the glycan probe is a lectin, an antibody, or a phage antibody.

(11) The method according to (10), wherein the lectin is AAL, ConA, PWM, or PNA.

(12) The method according to (11), wherein the lung cancer differential marker glycoprotein is neural cell adhesion molecule (NCAM1), and the detection of the binding thereof to AAL determines the histological type of the lung cancer as small cell cancer.

(13) The method according to (11), wherein the lung cancer differential marker glycoprotein is secretogranin III, and the detection of the binding thereof to AAL and/or ConA determines the histological type of the lung cancer as small cell cancer.

(14) The method according to (11), wherein the lung cancer differential marker glycoprotein is insulin-like growth factor-binding protein-L1 (IGFBP-L1), and the detection of the binding thereof to ConA and/or PWM determines the histological type of the lung cancer as small cell cancer.

(15) The method according to (11), wherein the lung cancer differential marker glycoprotein is fibronectin 1, and the detection of the binding thereof to AAL and/or PNA determines the histological type of the lung cancer as adenocarcinoma.

(16) The method according to any of (8) to (11), wherein the histological type of lung cancer is determined as small cell cancer or adenocarcinoma on the basis of a result of binding of the glycan probe to the glycan in the lung cancer differential marker glycoprotein and/or the fragment thereof, and a manner of binding of the lung cancer differential marker glycoprotein shown in Table 1 or 2 and/or the fragment thereof to the glycan probe.

(17) The method according to any of (7) to (16), wherein the sample is a body fluid, a cell, or a lung lavage.

(18) The method according to (17), wherein the body fluid is blood (including serum, plasma, and interstitial fluid), lymph, a cell extract, sputum, or pleural effusion.

(19) A lung cancer cell-identifying antibody for histological staining, binding to a lung cancer differential marker glycoprotein listed in Table 1 or 2 being glycosylated with a glycan at the asparagine residue(s) at the glycosylation site(s) shown in Table 1 or 2, and/or a fragment thereof, the fragment comprising at least one asparagine residue at a glycosylation site shown in Table 1 or 2 being glycosylated with a glycan, and thereby diagnosing lung cancer.

(20) The antibody according to (19), wherein the antibody is capable of determining a histological type of a lung cancer cell.

(21) The antibody according to (20), wherein the lung cancer differential marker glycoprotein is neuronal pentraxin receptor (NPR), and the histological type of the lung cancer cell is determined as small cell cancer.

The present specification encompasses the contents described in the specification and/or drawings of Japanese Patent Application No. 2010-209932 on which the priority of the present application is based.

Advantageous Effects of Invention

According to the lung cancer differential marker of the present invention, the presence or absence of lung cancer can be determined conveniently and highly reliably by the testing of a body fluid, a cell, or a lung lavage. According to the lung cancer differential marker of the present invention, the histological type of lung cancer can be further determined.

The method of the present invention can determine the presence or absence of lung cancer and the histological type of lung cancer using a body fluid, a cell, or a lung lavage with a more highly proper differential rate and lower invasiveness than existing tumor markers.

According to the glycan probe for differential diagnosis of lung cancer of the present invention, the presence or absence of lung cancer and further, its histological type can be determined by histological staining.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a Western blot image showing the expression of small cell lung cancer differential marker glycoproteins in the culture supernatants of cultured small cell lung cancer cells (Sc) and cultured lung adenocarcinoma cells (Ad).

FIG. 2 is a Western blot image showing the expression of lung adenocarcinoma differential marker glycoproteins in the culture supernatants of cultured small cell lung cancer cells (Sc) and cultured lung adenocarcinoma cells (Ad).

FIG. 3 is a Western blot image in which lung cancer differential marker glycoproteins in the culture supernatants of cultured small cell lung cancer cells (Sc) and cultured lung adenocarcinoma cells (Ad) fractionated with each lectin were detected with their respective anti-lung cancer differential marker protein antibodies.

FIG. 4 is a blot image in which a lung cancer differential marker glycoprotein (fibronectin 1) in the culture supernatants of cultured small cell lung cancer cells (Sc) and cultured lung adenocarcinoma cells (Ad) fractionated with an antibody (anti-fibronectin 1 antibody) was detected with AAL lectin.

FIG. 5 is a Western blot image in which lung cancer differential marker glycoproteins (NCAM and fibronectin 1) in the sera of small cell lung cancer patients (Sc) and lung adenocarcinoma patients (Ad) fractionated with AAL lectin were detected with their respective anti-lung cancer differential marker glycoprotein antibodies

FIG. 6 is a Western blot image in which a lung cancer differential marker glycoprotein (secretogranin III) in the sera of small cell lung cancer patients (Sc) and lung adenocarcinoma patients (Ad) fractionated by the serial chromatography of the sera was detected with an antibody (anti-secretogranin III antibody).

FIG. 7 is an image of histological staining of each histological type of lung cancer with an anti-neuronal pentraxin receptor (NPR) antibody.

FIG. 8 is a Western blot image in which a lung cancer differential marker glycoprotein (secretogranin III) in the sera of small cell lung cancer patients (Sc) and lung adenocarcinoma patients (Ad) fractionated by the multisample serial chromatography of the sera was detected with an antibody (anti-secretogranin III antibody).

FIG. 9 is a diagram showing the comparison of fluorescence signal intensities on PWM lectin spots, from a lung cancer differential marker glycoprotein (insulin-like growth factor-binding protein-L1), in an antibody-overlay lectin array using eluates purified using an antibody (anti-insulin-like growth factor-binding protein-L1 antibody) from the culture supernatants of cultured small cell lung cancer cells (Sc) and cultured lung adenocarcinoma cells (Ad).

FIG. 10 is a diagram showing the comparison of fluorescence signal intensities on PNA lectin spots, from a lung cancer differential marker glycoprotein (fibronectin 1), in an antibody-overlay lectin array using eluates purified using an antibody (anti-fibronectin 1 antibody) from the culture supernatants of cultured small cell lung cancer cells (Sc) and cultured lung adenocarcinoma cells (Ad).

DESCRIPTION OF EMBODIMENTS

1. Lung Cancer Differential Marker Glycoprotein and Fragment Thereof

The first embodiment of the present invention provides lung cancer differential marker glycoproteins shown in Table 1 or 2 above and fragment thereof.

1-1. Lung Cancer Differential Marker Glycoprotein

The "lung cancer differential marker glycoprotein" of this embodiment corresponds to a protein represented by each of Protein #1 to #31 in Table 1 and Protein #1 to #48 in Table 2. All of these proteins are lung cancer-specific glycoproteins comprising, in their amino acid sequences, a glycosylated asparagine residue at least at a position (counted from the initiating amino acid residue as the first position) represented by "Glycosylation site" in each table. In the case of, for example, acid alpha-glucosidase represented by Protein #1 in Table 1, glycans are linked to asparagine residues at least at positions 390, 470, and 882 in the amino acid sequence of this protein. Hereinafter, in the present specification, such a glycosylated protein is referred to as a "glycoprotein".

In each table, "gi(ID)" represents the ID number of each glycoprotein of this embodiment. A plurality of gi(ID) numbers registered for one protein are all described in the table. Also, a plurality of isoforms of one protein are indicated by isoform numbers together with their gi(ID) numbers in the table.

The glycan linked to the asparagine residue is not particularly limited as long as the glycan is linked in a lung cancer-specific manner. Examples thereof include fucosylated glycans, high mannose-type glycans, hybrid-type glycans, biantennary complex-type glycans, chitin, polylactosamine, and .beta.1,3-galactose epitopes. In this context, the "glycan linked in a lung cancer-specific manner" refers to a glycan linked to the asparagine residue represented by "Glycosylation site" in the table, only in the lung cancer cell-derived protein. Hence, as a rule, the lung cancer differential marker glycoprotein of this embodiment is a glycoprotein produced from a lung cancer cell. Thus, the detection of presence or absence of the glycoprotein, for example, in the serum of a test subject, using a glycan probe recognizing this glycoprotein can determine an individual having the glycoprotein as suffering lung cancer.

In the present specification, the "glycan probe" refers to a determinant that specifically recognizes a particular glycan and/or glycoconjugate such as a glycoprotein and binds thereto. Examples thereof include lectins, antibodies, and phage antibodies.

As described above, the histological types of "lung cancer" are known to consist of: small cell cancer; and non-small cell cancer further including adenocarcinoma, squamous cell carcinoma, and large cell cancer. Neuroendocrine cancer in the lung is also known. Most of the neuroendocrine cancer types are classified into small cell cancer, whereas the other histological types of this cancer are also known. The lung cancer differential marker glycoprotein of this embodiment can determine the histological type of lung cancer, depending on the type of the glycan linked to the prescribed asparagine residue. In the case of, for example, acid alpha-glucosidase represented by Protein #1 in Table 1, fucosylated glycans are linked to the prescribed asparagine residues only in a small cell lung cancer-derived glycoprotein. Thus, use of a lectin or an antibody that can bind to and recognize any of the fucosylated glycans can diagnose lung cancer with acid alpha-glucosidase as a lung cancer differential marker glycoprotein. In this case, the acid alpha-glucosidase can also serve as a marker that confirms the lung cancer as small cell cancer. Alternatively, in the case of biotimidase represented by Protein #2 in Table 1, fucosylated glycans are linked to the prescribed asparagine residues only in a lung adenocarcinoma-derived glycoprotein. Thus, use of a lectin or an antibody that can bind to and recognize any of the fucosylated glycans can diagnose lung cancer with biotimidase as a lung cancer differential marker glycoprotein. In this case, the biotimidase can also serve as a marker that confirms the lung cancer as adenocarcinoma.

The fucosylated glycan can be detected using AAL lectin. The high mannose-type glycan, the hybrid-type glycan, and the biantennary complex-type glycan can be detected using ConA lectin. The chitin and the polylactosamine can be detected using PWM lectin. The .beta.1,3-galactose epitope can be detected using PNA lectin. In Table 1, the presence and absence of the binding between each lung cancer differential marker glycoprotein and AAL lectin or ConA lectin are indicated by ".largecircle." and "x", respectively. Taking acid alpha-glucosidase represented by Protein #1 as an example, only a small cell lung cancer-derived glycoprotein has fucosylated glycans at the prescribed asparagine residues and is thus indicated by ".largecircle." in "AAL" of "Small cell cancer" and by "x" in the other boxes. The same holds true for the other lung cancer differential marker glycoproteins in Tables 1 and 2. Accordingly, each lung cancer differential marker glycoprotein (including a fragment of the lung cancer differential marker glycoprotein) shown in Tables 1 and 2 can serve as a marker for differentiation of small cell lung cancer or lung adenocarcinoma based on the manner of its binding to AAL lectin and ConA lectin for "small cell cancer" and "adenocarcinoma" in Tables 1 and 2.

1-2. Fragment of Lung Cancer Differential Marker Glycoprotein

The "lung cancer differential marker glycoprotein fragment" or the "fragment of the lung cancer differential marker glycoprotein" of this embodiment refers to an oligopeptide or polypeptide fragment consisting of a portion of the lung cancer differential marker glycoprotein. This fragment comprises, in its amino acid sequence, at least one asparagine residue at the glycosylation site shown in Table 1 or 2, wherein the lung cancer-specific glycan described in the paragraph "1-1. Lung cancer differential marker glycoprotein" is linked to this asparagine residue.

The amino acid length of the lung cancer differential marker glycoprotein fragment is not particularly limited and is preferably 5 to 100 amino acids, 8 to 80 amino acids, or 8 to 50 amino acids. Hereinafter, in the present specification, the lung cancer differential marker glycoprotein fragment is also referred to as a "glycoprotein fragment" or a "glycopeptide". Hereinafter, the lung cancer differential marker glycoprotein and its glycoprotein fragment are also collectively referred to as a "lung cancer differential marker".

Specific examples of the lung cancer differential marker glycoprotein fragment include glycopeptides consisting of amino acid sequences represented by SEQ ID NOs: 1 to 223 shown in Tables 1 and 2. These are glycoprotein fragments that were obtained by an IGOT method (described later) in identifying the lung cancer differential marker glycoproteins of this embodiment. Any of these glycoprotein fragments can be used as a lung cancer differential marker, as in the lung cancer differential marker glycoproteins, to determine the presence or absence of lung cancer and determine the histological type of the detected lung cancer, depending on the type of glycan linked to the prescribed asparagine residue. In each amino acid sequence shown in Tables 1 and 2, the underlined asparagine residue (N) represents a glycan-linked asparagine residue.

1-3. Obtainment of Lung Cancer Diagnosis Marker Glycoprotein

Methods by which the lung cancer differential marker glycoprotein fragments and the lung cancer differential marker glycoproteins of this embodiment were obtained and identified will be described below.

1-3-1. Large-Scale Identification of Lung Cancer-Specific Candidate Glycoprotein

The large-scale selective collection and concentration of the lung cancer-specific glycoproteins can adopt any of methods broadly classified into, for example, a method using probes having affinity for glycans, a method using chemical reaction with glycans (Zhang H. et al., Nat Biotechnol 21, 660-666 (2003)), and a method of introducing affinity tags to glycans. Here, the method using probes, which were used in the obtainment of the lung cancer differential marker glycoproteins in Tables 1 and 2, will be described.

First, lectins or anti-glycan antibodies reactive with glycans characteristically produced by cancer cells are selected as probes.

The probe lectins can be selected by the statistical analysis of glycan profiles using a lectin microarray. Alternatively, the probe lectins may be selected in consideration of literature information (some probe lectins to be used can be expected on the basis of, for example, increased fucosylation associated with malignant transformation) or the resulting determination performance of the histological type. Basically, lectins are selected by the statistical analysis of profile, and the selected lectins are validated on the basis of binding specificity. In the case of targeting lung cancer, for example, Aleuria aurantia-derived AAL lectin capable of detecting fucosylation, Canavalia ensiformis-derived ConA lectin capable of detecting high mannose-type, hybrid-type, or biantennary complex-type glycans, Phytolacca americana-derived PWM lectin capable of detecting chitin or polylactosamine, and/or Arachis hypogaea-derived PNA lectin capable of detecting .beta.1,3-galactose epitopes can be used.

The antibody probes or phage antibody probes may be prepared after structural determination of antigens (glycans). This is not a necessary condition, and these probes can also be prepared against the unknown structures of the antigenic glycans or glycopeptides.

For the large-scale identification of candidate glycoproteins, specifically, candidate glycoproteins are first collected from the culture supernatant of a lung cancer-derived cell line in a medium using the glycan probes (probe lectins and/or antibody probes or phage antibody probes). Use of the lung cancer-derived cell culture supernatant enables cancer cell-derived glycoproteins to be identified and can thus facilitate the detection of difference from glycans present on the serum-derived glycoproteins of healthy persons. This information can be useful in selectively obtaining candidate molecules from serum. The cells used may be derived from any histological type of lung cancer, i.e., small cell cancer or non-small cell cancer (adenocarcinoma, squamous cell carcinoma, or large cell cancer). Candidate glycoproteins may be collected from each histological type, and the results can be analyzed by comparison to obtain histological type-nonspecific lung cancer differential markers or histological type-specific lung cancer differential markers.

1-3-2. Identification of Glycopeptide by IGOT Method

The collected lung cancer differential marker candidate glycoproteins are then treated by a method based on an isotope-coded glycosylation site-specific tagging (IGOT) method and mass spectrometry to identify core glycopeptides in the candidate glycoproteins. The glycopeptides can be analyzed by a Lec-IGOT-LC/MS method described in, for example, JP Patent Publication (Kokai) No. 2004-233303 A (2004) (JP Patent No. 4220257) and Kaji H, et al., Mass spectrometric identification of N-linked glycopeptides using lectin-mediated affinity capture and glycosylation site-specific stable isotope tagging. Nature Protocols 1, 3019-3027 (2006). An example of the specific method will be described below.

(1) Glycan Cleavage and IGOT Method

The candidate glycoprotein groups collected with the probe lectins or the probe antibodies are digested with protease. From the obtained peptide groups, candidate glycopeptide groups are sampled and re-collected using the same probes as above. Alternatively, crude sample protein mixtures may be digested with protease without separating candidate glycoprotein groups from the crude sample protein mixtures, and candidate glycopeptide groups can be collected directly from the obtained crude peptide groups using the probe lectins or the probe antibodies. Subsequently, the obtained candidate glycopeptides are labeled by the IGOT method. The IGOT method involves dissociating glycans from the glycopeptides by treatment with enzymes such as glycopeptidase in oxygen isotope-labeled water, while causing the conversion of glycosylated asparagine to aspartic acid, during which the oxygen isotope (.sup.18O) in the water is incorporated into the candidate glycopeptides.

(2) LC/MS Shotgun Analysis of Labeled Peptide

The candidate glycopeptides labeled by the IGOT method are separated by liquid column chromatography (LC) and applied to mass spectrometry (MS). Their amino acid sequences are exhaustively identified by tandem mass spectrometry.

(3) Identification of Candidate Glycopeptide

According to, for example, an MS/MS ion search method described in the Standard Technology Collection (edited by the Japan Patent Office), Mass spectrometry, 3-6-2-2 Amino acid sequence analysis, a database can be searched by comparing the respective MS/MS peptide measurement results of the obtained candidate glycopeptides with the hypothetical MS/MS spectra of all peptides predicted according to the specificity of the protease actually used from the amino acid sequences of all proteins registered in the database. In this search, the following amino acid modifications are taken into consideration: oxidation of methionine residue side chain, pyroglutamic acid conversion (deamination or cyclization) of ammo-terminal glutamine, deamination of an amino terminus (carbamidomethylation of cysteine), deamidation of asparagine residue side chain (provided that the stable isotope oxygen is already incorporated therein). The candidate glycoproteins including the candidate glycopeptide are identified using search software Mascot.

(4) Identification of Glycosylation Site

Of the candidate glycopeptides identified by the MS/MS ion search method, each candidate glycopeptide that has an asparagine residue with deamidated (stable isotope-incorporated) side chain and contains an N-linked glycosylation consensus sequence (Asn-Xaa-[Ser/Thr], provided that Xaa is not Pro) is regarded as the glycoprotein fragment of this embodiment. Also, the asparagine residue in the consensus sequence of the obtained glycoprotein fragment is defined as the glycosylation site. When Xaa is Lys/Arg and the identified peptide sequence is cleaved at this site, the candidate glycopeptide in which a residue subsequent to Xaa is [Ser/Thr] with reference to the amino acid sequence of the whole protein thereof is also included in the glycoprotein fragment. As a rule, the glycosylation site is contained in the consensus sequence, although some reports suggest different glycosylation sites. In this specification, a glycosylation site cleaved with peptide-N-glycanase (glycopeptidase F, PNGase) can be identified.

The lung cancer differential marker glycoprotein fragment groups selected by the method described above and the lung cancer differential marker glycoproteins (comprising these fragments) identified on the basis of their amino acid sequences are defined as the glycoprotein fragment groups and the glycoproteins shown in Tables 1 and 2.

The glycoprotein fragment groups and the glycoproteins can serve as lung cancer differential markers for diagnosing lung cancer in a test subject, for example, by testing the presence or absence thereof in the serum of the test subject using lectins or antibodies recognizing them.

1-3-3. Validation of Lung Cancer Differential Marker

The significance of the lung cancer differential markers selected in the paragraph 1-3-2 can be further validated. For example, from among the glycoproteins and/or the glycoprotein fragments collected with the probe lectins from the lung cancer cell culture supernatant, significant lung cancer differential markers can be detected and confirmed by Western blot or immunoprecipitation using antibodies specifically recognizing the lung cancer differential markers separated and identified above. In the present specification, 31 lung cancer differential marker glycoproteins shown in Table 1 and fragments of the glycoproteins correspond to the lung cancer differential markers further selected by the detection with the antibodies.

Also, the lung cancer differential markers further selected by the detection with the antibodies may be further validated. For example, lung cancer differential markers are collected using the probe lectins from the sera of lung cancer patients, and significant markers can be further detected using the specific antibodies. As an example, neural cell adhesion molecule (NCAM 1) (represented by Protein #19 in Table 1) and fibronectin 1 (represented by Protein #8 in Table 1) shown in FIG. 5, and secretogranin III (represented by Protein #23 in Table 1) shown in FIGS. 6 and 8 correspond to the lung cancer differential marker glycoproteins confirmed to be useful by this further validation. In this context, the neural cell adhesion molecule binds to AAL lectin only in small cell lung cancer as shown in Table 1 and can thus serve as a lung cancer differential marker that allows differential diagnosis of small cell lung cancer. Likewise, the secretogranin III binds to AAL lectin and ConA lectin only in small cell lung cancer as shown in Table 1 and can thus serve as a lung cancer differential marker that allows differential diagnosis of small cell lung cancer. By contrast, the fibronectin 1 binds to AAL lectin only in lung adenocarcinoma as shown in Table 1 and can thus serve as a lung cancer differential marker that allows differential diagnosis of lung adenocarcinoma. Alternatively, for example, lung cancer differential markers may be collected from the culture supernatant of a lung cancer cell line using specific antibodies, and useful markers can be detected using a lectin array. As an example, insulin-like growth factor-binding protein-L1 (IGFBP-L1) (represented by Protein #12 in Table 1) shown in FIG. 9 and fibronectin 1 (represented by Protein #8 in Table 1) shown in FIG. 10 correspond to the lung cancer differential marker glycoproteins confirmed to be useful by the further validation. In this context, the insulin-like growth factor-binding protein-L1 (IGFBP-L1) produces a strong detectable fluorescence signal on a PWM lectin spot in small cell lung cancer as shown in FIG. 9 and can thus serve as a lung cancer differential marker that allows differential diagnosis of small cell lung cancer. By contrast, the fibronectin 1 produces a detectable fluorescence signal on a PNA lectin spot only in lung adenocarcinoma as shown in FIG. 10 and can thus serve as a lung cancer differential marker that allows differential diagnosis of lung adenocarcinoma.

1-4. Detection of Lung Cancer Differential Marker Glycopeptide and Glycoprotein

1-4-1. Mass Spectrometry

The lung cancer differential marker glycopeptides and glycoproteins can be detected with a mass spectrometer as a detector from samples collected with the probe lectins or the like binding to glycans.

The collected lung cancer differential marker glycopeptides can be detected, preferably, by the cleavage of their glycans followed by liquid chromatography (LC) to separate and elute peptides, which are then introduced in order directly to a mass spectrometer (MS) on line. The mass spectrometry is not only available for obtaining mass spectra, but also available for obtaining MS/MS spectra using a fragmentation method such as collision-induced dissociation (CID) and further detect a plurality of fragment ions generated by CID or the like only when pre-selected ions are detected (this approach is also called single reaction monitoring or multiple reaction monitoring). Furthermore, analyte peptides differing in mass by the incorporation of stable isotopes into some of synthesized core peptide moieties of lung cancer differential marker glycopeptides may be added to analysis samples, and their respective signal intensities can be compared to thereby achieve relative or absolute quantitative analysis. More simply, the signal intensity of the detected ion may be compared between two or more samples or with that of a reference sample to achieve simplified quantification.

The lung cancer differential marker glycoproteins can be detected using various proteomics approaches known in the art. For example, the collected proteins can be separated by one-dimensional or two-dimensional gel electrophoresis and quantified by the comparison of signal intensities (dye, fluorescence, etc.) between the target spot and a reference sample. In the case of a detection method using a mass spectrometer, the collected protein groups are digested with protease, and the formed peptides can be detected by LC/MS analysis. For such quantification, various methods based on stable isotope labeling (ICAT, MCAT, iTRAQ, SILAC, etc.) can be used in combination with a non-labeled simple quantification method (peptide counting method, area integration method, etc.). As further described later, the glycoproteins may be quantified by ELISA.

1-4-2. Lectin Microarray

(1) Glycan Profiling Using Lectin Microarray

(a) Lectin Microarray (Also Simply Referred to as a Lectin Array)

The lectin array refers to a substrate on which plural types of lectins differing in specificity are immobilized in parallel (i.e., in the form of array) as glycan probes. The lectin array allows concurrent analysis on the types of lectins interacted with analyte glycoconjugates and the degrees of these interactions. By use of this lectin array, information required for estimating glycan structures can be obtained by single analysis, while the steps from sample preparation to scanning can be carried out quickly and conveniently. In a glycan profiling system such as mass spectrometry, glycoproteins cannot be analyzed directly and must thus be decomposed into glycopeptides or free glycans in advance. In the lectin microarray, advantageously, glycoproteins can be analyzed directly by merely introducing, for example, fluorescent materials directly into the core protein moieties thereof. The lectin microarray technique has been developed by the present inventors, and the fundamental principles of this technique are described in, for example, Kuno A., et al. Nat. Methods 2, 851-856 (2005).

Typical examples of the lectins used in the lectin array include those described in the following Table 3:

[Table 3]

#1 Sugar Binding Specificity

For example, a lectin array comprising 45 types of lectins (including the 43 types of lectins described above) immobilized on a substrate is already commercially available under the product name of LecChip from GP Biosciences Ltd.

(2) Statistical Analysis of Glycan Profiles Using Lectin Array

The lectin array has already evolved into a practical technique by which a quantitative comparative glycan profiling can be realized not only for purified preparations but also for mixed samples such as serum or cell lysates. Particularly, the comparative glycan profiling of cell surface glycans has achieved remarkable development (Ebe, Y et al. J. Biochem. 139, 323-327 (2006); Pilobello, K. T. et al. Proc Natl Acad Sci USA. 104, 11534-11539 (2007); and Tateno, H. et al. Glycobiology 17, 1138-1146 (2007)).

Also, data mining by the statistical analysis of glycan profiles can be carried out by a method shown in, for example, "Kuno A, et al. J Proteomics Bioinform. 1. 68-72 (2008)" or "the Japanese Society of Carbohydrate Research 2008/8/18, Development of Application Technique for Lectin Microarray--Comparative Glycan Profiling and Statistical Analysis of Biological Sample-, Atsushi Kuno, Atsushi Matsuda, Yoko Itakura, Hideki Matsuzaki, Hisashi Narimatsu, Jun Hirabayashi" and "Matsuda A, et al. Biochem Biophys Res Commun. 370, 259-263 (2008)".

(3) Antibody-Overlay Lectin Microarray Method

The platform of the lectin microarray in this method is basically the same as above. For detection, the sample of the test subject is not directly labeled with a fluorescent material or the like but is indirectly labeled by the introduction of a fluorescent group or the like into the sample of the test subject via an antibody. This application method can realize concurrent multisample analysis more conveniently and quickly (see "Kuno A, Kato Y, Matsuda A, Kaneko M K, Ito H, Amano K, Chiba Y, Narimatsu H, Hirabayashi, J. Mol. Cell Proteomics. 8, 99-108 (2009)", "Jun Hirabayashi, Atsushi Kuno, Noboru Uchiyama, "Development of Application Technology for Glycan Profiling Using Lectin Microarray", Experimental Medicine, extra number "Study on Cancer Diagnosis at Molecular Level--Challenge to Clinical Application", Yodosha Co., Ltd., Vol. 25 (17) 164-171 (2007)", Atsushi Kuno, Jun Hirabayashi, "Application of Glycan Profiling System Based On Lectin Microarray to Glycan Biomarker Search", and Genetic Medicine MOOK No. 11 "Development of Clinical Glycan Biomarker and Elucidation of Glycan Function", pp. 34-39, Medical Do, Inc. (2008)).

For example, glycan moieties in glycoprotein samples obtained from a test subject are recognized by lectins on the lectin microarray. Thus, antibodies against the core protein moieties thereof can be overlaid on the glycoproteins to thereby specifically and highly sensitively detect the glycoproteins without labeling the test glycoproteins or highly purifying them.

(4) Lectin-Overlay Antibody Microarray Method

This method employs, instead of the lectin microarray, an antibody microarray in which antibodies against core proteins are immobilized in parallel (i.e., in the form of array) on a substrate such as a glass substrate. This method requires the same numbers of antibodies as the number of markers to be examined and also requires determining lectins in advance for detecting the alteration of glycans.

1-4-3 Lectin-Antibody Sandwich Immunological Detection

A simple and inexpensive sandwich detection method can be designed on the basis of the results obtained using the lectin array. Basically, this method can adopt the protocol of the sandwich detection method using two types of antibodies except that one of the antibodies is replaced by lectins. Thus, this approach is also applicable to automatization using an existing automatic immunodetection apparatus. The point to be noted is only the reaction between antibodies and lectins to be used for sandwiching antigens. Each antibody has at least two N-linked glycans. When the lectins used recognize glycans on the antibodies, background noise inevitably occurs during sandwich detection due to the binding reaction therebetween. A possible approach for preventing the generation of this noise signal involves modifying the glycan moieties on the antibodies or using only Fab fragments, which contain no such glycan moieties. These approaches have already been known in the art. For the method of modifying the glycan moieties, for example, Chen S. et al., Nat. Methods. 4, 437-44 (2007) and Comunale M A, et al., J Proteome Res. 8, 595-602 (2009) can be referred to. For the method using Fab fragments, for example, Matsumoto H., et al., Clin Chem Lab Med 48, 505-512 (2010) can be referred to.

1-4-4. Method Using Serial Chromatography

The antibody-overlay lectin array is the best approach for statistically finding lectins that most precisely reflect the disease-specific alteration of glycans on the lung cancer differential marker glycoproteins. This approach, however, inevitably requires antibodies that permit immunoprecipitation and overlay detection. Nevertheless, such antibodies are not always available. In this regard, the immunological quantitative detection of target glycoproteins from among glycoproteins collected with probe lectins is generally carried out as means for using a larger number of candidate molecules in the detection of lung cancer. Specifically, SDS-PAGE is performed, and target glycoproteins are immunologically detected by Western blot after membrane transfer. The signal intensities of the obtained bands can be compared to thereby quantitatively estimate the change between samples. The significance of each marker candidate can be validated on the basis of change in the amount of the protein having the cancer-specific alteration of a glycan, to thereby narrow down the candidate glycoproteins. In this embodiment, generally, the AAL lectin used in the step of identifying candidate molecules is also used as a probe protein in this validation. Examples of practice under such a strategy include the report of Liu Y et al., J Proteome Res. 9, 798-805 (2010). Serum proteins are known to differ in the structures (the degree of branching, etc.) or fucosylation (core fucose, blood group antigen, etc.) of their N-linked glycans, depending on the types thereof. Reportedly, even the same molecules may therefore be differently fucosylated. For example, Nakagawa T. et al., J. Biol. Chem. 281, 29797-29806 (2006) discloses that al antitrypsin molecules are differently fucosylated. Such proteins may or may not be increased at different times depending on the type of disease and the stage of its progression. Thus, the approach is not ideal, in which all fucose-containing glycoproteins are collected using AAL probes, which can recognize almost all fucosylation patterns and collect the glycoproteins, and the collected glycoproteins are quantitatively compared. Hence, we have conceived that proteins are separated and fractionated by serial chromatography using two different fucose-recognizing lectins and these fractions are quantitatively compared and analyzed. The lectins used in this approach are LCA and AAL. Previous lectin specificity analysis has revealed that the LCA lectin recognizes core-fucosylated glycans with fewer branches, among N-linked glycans. The AAL lectin is known to be capable of recognizing every core fucose with any number of branches in N-linked glycans as well as even fucosylation at the non-reducing end typified by ABO, Lewis antigens, or the like. This means that LCA has high specificity while AAL has low specificity. Thus, in the first step, fucose-containing glycoproteins binding to LCA are captured by LCA column chromatography. These glycoproteins are regarded as LCA-bound fucose-containing glycoproteins. In this chromatography, fucose-containing glycoproteins having no core-fucosylated N-linked glycan with fewer branches are fractionated into unbound fractions without binding to the LCA column. In order to capture such fucose-containing glycoprotein groups from the LCA-unbound fractions, the LCA-unbound fractions are subjected to AAL column chromatography. The glycoprotein groups captured by AAL in this step are regarded as LCA-unbound/AAL-bound fucose-containing glycoproteins. These procedures can presumably evaluate increase or decrease in fucosylation of the same protein associated with the disease, on the basis of the type of modification.

2. Method for Determining Lung Cancer

The second embodiment of the present invention provides a method for determining lung cancer. The method of this embodiment comprises detecting the lung cancer differential marker of embodiment 1 from a sample obtained from a test subject, wherein the detection of the glycoprotein or fragment determines that the test subject suffers lung cancer.

2-1. Definition and Summary

In the present specification, the "test subject" refers to a person to be subjected to a test, i.e., a human donor of a sample described later. The test subject may be any of patients having a certain disease or healthy persons. The test subject is preferably a person possibly having lung cancer or a lung cancer patient.

The "sample" refers to a part that is obtained from the test subject and subjected to the differential method of this embodiment. For example, a body fluid, a cell from cancer tissue or the like, or a lung lavage obtained during operation applies to the sample.

The "body fluid" refers to a biological sample in a liquid state obtained from the test subject. Examples thereof include blood (including serum, plasma, and interstitial fluid), lymph, extracts of each tissue or cell, pleural effusion, sputum, spinal fluid, lacrimal fluid, nasal discharge, saliva, urine, vaginal fluid, and seminal fluid. The body fluid is preferably blood, lymph, pleural effusion, or sputum. The body fluid may be used, if necessary, after treatment such as the dilution or concentration of the sample obtained from the test subject or the addition of an anticoagulant such as heparin thereto. Alternatively, the body fluid may be used directly without such pretreatment. The body fluid can be obtained by a method known in the art. For example, blood or lymph can be obtained according to a blood collection method known in the art. Specifically, peripheral blood can be obtained from the vein or the like of a peripheral site by injection. The body fluid may be used immediately after obtainment. Alternatively, the body fluid may be cryopreserved or refrigerated for a given period and then treated, if necessary, by thawing or the like before use. In this embodiment, in the case of using serum, sufficient amounts of lung cancer differential markers can be detected using a volume of 10 .mu.L, to 100 .mu.L, 20 .mu.L, to 80 .mu.L, to 70 .mu.L to 60 .mu.L, or 45 .mu.L to 55 .mu.L.

In the differential method of this embodiment, the lung cancer diagnosis marker used in detection can be any lung cancer diagnosis marker as long as the marker is a lung cancer marker glycoprotein listed in Table 1 or 2 being glycosylated with a glycan at the asparagine residue(s) at the glycosylation site(s) shown in Table 1 or 2, or a glycoprotein fragment thereof comprising at least one asparagine residue at the glycosylation site shown in Table 1 or 2 being glycosylated with a glycan. These lung cancer diagnosis markers may be used alone or in combination of two or more thereof in the diagnosis method of this embodiment. For example, two or more different lung cancer diagnosis marker glycoproteins may be used. Alternatively, two or more different fragments of the same lung cancer diagnosis marker glycoprotein may be used. Preferably, the lung cancer diagnosis marker(s) shown in Table 1 is used. More preferably, lung cancer diagnosis markers having the same or different diagnostic characteristics for small cell lung cancer and lung adenocarcinoma are used. Examples of the use method include a method using neural cell adhesion molecule (NCAM1), secretogranin III, and/or insulin-like growth factor-binding protein-L1 (IGFBP-L1) shown in Table 1 for differential diagnosis of small cell lung cancer, in combination with fibronectin 1 shown in Table 1 for differential diagnosis of lung adenocarcinoma as the lung cancer differential markers of this embodiment. This method is convenient because it can determine the presence or absence of lung cancer in the test subject while also determining the histological type (small cell cancer or adenocarcinoma) of the lung cancer. When the lung cancer differential marker shown in Table 1 or 2 is detected from the sample of the test subject by any method using, alone or in combination, lectins or antibodies binding to a fucosylated glycan, a high mannose-type glycan, a hybrid-type glycan, a biantennary complex-type glycan, chitin, polylactosamine, and/or a .beta.1,3-galactose epitope, this test subject can be confirmed to have lung cancer or to be highly likely to have lung cancer. For example, the fucosylated glycan can be detected using AAL lectin specifically binding thereto. The high mannose-type glycan, the hybrid-type glycan, and the biantennary complex-type glycan can be detected using ConA lectin specifically binding thereto. The chitin and the polylactosamine can be detected using PWM lectin specifically binding thereto. The .beta.1,3-galactose epitope can be detected using PNA lectin specifically binding thereto.

Alternatively, the histological type of lung cancer in the test subject may be confirmed as small cell cancer or adenocarcinoma on the basis of results of binding of the lectin to the glycan in the lung cancer differential marker in the sample obtained from the test subject, and the manner of binding of the lung cancer differential marker glycoprotein shown in Table 1 or 2 and/or the fragment thereof to the lectin. According to the manner of lectin binding of the glycoprotein shown in Table 1, for example, the glycans of acid alpha-glucosidase represented by Protein #1 bind to AAL lectin only in small cell lung cancer. Thus, when acid alpha-glucosidase bound to AAL lectin is detected from the sample obtained from a test subject, this test subject can be confirmed to have lung cancer whose histological type is small cell cancer. Alternatively, when neural cell adhesion molecule (NCAM1) as the lung cancer differential marker glycoprotein or its glycoprotein fragment bound with AAL lectin is detected, the histological type of lung cancer can be confirmed as small cell cancer. When fibronectin 1 as the lung cancer differential marker glycoprotein or its glycoprotein fragment bound with AAL lectin or an anti-fibronectin 1 (fragment) antibody is detected, the histological type of lung cancer can be confirmed as adenocarcinoma.

2-2. Method for Detecting Lung Cancer Differential Marker

Examples of the method for detecting the lung cancer differential marker can include the combination of two methods: a method involving using a lectin specifically binding to a glycan in each lung cancer differential marker (hereinafter, in the present specification, this lectin is referred to as lectin A for the sake of convenience) to select a lung cancer differential marker having the glycan; and a method involving detecting a moiety (core protein) other than the glycan of each lung cancer differential marker to thereby detect the lung cancer differential marker. Alternative examples of the method can include a method involving using an antibody to detect the lung cancer differential marker of interest, the antibody being specific for the lung cancer differential marker having a glycan specifically binding to lectin A and recognizing an epitope located in proximity to the glycosylation site.

In this context, the method of detecting a glycan specifically binding to lectin A and the method of detecting a core protein may be a method of assaying a glycan specifically binding to lectin A and a method of assaying a core protein, respectively. For example, the lung cancer differential marker can be detected using an antibody against the core protein and lectin A to thereby differentiate between lung cancer patients and healthy persons. Preferably, the antibody overlay method using a lectin array (Kuno A, Kato Y, Matsuda A, Kaneko M K, Ito H, Amano K, Chiba Y, Narimatsu H, Hirabayashi J. Mol. Cell Proteomics. 8, 99-108 (2009)) can be used. More simply, the lectin-antibody sandwich immunological detection method can be used for detection.

Examples of the specific method for determining lung cancer using the lung cancer differential marker having a glycan specifically binding to lectin A include, a method comprising the following steps:

(1) separating lung cancer differential markers using lectin A from serum obtained from a test subject to thereby select a protein group having the glycan specifically binding to lectin A; and

(2) subsequently screening the lung cancer differential markers using an anti-lung cancer differential marker glycoprotein antibody specifically recognizing the moiety other than the glycan specifically binding to lectin A, to thereby detect the lung cancer differential marker of interest having the glycan specifically binding to lectin A, wherein when this marker is detected, this test subject is determined to be afflicted with lung cancer.

The lung cancer differential marker having the glycan specifically reacting with lectin A can be selected using, for example, the method of assaying a glycan specifically binding to lectin A using, specifically, a lectin A-immobilized column or array, and lung cancer differential marker assaying means, specifically, an antibody against the novel lung cancer differential marker glycoprotein or the fragment thereof. Preferably, the lectin-antibody sandwich ELISA or the antibody-overlay lectin array method can be used.

Also, the concentration of the lung cancer differential marker having the glycan specifically reacting with lectin A can be measured. Examples of the measurement method include the antibody-overlay lectin array method using a lectin array, LC-MS, immunoassay, enzymatic activity assay, and capillary electrophoresis. Preferably, a qualitative or quantitative approach can be used, which includes: LC-MS; and enzyme immunoassay, two-antibody sandwich ELISA, gold colloid method, radioimmunoassay, latex agglutination immunoassay, fluorescent immunoassay, Western blot, immunohistochemical method, surface plasmon resonance spectroscopy (SPR method), and quartz crystal microbalance (QCM) method, using a monoclonal or polyclonal antibody specific for the novel lung cancer differential marker glycoprotein having the glycan specifically reacting with lectin A or the fragment thereof.

2-3. Preparation of Specific Polyclonal and/or Monoclonal Antibodies Using Lung Cancer Differential Marker Glycoprotein or its Glycopeptide

In the method for detecting lung cancer using the novel lung cancer differential marker glycoprotein or its fragment glycopeptide, polyclonal and/or monoclonal antibodies specific for the differential marker glycoprotein or its glycopeptide may be used. For example, an easily obtainable antibody, such as a commercially available antibody, specific for the glycoprotein or the like can be used. If such an antibody is not easily obtainable, it can be prepared, for example, by the following method:

For example, the anti-lung cancer differential marker glycopeptide polyclonal antibody can be prepared using a well-known method. Specifically, an adjuvant is added to an obtained antigenic lung cancer differential marker glycoprotein or glycopeptide. The antigen used may be synthesized as a lung cancer differential marker glycopeptide containing glycosylation site(s) (asparagine residue(s)). Examples of the adjuvant include complete Freund's adjuvant and incomplete Freund's adjuvant. These adjuvants may be used as a mixture. The antigen may be inoculated, together with the adjuvant, to an antibody-producing animal to thereby boost antibody production. Alternatively, this peptide may be covalently bonded to commercially available keyhole limpet hemocyanin (KLH) and inoculated to an antibody-producing animal. In this procedure, granulocyte-macrophage colony stimulating factor (GM-CSF) may also be administered to the animal simultaneously therewith to thereby boost antibody production. Examples of the antibody-producing animal that can be used in antigen inoculation include mammals, for example, mice, rats, horses, monkeys, rabbits, goats, and sheep. Immunization can employ any of existing methods and is performed mainly by intravenous injection, hypodermic injection, intraperitoneal injection, or the like. The interval between immunization doses is not particularly limited and is an interval of several days to several weeks, preferably 4 to 21 days.

2 to 3 days after the final immunization date, whole blood is obtained from the immunized animal. After serum separation, the polyclonal antibody can be prepared.

Alternatively, for example, the anti-lung cancer marker glycopeptide monoclonal antibody can be prepared by the method of Kohler and Milstein (Nature Vol. 256, pp. 495-497 (1975)). For example, antibody-producing cells obtained from the antigen-immunized animal are fused with myeloma cells to prepare hybridomas. From the obtained hybridomas, clones producing the anti-lung cancer differential marker glycopeptide monoclonal antibody can be selected to thereby prepare the monoclonal antibody.

Specifically, 2 to 3 days after the final immunization date in the preparation of the polyclonal antibody, antibody-producing cells are collected. Examples of the antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells.

Cell lines that are derived from various animals (e.g., mice, rats, and humans) and are generally obtainable by those skilled in the art are used as the myeloma cells to be fused with the antibody-producing cells. The cell line used is a drug resistance cell line that cannot survive in a selective medium (e.g., HAT medium) in an unfused state, but can characteristically survive therein only in a fused state. In general, 8-azaguanine-resistant line is used. This cell line is deficient in hypoxanthine-guanine-phosphoribosyl transferase and cannot grow in a hypoxanthine-aminopterin-thymidine (HAT) medium.

The myeloma cells have already been known in the art, and various cell lines can be used preferably, for example, P3(P3x63Ag8.653) (J. Immunol. 123, 1548-1550 1979)), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology 81, 1-7 (1978)), NS-1 (Kohler, G. and Milstein, C., Eur. J. Immunol. 6, 511-519 (1976)), MPC-11 (Margulies, D. H. et al., Cell 8, 405-415 (1976)), SP2/0 (Shulman, M. et al., Nature 276, 269-270 (1978)), FO (de St. Groth, S. F. et al., J. Immunol. Methods 35, 1-21 (1980)), 5194 (Trowbridge, I. S., J. Exp. Med. 148, 313-323 (1978)), and 8210 (Galfre, G. et al., Nature 277, 131-133 (1979)).

Next, the myeloma cells and the antibody-producing cells are fused with each other. This cell fusion is performed by the contact between the myeloma cells and the antibody-producing cells at a mixing ratio of 1:1 to 1:10 at 30 to 37.degree. C. for 1 to 15 minutes in the presence of a fusion promoter in a medium for animal cell culture such as MEM, DMEM, or RPMI-1640 medium. A fusion promoter or a fusion virus, such as polyethylene glycol or polyvinyl alcohol having an average molecular weight of 1,000 to 6,000 or Sendai virus can be used for promoting the cell fusion. Alternatively, the antibody-producing cells and the myeloma cells may be fused with each other using a commercially available cell fusion apparatus based on electrical stimulation (e.g., electroporation).

After the cell fusion, hybridomas of interest are selected from the fused cells. Examples of the method therefor include a method using selective growth of the cells in a selective medium. Specifically, the cell suspension is diluted with an appropriate medium and then seeded over a microtiter plate. A selective medium (e.g., HAT medium) is added to each well, and the cells are subsequently cultured with the selective medium appropriately replaced by a fresh one. As a result, the cells that have grown can be obtained as hybridomas.

The hybridoma screening is performed by, for example, a limiting dilution method or a fluorescence excitation method using a cell sorter. Finally, monoclonal antibody-producing hybridomas are obtained. Examples of the method for obtaining the monoclonal antibody from the obtained hybridomas include ordinary cell culture and ascitic fluid formation methods.

3. Lung Cancer Cell-Identifying Antibody for Histological Staining

The third embodiment of the present invention provides a lung cancer cell-identifying antibody for histological staining. The antibody of this embodiment is an antibody that can specifically recognize and bind to the lung cancer differential marker according to embodiment 1, i.e., at least one lung cancer differential marker glycoprotein shown in Table 1 or 2 being glycosylated with a glycan at the asparagine residue(s) at the glycosylation site(s) shown in Table 1 or 2, and/or at least one fragment thereof, the fragment comprising at least one asparagine residue at the glycosylation site shown in Table 1 or 2 being glycosylated with a glycan, and thereby specifically identifying a lung cancer cell in histological staining.

The epitope of the lung cancer differential marker according to embodiment 1 recognized by the antibody of this embodiment is not particularly limited and is preferably a core protein or a fragment of the core protein, more preferably a moiety encompassing both of a glycan and its neighboring peptide sequence. In this case, the length of the peptide sequence is 5 to 15 amino acids, 5 to 10 amino acids, or 5 to 8 amino acids. In addition to this antibody probe, a phage antibody probe prepared using a phage can also be used as a glycan probe for identification of a lung cancer cell.

The antibody of this embodiment is capable of determining the histological type of a lung cancer cell. Examples of such an antibody include an antibody (R&D Systems, Inc., Anti-human Neuronal Pentraxin Receptor Antibody) against neuronal pentraxin receptor (NPR) represented by Protein #21 in Table 1. This anti-neuronal pentraxin receptor antibody is capable of specifically identifying a small cell cancer-derived cell, as shown in FIG. 7, and as such, can serve as an antibody for identification of a small cell cancer cell. Thus, the proteins collected and identified with cancer-specific glycan probe lectins are usually expressed at an exceedingly low level at other tissues or normal sites and thus include tissue markers that can function by means of only the antibody or the phage antibody without the need of differentiating between small cell cancer-derived cells and lung adenocarcinoma-derived cells using lectins or the like.

EXAMPLES

Example 1

Selection of Glycopeptide Marker by Glycoproteomics (IGOT-LC/MS method)

1. Method for Preparing Culture Supernatant of Human Lung Cancer Cell

Three lung adenocarcinoma cell lines (H358, H1975, and LX-1) and three small cell lung cancer cell lines (H2171, H524, and H526) were separately cultured for 3 days using a high-glucose medium containing 90% DMEM and 10% FBS (PS+) in a dish having a diameter of 14 cm to achieve 80 to 90% confluence. The FBS-containing medium was discarded by aspiration, and the cells of each line were washed with 10 mL/dish of a serum-free medium (100% DMEM-high glucose, no additive). After addition of a serum-free medium at a concentration of 30 mL/dish, the cells were cultured for 48 hours. The cells thus cultured were centrifuged at 1000 rpm for 30 minutes to recover a supernatant. The supernatant (culture supernatant) was further recovered by centrifugation at 3000 rpm for 30 minutes and cryopreserved at -80.degree. C. The culture supernatant stored at this temperature was thawed before use, filtered through a 0.45-.mu.m filter, and then used in Examples below. NaN.sub.3 was added to each culture supernatant to adjust the final concentration to 0.1%.

2. Method for Identifying Glycoprotein at Large Scale

(1) Preparation of Peptide Sample

Trichloroacetic acid (TCA, 100% saturated aqueous solution) was added at a final concentration of 10% to each culture supernatant thus prepared. The mixture was cooled on ice for 10 to 60 minutes to precipitate proteins. The precipitates were recovered by centrifugation at a high speed at 4.degree. C. The precipitates were suspended in ice-cold acetone and washed twice to remove TCA. A lysis buffer solution (containing 0.5 M tris-HCl buffer solution, pH 8-8.5, 7 M guanidine hydrochloride, and 10 mM EDTA) was added to the precipitates to adjust the protein concentration to 5 to 10 mg/mL, while the proteins were lysed therein. In another method, each culture supernatant was applied to an ultrafiltration membrane having a molecular weight cutoff of 10,000 at 4.degree. C. to concentrate proteins, to which a lysis buffer solution was then added. The protein solution was filtered again to prepare a sample protein solution.

Extracts were recovered from each supernatant by centrifugation at a high speed. Nitrogen gas was passed through or sprayed to the extracts to remove dissolved oxygen. Then, dithiothreitol (DTT) in the form of a powder or dissolved in a small amount of a lysis buffer solution was added thereto in an amount equal to the protein weight. The mixture was reacted at room temperature for 1 to 2 hours with nitrogen gas passed therethrough or in a nitrogen gas atmosphere to reduce the disulfide bond. Subsequently, iodoacetamide for S-alkylation was added thereto in an amount of 2.5 times the protein weight. The mixture was reacted at room temperature for 1 to 2 hours in the dark. The reaction solution was dialyzed at 4.degree. C. (cooling room) against a 50- to 100-fold amount of a buffer solution (in general, 10 mM ammonium bicarbonate buffer solution, pH 8.6) as an external solution. The external solution was replaced by a fresh one three to five times at appropriate time intervals to remove the denaturant (guanidine hydrochloride) or an excess of the reagents. Although proteins were partially precipitated, this suspension was subjected directly to protein quantification. Trypsin (sequencing grade or higher) with a weight of 1/100 to 1/50 of the protein amount was added thereto to digest the proteins overnight (approximately 16 hours) at 37.degree. C. The progression of the digestion was confirmed by SDS-gel electrophoresis. When sufficient digestion was confirmed, the reaction was terminated by the addition of phenylmethanesulfonyl fluoride (PMSF) at a final concentration of 5 mM.

(2) Collection and Purification of Candidate Glycopeptide

The sample peptides prepared in the preceding step were applied to probe lectin (AAL lectin or ConA lectin)-immobilized columns. After washing, candidate glycopeptides were eluted by a method appropriate for the specificity of each lectin, i.e., using a buffer solution containing 5 mM fucose as to the AAL lectin and using a buffer solution containing 0.2 M methylmannoside as to the ConA lectin. To the obtained candidate glycopeptide solution, an equal volume of ethanol and a 4-fold volume of 1-butanol were added, and the mixture was applied to a Sepharose column equilibrated in advance with water:ethanol:1-butanol (1:1:4 (v/v)). The column was washed with this equilibrating solvent, and candidate glycopeptides were then eluted with 50% ethanol (v/v). Each candidate glycopeptide fraction was transferred in small portions to a microtube containing 2 .mu.l, of glycerol and concentrated by centrifugation under reduced pressure (i.e., water was removed by centrifugation under reduced pressure). This procedure was repeated to concentrate all candidate glycopeptide fractions.

(3) Glycan Cleavage and Isotope-Coded Glycosylation Site-Specific Tagging (IGOT) Method

A necessary amount of a buffer solution was added to the purified candidate glycopeptides (glycerol solution), and the mixture was concentrated again by centrifugation under reduced pressure. Then, stable oxygen isotope-18 (.sup.18O)-labeled water (H.sub.2.sup.18O) was added thereto to dissolve the concentrate (glycerol concentration: 10% or lower). Peptide-N-glycanase (glycopeptidase F, PNGase) prepared with labeled water was added thereto and reacted overnight at 37.degree. C. This reaction causes the conversion of the glycosylated asparagine to aspartic acid, during which the oxygen isotope (.sup.18O) in the water is incorporated into the candidate glycopeptide to label the candidate glycopeptide.

(4) LC/MS Shotgun Analysis of Labeled Peptide

The IGOT reaction solution was diluted with 0.1% formic acid and subjected to LC/MS shotgun analysis. In this analysis, a nano-LC system based on a direct nano-flow pump was used for high-separation, high-reproducibility, and high-sensitivity detection. The injected candidate glycopeptides were temporarily collected onto a trap column (reverse-phase C18 silica gel carrier) intended for desalting. After washing, the glycopeptides were separated by the concentration gradient of acetonitrile using frit-less spray tip nano-columns (inside diameter: 150 .mu.m.times.50 mL) packed with the same resins. The eluate was ionized via an electrospray interface and directly introduced into a mass spectrometer. The glycopeptides were analyzed by collision-induced dissociation (CID)-tandem mass spectrometry in a data-dependent mode in which two ions at the maximum to be analyzed were selected.

(5) Search for Candidate Glycopeptide by MS/MS Ion Search Method

Thousands of MS/MS spectra thus obtained were individually smoothed and converted to centroid spectra to prepare peak lists. On the basis of the peak lists, each candidate glycopeptide was identified by the MS/MS ion search method using a protein amino acid sequence database. The search engine used was Mascot (Matrix Science Ltd.). The following parameters were used for search conditions: a fragmentation method used: trypsin digestion, the maximum number of missed cleavage: 2, fixed modification: carbamidomethylation of cysteine, variable modifications: deamination of an N-terminal amino group (N-terminal glutamine), oxidation of methionine, .sup.18O-incorporating deamidation of asparagine (glycosylation site), error tolerance of MS spectrum: 500 ppm, and error tolerance of MS/MS spectrum: 0.5 Da.

(6) Identification of Candidate Glycopeptide

The database was searched under the conditions described above. The obtained identification results were validated according to criteria shown below. The obtained candidate glycopeptides were regarded as lung cancer differential marker glycopeptides (lung cancer differential marker glycoprotein fragments).

(i) The probability score (coincidence probability: Expectation value) of identification is 0.05 or less.

(ii) The number of fragment ions contributing to identification is 4 or more.

(iii) Error (ppm) is not significantly deviated from systematic error (mass error being 0.5 Da or less).

(iv) Each identified peptide has consensus sequence(s) with the number of Asn modifications (conversion to Asp and incorporation of .sup.18O) equal to or fewer than the number of the consensus sequence(s).

(7) Identification of Lung Cancer Differential Marker Glycoprotein

Glycoproteins containing the sequences of the selected lung cancer differential marker glycopeptides can be identified using these sequences. More specifically, the whole amino acid sequences of corresponding lung cancer differential marker glycoproteins were identified from the amino acid sequence database NCBI-Refseq on the basis of the "peptide sequences" of lung cancer differential marker glycopeptides represented by SEQ ID NOs: 1 to 223 in Tables 1 and 2.

(Results)

The lung cancer differential marker glycoproteins obtained by the steps described above are shown in Table 2.

3. Validation of Culture Supernatant-Derived Protein by Immunoblot

Each of the culture supernatants of lung cancer cell lines (three lung adenocarcinoma cell lines: H358, H1975, and LX-1, and three small cell lung cancer cell lines: H2171, H524, and H526) separately cultured for 24 hours in a serum-free medium was concentrated 10-fold through Amicon Ultra-154 Centrifugal Filter Units (cutoff: 10 kDa, Millipore Corp.). Each sample (10 .mu.L) was developed by SDS-PAGE on a 10%, 12.5%, or 17% acrylamide gel of XV PANTERA SYSTEM (Maruko Shokai Co., Ltd.) and then transferred to a PVDF membrane (GE Healthcare Japan Corp.) at 200 mA for 40 min. The blocking agent used was PBSTx (Dulbecco's PBS supplemented with 1% Triton X-100) containing 5% skimmed milk or 5% BSA dissolved therein. The membrane was blocked at room temperature for 1 hour. After 10-minute washing three times with PBSTx, the membrane was reacted for 1 hour with primary antibodies (Table 4) biotinylated in advance with Biotin Labeling Kit-NH.sub.2 (Dojindo Laboratories). After 10-minute washing three times with PBSTx again, the membrane was reacted for 1 hour with secondary antibody-HRP-conjugated streptavidin (1:3000 dilution, GE Healthcare Japan Corp.). After 10-minute washing three times with PBSTx, the enzymatic reaction of HRP was caused using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Inc.). The obtained signals were developed onto Amersham Hyperfilm ECL (GE Healthcare Japan Corp.). The amounts of proteins present in the culture supernatants derived from the lung adenocarcinoma and small cell lung cancer cell lines were analyzed by comparison.

TABLE-US-00003 TABLE 4 Antibody # Antibody name [Manufacturer: Catalog No./Distributer] 01 neural cell adhesion molecule 1 (NCAM-1/CD56) [LSP: MAB24081/Funakoshi Corp.] 02 neuronal pentraxin II (NPTX2) [LSP: LS-C53292-50/Funakoshi Corp.] 03 Thy-1 cell surface antigen [ABV: H00007070-M01/Cosmo Bio Co., Ltd.] 04 insulin-like growth factor 2 receptor (IGF-IIR) [RSD: AF2447/Cosmo Bio Co., Ltd.] 05 insulin-like growth factor 2 receptor (IGF-IIR) [RSD: MAB2447/Funakoshi Corp.] 06 acid alpha-glucosidase preproprotein (GAA) [LSP: LS-C80648-50/Cosmo Bio Co., Ltd.] 07 sparc/osteonectin, cwcv and kazal-like domains proteoglycan 2 (testican2/SPOCK2) [RSD: MAB2328/Funakoshi Corp.] 08 sparc/osteonectin, cwcv and kazal-like domains proteoglycan 2 (testican2/SPOCK2) [ABV: H00009806-B01P/Funakoshi Corp.] 09 cell adhesion molecule 4 (CADM4) [LSP: LS-C36881/Cosmo Bio Co., Ltd.] 10 secretogranin III (SgIII) [SCB: SC-1492/Cosmo Bio Co., Ltd.] 11 melanoma-associated antigen p97 (MFI2) [ABV: H00004241-B01P/Cosmo Bio Co., Ltd.] 12 cathepsin L2 preproprotein (CTSL2) [ABV: PAB8639/Cosmo Bio Co., Ltd.] 13 cathepsin L2 preproprotein (CTSL2) [DFK: F-106/Cosmo Bio Co., Ltd.] 14 lysosomal acid phosphatase 2 precursor (ACP2) [ABV: PAB7218/Cosmo Bio Co., Ltd.] 15 lysosomal acid phosphatase 2 precursor (ACP2) [ABV: H00000053-M01/Cosmo Bio Co., Ltd.] 16 cathepsin L1 (CTSL1) [ABV: PAB8638/Cosmo Bio Co., Ltd.] 17 cathepsin L1 (CTSL1) [ABV: MAB1432/Cosmo Bio Co., Ltd.] 18 deoxyribonuclease II, lysosomal precursor (DNASE2) [ABV: H00001777-B01P/Cosmo Bio Co., Ltd.] 19 integral membrane protein 1 (ITM1) [SCB: SC-100796/Funakoshi Corp.] 20 integral membrane protein 1 (ITM1) [ABV: H00003703-M02/Cosmo Bio Co., Ltd.] 21 neuronal pentraxin receptor (NPTXR/NPR) [RSD: AF4414/Funakoshi Corp.] 22 neuronal pentraxin receptor (NPTXR/NPR) [LSP: LS-C73727-100/Funakoshi Corp.] 23 neuronal pentraxin receptor (NPTXR/NPR) [SCB: SC-12483/Cosmo Bio Co., Ltd.] 24 sushi domain containing 2 (SUSD2) [ABV: H00056241-B01/Cosmo Bio Co., Ltd.] 25 sel-1 suppressor of lin-12 like (SEL1L) [LSP: LS-B2253-50/Funakoshi Corp.] 26 sel-1 suppressor of lin-12 like (SEL1L) [LSP: LS-C55443-100/Funakoshi Corp.] 27 sel-1 suppressor of lin-12 like (SEL1L) [ABV: PAB7473/Cosmo Bio Co., Ltd.] 28 source of immunodominant MHC-associated peptides (STT3B) [PG: 15323-1-AP/Cosmo Bio Co., Ltd.] 29 IGFBP-L1 [RSD: AF3877/Cosmo Bio Co., Ltd.] 30 neural cell adhesion molecule 1 (NCAM-1/CD56) [SCB: sc-71647/Cosmo Bio Co., Ltd.] 31 Fibronectin (H-300) [SCB: sc-9068/Cosmo Bio Co., Ltd.] 32 Galectin-3BP/MAC-2BP (MAC2BP) [RSD: AF2226/Cosmo Bio Co., Ltd.] 33 Cathepsin D [RSD: AF1014/Cosmo Bio Co., Ltd.] 34 Fibronectin (C-20) [SCB: sc-6952/Cosmo Bio Co., Ltd.] 35 neogenin homolog 1 (NGN) [SCB: SC-15337]/Cosmo Bio Co., Ltd.] 36 laminin alpha 5 [SCB: SC-20145/Cosmo Bio Co., Ltd.] 37 laminin, beta 1 precursor [SCB: sc-17763/Cosmo Bio Co., Ltd.] 38 phospholipid transfer protein isoform b precursor (PLTP) [RSD: AF5109/Cosmo Bio Co., Ltd.] 39 melanoma cell adhesion molecule (MCAM) [Millipore: MAB16985/] 40 L1 cell adhesion molecule isoform 2 precursor (L1CAM) [LSP: LS-C49042/Cosmo Bio Co., Ltd.] 41 biotinidase precursor (BTD) [ABV: H00000686-M01/Funakoshi Corp.] 42 ribonuclease T2 precursor (RNASET2) [ABV: H00008635-B01/Cosmo Bio Co., Ltd.] 43 tubulointerstitial nephritis antigen-like 1 (TINAGL1) [ABV: H00064129-B02P/Cosmo Bio Co., Ltd.] 44 v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog isoform 2 precursor (KIT) [LSP: LS-C40873-100/Cosmo Bio Co., Ltd.] 45 secretogranin III (SgIII) [SCB: SC-50289] 46 secretogranin III (SgIII) [SIGMA: HPA-006880] 47 IGFBP-L1 [RSD: BAF3877/Cosmo Bio Co., Ltd.]

(Results)

The lung cancer differential marker glycoproteins obtained by the steps described above are shown in Table 1 and FIGS. 1 and 2.

4. Batch Fractionation of Culture Supernatant Using AAL Lectin

The culture supernatants were fractionated using AAL lectin. Specifically, an AAL-conjugated resin was washed five times with a 3-fold amount of PBS and then prepared into a 50% slurry solution. To 30 .mu.L of the prepared AAL-conjugated resin, 30 .mu.L of each culture supernatant was added, and the resin was shaken at 4.degree. C. for 5 hours. After centrifugation (2,000 rpm, 2 min.), the supernatant was removed, and 50 .mu.L of a wash buffer (0.1% SDS in PBST, and 0.1% Triton X-100) was then added to the resulting resin. After centrifugation (2,000 rpm, 2 min.) two times, 500 .mu.L of a wash buffer was added thereto. The supernatant was removed by centrifugation (2,000 rpm, 2 min.), and the resin was washed. For elution from the washed resin, 15 .mu.L of 0.2 M fucose in PBS containing 0.02% SDS was added thereto, and the resin was shaken at 4.degree. C. for 5 hours. After centrifugation (2,000 rpm, 2 min.), the supernatant was recovered. 15 .mu.L of an elution buffer was further added thereto, and the resin was centrifuged (2,000 rpm, 2 min.) to elute AAL-bound fractions. 10 .mu.L each of the eluted fractions was developed by SDS-PAGE, and proteins were detected by Western blot. Similar procedures were performed using ConA lectin except that 0.5 M methylmannoside was used in elution.

(Results)

The lung cancer differential marker glycoproteins obtained by the steps described above are shown in FIG. 3.

5. Fractionation by Immunoprecipitation

Antibodies against all the candidate glycoproteins shown in Table 1 were biotinylated, if unbiotinylated, using Biotin Labeling Kit-NH.sub.2 (Dojindo Laboratories) according to the manual. 1 .mu.g of the biotinylated antibodies was added to 100 .mu.L of each culture supernatant and shaken at 1,400 rpm at 20.degree. C. for 2 hours. For washing of magnetic beads (Invitrogen Corp.), 100 .mu.L of TBSTx (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1% Triton X-100) was added to 20 .mu.L of the magnetic beads and stirred, followed by supernatant removal three times by centrifugation (10,000 rpm, 3 sec.). The reacted culture supernatant and antibodies were transferred to the magnetic beads and shaken at 1,400 rpm at 20.degree. C. for 1 hour. The antibodies bound with the magnetic beads were recovered using a magnetic stand. The recovered magnetic beads were washed three times with 1 mL of TBSTx. After addition of 20 ul of an elution buffer (0.2% SDS in TBS), the mixture was stirred and then heated two times at 98.degree. C. or 60.degree. C. for 5 minutes for elution. In order to remove the antibodies on the eluted sample, 40 .mu.L of magnetic beads washed in the same way as above was added to the eluted sample and shaken at 1,400 rpm at 20.degree. C. for 2 hours. The antibody-bound magnetic beads were removed using a magnetic stand, and the remaining portion was used as a sample. The prepared sample was developed by SDS-PAGE and then subjected to lectin blot.

(Results)

The lung cancer differential marker glycoproteins obtained by the steps described above are shown in FIG. 4.

6. Evaluation by Lectin Blot

10 .mu.L of the immunoprecipitated sample was developed on a 10% gel by SDS-PAGE. After SDS-PAGE, the proteins were transferred to a PVDF membrane (GE Healthcare Japan Corp.). The PVDF membrane was blocked with 5% BSA at room temperature for 1 hour and then reacted with already biotinylated AAL lectin (Seikagaku Corp.) at room temperature for 1 hour. Then, the membrane was reacted with secondary antibody-HRP-conjugated streptavidin at room temperature for 1 hour. The proteins were detected using Western Lightning. PBS-T was used in the dilution of lectin.

(Results)

The lung cancer differential marker glycoproteins obtained by the steps described above are shown in FIG. 4.

7. Column Fractionation of Serum Using AAL Lectin

Serum was fractionated using AAL lectin. Specifically, a commercially available column was packed with 1 mL of an AAL-conjugated resin and washed with TBS in an amount of 10 times the amount of the resin, with 0.5 M NaCl in an amount of twice the amount of the resin, and with TBS in an amount of 10 times the amount of the resin to prepare an AAL column. Serum was added to the AAL column and then reacted for 1 hour in the resin. Then, the column was washed with TBS in an amount of 5 times the amount of the resin. For elution, 1 mL of 20 mM fucose was added thereto and reacted for 1 hour in the resin. Then, 4 mL of 20 mM fucose was further added thereto (a total of 5 mL) to elute the proteins.

(Results)

The lung cancer differential marker glycoproteins obtained by the steps described above are shown in FIG. 5.

8. Fractionation of Serum by Serial Chromatography

Serum was fractionated at a low temperature by serial chromatography using LCA agarose (J-Oil Mills, Inc.) and AAL agarose (J-Oil Mills, Inc.). Specifically, 100 .mu.L of serum was diluted 4-fold with PBS. The serum sample was first applied to a column packed with 5 mL of LCA agarose (0.7.times.13 cm). Next, the obtained unbound serum fractions were applied to a column packed with 2.5 mL of AAL agarose (0.7.times.5.5 cm). After sufficient washing with PBS, the proteins were eluted with PBS containing 0.2 M fucose. The eluted fractions were concentrated through Ultrafree.TM. Centrifugal Filter Device (cutoff: 30 kDa, Millipore Corp.). The candidate molecules in the obtained samples were analyzed by comparison by immunoblot in the same way as above.

(Results)

The lung cancer differential marker glycoproteins obtained by the steps described above are shown in FIG. 6.

9. Multisample Fractionation of Serum by Serial Chromatography

The multisample comparative analysis of sera requires fractionating a small amount of a serum sample in a short time. For this purpose, high-throughput serial chromatography was established. Sera were fractionated by serial chromatography using LCA agarose (J-Oil Mills, Inc.) and AAL agarose (Vector Laboratories, Inc.). 50 .mu.L of a serum sample was applied to a tip column packed with 300 .mu.L of LCA agarose (0.37.times.2.3 cm). Next, the obtained unbound serum fractions were applied to an open column packed with 250 .mu.L of AAL agarose (0.8.times.0.5 cm). After sufficient washing with PBS, the proteins were eluted with PBS containing 0.02 M fucose. The eluted fractions were concentrated through Ultrafree.TM. Centrifugal Filter Device (cutoff: 30 kDa, Millipore Corp.). The candidate molecules in the obtained samples (corresponding to 10 .mu.L of sera) were analyzed by comparison by immunoblot in the same way as above.

(Results)

The lung cancer differential marker glycoproteins obtained by the steps described above are shown in FIG. 8.

10. Antibody-Overlay Lectin Array

An appropriate amount of the glycoprotein solution obtained in the paragraph "5. Fractionation by immunoprecipitation" was adjusted to 60 .mu.L with PBSTx (phosphate-buffered saline containing 1% Triton X-100) as a lectin array reaction buffer. This solution was added to each reaction vessel of a lectin array composed of 8 reaction vessels per a single glass plate, and reacted at 20.degree. C. for 10 hours or longer. This lectin array substrate composed of 8 reaction vessels was prepared according to the approach of Uchiyama et al. (Proteomics 8, 3042-3050 (2008)). In this way, the binding reaction between the glycans on the glycoproteins and 43 types of lectins immobilized on the array substrate reaches an equilibrium state. In order to prevent noise from being generated by the binding of the glycans of antibodies for detection to unreacted lectins on the substrate, 2 .mu.L of a human serum-derived IgG solution (manufactured by Sigma-Aldrich Corp.) was then added and reacted for 30 minutes. Each reaction vessel was washed three times with 60 .mu.L of PBSTx. Then, 2 .mu.L of a human serum-derived IgG solution was added thereto again and slightly stirred. Subsequently, biotinylated antibodies for detection against the glycoproteins were added in an amount corresponding to 100 ng and reacted at 20.degree. C. for 1 hour. After this antigen-antibody reaction, each reaction vessel was washed three times with 60 .mu.l, of PBSTx. Then, the array was scanned with an array scanner GlycoStation manufactured by Moritex Corporation to compare fluorescence intensities on the lectin spots reacted in a lung cancer tissue-specific manner.

(Results)

The lung cancer differential marker glycoproteins obtained by the steps described above are shown in FIGS. 9 and 10.

The results of these experiments revealed that some lectins, such as PNA and PWM, other than AAL or ConA and lectins having specificity related to these ligands are effective for the differential diagnosis of lung cancer. This means that a plurality of lectin signals derived from glycoproteins may be combined in various ways to thereby more accurately determine the histological type of lung cancer.

Example 2

Histological Staining of Lung Cancer Cell Using Lung Cancer Differential Marker

Histological staining with antibodies was tested using lung cancer differential markers.

First, paraffin that covered a formalin-fixed lung cancer tissue section (5 .mu.m thick) was removed according to a standard method. The deparaffinized tissue section was washed with PBS, dried in air, and then dipped in a 10 mM citrate buffer. The intermolecular (intramolecular) bridges derived from formalin fixation were dissociated by autoclaving at 121.degree. C. for 15 minutes. The section thus treated was left standing at room temperature for a while and then dipped three repetitive times in PBS for 5 minutes to wash the surface of the tissue. Subsequently, the section was treated with 0.3% H.sub.2O.sub.2-MeOH at room temperature for 10 minutes for the blocking reaction of endogenous peroxidase. After washing with PBS (5 min..times.3), a primary antibody solution (R&D Systems, Inc.; anti-NPR antibody suspended at a concentration of 3 .mu.g/mL in PBS) was added onto the tissue section to cause binding reaction at 20.degree. C. for 2 hours in a humidifying box. After washing with PBS (5 min..times.3), chromogenic reaction was initiated using an anti-sheep FITC conjugate (Santa Cruz Biotechnology, Inc.) as a secondary antibody and an anti-FITC HRP conjugate (Takara Bio Inc.) as an enzymatically labeled antibody and terminated by dipping in Milli-Q water for 5 minutes three times. Finally, the nucleic acids were stained with hematoxylin at room temperature for 1 minute, followed by washing in running water.

(Results)

The lung cancer differential marker glycoproteins obtained by the steps described above are shown in FIG. 7. Staining specific only for small cell cancer was confirmed.

All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

SEQUENCE LISTINGS

1

23518PRTHomo sapiens 1Gln Val Val Glu Asn Met Thr Arg 1 5 215PRTHomo sapiens 2Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys 1 5 10 15 321PRTHomo sapiens 3Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile Val 1 5 10 15 Asn Glu Leu Val Arg 20 419PRTHomo sapiens 4Asp Val Gln Ile Ile Val Phe Pro Glu Asp Gly Ile His Gly Phe Asn 1 5 10 15 Phe Thr Arg 59PRTHomo sapiens 5Phe Asn Asp Thr Glu Val Leu Gln Arg 1 5 631PRTHomo sapiens 6Ser His Leu Ile Ile Ala Gln Val Ala Lys Asn Pro Val Gly Leu Ile 1 5 10 15 Gly Ala Glu Asn Ala Thr Gly Glu Thr Asp Pro Ser His Ser Lys 20 25 30 724PRTHomo sapiens 7Asn Pro Val Gly Leu Ile Gly Ala Glu Asn Ala Thr Gly Glu Thr Asp 1 5 10 15 Pro Ser His Ser Lys Phe Leu Lys 20 813PRTHomo sapiens 8Tyr Tyr Lys Gly Ser Leu Ser Tyr Leu Asn Val Thr Arg 1 5 10 921PRTHomo sapiens 9Tyr Asn Pro Lys Tyr Ser Val Ala Asn Asp Thr Gly Phe Val Asp Ile 1 5 10 15 Pro Lys Gln Glu Lys 20 1014PRTHomo sapiens 10Tyr Ser Val Ala Asn Asp Thr Gly Phe Val Asp Ile Pro Lys 1 5 10 1122PRTHomo sapiens 11Tyr Arg Pro Glu Asn Ser Val Ala Asn Asp Thr Gly Phe Thr Val Val 1 5 10 15 Ala Pro Gly Lys Glu Lys 20 1222PRTHomo sapiens 12Asn Leu Asp His Gly Val Leu Val Val Gly Tyr Gly Phe Glu Gly Ala 1 5 10 15 Asn Ser Asn Asn Ser Lys 20 139PRTHomo sapiens 13Gln Thr Leu Phe Phe Asn Gly Thr Arg 1 5 1420PRTHomo sapiens 14Ser Asn Thr Ser Gln Leu Ala Phe Leu Leu Tyr Asn Asp Gln Pro Pro 1 5 10 15 Gln Pro Ser Lys 20 1528PRTHomo sapiens 15Gly Gly Asn Ser Asn Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn 1 5 10 15 Asn His Asn Tyr Thr Asp Cys Thr Ser Glu Gly Arg 20 25 1618PRTHomo sapiens 16Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn Asp Thr Phe 1 5 10 15 His Lys 1717PRTHomo sapiens 17Arg His Glu Glu Gly His Met Leu Asn Cys Thr Cys Phe Gly Gln Gly 1 5 10 15 Arg 1816PRTHomo sapiens 18His Glu Glu Gly His Met Leu Asn Cys Thr Cys Phe Gly Gln Gly Arg 1 5 10 15 1932PRTHomo sapiens 19Glu Ser Lys Pro Leu Thr Ala Gln Gln Thr Thr Lys Leu Asp Ala Pro 1 5 10 15 Thr Asn Leu Gln Phe Val Asn Glu Thr Asp Ser Thr Val Leu Val Arg 20 25 30 2020PRTHomo sapiens 20Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu Thr Asp Ser Thr 1 5 10 15 Val Leu Val Arg 20 2113PRTHomo sapiens 21Trp Thr Pro Leu Asn Ser Ser Thr Ile Ile Gly Tyr Arg 1 5 10 2221PRTHomo sapiens 22Ala Asp Val Gly Gly Glu Ala Ala Gly Thr Ser Ile Asn His Ser Gln 1 5 10 15 Ala Val Leu Gln Arg 20 2310PRTHomo sapiens 23Gln Gly Asn Ala Ser Asp Val Val Leu Arg 1 5 10 2412PRTHomo sapiens 24Phe Phe Asp Val Asn Gly Ser Ala Phe Leu Pro Arg 1 5 10 2519PRTHomo sapiens 25Ser Leu Leu Glu Phe Asn Thr Thr Val Ser Cys Asp Gln Gln Gly Thr 1 5 10 15 Asn His Arg 2617PRTHomo sapiens 26Met Ser Val Ile Asn Phe Glu Cys Asn Lys Thr Ala Gly Asn Asp Gly 1 5 10 15 Lys 2719PRTHomo sapiens 27Thr Asn Ile Thr Leu Val Cys Lys Pro Gly Asp Leu Glu Ser Ala Pro 1 5 10 15 Val Leu Arg 2820PRTHomo sapiens 28Gly Tyr Pro Cys Gly Gly Asn Lys Thr Ala Ser Ser Val Ile Glu Leu 1 5 10 15 Thr Cys Thr Lys 20 2911PRTHomo sapiens 29Gly Leu Cys Val Asn Ala Ser Ala Val Ser Arg 1 5 10 3021PRTHomo sapiens 30Tyr Lys Val Asp Tyr Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe Ser 1 5 10 15 Ser Glu Ser Lys Arg 20 3132PRTHomo sapiens 31Asp Gly Pro Cys Glu Phe Ala Pro Val Val Val Val Pro Pro Arg Ser 1 5 10 15 Val His Asn Val Thr Gly Ala Gln Val Gly Leu Ser Cys Glu Val Arg 20 25 30 3217PRTHomo sapiens 32Ser Val His Asn Val Thr Gly Ala Gln Val Gly Leu Ser Cys Glu Val 1 5 10 15 Arg 3316PRTHomo sapiens 33Thr Ile Leu Val Asp Asn Asn Thr Trp Asn Asn Thr His Ile Ser Arg 1 5 10 15 3422PRTHomo sapiens 34Ile Leu Thr Ala Asp Asn Gln Thr Tyr Met Ala Val Gln Gly Ser Thr 1 5 10 15 Ala Tyr Leu Leu Cys Lys 20 3517PRTHomo sapiens 35Trp Tyr Ser Leu Gly Lys Val Pro Gly Asn Gln Thr Ser Thr Thr Leu 1 5 10 15 Lys 3636PRTHomo sapiens 36Val Gln Trp Arg Pro Gln Gly Thr Arg Gly Pro Trp Gln Glu Gln Ile 1 5 10 15 Val Ser Asp Pro Phe Leu Val Val Ser Asn Thr Ser Thr Phe Val Pro 20 25 30 Tyr Glu Ile Lys 35 3712PRTHomo sapiens 37Thr His Asn Leu Thr Asp Leu Ser Pro His Leu Arg 1 5 10 3821PRTHomo sapiens 38Phe Asn Pro Asn Ile Ser Trp Gln Pro Ile Pro Val His Thr Val Pro 1 5 10 15 Ile Thr Glu Asp Arg 20 3920PRTHomo sapiens 39Tyr Glu Gln Leu Gln Asn Glu Thr Arg Gln Thr Pro Glu Tyr Gln Asn 1 5 10 15 Glu Ser Ser Arg 20 4011PRTHomo sapiens 40Gln Thr Pro Glu Tyr Gln Asn Glu Ser Ser Arg 1 5 10 4120PRTHomo sapiens 41Cys Gly Leu Ser Gln Ser Gln Gly Asn Leu Ser His Val Asp Trp Phe 1 5 10 15 Ser Val His Lys 20 4213PRTHomo sapiens 42Cys Val Ala Ser Val Pro Ser Ile Pro Gly Leu Asn Arg 1 5 10 4320PRTHomo sapiens 43Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys Cys Gly Asn Met Ser 1 5 10 15 Glu Ala Phe Arg 20 4423PRTHomo sapiens 44Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn Ala Ser Cys 1 5 10 15 Val Pro Val Asn Asn Pro Lys 20 4517PRTHomo sapiens 45Gly Ser Ser Val Ile Leu Asn Cys Ser Ala Tyr Ser Glu Pro Ser Pro 1 5 10 15 Lys 4623PRTHomo sapiens 46Val Ile Lys Leu Pro Ser Gly Met Leu Val Ile Ser Asn Ala Thr Glu 1 5 10 15 Gly Asp Gly Gly Leu Tyr Arg 20 4719PRTHomo sapiens 47Thr Pro Ala Ser Asp Pro His Gly Asp Asn Leu Thr Tyr Ser Val Phe 1 5 10 15 Tyr Thr Lys 4811PRTHomo sapiens 48Thr Ser Thr Arg Asn Ile Ser Ser Glu Glu Lys 1 5 10 4914PRTHomo sapiens 49Asp Gly Gln Leu Leu Pro Ser Ser Asn Tyr Ser Asn Ile Lys 1 5 10 5030PRTHomo sapiens 50Ile Tyr Asn Thr Pro Ser Ala Ser Tyr Leu Glu Val Thr Pro Asp Ser 1 5 10 15 Glu Asn Asp Phe Gly Asn Tyr Asn Cys Thr Ala Val Asn Arg 20 25 30 5113PRTHomo sapiens 51Ala Asn Val Ser Asn Ala Gly Leu Pro Gly Asp Phe Arg 1 5 10 5219PRTHomo sapiens 52Val Ala Glu Leu Glu Asp Glu Lys Ser Leu Leu His Asn Glu Thr Ser 1 5 10 15 Ala His Arg 5323PRTHomo sapiens 53Ala Leu Pro Gly Gly Ala Asp Asn Ala Ser Val Ala Ser Gly Ala Ala 1 5 10 15 Ala Ser Pro Gly Pro Gln Arg 20 5413PRTHomo sapiens 54Ala Tyr Trp Pro Asp Val Ile His Ser Phe Pro Asn Arg 1 5 10 5518PRTHomo sapiens 55Gln Asp Gln Gln Leu Gln Asn Cys Thr Glu Pro Gly Glu Gln Pro Ser 1 5 10 15 Pro Lys 5624PRTHomo sapiens 56Lys Thr Tyr Pro Pro Glu Asn Lys Pro Gly Gln Ser Asn Tyr Ser Phe 1 5 10 15 Val Asp Asn Leu Asn Leu Leu Lys 20 5713PRTHomo sapiens 57Asn Lys Leu Glu Lys Asn Ala Thr Asp Asn Ile Ser Lys 1 5 10 5813PRTHomo sapiens 58Glu Ala Ser Ile Val Gly Glu Asn Glu Thr Tyr Pro Arg 1 5 10 5930PRTHomo sapiens 59Leu Arg Asp Trp Phe Gln Leu Leu His Glu Asn Ser Lys Gln Asn Gly 1 5 10 15 Ser Ala Ser Ser Val Ala Gly Pro Ala Ser Gly Leu Asp Lys 20 25 30 6017PRTHomo sapiens 60Gln Asn Gly Ser Ala Ser Ser Val Ala Gly Pro Ala Ser Gly Leu Asp 1 5 10 15 Lys 6121PRTHomo sapiens 61Leu Asp Cys Arg His Glu Asn Thr Ser Ser Ser Pro Ile Gln Tyr Glu 1 5 10 15 Phe Ser Leu Thr Arg 20 6223PRTHomo sapiens 62Gly Arg Ala Asp Asp Cys Ala Leu Pro Tyr Leu Gly Ala Ile Cys Tyr 1 5 10 15 Cys Asp Leu Phe Cys Asn Arg 20 6327PRTHomo sapiens 63Ala Ile Asn Gln Gly Asn Tyr Gly Trp Gln Ala Gly Asn His Ser Ala 1 5 10 15 Phe Trp Gly Met Thr Leu Asp Glu Gly Ile Arg 20 25 6413PRTHomo sapiens 64Ser Leu Tyr Gly Lys Glu Asp Asn Asp Thr Leu Val Arg 1 5 10 6519PRTHomo sapiens 65Thr Phe Thr Asp Lys Trp Glu Asp Tyr Pro Lys Ser Glu Asn Glu Ser 1 5 10 15 Asn Ile Arg 6622PRTHomo sapiens 66Cys Ser Ala Ser Val Leu Pro Val Asp Val Gln Thr Leu Asn Ser Ser 1 5 10 15 Gly Pro Pro Phe Gly Lys 20 6721PRTHomo sapiens 67Leu Val Val Gln Ser Ser Ile Asp Ser Ser Ala Phe Lys His Asn Gly 1 5 10 15 Thr Val Glu Cys Lys 20 6816PRTHomo sapiens 68Leu Val Gly Gly Pro Val Ala Gly Gly Asp Pro Asn Gln Thr Ile Arg 1 5 10 15 6914PRTHomo sapiens 69Leu Asn Leu Thr Ser Pro Asp Leu Phe Trp Leu Val Phe Arg 1 5 10 7018PRTHomo sapiens 70Val Trp Gln Gly His Ala Asn Ala Ser Phe Cys Pro His Gly Tyr Gly 1 5 10 15 Cys Arg 7148PRTHomo sapiens 71Thr Ile Pro Pro Asp Cys Leu Leu Cys Gln Pro Gln Thr Phe Gly Cys 1 5 10 15 His Pro Leu Val Gly Cys Glu Glu Cys Asn Cys Ser Gly Pro Gly Ile 20 25 30 Gln Glu Leu Thr Asp Pro Thr Cys Asp Thr Asp Ser Gly Gln Cys Lys 35 40 45 7220PRTHomo sapiens 72Cys Glu Ile Cys Ala Pro Gly Phe Tyr Gly Asn Ala Leu Leu Pro Gly 1 5 10 15 Asn Cys Thr Arg 20 7311PRTHomo sapiens 73Gly Ile Asn Ala Ser Ser Met Ala Trp Ala Arg 1 5 10 7416PRTHomo sapiens 74Leu His Arg Leu Asn Ala Ser Ile Ala Asp Leu Gln Ser Gln Leu Arg 1 5 10 15 7527PRTHomo sapiens 75Thr Leu Ser Glu Leu Met Ser Gln Thr Gly His Leu Gly Leu Ala Asn 1 5 10 15 Ala Ser Ala Pro Ser Gly Glu Gln Leu Leu Arg 20 25 7613PRTHomo sapiens 76Asp Asn Ala Thr Leu Gln Ala Thr Leu His Ala Ala Arg 1 5 10 7734PRTHomo sapiens 77Leu Val Glu Ala Ala Glu Ala His Ala Gln Gln Leu Gly Gln Leu Ala 1 5 10 15 Leu Asn Leu Ser Ser Ile Ile Leu Asp Val Asn Gln Asp Arg Leu Thr 20 25 30 Gln Arg 7827PRTHomo sapiens 78Gln Gly Leu Val Asp Arg Ala Gln Gln Leu Leu Ala Asn Ser Thr Ala 1 5 10 15 Leu Glu Glu Ala Met Leu Gln Glu Gln Gln Arg 20 25 7915PRTHomo sapiens 79Gly Val His Asn Ala Ser Leu Ala Leu Ser Ala Ser Ile Gly Arg 1 5 10 15 8018PRTHomo sapiens 80Leu Asn Thr Thr Gly Val Ser Ala Gly Cys Thr Ala Asp Leu Leu Val 1 5 10 15 Gly Arg 8131PRTHomo sapiens 81Val Phe Asp Leu Gln Gln Asn Leu Gly Ser Val Asn Val Ser Thr Gly 1 5 10 15 Cys Ala Pro Ala Leu Gln Ala Gln Thr Pro Gly Leu Gly Pro Arg 20 25 30 8223PRTHomo sapiens 82Lys Cys Val Cys Asn Tyr Leu Gly Thr Val Gln Glu His Cys Asn Gly 1 5 10 15 Ser Asp Cys Gln Cys Asp Lys 20 8313PRTHomo sapiens 83Leu Ser Asp Thr Thr Ser Gln Ser Asn Ser Thr Ala Lys 1 5 10 8414PRTHomo sapiens 84Gln Ser Ala Glu Asp Ile Leu Leu Lys Thr Asn Ala Thr Lys 1 5 10 8536PRTHomo sapiens 85Ala Ile Lys Gln Ala Asp Glu Asp Ile Gln Gly Thr Gln Asn Leu Leu 1 5 10 15 Thr Ser Ile Glu Ser Glu Thr Ala Ala Ser Glu Glu Thr Leu Phe Asn 20 25 30 Ala Ser Gln Arg 35 8614PRTHomo sapiens 86Gly Lys Glu Gly His Phe Tyr Tyr Asn Ile Ser Glu Val Lys 1 5 10 8714PRTHomo sapiens 87Met Lys Val Ser Asn Val Ser Cys Gln Ala Ser Val Ser Arg 1 5 10 8812PRTHomo sapiens 88Val Ser Asn Val Ser Cys Gln Ala Ser Val Ser Arg 1 5 10 8916PRTHomo sapiens 89Gly Ala Phe Phe Pro Leu Thr Glu Arg Asn Trp Ser Leu Pro Asn Arg 1 5 10 15 9023PRTHomo sapiens 90Phe Arg Ile Tyr Ser Asn His Ser Ala Leu Glu Ser Leu Ala Leu Ile 1 5 10 15 Pro Leu Gln Ala Pro Leu Lys 20 9116PRTHomo sapiens 91Gly Ala Phe Phe Pro Leu Thr Glu Arg Asn Trp Ser Leu Pro Asn Arg 1 5 10 15 9220PRTHomo sapiens 92Lys Leu Gly Asp Cys Ile Ser Glu Asp Ser Tyr Pro Asp Gly Asn Ile 1 5 10 15 Thr Trp Tyr Arg 20 9312PRTHomo sapiens 93Asn Ala Ile Lys Glu Gly Asp Asn Ile Thr Leu Lys 1 5 10 948PRTHomo sapiens 94Glu Gly Asp Asn Ile Thr Leu Lys 1 5 9512PRTHomo sapiens 95Asn Ala Thr Val Val Trp Met Lys Asp Asn Ile Arg 1 5 10 9625PRTHomo sapiens 96Tyr Tyr Ser Lys Ile Ile Ile Ser Pro Glu Glu Asn Val Thr Leu Thr 1 5 10 15 Cys Thr Ala Glu Asn Gln Leu Glu Arg 20 25 9711PRTHomo sapiens 97Glu Ala Ser Gln Asn Ile Thr Tyr Ile Cys Lys 1 5 10 9811PRTHomo sapiens 98Leu Glu Asn Ile Thr Asn Pro Trp Ser Pro Arg 1 5 10 9930PRTHomo sapiens 99Leu Val Gln Leu Phe Pro Asn Asp Thr Ser Leu Lys Asn Asp Leu Gly 1 5 10 15 Val Gly Tyr Leu Leu Ile Gly Asp Asn Asp Asn Ala Lys Lys 20 25 30 10013PRTHomo sapiens 100Asp Phe Arg Tyr Asn Leu Ser Glu Val Leu Gln Gly Lys 1 5 10 10117PRTHomo sapiens 101His Leu Glu Leu Ala Gly Glu Asn Pro Ser Ser Asp Ile Asn Cys Thr 1 5 10 15 Lys 10218PRTHomo sapiens 102Trp Thr Pro Asp Asp Tyr Ile Asn Met Thr Ser Asp Cys Ser Ser Phe 1 5 10 15 Ile Lys 10328PRTHomo sapiens 103Ala Ala Thr Ser Arg Pro Glu Arg Val Trp Pro Asp Gly Val Ile Pro 1 5 10 15 Phe Val Ile Gly Gly Asn Phe Thr Gly Ser Gln Arg 20 25 10420PRTHomo sapiens 104Ile Ser Val Thr Pro Gly Glu Lys Ile Ile Leu Asn Phe Thr Ser Leu 1 5 10 15 Asp Leu Tyr Arg 20 10512PRTHomo sapiens 105Leu Asn Gly Ser Ile Thr Ser Pro Gly Trp Pro Lys 1 5 10 10621PRTHomo sapiens 106Ile His Gly Gln Glu Leu Pro Phe Glu Ala Val Val Leu Asn Lys Thr 1 5 10 15 Ser Gly Glu Gly Arg 20 10713PRTHomo

sapiens 107Asn Leu Thr Asp Gln Phe Thr Ile Thr Met Trp Met Lys 1 5 10 10814PRTHomo sapiens 108Gln Glu Cys Leu Glu Leu Asn His Ser Glu Leu His Gln Arg 1 5 10 10921PRTHomo sapiens 109His Leu Asp Ala Thr Asn Ser Thr Ala Gly Tyr Ser Ile Tyr Gly Val 1 5 10 15 Gly Ser Met Ser Arg 20 11022PRTHomo sapiens 110Leu Leu Asn Thr Thr Asp Val Tyr Leu Leu Pro Ser Leu Asn Pro Asp 1 5 10 15 Gly Phe Glu Arg Ala Arg 20 11111PRTHomo sapiens 111Arg Phe Ala Asn Glu Tyr Pro Asn Ile Thr Arg 1 5 10 11222PRTHomo sapiens 112His Ile Trp Ser Leu Glu Ile Ser Asn Lys Pro Asn Val Ser Glu Pro 1 5 10 15 Glu Glu Pro Lys Ile Arg 20 11318PRTHomo sapiens 113Gly Lys Asp Leu Asp Thr Asp Phe Thr Asn Asn Ala Ser Gln Pro Glu 1 5 10 15 Thr Lys 11413PRTHomo sapiens 114Gly Arg Asp Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys 1 5 10 11522PRTHomo sapiens 115Asp Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr 1 5 10 15 Asp Phe Ser Ser Ala Lys 20 11622PRTHomo sapiens 116Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys Glu 1 5 10 15 Val Thr Glu Leu Thr Arg 20 11718PRTHomo sapiens 117Gln Gln Met Glu Asn Tyr Pro Lys Asn Asn His Thr Ala Ser Ile Leu 1 5 10 15 Asp Arg 11810PRTHomo sapiens 118Asn Asn His Thr Ala Ser Ile Leu Asp Arg 1 5 10 11922PRTHomo sapiens 119Asn Arg Val Pro Asp Ser Cys Cys Ile Asn Val Thr Val Gly Cys Gly 1 5 10 15 Ile Asn Phe Asn Glu Lys 20 12021PRTHomo sapiens 120Thr Phe Lys Asn Glu Ser Glu Asn Thr Cys Gln Asp Val Asp Glu Cys 1 5 10 15 Gln Gln Asn Pro Arg 20 12116PRTHomo sapiens 121Arg Leu Ser Ala Val Asn Ser Ile Phe Leu Ser His Asn Asn Thr Lys 1 5 10 15 12216PRTHomo sapiens 122Arg Leu Ser Ala Val Asn Ser Ile Phe Leu Ser His Asn Asn Thr Lys 1 5 10 15 12321PRTHomo sapiens 123Thr Phe Lys Asn Glu Ser Glu Asn Thr Cys Gln Asp Val Asp Glu Cys 1 5 10 15 Gln Gln Asn Pro Arg 20 12416PRTHomo sapiens 124Arg Leu Ser Ala Val Asn Ser Ile Phe Leu Ser His Asn Asn Thr Lys 1 5 10 15 12512PRTHomo sapiens 125Phe Leu Asn Asn Gly Thr Cys Thr Ala Glu Gly Lys 1 5 10 12619PRTHomo sapiens 126Phe Lys Leu Ser Asp Leu Ser Ile Asn Ser Thr Glu Cys Leu His Val 1 5 10 15 His Cys Arg 12717PRTHomo sapiens 127Leu Ser Asp Leu Ser Ile Asn Ser Thr Glu Cys Leu His Val His Cys 1 5 10 15 Arg 12826PRTHomo sapiens 128Ser Ile Pro Ala Cys Val Pro Trp Ser Pro Tyr Leu Phe Gln Pro Asn 1 5 10 15 Asp Thr Cys Ile Val Ser Gly Trp Gly Arg 20 25 12912PRTHomo sapiens 129Leu Ile Ser Asn Cys Ser Lys Phe Tyr Gly Asn Arg 1 5 10 13017PRTHomo sapiens 130Tyr Ser Val Glu Lys Phe Asn Asn Cys Thr Asn Asp Met Phe Leu Phe 1 5 10 15 Lys 13115PRTHomo sapiens 131Phe Asn Asn Cys Thr Asn Asp Met Phe Leu Phe Lys Glu Ser Arg 1 5 10 15 13213PRTHomo sapiens 132Leu Asp Asp Cys Asp Phe Gln Thr Asn His Thr Leu Lys 1 5 10 13326PRTHomo sapiens 133Ser Leu Asp Arg Glu Ala Glu Thr Ile Lys Asn Gly Ile Tyr Asn Ile 1 5 10 15 Thr Val Leu Ala Ser Asp Gln Gly Gly Arg 20 25 1348PRTHomo sapiens 134Ala Ile Asn Asp Thr Ala Ala Arg 1 5 13511PRTHomo sapiens 135Val Leu Glu Ala Val Asn Gly Thr Asp Ala Arg 1 5 10 13614PRTHomo sapiens 136Leu Gln Phe Asp Asp Asn Gly Thr Tyr Thr Cys Gln Val Lys 1 5 10 13721PRTHomo sapiens 137Ala Ile Gly Gly Ile Asp Trp Asp Ser His Pro Phe Gly Tyr Asn Leu 1 5 10 15 Thr Leu Gln Ala Lys 20 1388PRTHomo sapiens 138Gln Val Tyr Asn Leu Thr Val Arg 1 5 13921PRTHomo sapiens 139Ile Val Val Asn Val Ser Asp Thr Asn Asp His Ala Pro Trp Phe Thr 1 5 10 15 Ala Ser Ser Tyr Lys 20 14020PRTHomo sapiens 140Gln Leu Leu His Lys Ile Asn Ser Ser Val Thr Asp Ile Glu Glu Ile 1 5 10 15 Ile Gly Val Arg 20 1419PRTHomo sapiens 141Leu Asp Gly Ser Thr Asn Phe Thr Arg 1 5 14215PRTHomo sapiens 142Leu His Val Gly Asn Tyr Asn Gly Thr Ala Gly Asp Ala Leu Arg 1 5 10 15 14335PRTHomo sapiens 143Ile His Thr Pro Ser Leu His Val Asn Gly Ser Leu Ile Leu Pro Ile 1 5 10 15 Gly Ser Ile Lys Pro Leu Asp Phe Ser Leu Leu Asn Val Gln Asp Gln 20 25 30 Glu Gly Arg 35 14419PRTHomo sapiens 144Ile Val Tyr Asn Ile Thr Leu Pro Leu His Pro Asn Gln Gly Ile Ile 1 5 10 15 Glu His Arg 14524PRTHomo sapiens 145Ile Ser Gly Ser Glu Val Glu Glu Leu Ser Glu Val Ser Asn Phe Thr 1 5 10 15 Met Glu Asp Ile Asn Asn Lys Lys 20 1469PRTHomo sapiens 146Tyr Thr Ser Tyr Asn Val Ser Glu Lys 1 5 14717PRTHomo sapiens 147Val Ile Ile Asn Asp Thr Glu Asp Glu Pro Thr Leu Glu Phe Asp Lys 1 5 10 15 Lys 14821PRTHomo sapiens 148Leu Arg Ala Asn Gln Ser Trp Glu Asp Ser Asn Thr Asp Leu Val Pro 1 5 10 15 Ala Pro Ala Val Arg 20 14926PRTHomo sapiens 149Ala Asn Gln Ser Trp Glu Asp Ser Asn Thr Asp Leu Val Pro Ala Pro 1 5 10 15 Ala Val Arg Ile Leu Thr Pro Glu Val Arg 20 25 15012PRTHomo sapiens 150Phe Gln Leu Leu Asn Phe Ser Ser Ser Glu Leu Lys 1 5 10 15113PRTHomo sapiens 151Val Ser Leu Thr Asn Val Ser Ile Ser Asp Glu Gly Arg 1 5 10 15210PRTHomo sapiens 152Asn Thr Thr Phe Asn Val Glu Ser Thr Lys 1 5 10 15312PRTHomo sapiens 153Asp Ser Cys Glu Ser Asn His Asn Ile Thr Cys Lys 1 5 10 15414PRTHomo sapiens 154Ser Glu Asn Ala Ser Leu Val Leu Ser Ser Ser Asn Gln Lys 1 5 10 15538PRTHomo sapiens 155Thr Glu Leu Asp Leu Arg Pro Gln Gly Leu Glu Leu Phe Glu Asn Thr 1 5 10 15 Ser Ala Pro Tyr Gln Leu Gln Thr Phe Val Leu Pro Ala Thr Pro Pro 20 25 30 Gln Leu Val Ser Pro Arg 35 15615PRTHomo sapiens 156Leu Asn Pro Thr Val Thr Tyr Gly Asn Asp Ser Phe Ser Ala Lys 1 5 10 15 15714PRTHomo sapiens 157Ser Val Gln Leu His Asp Ser Gly Asn Tyr Ser Cys Tyr Arg 1 5 10 15828PRTHomo sapiens 158Asp Ser Cys Gln Gln Gly Ser Asn Met Thr Leu Ile Gly Glu Asn Gly 1 5 10 15 His Ser Thr Asp Thr Leu Thr Gly Ser Gly Phe Arg 20 25 15920PRTHomo sapiens 159Arg Leu Asn Ser Thr His Cys Gln Asp Ile Asn Glu Cys Ala Met Pro 1 5 10 15 Gly Val Cys Arg 20 16030PRTHomo sapiens 160Asp Arg Ser His Cys Glu Asp Ile Asp Glu Cys Asp Phe Pro Ala Ala 1 5 10 15 Cys Ile Gly Gly Asp Cys Ile Asn Thr Asn Gly Ser Tyr Arg 20 25 30 16112PRTHomo sapiens 161Asn Thr Ser Val Gly Leu Leu Tyr Ser Gly Cys Arg 1 5 10 16218PRTHomo sapiens 162Lys Phe Asn Ile Thr Glu Ser Val Leu Gln Gly Leu Leu Lys Pro Leu 1 5 10 15 Phe Lys 16316PRTHomo sapiens 163Asn Ile Glu Asp Ala Leu Asn Gln Leu Phe Arg Asn Ser Ser Ile Lys 1 5 10 15 16412PRTHomo sapiens 164Asn Gly Thr Gln Leu Gln Asn Phe Thr Leu Asp Arg 1 5 10 16531PRTHomo sapiens 165Lys Leu Trp Ala Asp Thr Thr Cys Gly Gln Asn Ala Thr Glu Leu Tyr 1 5 10 15 Cys Phe Tyr Ser Glu Asn Thr Asp Leu Thr Cys Arg Gln Pro Lys 20 25 30 16619PRTHomo sapiens 166Tyr Phe Ala Thr Asn Cys Ser Ala Thr Phe Gly Leu Glu Asp Asp Val 1 5 10 15 Val Lys Lys 16710PRTHomo sapiens 167Phe Asn His Thr Gln Thr Ile Gln Gln Lys 1 5 10 16824PRTHomo sapiens 168Val Ile Ser Val Asp Glu Leu Asn Asp Thr Ile Ala Ala Asn Leu Ser 1 5 10 15 Asp Thr Glu Phe Tyr Gly Ala Lys 20 16920PRTHomo sapiens 169Glu Arg Pro Pro Thr Phe Leu Thr Pro Glu Gly Asn Ala Ser Asn Lys 1 5 10 15 Glu Glu Leu Arg 20 1709PRTHomo sapiens 170Glu Asp Gly Met Leu Pro Lys Asn Arg 1 5 17135PRTHomo sapiens 171Gly Ser Ala Leu His Glu Asp Ile Tyr Val Leu His Glu Asn Gly Thr 1 5 10 15 Leu Glu Ile Pro Val Ala Gln Lys Asp Ser Thr Gly Thr Tyr Thr Cys 20 25 30 Val Ala Arg 35 17219PRTHomo sapiens 172Val Asn Val Val Asn Ser Thr Leu Ala Glu Val His Trp Asp Pro Val 1 5 10 15 Pro Leu Lys 17318PRTHomo sapiens 173Gln Leu Leu Glu Lys Val Gln Asn Met Ser Gln Ser Ile Glu Val Leu 1 5 10 15 Asp Arg 17414PRTHomo sapiens 174Val Gln Asn Met Ser Gln Ser Ile Glu Val Leu Asp Arg Arg 1 5 10 17515PRTHomo sapiens 175Ser Met Val Asp Phe Met Asn Thr Asp Asn Phe Thr Ser His Arg 1 5 10 15 17619PRTHomo sapiens 176Leu Asp Pro Val Ser Leu Gln Thr Leu Gln Thr Trp Asn Thr Ser Tyr 1 5 10 15 Pro Lys Arg 17718PRTHomo sapiens 177Gln Leu Leu Glu Lys Val Gln Asn Met Ser Gln Ser Ile Glu Val Leu 1 5 10 15 Asp Arg 17811PRTHomo sapiens 178Glu Leu Gln Tyr Val Lys Gln Glu Cys Asn Arg 1 5 10 17917PRTHomo sapiens 179Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys 1 5 10 15 Arg 18018PRTHomo sapiens 180Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr 1 5 10 15 Gln Lys 18113PRTHomo sapiens 181His Ile Gly His Ala Asn Leu Thr Phe Glu Gln Leu Arg 1 5 10 1828PRTHomo sapiens 182Gly Ile Asn Glu Ser Tyr Lys Lys 1 5 18319PRTHomo sapiens 183Phe Leu Asn Asp Ser Ile Val Asp Pro Val Asp Ser Glu Trp Phe Gly 1 5 10 15 Phe Tyr Arg 18427PRTHomo sapiens 184His Tyr Gly Pro Gly Trp Val Ser Met Ala Asn Ala Gly Lys Asp Thr 1 5 10 15 Asn Gly Ser Gln Phe Phe Ile Thr Thr Val Lys 20 25 18528PRTHomo sapiens 185Gly Thr Trp Ser Thr Ala Glu Ser Gly Ala Glu Cys Thr Asn Trp Asn 1 5 10 15 Ser Ser Ala Leu Ala Gln Lys Pro Tyr Ser Gly Arg 20 25 18630PRTHomo sapiens 186Ala Gly Lys Tyr Ser Ser Glu Phe Cys Ser Thr Pro Ala Cys Ser Glu 1 5 10 15 Gly Asn Ser Asp Cys Tyr Phe Gly Asn Gly Ser Ala Tyr Arg 20 25 30 18727PRTHomo sapiens 187Tyr Ser Ser Glu Phe Cys Ser Thr Pro Ala Cys Ser Glu Gly Asn Ser 1 5 10 15 Asp Cys Tyr Phe Gly Asn Gly Ser Ala Tyr Arg 20 25 18822PRTHomo sapiens 188Cys Thr Ser Gln His Leu Leu Asn Arg Thr Val Thr Asp Asn Met Leu 1 5 10 15 Cys Ala Gly Asp Thr Arg 20 18919PRTHomo sapiens 189Gln His Thr Val Thr Thr Thr Thr Lys Gly Glu Asn Phe Thr Glu Thr 1 5 10 15 Asp Val Lys 19010PRTHomo sapiens 190Gly Glu Asn Phe Thr Glu Thr Asp Val Lys 1 5 10 19114PRTHomo sapiens 191Trp Phe Ser Ala Gly Leu Ala Ser Asn Ser Ser Trp Leu Arg 1 5 10 19219PRTHomo sapiens 192Ser Val Val Ala Pro Ala Thr Asp Gly Gly Leu Asn Leu Thr Ser Thr 1 5 10 15 Phe Leu Arg 19317PRTHomo sapiens 193Lys Val Glu Val Glu Pro Leu Asn Ser Thr Ala Val His Val Tyr Trp 1 5 10 15 Lys 19431PRTHomo sapiens 194Asp Ile Asn Ser Gln Gln Glu Leu Gln Asn Ile Thr Thr Asp Thr Arg 1 5 10 15 Phe Thr Leu Thr Gly Leu Lys Pro Asp Thr Thr Tyr Asp Ile Lys 20 25 30 19517PRTHomo sapiens 195Lys Val Glu Val Glu Pro Leu Asn Ser Thr Ala Val His Val Tyr Trp 1 5 10 15 Lys 19631PRTHomo sapiens 196Asp Ile Asn Ser Gln Gln Glu Leu Gln Asn Ile Thr Thr Asp Thr Arg 1 5 10 15 Phe Thr Leu Thr Gly Leu Lys Pro Asp Thr Thr Tyr Asp Ile Lys 20 25 30 19715PRTHomo sapiens 197Gln Leu Thr Leu Gln Trp Glu Pro Leu Gly Tyr Asn Val Thr Arg 1 5 10 15 19815PRTHomo sapiens 198Leu Leu Ala Asn Ser Ser Met Leu Gly Glu Gly Gln Val Leu Arg 1 5 10 15 19917PRTHomo sapiens 199Ile Val Ser Pro Glu Pro Gly Gly Ala Val Gly Pro Asn Leu Thr Cys 1 5 10 15 Arg 20015PRTHomo sapiens 200Leu Leu Ala Asn Ser Ser Met Leu Gly Glu Gly Gln Val Leu Arg 1 5 10 15 20117PRTHomo sapiens 201Ile Val Ser Pro Glu Pro Gly Gly Ala Val Gly Pro Asn Leu Thr Cys 1 5 10 15 Arg 20213PRTHomo sapiens 202Ser Val Asn Leu Ser Asp Gly Glu Leu Leu Ser Ile Arg 1 5 10 20334PRTHomo sapiens 203His Leu Thr Cys Leu Asn Ala Thr Gln Pro Phe Trp Asp Ser Lys Glu 1 5 10 15 Pro Val Cys Ile Ala Ala Cys Gly Gly Val Ile Arg Asn Ala Thr Thr 20 25 30 Gly Arg 20427PRTHomo sapiens 204Ile Val Ser Pro Gly Phe Pro Gly Asn Tyr Ser Asn Asn Leu Thr Cys 1 5 10 15 His Trp Leu Leu Glu Ala Pro Glu Gly Gln Arg 20 25 20518PRTHomo sapiens 205Gln Ala Ile His Val Gly Asn Gln Thr Phe Asn Asp Gly Thr Ile Val 1 5 10 15 Glu Lys 20623PRTHomo sapiens 206Leu His His His Leu Asp His Asn Asn Thr His His Phe His Asn Asp 1 5 10 15 Ser Ile Thr Pro Ser Glu Arg 20 20724PRTHomo sapiens 207Gly Glu Pro Ser Asn Glu Pro Ser Thr Glu Thr Asn Lys Thr Gln Glu 1 5 10 15 Gln Ser Asp Val Lys Leu Pro Lys 20 20820PRTHomo sapiens 208Lys Asp Leu Asn Glu Asp Asp His His His Glu Cys Leu Asn Val Thr 1 5 10 15 Gln Leu Leu Lys 20 20924PRTHomo sapiens 209Gln Ser Gly Val Asn Ala Thr Leu Pro Glu Glu Asn Gln Pro Val Val 1 5 10 15 Phe Asn His Val Tyr Asn Ile Lys 20 21031PRTHomo sapiens 210Cys Ile Asn Gly Thr Cys Tyr Cys Glu Glu Gly Phe Thr Gly Glu Asp 1 5 10 15 Cys Gly Lys Pro Thr Cys Pro His Ala Cys His Thr Gln Gly Arg 20 25 30 21127PRTHomo sapiens 211Gln Thr Gly Leu Ala Pro Gly Gln Glu Tyr Glu Ile Ser Leu His Ile 1 5 10 15 Val Lys Asn Asn Thr Arg Gly Pro Gly Leu Lys 20 25 21217PRTHomo sapiens 212Leu Asn Tyr Ser Leu Pro Thr Gly Gln Trp Val Gly Val Gln Leu Pro 1 5 10 15 Arg

21324PRTHomo sapiens 213Asn Thr Thr Ser Tyr Val Leu Arg Gly Leu Glu Pro Gly Gln Glu Tyr 1 5 10 15 Asn Val Leu Leu Thr Ala Glu Lys 20 21425PRTHomo sapiens 214Val Lys Ala Ser Thr Glu Gln Ala Pro Glu Leu Glu Asn Leu Thr Val 1 5 10 15 Thr Glu Val Gly Trp Asp Gly Leu Arg 20 25 21527PRTHomo sapiens 215Leu Asn Trp Thr Ala Ala Asp Gln Ala Tyr Glu His Phe Ile Ile Gln 1 5 10 15 Val Gln Glu Ala Asn Lys Val Glu Ala Ala Arg 20 25 21614PRTHomo sapiens 216Leu Leu Glu Thr Val Glu Tyr Asn Ile Ser Gly Ala Glu Arg 1 5 10 2178PRTHomo sapiens 217Tyr Phe Tyr Asn Asn Gln Thr Lys 1 5 21829PRTHomo sapiens 218Leu Arg Leu Ala Ser Pro Pro Ser Gln Gly Glu Val Pro Pro Gly Pro 1 5 10 15 Leu Pro Glu Ala Val Leu Ala Leu Tyr Asn Ser Thr Arg 20 25 21917PRTHomo sapiens 219Gln Cys Asn Gly Thr Ser Thr Cys Trp Cys Val Asn Thr Ala Gly Val 1 5 10 15 Arg 22018PRTHomo sapiens 220His Arg Pro Thr Ala Gly Ala Phe Asn His Ser Asp Leu Asp Ala Glu 1 5 10 15 Leu Arg 22115PRTHomo sapiens 221Leu Ser Asn Ile Ser His Leu Asn Tyr Cys Glu Pro Asp Leu Arg 1 5 10 15 22216PRTHomo sapiens 222Glu Ser Trp Gly Gln Glu Ser Asn Ala Gly Asn Gln Thr Val Val Arg 1 5 10 15 22315PRTHomo sapiens 223Asn Ala Ser Val Pro Gln Ile Leu Ile Ile Val Thr Asp Gly Lys 1 5 10 15 224468PRTHomo sapienssecretogranin III 224Met Gly Phe Leu Gly Thr Gly Thr Trp Ile Leu Val Leu Val Leu Pro 1 5 10 15 Ile Gln Ala Phe Pro Lys Pro Gly Gly Ser Gln Asp Lys Ser Leu His 20 25 30 Asn Arg Glu Leu Ser Ala Glu Arg Pro Leu Asn Glu Gln Ile Ala Glu 35 40 45 Ala Glu Glu Asp Lys Ile Lys Lys Thr Tyr Pro Pro Glu Asn Lys Pro 50 55 60 Gly Gln Ser Asn Tyr Ser Phe Val Asp Asn Leu Asn Leu Leu Lys Ala 65 70 75 80 Ile Thr Glu Lys Glu Lys Ile Glu Lys Glu Arg Gln Ser Ile Arg Ser 85 90 95 Ser Pro Leu Asp Asn Lys Leu Asn Val Glu Asp Val Asp Ser Thr Lys 100 105 110 Asn Arg Lys Leu Ile Asp Asp Tyr Asp Ser Thr Lys Ser Gly Leu Asp 115 120 125 His Lys Phe Gln Asp Asp Pro Asp Gly Leu His Gln Leu Asp Gly Thr 130 135 140 Pro Leu Thr Ala Glu Asp Ile Val His Lys Ile Ala Ala Arg Ile Tyr 145 150 155 160 Glu Glu Asn Asp Arg Ala Val Phe Asp Lys Ile Val Ser Lys Leu Leu 165 170 175 Asn Leu Gly Leu Ile Thr Glu Ser Gln Ala His Thr Leu Glu Asp Glu 180 185 190 Val Ala Glu Val Leu Gln Lys Leu Ile Ser Lys Glu Ala Asn Asn Tyr 195 200 205 Glu Glu Asp Pro Asn Lys Pro Thr Ser Trp Thr Glu Asn Gln Ala Gly 210 215 220 Lys Ile Pro Glu Lys Val Thr Pro Met Ala Ala Ile Gln Asp Gly Leu 225 230 235 240 Ala Lys Gly Glu Asn Asp Glu Thr Val Ser Asn Thr Leu Thr Leu Thr 245 250 255 Asn Gly Leu Glu Arg Arg Thr Lys Thr Tyr Ser Glu Asp Asn Phe Glu 260 265 270 Glu Leu Gln Tyr Phe Pro Asn Phe Tyr Ala Leu Leu Lys Ser Ile Asp 275 280 285 Ser Glu Lys Glu Ala Lys Glu Lys Glu Thr Leu Ile Thr Ile Met Lys 290 295 300 Thr Leu Ile Asp Phe Val Lys Met Met Val Lys Tyr Gly Thr Ile Ser 305 310 315 320 Pro Glu Glu Gly Val Ser Tyr Leu Glu Asn Leu Asp Glu Met Ile Ala 325 330 335 Leu Gln Thr Lys Asn Lys Leu Glu Lys Asn Ala Thr Asp Asn Ile Ser 340 345 350 Lys Leu Phe Pro Ala Pro Ser Glu Lys Ser His Glu Glu Thr Asp Ser 355 360 365 Thr Lys Glu Glu Ala Ala Lys Met Glu Lys Glu Tyr Gly Ser Leu Lys 370 375 380 Asp Ser Thr Lys Asp Asp Asn Ser Asn Pro Gly Gly Lys Thr Asp Glu 385 390 395 400 Pro Lys Gly Lys Thr Glu Ala Tyr Leu Glu Ala Ile Arg Lys Asn Ile 405 410 415 Glu Trp Leu Lys Lys His Asp Lys Lys Gly Asn Lys Glu Asp Tyr Asp 420 425 430 Leu Ser Lys Met Arg Asp Phe Ile Asn Lys Gln Ala Asp Ala Tyr Val 435 440 445 Glu Lys Gly Ile Leu Asp Lys Glu Glu Ala Glu Ala Ile Lys Arg Ile 450 455 460 Tyr Ser Ser Leu 465 2252477PRTHomo sapiensFibronectin 1 isoform 1 225Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln 20 25 30 Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser 35 40 45 Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 50 55 60 Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly 65 70 75 80 Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr 85 90 95 Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr 100 105 110 Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala 115 120 125 Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly 130 135 140 Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr 145 150 155 160 Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu 165 170 175 Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly 180 185 190 Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp 195 200 205 Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 210 215 220 Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr 225 230 235 240 Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu 245 250 255 Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg 260 265 270 His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp 275 280 285 Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro 290 295 300 Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met 305 310 315 320 Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu 325 330 335 Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly 340 345 350 Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly 355 360 365 Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu 370 375 380 Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe 385 390 395 400 Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn 405 410 415 Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr 420 425 430 Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr 435 440 445 Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 450 455 460 Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile 465 470 475 480 Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys 485 490 495 Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser 500 505 510 Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn 515 520 525 Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr 530 535 540 Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln 545 550 555 560 Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp 565 570 575 Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg 580 585 590 Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser 595 600 605 Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 610 615 620 Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys 625 630 635 640 Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu 645 650 655 Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys 660 665 670 Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly 675 680 685 His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr 690 695 700 Pro Val Thr Ser Asn Thr Val Thr Gly Glu Thr Thr Pro Phe Ser Pro 705 710 715 720 Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser Ser Phe 725 730 735 Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe Arg Val 740 745 750 Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp Leu 755 760 765 Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly Arg 770 775 780 Lys Tyr Ile Val Asn Val Tyr Gln Ile Ser Glu Asp Gly Glu Gln Ser 785 790 795 800 Leu Ile Leu Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro Asp 805 810 815 Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp Ser 820 825 830 Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro Ser 835 840 845 Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn Ser 850 855 860 Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile 865 870 875 880 Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln Gln 885 890 895 Glu Thr Thr Gly Thr Pro Arg Ser Asp Thr Val Pro Ser Pro Arg Asp 900 905 910 Leu Gln Phe Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp Thr 915 920 925 Pro Pro Glu Ser Ala Val Thr Gly Tyr Arg Val Asp Val Ile Pro Val 930 935 940 Asn Leu Pro Gly Glu His Gly Gln Arg Leu Pro Ile Ser Arg Asn Thr 945 950 955 960 Phe Ala Glu Val Thr Gly Leu Ser Pro Gly Val Thr Tyr Tyr Phe Lys 965 970 975 Val Phe Ala Val Ser His Gly Arg Glu Ser Lys Pro Leu Thr Ala Gln 980 985 990 Gln Thr Thr Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu 995 1000 1005 Thr Asp Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln 1010 1015 1020 Ile Thr Gly Tyr Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln 1025 1030 1035 Pro Arg Gln Tyr Asn Val Gly Pro Ser Val Ser Lys Tyr Pro Leu 1040 1045 1050 Arg Asn Leu Gln Pro Ala Ser Glu Tyr Thr Val Ser Leu Val Ala 1055 1060 1065 Ile Lys Gly Asn Gln Glu Ser Pro Lys Ala Thr Gly Val Phe Thr 1070 1075 1080 Thr Leu Gln Pro Gly Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val 1085 1090 1095 Thr Glu Thr Thr Ile Val Ile Thr Trp Thr Pro Ala Pro Arg Ile 1100 1105 1110 Gly Phe Lys Leu Gly Val Arg Pro Ser Gln Gly Gly Glu Ala Pro 1115 1120 1125 Arg Glu Val Thr Ser Asp Ser Gly Ser Ile Val Val Ser Gly Leu 1130 1135 1140 Thr Pro Gly Val Glu Tyr Val Tyr Thr Ile Gln Val Leu Arg Asp 1145 1150 1155 Gly Gln Glu Arg Asp Ala Pro Ile Val Asn Lys Val Val Thr Pro 1160 1165 1170 Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr 1175 1180 1185 Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile 1190 1195 1200 Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly 1205 1210 1215 Asn Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr 1220 1225 1230 Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr 1235 1240 1245 Thr Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile 1250 1255 1260 Ile Pro Glu Val Pro Gln Leu Thr Asp Leu Ser Phe Val Asp Ile 1265 1270 1275 Thr Asp Ser Ser Ile Gly Leu Arg Trp Thr Pro Leu Asn Ser Ser 1280 1285 1290 Thr Ile Ile Gly Tyr Arg Ile Thr Val Val Ala Ala Gly Glu Gly 1295 1300 1305 Ile Pro Ile Phe Glu Asp Phe Val Asp Ser Ser Val Gly Tyr Tyr 1310 1315 1320 Thr Val Thr Gly Leu Glu Pro Gly Ile Asp Tyr Asp Ile Ser Val 1325 1330 1335 Ile Thr Leu Ile Asn Gly Gly Glu Ser Ala Pro Thr Thr Leu Thr 1340 1345 1350 Gln Gln Thr Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn 1355 1360 1365 Ile Gly Pro Asp Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser 1370 1375 1380 Ile Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser Pro Val Lys Asn 1385 1390 1395 Glu Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser Asp Asn Ala 1400 1405 1410 Val Val Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val Val Ser 1415 1420 1425 Val Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg Gly 1430 1435 1440 Arg Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser 1445 1450 1455 Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg 1460 1465 1470 Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe 1475 1480 1485 Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser 1490 1495 1500 Ile Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser 1505 1510 1515 Ile Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly 1520 1525 1530 Gln Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val 1535 1540 1545 Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala

1550 1555 1560 Val Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly 1565 1570 1575 Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr 1580 1585 1590 Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr 1595 1600 1605 Val Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys 1610 1615 1620 Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 1625 1630 1635 Met Gln Val Thr Asp Val Gln Asp Asn Ser Ile Ser Val Lys Trp 1640 1645 1650 Leu Pro Ser Ser Ser Pro Val Thr Gly Tyr Arg Val Thr Thr Thr 1655 1660 1665 Pro Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys Thr Ala Gly Pro 1670 1675 1680 Asp Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro Thr Val Glu 1685 1690 1695 Tyr Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu Ser Gln 1700 1705 1710 Pro Leu Val Gln Thr Ala Val Thr Asn Ile Asp Arg Pro Lys Gly 1715 1720 1725 Leu Ala Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile Ala Trp 1730 1735 1740 Glu Ser Pro Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser 1745 1750 1755 Ser Pro Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro Asp Gly 1760 1765 1770 Glu Glu Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu 1775 1780 1785 Tyr Thr Val Ser Val Val Ala Leu His Asp Asp Met Glu Ser Gln 1790 1795 1800 Pro Leu Ile Gly Thr Gln Ser Thr Ala Ile Pro Ala Pro Thr Asp 1805 1810 1815 Leu Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp 1820 1825 1830 Thr Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr 1835 1840 1845 Pro Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro 1850 1855 1860 Asp Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys 1865 1870 1875 Tyr Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg 1880 1885 1890 Pro Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro 1895 1900 1905 Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile 1910 1915 1920 Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp 1925 1930 1935 Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys 1940 1945 1950 Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr 1955 1960 1965 Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser 1970 1975 1980 Ser Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser 1985 1990 1995 Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser 2000 2005 2010 Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr 2015 2020 2025 Glu Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg 2030 2035 2040 Pro Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr 2045 2050 2055 Glu Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser 2060 2065 2070 Glu Pro Leu Ile Gly Arg Lys Lys Thr Asp Glu Leu Pro Gln Leu 2075 2080 2085 Val Thr Leu Pro His Pro Asn Leu His Gly Pro Glu Ile Leu Asp 2090 2095 2100 Val Pro Ser Thr Val Gln Lys Thr Pro Phe Val Thr His Pro Gly 2105 2110 2115 Tyr Asp Thr Gly Asn Gly Ile Gln Leu Pro Gly Thr Ser Gly Gln 2120 2125 2130 Gln Pro Ser Val Gly Gln Gln Met Ile Phe Glu Glu His Gly Phe 2135 2140 2145 Arg Arg Thr Thr Pro Pro Thr Thr Ala Thr Pro Ile Arg His Arg 2150 2155 2160 Pro Arg Pro Tyr Pro Pro Asn Val Gly Glu Glu Ile Gln Ile Gly 2165 2170 2175 His Ile Pro Arg Glu Asp Val Asp Tyr His Leu Tyr Pro His Gly 2180 2185 2190 Pro Gly Leu Asn Pro Asn Ala Ser Thr Gly Gln Glu Ala Leu Ser 2195 2200 2205 Gln Thr Thr Ile Ser Trp Ala Pro Phe Gln Asp Thr Ser Glu Tyr 2210 2215 2220 Ile Ile Ser Cys His Pro Val Gly Thr Asp Glu Glu Pro Leu Gln 2225 2230 2235 Phe Arg Val Pro Gly Thr Ser Thr Ser Ala Thr Leu Thr Gly Leu 2240 2245 2250 Thr Arg Gly Ala Thr Tyr Asn Ile Ile Val Glu Ala Leu Lys Asp 2255 2260 2265 Gln Gln Arg His Lys Val Arg Glu Glu Val Val Thr Val Gly Asn 2270 2275 2280 Ser Val Asn Glu Gly Leu Asn Gln Pro Thr Asp Asp Ser Cys Phe 2285 2290 2295 Asp Pro Tyr Thr Val Ser His Tyr Ala Val Gly Asp Glu Trp Glu 2300 2305 2310 Arg Met Ser Glu Ser Gly Phe Lys Leu Leu Cys Gln Cys Leu Gly 2315 2320 2325 Phe Gly Ser Gly His Phe Arg Cys Asp Ser Ser Arg Trp Cys His 2330 2335 2340 Asp Asn Gly Val Asn Tyr Lys Ile Gly Glu Lys Trp Asp Arg Gln 2345 2350 2355 Gly Glu Asn Gly Gln Met Met Ser Cys Thr Cys Leu Gly Asn Gly 2360 2365 2370 Lys Gly Glu Phe Lys Cys Asp Pro His Glu Ala Thr Cys Tyr Asp 2375 2380 2385 Asp Gly Lys Thr Tyr His Val Gly Glu Gln Trp Gln Lys Glu Tyr 2390 2395 2400 Leu Gly Ala Ile Cys Ser Cys Thr Cys Phe Gly Gly Gln Arg Gly 2405 2410 2415 Trp Arg Cys Asp Asn Cys Arg Arg Pro Gly Gly Glu Pro Ser Pro 2420 2425 2430 Glu Gly Thr Thr Gly Gln Ser Tyr Asn Gln Tyr Ser Gln Arg Tyr 2435 2440 2445 His Gln Arg Thr Asn Thr Asn Val Asn Cys Pro Ile Glu Cys Phe 2450 2455 2460 Met Pro Leu Asp Val Gln Ala Asp Arg Glu Asp Ser Arg Glu 2465 2470 2475 2262421PRTHomo sapiensFibronectin 1 isoform 2 226Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln 20 25 30 Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser 35 40 45 Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 50 55 60 Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly 65 70 75 80 Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr 85 90 95 Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr 100 105 110 Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala 115 120 125 Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly 130 135 140 Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr 145 150 155 160 Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu 165 170 175 Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly 180 185 190 Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp 195 200 205 Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 210 215 220 Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr 225 230 235 240 Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu 245 250 255 Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg 260 265 270 His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp 275 280 285 Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro 290 295 300 Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met 305 310 315 320 Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu 325 330 335 Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly 340 345 350 Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly 355 360 365 Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu 370 375 380 Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe 385 390 395 400 Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn 405 410 415 Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr 420 425 430 Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr 435 440 445 Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 450 455 460 Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile 465 470 475 480 Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys 485 490 495 Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser 500 505 510 Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn 515 520 525 Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr 530 535 540 Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln 545 550 555 560 Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp 565 570 575 Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg 580 585 590 Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser 595 600 605 Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 610 615 620 Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys 625 630 635 640 Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu 645 650 655 Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys 660 665 670 Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly 675 680 685 His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr 690 695 700 Pro Val Thr Ser Asn Thr Val Thr Gly Glu Thr Thr Pro Phe Ser Pro 705 710 715 720 Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser Ser Phe 725 730 735 Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe Arg Val 740 745 750 Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp Leu 755 760 765 Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly Arg 770 775 780 Lys Tyr Ile Val Asn Val Tyr Gln Ile Ser Glu Asp Gly Glu Gln Ser 785 790 795 800 Leu Ile Leu Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro Asp 805 810 815 Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp Ser 820 825 830 Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro Ser 835 840 845 Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn Ser 850 855 860 Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile 865 870 875 880 Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln Gln 885 890 895 Glu Thr Thr Gly Thr Pro Arg Ser Asp Thr Val Pro Ser Pro Arg Asp 900 905 910 Leu Gln Phe Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp Thr 915 920 925 Pro Pro Glu Ser Ala Val Thr Gly Tyr Arg Val Asp Val Ile Pro Val 930 935 940 Asn Leu Pro Gly Glu His Gly Gln Arg Leu Pro Ile Ser Arg Asn Thr 945 950 955 960 Phe Ala Glu Val Thr Gly Leu Ser Pro Gly Val Thr Tyr Tyr Phe Lys 965 970 975 Val Phe Ala Val Ser His Gly Arg Glu Ser Lys Pro Leu Thr Ala Gln 980 985 990 Gln Thr Thr Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu 995 1000 1005 Thr Asp Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln 1010 1015 1020 Ile Thr Gly Tyr Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln 1025 1030 1035 Pro Arg Gln Tyr Asn Val Gly Pro Ser Val Ser Lys Tyr Pro Leu 1040 1045 1050 Arg Asn Leu Gln Pro Ala Ser Glu Tyr Thr Val Ser Leu Val Ala 1055 1060 1065 Ile Lys Gly Asn Gln Glu Ser Pro Lys Ala Thr Gly Val Phe Thr 1070 1075 1080 Thr Leu Gln Pro Gly Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val 1085 1090 1095 Thr Glu Thr Thr Ile Val Ile Thr Trp Thr Pro Ala Pro Arg Ile 1100 1105 1110 Gly Phe Lys Leu Gly Val Arg Pro Ser Gln Gly Gly Glu Ala Pro 1115 1120 1125 Arg Glu Val Thr Ser Asp Ser Gly Ser Ile Val Val Ser Gly Leu 1130 1135 1140 Thr Pro Gly Val Glu Tyr Val Tyr Thr Ile Gln Val Leu Arg Asp 1145 1150 1155 Gly Gln Glu Arg Asp Ala Pro Ile Val Asn Lys Val Val Thr Pro 1160 1165 1170 Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr 1175 1180 1185 Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile 1190 1195 1200 Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly 1205 1210 1215 Asn Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr 1220 1225 1230 Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr 1235 1240 1245 Thr Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile 1250 1255 1260 Ile Pro Glu Val Pro Gln Leu Thr Asp Leu Ser Phe Val Asp Ile 1265 1270 1275 Thr Asp Ser Ser Ile Gly Leu Arg Trp Thr Pro Leu Asn Ser Ser 1280 1285 1290 Thr Ile Ile Gly Tyr Arg Ile Thr Val Val Ala Ala Gly Glu Gly 1295 1300 1305 Ile Pro Ile Phe Glu Asp Phe Val Asp Ser Ser Val Gly Tyr Tyr 1310

1315 1320 Thr Val Thr Gly Leu Glu Pro Gly Ile Asp Tyr Asp Ile Ser Val 1325 1330 1335 Ile Thr Leu Ile Asn Gly Gly Glu Ser Ala Pro Thr Thr Leu Thr 1340 1345 1350 Gln Gln Thr Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn 1355 1360 1365 Ile Gly Pro Asp Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser 1370 1375 1380 Ile Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser Pro Val Lys Asn 1385 1390 1395 Glu Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser Asp Asn Ala 1400 1405 1410 Val Val Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val Val Ser 1415 1420 1425 Val Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg Gly 1430 1435 1440 Arg Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser 1445 1450 1455 Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg 1460 1465 1470 Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe 1475 1480 1485 Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser 1490 1495 1500 Ile Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser 1505 1510 1515 Ile Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly 1520 1525 1530 Gln Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val 1535 1540 1545 Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala 1550 1555 1560 Val Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly 1565 1570 1575 Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr 1580 1585 1590 Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr 1595 1600 1605 Val Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys 1610 1615 1620 Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 1625 1630 1635 Met Gln Val Thr Asp Val Gln Asp Asn Ser Ile Ser Val Lys Trp 1640 1645 1650 Leu Pro Ser Ser Ser Pro Val Thr Gly Tyr Arg Val Thr Thr Thr 1655 1660 1665 Pro Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys Thr Ala Gly Pro 1670 1675 1680 Asp Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro Thr Val Glu 1685 1690 1695 Tyr Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu Ser Gln 1700 1705 1710 Pro Leu Val Gln Thr Ala Val Thr Asn Ile Asp Arg Pro Lys Gly 1715 1720 1725 Leu Ala Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile Ala Trp 1730 1735 1740 Glu Ser Pro Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser 1745 1750 1755 Ser Pro Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro Asp Gly 1760 1765 1770 Glu Glu Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu 1775 1780 1785 Tyr Thr Val Ser Val Val Ala Leu His Asp Asp Met Glu Ser Gln 1790 1795 1800 Pro Leu Ile Gly Thr Gln Ser Thr Ala Ile Pro Ala Pro Thr Asp 1805 1810 1815 Leu Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp 1820 1825 1830 Thr Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr 1835 1840 1845 Pro Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro 1850 1855 1860 Asp Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys 1865 1870 1875 Tyr Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg 1880 1885 1890 Pro Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro 1895 1900 1905 Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile 1910 1915 1920 Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp 1925 1930 1935 Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys 1940 1945 1950 Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr 1955 1960 1965 Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser 1970 1975 1980 Ser Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser 1985 1990 1995 Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser 2000 2005 2010 Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr 2015 2020 2025 Glu Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg 2030 2035 2040 Pro Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr 2045 2050 2055 Glu Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser 2060 2065 2070 Glu Pro Leu Ile Gly Arg Lys Lys Thr Val Gln Lys Thr Pro Phe 2075 2080 2085 Val Thr His Pro Gly Tyr Asp Thr Gly Asn Gly Ile Gln Leu Pro 2090 2095 2100 Gly Thr Ser Gly Gln Gln Pro Ser Val Gly Gln Gln Met Ile Phe 2105 2110 2115 Glu Glu His Gly Phe Arg Arg Thr Thr Pro Pro Thr Thr Ala Thr 2120 2125 2130 Pro Ile Arg His Arg Pro Arg Pro Tyr Pro Pro Asn Val Gly Gln 2135 2140 2145 Glu Ala Leu Ser Gln Thr Thr Ile Ser Trp Ala Pro Phe Gln Asp 2150 2155 2160 Thr Ser Glu Tyr Ile Ile Ser Cys His Pro Val Gly Thr Asp Glu 2165 2170 2175 Glu Pro Leu Gln Phe Arg Val Pro Gly Thr Ser Thr Ser Ala Thr 2180 2185 2190 Leu Thr Gly Leu Thr Arg Gly Ala Thr Tyr Asn Ile Ile Val Glu 2195 2200 2205 Ala Leu Lys Asp Gln Gln Arg His Lys Val Arg Glu Glu Val Val 2210 2215 2220 Thr Val Gly Asn Ser Val Asn Glu Gly Leu Asn Gln Pro Thr Asp 2225 2230 2235 Asp Ser Cys Phe Asp Pro Tyr Thr Val Ser His Tyr Ala Val Gly 2240 2245 2250 Asp Glu Trp Glu Arg Met Ser Glu Ser Gly Phe Lys Leu Leu Cys 2255 2260 2265 Gln Cys Leu Gly Phe Gly Ser Gly His Phe Arg Cys Asp Ser Ser 2270 2275 2280 Arg Trp Cys His Asp Asn Gly Val Asn Tyr Lys Ile Gly Glu Lys 2285 2290 2295 Trp Asp Arg Gln Gly Glu Asn Gly Gln Met Met Ser Cys Thr Cys 2300 2305 2310 Leu Gly Asn Gly Lys Gly Glu Phe Lys Cys Asp Pro His Glu Ala 2315 2320 2325 Thr Cys Tyr Asp Asp Gly Lys Thr Tyr His Val Gly Glu Gln Trp 2330 2335 2340 Gln Lys Glu Tyr Leu Gly Ala Ile Cys Ser Cys Thr Cys Phe Gly 2345 2350 2355 Gly Gln Arg Gly Trp Arg Cys Asp Asn Cys Arg Arg Pro Gly Gly 2360 2365 2370 Glu Pro Ser Pro Glu Gly Thr Thr Gly Gln Ser Tyr Asn Gln Tyr 2375 2380 2385 Ser Gln Arg Tyr His Gln Arg Thr Asn Thr Asn Val Asn Cys Pro 2390 2395 2400 Ile Glu Cys Phe Met Pro Leu Asp Val Gln Ala Asp Arg Glu Asp 2405 2410 2415 Ser Arg Glu 2420 2272355PRTHomo sapiensFibronectin 1 isoform 3 227Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln 20 25 30 Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser 35 40 45 Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 50 55 60 Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly 65 70 75 80 Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr 85 90 95 Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr 100 105 110 Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala 115 120 125 Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly 130 135 140 Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr 145 150 155 160 Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu 165 170 175 Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly 180 185 190 Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp 195 200 205 Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 210 215 220 Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr 225 230 235 240 Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu 245 250 255 Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg 260 265 270 His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp 275 280 285 Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro 290 295 300 Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met 305 310 315 320 Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu 325 330 335 Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly 340 345 350 Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly 355 360 365 Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu 370 375 380 Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe 385 390 395 400 Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn 405 410 415 Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr 420 425 430 Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr 435 440 445 Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 450 455 460 Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile 465 470 475 480 Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys 485 490 495 Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser 500 505 510 Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn 515 520 525 Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr 530 535 540 Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln 545 550 555 560 Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp 565 570 575 Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg 580 585 590 Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser 595 600 605 Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 610 615 620 Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys 625 630 635 640 Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu 645 650 655 Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys 660 665 670 Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly 675 680 685 His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr 690 695 700 Pro Val Thr Ser Asn Thr Val Thr Gly Glu Thr Thr Pro Phe Ser Pro 705 710 715 720 Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser Ser Phe 725 730 735 Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe Arg Val 740 745 750 Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp Leu 755 760 765 Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly Arg 770 775 780 Lys Tyr Ile Val Asn Val Tyr Gln Ile Ser Glu Asp Gly Glu Gln Ser 785 790 795 800 Leu Ile Leu Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro Asp 805 810 815 Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp Ser 820 825 830 Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro Ser 835 840 845 Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn Ser 850 855 860 Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile 865 870 875 880 Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln Gln 885 890 895 Glu Thr Thr Gly Thr Pro Arg Ser Asp Thr Val Pro Ser Pro Arg Asp 900 905 910 Leu Gln Phe Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp Thr 915 920 925 Pro Pro Glu Ser Ala Val Thr Gly Tyr Arg Val Asp Val Ile Pro Val 930 935 940 Asn Leu Pro Gly Glu His Gly Gln Arg Leu Pro Ile Ser Arg Asn Thr 945 950 955 960 Phe Ala Glu Val Thr Gly Leu Ser Pro Gly Val Thr Tyr Tyr Phe Lys 965 970 975 Val Phe Ala Val Ser His Gly Arg Glu Ser Lys Pro Leu Thr Ala Gln 980 985 990 Gln Thr Thr Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu 995 1000 1005 Thr Asp Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln 1010 1015 1020 Ile Thr Gly Tyr Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln 1025 1030 1035 Pro Arg Gln Tyr Asn Val Gly Pro Ser Val Ser Lys Tyr Pro Leu 1040 1045 1050 Arg Asn Leu Gln Pro Ala Ser Glu Tyr Thr Val Ser Leu Val Ala 1055 1060 1065 Ile Lys Gly Asn Gln Glu Ser Pro Lys Ala Thr Gly Val Phe Thr 1070 1075 1080 Thr Leu Gln Pro Gly Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val 1085 1090 1095 Thr Glu Thr Thr Ile Val Ile Thr Trp Thr Pro Ala Pro Arg Ile 1100 1105 1110 Gly Phe Lys Leu Gly Val Arg Pro Ser Gln Gly Gly Glu Ala Pro 1115 1120 1125 Arg Glu Val Thr Ser Asp Ser Gly Ser Ile

Val Val Ser Gly Leu 1130 1135 1140 Thr Pro Gly Val Glu Tyr Val Tyr Thr Ile Gln Val Leu Arg Asp 1145 1150 1155 Gly Gln Glu Arg Asp Ala Pro Ile Val Asn Lys Val Val Thr Pro 1160 1165 1170 Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr 1175 1180 1185 Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile 1190 1195 1200 Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly 1205 1210 1215 Asn Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr 1220 1225 1230 Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr 1235 1240 1245 Thr Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile 1250 1255 1260 Ile Pro Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile 1265 1270 1275 Gly Pro Asp Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile 1280 1285 1290 Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu 1295 1300 1305 Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser Asp Asn Ala Val 1310 1315 1320 Val Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val Val Ser Val 1325 1330 1335 Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg Gly Arg 1340 1345 1350 Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser Asp 1355 1360 1365 Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala 1370 1375 1380 Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe Ser 1385 1390 1395 Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile 1400 1405 1410 Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile 1415 1420 1425 Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln 1430 1435 1440 Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala 1445 1450 1455 Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val 1460 1465 1470 Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn 1475 1480 1485 Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala 1490 1495 1500 Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 1505 1510 1515 Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro 1520 1525 1530 Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln Met 1535 1540 1545 Gln Val Thr Asp Val Gln Asp Asn Ser Ile Ser Val Lys Trp Leu 1550 1555 1560 Pro Ser Ser Ser Pro Val Thr Gly Tyr Arg Val Thr Thr Thr Pro 1565 1570 1575 Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys Thr Ala Gly Pro Asp 1580 1585 1590 Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro Thr Val Glu Tyr 1595 1600 1605 Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu Ser Gln Pro 1610 1615 1620 Leu Val Gln Thr Ala Val Thr Asn Ile Asp Arg Pro Lys Gly Leu 1625 1630 1635 Ala Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile Ala Trp Glu 1640 1645 1650 Ser Pro Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser Ser 1655 1660 1665 Pro Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro Asp Gly Glu 1670 1675 1680 Glu Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu Tyr 1685 1690 1695 Thr Val Ser Val Val Ala Leu His Asp Asp Met Glu Ser Gln Pro 1700 1705 1710 Leu Ile Gly Thr Gln Ser Thr Ala Ile Pro Ala Pro Thr Asp Leu 1715 1720 1725 Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp Thr 1730 1735 1740 Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr Pro 1745 1750 1755 Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp 1760 1765 1770 Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys Tyr 1775 1780 1785 Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg Pro 1790 1795 1800 Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro Arg 1805 1810 1815 Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser 1820 1825 1830 Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala 1835 1840 1845 Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro 1850 1855 1860 Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp 1865 1870 1875 Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser 1880 1885 1890 Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn 1895 1900 1905 Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp 1910 1915 1920 Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu 1925 1930 1935 Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro 1940 1945 1950 Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu 1955 1960 1965 Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu 1970 1975 1980 Pro Leu Ile Gly Arg Lys Lys Thr Asp Glu Leu Pro Gln Leu Val 1985 1990 1995 Thr Leu Pro His Pro Asn Leu His Gly Pro Glu Ile Leu Asp Val 2000 2005 2010 Pro Ser Thr Val Gln Lys Thr Pro Phe Val Thr His Pro Gly Tyr 2015 2020 2025 Asp Thr Gly Asn Gly Ile Gln Leu Pro Gly Thr Ser Gly Gln Gln 2030 2035 2040 Pro Ser Val Gly Gln Gln Met Ile Phe Glu Glu His Gly Phe Arg 2045 2050 2055 Arg Thr Thr Pro Pro Thr Thr Ala Thr Pro Ile Arg His Arg Pro 2060 2065 2070 Arg Pro Tyr Pro Pro Asn Val Gly Gln Glu Ala Leu Ser Gln Thr 2075 2080 2085 Thr Ile Ser Trp Ala Pro Phe Gln Asp Thr Ser Glu Tyr Ile Ile 2090 2095 2100 Ser Cys His Pro Val Gly Thr Asp Glu Glu Pro Leu Gln Phe Arg 2105 2110 2115 Val Pro Gly Thr Ser Thr Ser Ala Thr Leu Thr Gly Leu Thr Arg 2120 2125 2130 Gly Ala Thr Tyr Asn Ile Ile Val Glu Ala Leu Lys Asp Gln Gln 2135 2140 2145 Arg His Lys Val Arg Glu Glu Val Val Thr Val Gly Asn Ser Val 2150 2155 2160 Asn Glu Gly Leu Asn Gln Pro Thr Asp Asp Ser Cys Phe Asp Pro 2165 2170 2175 Tyr Thr Val Ser His Tyr Ala Val Gly Asp Glu Trp Glu Arg Met 2180 2185 2190 Ser Glu Ser Gly Phe Lys Leu Leu Cys Gln Cys Leu Gly Phe Gly 2195 2200 2205 Ser Gly His Phe Arg Cys Asp Ser Ser Arg Trp Cys His Asp Asn 2210 2215 2220 Gly Val Asn Tyr Lys Ile Gly Glu Lys Trp Asp Arg Gln Gly Glu 2225 2230 2235 Asn Gly Gln Met Met Ser Cys Thr Cys Leu Gly Asn Gly Lys Gly 2240 2245 2250 Glu Phe Lys Cys Asp Pro His Glu Ala Thr Cys Tyr Asp Asp Gly 2255 2260 2265 Lys Thr Tyr His Val Gly Glu Gln Trp Gln Lys Glu Tyr Leu Gly 2270 2275 2280 Ala Ile Cys Ser Cys Thr Cys Phe Gly Gly Gln Arg Gly Trp Arg 2285 2290 2295 Cys Asp Asn Cys Arg Arg Pro Gly Gly Glu Pro Ser Pro Glu Gly 2300 2305 2310 Thr Thr Gly Gln Ser Tyr Asn Gln Tyr Ser Gln Arg Tyr His Gln 2315 2320 2325 Arg Thr Asn Thr Asn Val Asn Cys Pro Ile Glu Cys Phe Met Pro 2330 2335 2340 Leu Asp Val Gln Ala Asp Arg Glu Asp Ser Arg Glu 2345 2350 2355 2282330PRTHomo sapiensFibronectin 1 isoform 4 228Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln 20 25 30 Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser 35 40 45 Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 50 55 60 Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly 65 70 75 80 Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr 85 90 95 Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr 100 105 110 Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala 115 120 125 Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly 130 135 140 Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr 145 150 155 160 Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu 165 170 175 Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly 180 185 190 Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp 195 200 205 Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 210 215 220 Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr 225 230 235 240 Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu 245 250 255 Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg 260 265 270 His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp 275 280 285 Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro 290 295 300 Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met 305 310 315 320 Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu 325 330 335 Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly 340 345 350 Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly 355 360 365 Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu 370 375 380 Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe 385 390 395 400 Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn 405 410 415 Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr 420 425 430 Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr 435 440 445 Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 450 455 460 Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile 465 470 475 480 Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys 485 490 495 Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser 500 505 510 Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn 515 520 525 Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr 530 535 540 Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln 545 550 555 560 Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp 565 570 575 Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg 580 585 590 Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser 595 600 605 Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 610 615 620 Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys 625 630 635 640 Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu 645 650 655 Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys 660 665 670 Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly 675 680 685 His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr 690 695 700 Pro Val Thr Ser Asn Thr Val Thr Gly Glu Thr Thr Pro Phe Ser Pro 705 710 715 720 Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser Ser Phe 725 730 735 Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe Arg Val 740 745 750 Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp Leu 755 760 765 Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly Arg 770 775 780 Lys Tyr Ile Val Asn Val Tyr Gln Ile Ser Glu Asp Gly Glu Gln Ser 785 790 795 800 Leu Ile Leu Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro Asp 805 810 815 Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp Ser 820 825 830 Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro Ser 835 840 845 Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn Ser 850 855 860 Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile 865 870 875 880 Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln Gln 885 890 895 Glu Thr Thr Gly Thr Pro Arg Ser Asp Thr Val Pro Ser Pro Arg Asp 900 905 910 Leu Gln Phe Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp Thr 915 920 925 Pro Pro Glu Ser Ala Val Thr Gly Tyr Arg Val Asp Val Ile Pro Val 930 935 940 Asn Leu Pro Gly Glu His Gly Gln Arg Leu Pro Ile Ser Arg Asn Thr 945 950 955 960 Phe Ala Glu Val Thr Gly Leu Ser Pro Gly Val Thr Tyr Tyr Phe Lys 965 970 975 Val Phe Ala Val Ser His Gly Arg Glu Ser Lys Pro Leu Thr Ala Gln 980 985 990 Gln Thr Thr Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu 995 1000 1005 Thr Asp Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln

1010 1015 1020 Ile Thr Gly Tyr Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln 1025 1030 1035 Pro Arg Gln Tyr Asn Val Gly Pro Ser Val Ser Lys Tyr Pro Leu 1040 1045 1050 Arg Asn Leu Gln Pro Ala Ser Glu Tyr Thr Val Ser Leu Val Ala 1055 1060 1065 Ile Lys Gly Asn Gln Glu Ser Pro Lys Ala Thr Gly Val Phe Thr 1070 1075 1080 Thr Leu Gln Pro Gly Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val 1085 1090 1095 Thr Glu Thr Thr Ile Val Ile Thr Trp Thr Pro Ala Pro Arg Ile 1100 1105 1110 Gly Phe Lys Leu Gly Val Arg Pro Ser Gln Gly Gly Glu Ala Pro 1115 1120 1125 Arg Glu Val Thr Ser Asp Ser Gly Ser Ile Val Val Ser Gly Leu 1130 1135 1140 Thr Pro Gly Val Glu Tyr Val Tyr Thr Ile Gln Val Leu Arg Asp 1145 1150 1155 Gly Gln Glu Arg Asp Ala Pro Ile Val Asn Lys Val Val Thr Pro 1160 1165 1170 Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr 1175 1180 1185 Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile 1190 1195 1200 Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly 1205 1210 1215 Asn Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr 1220 1225 1230 Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr 1235 1240 1245 Thr Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile 1250 1255 1260 Ile Pro Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile 1265 1270 1275 Gly Pro Asp Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile 1280 1285 1290 Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu 1295 1300 1305 Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser Asp Asn Ala Val 1310 1315 1320 Val Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val Val Ser Val 1325 1330 1335 Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg Gly Arg 1340 1345 1350 Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser Asp 1355 1360 1365 Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala 1370 1375 1380 Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe Ser 1385 1390 1395 Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile 1400 1405 1410 Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile 1415 1420 1425 Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln 1430 1435 1440 Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala 1445 1450 1455 Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val 1460 1465 1470 Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn 1475 1480 1485 Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala 1490 1495 1500 Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 1505 1510 1515 Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro 1520 1525 1530 Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln Met 1535 1540 1545 Gln Val Thr Asp Val Gln Asp Asn Ser Ile Ser Val Lys Trp Leu 1550 1555 1560 Pro Ser Ser Ser Pro Val Thr Gly Tyr Arg Val Thr Thr Thr Pro 1565 1570 1575 Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys Thr Ala Gly Pro Asp 1580 1585 1590 Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro Thr Val Glu Tyr 1595 1600 1605 Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu Ser Gln Pro 1610 1615 1620 Leu Val Gln Thr Ala Val Thr Asn Ile Asp Arg Pro Lys Gly Leu 1625 1630 1635 Ala Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile Ala Trp Glu 1640 1645 1650 Ser Pro Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser Ser 1655 1660 1665 Pro Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro Asp Gly Glu 1670 1675 1680 Glu Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu Tyr 1685 1690 1695 Thr Val Ser Val Val Ala Leu His Asp Asp Met Glu Ser Gln Pro 1700 1705 1710 Leu Ile Gly Thr Gln Ser Thr Ala Ile Pro Ala Pro Thr Asp Leu 1715 1720 1725 Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp Thr 1730 1735 1740 Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr Pro 1745 1750 1755 Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp 1760 1765 1770 Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys Tyr 1775 1780 1785 Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg Pro 1790 1795 1800 Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro Arg 1805 1810 1815 Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser 1820 1825 1830 Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala 1835 1840 1845 Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro 1850 1855 1860 Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp 1865 1870 1875 Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser 1880 1885 1890 Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn 1895 1900 1905 Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp 1910 1915 1920 Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu 1925 1930 1935 Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro 1940 1945 1950 Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu 1955 1960 1965 Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu 1970 1975 1980 Pro Leu Ile Gly Arg Lys Lys Thr Val Gln Lys Thr Pro Phe Val 1985 1990 1995 Thr His Pro Gly Tyr Asp Thr Gly Asn Gly Ile Gln Leu Pro Gly 2000 2005 2010 Thr Ser Gly Gln Gln Pro Ser Val Gly Gln Gln Met Ile Phe Glu 2015 2020 2025 Glu His Gly Phe Arg Arg Thr Thr Pro Pro Thr Thr Ala Thr Pro 2030 2035 2040 Ile Arg His Arg Pro Arg Pro Tyr Pro Pro Asn Val Gly Gln Glu 2045 2050 2055 Ala Leu Ser Gln Thr Thr Ile Ser Trp Ala Pro Phe Gln Asp Thr 2060 2065 2070 Ser Glu Tyr Ile Ile Ser Cys His Pro Val Gly Thr Asp Glu Glu 2075 2080 2085 Pro Leu Gln Phe Arg Val Pro Gly Thr Ser Thr Ser Ala Thr Leu 2090 2095 2100 Thr Gly Leu Thr Arg Gly Ala Thr Tyr Asn Ile Ile Val Glu Ala 2105 2110 2115 Leu Lys Asp Gln Gln Arg His Lys Val Arg Glu Glu Val Val Thr 2120 2125 2130 Val Gly Asn Ser Val Asn Glu Gly Leu Asn Gln Pro Thr Asp Asp 2135 2140 2145 Ser Cys Phe Asp Pro Tyr Thr Val Ser His Tyr Ala Val Gly Asp 2150 2155 2160 Glu Trp Glu Arg Met Ser Glu Ser Gly Phe Lys Leu Leu Cys Gln 2165 2170 2175 Cys Leu Gly Phe Gly Ser Gly His Phe Arg Cys Asp Ser Ser Arg 2180 2185 2190 Trp Cys His Asp Asn Gly Val Asn Tyr Lys Ile Gly Glu Lys Trp 2195 2200 2205 Asp Arg Gln Gly Glu Asn Gly Gln Met Met Ser Cys Thr Cys Leu 2210 2215 2220 Gly Asn Gly Lys Gly Glu Phe Lys Cys Asp Pro His Glu Ala Thr 2225 2230 2235 Cys Tyr Asp Asp Gly Lys Thr Tyr His Val Gly Glu Gln Trp Gln 2240 2245 2250 Lys Glu Tyr Leu Gly Ala Ile Cys Ser Cys Thr Cys Phe Gly Gly 2255 2260 2265 Gln Arg Gly Trp Arg Cys Asp Asn Cys Arg Arg Pro Gly Gly Glu 2270 2275 2280 Pro Ser Pro Glu Gly Thr Thr Gly Gln Ser Tyr Asn Gln Tyr Ser 2285 2290 2295 Gln Arg Tyr His Gln Arg Thr Asn Thr Asn Val Asn Cys Pro Ile 2300 2305 2310 Glu Cys Phe Met Pro Leu Asp Val Gln Ala Asp Arg Glu Asp Ser 2315 2320 2325 Arg Glu 2330 2292296PRTHomo sapiensFibronectin 1 isoform 5 229Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln 20 25 30 Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser 35 40 45 Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 50 55 60 Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly 65 70 75 80 Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr 85 90 95 Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr 100 105 110 Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala 115 120 125 Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly 130 135 140 Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr 145 150 155 160 Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu 165 170 175 Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly 180 185 190 Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp 195 200 205 Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 210 215 220 Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr 225 230 235 240 Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu 245 250 255 Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg 260 265 270 His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp 275 280 285 Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro 290 295 300 Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met 305 310 315 320 Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu 325 330 335 Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly 340 345 350 Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly 355 360 365 Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu 370 375 380 Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe 385 390 395 400 Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn 405 410 415 Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr 420 425 430 Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr 435 440 445 Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 450 455 460 Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile 465 470 475 480 Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys 485 490 495 Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser 500 505 510 Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn 515 520 525 Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr 530 535 540 Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln 545 550 555 560 Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp 565 570 575 Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg 580 585 590 Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser 595 600 605 Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 610 615 620 Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys 625 630 635 640 Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu 645 650 655 Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys 660 665 670 Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly 675 680 685 His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr 690 695 700 Pro Val Thr Ser Asn Thr Val Thr Gly Glu Thr Thr Pro Phe Ser Pro 705 710 715 720 Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser Ser Phe 725 730 735 Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe Arg Val 740 745 750 Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp Leu 755 760 765 Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly Arg 770 775 780 Lys Tyr Ile Val Asn Val Tyr Gln Ile Ser Glu Asp Gly Glu Gln Ser 785 790 795 800 Leu Ile Leu Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro Asp 805 810 815 Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp Ser 820 825 830 Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro Ser 835 840 845 Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn Ser 850 855 860 Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile 865 870 875 880 Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln Gln 885 890 895 Glu Thr Thr Gly Thr Pro Arg Ser Asp Thr Val Pro Ser Pro Arg Asp 900 905 910 Leu Gln Phe Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp

Thr 915 920 925 Pro Pro Glu Ser Ala Val Thr Gly Tyr Arg Val Asp Val Ile Pro Val 930 935 940 Asn Leu Pro Gly Glu His Gly Gln Arg Leu Pro Ile Ser Arg Asn Thr 945 950 955 960 Phe Ala Glu Val Thr Gly Leu Ser Pro Gly Val Thr Tyr Tyr Phe Lys 965 970 975 Val Phe Ala Val Ser His Gly Arg Glu Ser Lys Pro Leu Thr Ala Gln 980 985 990 Gln Thr Thr Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu 995 1000 1005 Thr Asp Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln 1010 1015 1020 Ile Thr Gly Tyr Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln 1025 1030 1035 Pro Arg Gln Tyr Asn Val Gly Pro Ser Val Ser Lys Tyr Pro Leu 1040 1045 1050 Arg Asn Leu Gln Pro Ala Ser Glu Tyr Thr Val Ser Leu Val Ala 1055 1060 1065 Ile Lys Gly Asn Gln Glu Ser Pro Lys Ala Thr Gly Val Phe Thr 1070 1075 1080 Thr Leu Gln Pro Gly Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val 1085 1090 1095 Thr Glu Thr Thr Ile Val Ile Thr Trp Thr Pro Ala Pro Arg Ile 1100 1105 1110 Gly Phe Lys Leu Gly Val Arg Pro Ser Gln Gly Gly Glu Ala Pro 1115 1120 1125 Arg Glu Val Thr Ser Asp Ser Gly Ser Ile Val Val Ser Gly Leu 1130 1135 1140 Thr Pro Gly Val Glu Tyr Val Tyr Thr Ile Gln Val Leu Arg Asp 1145 1150 1155 Gly Gln Glu Arg Asp Ala Pro Ile Val Asn Lys Val Val Thr Pro 1160 1165 1170 Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr 1175 1180 1185 Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile 1190 1195 1200 Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly 1205 1210 1215 Asn Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr 1220 1225 1230 Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr 1235 1240 1245 Thr Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile 1250 1255 1260 Ile Pro Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile 1265 1270 1275 Gly Pro Asp Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile 1280 1285 1290 Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu 1295 1300 1305 Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser Asp Asn Ala Val 1310 1315 1320 Val Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val Val Ser Val 1325 1330 1335 Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg Gly Arg 1340 1345 1350 Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser Asp 1355 1360 1365 Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala 1370 1375 1380 Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe Ser 1385 1390 1395 Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile 1400 1405 1410 Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile 1415 1420 1425 Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln 1430 1435 1440 Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala 1445 1450 1455 Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val 1460 1465 1470 Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn 1475 1480 1485 Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala 1490 1495 1500 Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 1505 1510 1515 Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro 1520 1525 1530 Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln Met 1535 1540 1545 Gln Val Thr Asp Val Gln Asp Asn Ser Ile Ser Val Lys Trp Leu 1550 1555 1560 Pro Ser Ser Ser Pro Val Thr Gly Tyr Arg Val Thr Thr Thr Pro 1565 1570 1575 Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys Thr Ala Gly Pro Asp 1580 1585 1590 Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro Thr Val Glu Tyr 1595 1600 1605 Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu Ser Gln Pro 1610 1615 1620 Leu Val Gln Thr Ala Val Thr Thr Ile Pro Ala Pro Thr Asp Leu 1625 1630 1635 Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp Thr 1640 1645 1650 Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr Pro 1655 1660 1665 Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp 1670 1675 1680 Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys Tyr 1685 1690 1695 Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg Pro 1700 1705 1710 Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro Arg 1715 1720 1725 Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser 1730 1735 1740 Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala 1745 1750 1755 Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro 1760 1765 1770 Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp 1775 1780 1785 Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser 1790 1795 1800 Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn 1805 1810 1815 Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp 1820 1825 1830 Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu 1835 1840 1845 Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro 1850 1855 1860 Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu 1865 1870 1875 Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu 1880 1885 1890 Pro Leu Ile Gly Arg Lys Lys Thr Asp Glu Leu Pro Gln Leu Val 1895 1900 1905 Thr Leu Pro His Pro Asn Leu His Gly Pro Glu Ile Leu Asp Val 1910 1915 1920 Pro Ser Thr Val Gln Lys Thr Pro Phe Val Thr His Pro Gly Tyr 1925 1930 1935 Asp Thr Gly Asn Gly Ile Gln Leu Pro Gly Thr Ser Gly Gln Gln 1940 1945 1950 Pro Ser Val Gly Gln Gln Met Ile Phe Glu Glu His Gly Phe Arg 1955 1960 1965 Arg Thr Thr Pro Pro Thr Thr Ala Thr Pro Ile Arg His Arg Pro 1970 1975 1980 Arg Pro Tyr Pro Pro Asn Val Gly Glu Glu Ile Gln Ile Gly His 1985 1990 1995 Ile Pro Arg Glu Asp Val Asp Tyr His Leu Tyr Pro His Gly Pro 2000 2005 2010 Gly Leu Asn Pro Asn Ala Ser Thr Gly Gln Glu Ala Leu Ser Gln 2015 2020 2025 Thr Thr Ile Ser Trp Ala Pro Phe Gln Asp Thr Ser Glu Tyr Ile 2030 2035 2040 Ile Ser Cys His Pro Val Gly Thr Asp Glu Glu Pro Leu Gln Phe 2045 2050 2055 Arg Val Pro Gly Thr Ser Thr Ser Ala Thr Leu Thr Gly Leu Thr 2060 2065 2070 Arg Gly Ala Thr Tyr Asn Ile Ile Val Glu Ala Leu Lys Asp Gln 2075 2080 2085 Gln Arg His Lys Val Arg Glu Glu Val Val Thr Val Gly Asn Ser 2090 2095 2100 Val Asn Glu Gly Leu Asn Gln Pro Thr Asp Asp Ser Cys Phe Asp 2105 2110 2115 Pro Tyr Thr Val Ser His Tyr Ala Val Gly Asp Glu Trp Glu Arg 2120 2125 2130 Met Ser Glu Ser Gly Phe Lys Leu Leu Cys Gln Cys Leu Gly Phe 2135 2140 2145 Gly Ser Gly His Phe Arg Cys Asp Ser Ser Arg Trp Cys His Asp 2150 2155 2160 Asn Gly Val Asn Tyr Lys Ile Gly Glu Lys Trp Asp Arg Gln Gly 2165 2170 2175 Glu Asn Gly Gln Met Met Ser Cys Thr Cys Leu Gly Asn Gly Lys 2180 2185 2190 Gly Glu Phe Lys Cys Asp Pro His Glu Ala Thr Cys Tyr Asp Asp 2195 2200 2205 Gly Lys Thr Tyr His Val Gly Glu Gln Trp Gln Lys Glu Tyr Leu 2210 2215 2220 Gly Ala Ile Cys Ser Cys Thr Cys Phe Gly Gly Gln Arg Gly Trp 2225 2230 2235 Arg Cys Asp Asn Cys Arg Arg Pro Gly Gly Glu Pro Ser Pro Glu 2240 2245 2250 Gly Thr Thr Gly Gln Ser Tyr Asn Gln Tyr Ser Gln Arg Tyr His 2255 2260 2265 Gln Arg Thr Asn Thr Asn Val Asn Cys Pro Ile Glu Cys Phe Met 2270 2275 2280 Pro Leu Asp Val Gln Ala Asp Arg Glu Asp Ser Arg Glu 2285 2290 2295 2302176PRTHomo sapiensFibronectin 1 isoform 6 230Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln 20 25 30 Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser 35 40 45 Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 50 55 60 Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly 65 70 75 80 Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr 85 90 95 Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr 100 105 110 Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala 115 120 125 Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly 130 135 140 Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr 145 150 155 160 Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu 165 170 175 Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly 180 185 190 Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp 195 200 205 Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 210 215 220 Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr 225 230 235 240 Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu 245 250 255 Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg 260 265 270 His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp 275 280 285 Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro 290 295 300 Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met 305 310 315 320 Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu 325 330 335 Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly 340 345 350 Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly 355 360 365 Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu 370 375 380 Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe 385 390 395 400 Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn 405 410 415 Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr 420 425 430 Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr 435 440 445 Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 450 455 460 Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile 465 470 475 480 Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys 485 490 495 Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser 500 505 510 Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn 515 520 525 Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr 530 535 540 Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln 545 550 555 560 Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp 565 570 575 Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg 580 585 590 Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser 595 600 605 Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 610 615 620 Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys 625 630 635 640 Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu 645 650 655 Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys 660 665 670 Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly 675 680 685 His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr 690 695 700 Pro Val Thr Ser Asn Thr Val Thr Gly Glu Thr Thr Pro Phe Ser Pro 705 710 715 720 Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser Ser Phe 725 730 735 Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe Arg Val 740 745 750 Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp Leu 755 760 765 Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly Arg 770 775 780 Lys Tyr Ile Val Asn Val Tyr Gln Ile Ser Glu Asp Gly Glu Gln Ser 785 790 795 800 Leu Ile Leu Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro Asp 805 810 815 Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp Ser 820 825 830 Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro Ser 835 840 845 Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn Ser 850

855 860 Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile 865 870 875 880 Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln Gln 885 890 895 Glu Thr Thr Gly Thr Pro Arg Ser Asp Thr Val Pro Ser Pro Arg Asp 900 905 910 Leu Gln Phe Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp Thr 915 920 925 Pro Pro Glu Ser Ala Val Thr Gly Tyr Arg Val Asp Val Ile Pro Val 930 935 940 Asn Leu Pro Gly Glu His Gly Gln Arg Leu Pro Ile Ser Arg Asn Thr 945 950 955 960 Phe Ala Glu Val Thr Gly Leu Ser Pro Gly Val Thr Tyr Tyr Phe Lys 965 970 975 Val Phe Ala Val Ser His Gly Arg Glu Ser Lys Pro Leu Thr Ala Gln 980 985 990 Gln Thr Thr Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu 995 1000 1005 Thr Asp Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln 1010 1015 1020 Ile Thr Gly Tyr Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln 1025 1030 1035 Pro Arg Gln Tyr Asn Val Gly Pro Ser Val Ser Lys Tyr Pro Leu 1040 1045 1050 Arg Asn Leu Gln Pro Ala Ser Glu Tyr Thr Val Ser Leu Val Ala 1055 1060 1065 Ile Lys Gly Asn Gln Glu Ser Pro Lys Ala Thr Gly Val Phe Thr 1070 1075 1080 Thr Leu Gln Pro Gly Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val 1085 1090 1095 Thr Glu Thr Thr Ile Val Ile Thr Trp Thr Pro Ala Pro Arg Ile 1100 1105 1110 Gly Phe Lys Leu Gly Val Arg Pro Ser Gln Gly Gly Glu Ala Pro 1115 1120 1125 Arg Glu Val Thr Ser Asp Ser Gly Ser Ile Val Val Ser Gly Leu 1130 1135 1140 Thr Pro Gly Val Glu Tyr Val Tyr Thr Ile Gln Val Leu Arg Asp 1145 1150 1155 Gly Gln Glu Arg Asp Ala Pro Ile Val Asn Lys Val Val Thr Pro 1160 1165 1170 Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr 1175 1180 1185 Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile 1190 1195 1200 Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly 1205 1210 1215 Asn Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr 1220 1225 1230 Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr 1235 1240 1245 Thr Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile 1250 1255 1260 Ile Pro Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile 1265 1270 1275 Gly Pro Asp Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile 1280 1285 1290 Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu 1295 1300 1305 Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser Asp Asn Ala Val 1310 1315 1320 Val Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val Val Ser Val 1325 1330 1335 Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg Gly Arg 1340 1345 1350 Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser Asp 1355 1360 1365 Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala 1370 1375 1380 Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe Ser 1385 1390 1395 Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile 1400 1405 1410 Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile 1415 1420 1425 Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln 1430 1435 1440 Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala 1445 1450 1455 Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val 1460 1465 1470 Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn 1475 1480 1485 Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala 1490 1495 1500 Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 1505 1510 1515 Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro 1520 1525 1530 Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln Met 1535 1540 1545 Gln Val Thr Asp Val Gln Asp Asn Ser Ile Ser Val Lys Trp Leu 1550 1555 1560 Pro Ser Ser Ser Pro Val Thr Gly Tyr Arg Val Thr Thr Thr Pro 1565 1570 1575 Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys Thr Ala Gly Pro Asp 1580 1585 1590 Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro Thr Val Glu Tyr 1595 1600 1605 Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu Ser Gln Pro 1610 1615 1620 Leu Val Gln Thr Ala Val Thr Thr Ile Pro Ala Pro Thr Asp Leu 1625 1630 1635 Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp Thr 1640 1645 1650 Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr Pro 1655 1660 1665 Lys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp 1670 1675 1680 Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr Lys Tyr 1685 1690 1695 Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg Pro 1700 1705 1710 Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro Arg 1715 1720 1725 Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser 1730 1735 1740 Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala 1745 1750 1755 Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro 1760 1765 1770 Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp 1775 1780 1785 Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser 1790 1795 1800 Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn 1805 1810 1815 Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp 1820 1825 1830 Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu 1835 1840 1845 Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro 1850 1855 1860 Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu 1865 1870 1875 Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu 1880 1885 1890 Pro Leu Ile Gly Arg Lys Lys Thr Gly Gln Glu Ala Leu Ser Gln 1895 1900 1905 Thr Thr Ile Ser Trp Ala Pro Phe Gln Asp Thr Ser Glu Tyr Ile 1910 1915 1920 Ile Ser Cys His Pro Val Gly Thr Asp Glu Glu Pro Leu Gln Phe 1925 1930 1935 Arg Val Pro Gly Thr Ser Thr Ser Ala Thr Leu Thr Gly Leu Thr 1940 1945 1950 Arg Gly Ala Thr Tyr Asn Ile Ile Val Glu Ala Leu Lys Asp Gln 1955 1960 1965 Gln Arg His Lys Val Arg Glu Glu Val Val Thr Val Gly Asn Ser 1970 1975 1980 Val Asn Glu Gly Leu Asn Gln Pro Thr Asp Asp Ser Cys Phe Asp 1985 1990 1995 Pro Tyr Thr Val Ser His Tyr Ala Val Gly Asp Glu Trp Glu Arg 2000 2005 2010 Met Ser Glu Ser Gly Phe Lys Leu Leu Cys Gln Cys Leu Gly Phe 2015 2020 2025 Gly Ser Gly His Phe Arg Cys Asp Ser Ser Arg Trp Cys His Asp 2030 2035 2040 Asn Gly Val Asn Tyr Lys Ile Gly Glu Lys Trp Asp Arg Gln Gly 2045 2050 2055 Glu Asn Gly Gln Met Met Ser Cys Thr Cys Leu Gly Asn Gly Lys 2060 2065 2070 Gly Glu Phe Lys Cys Asp Pro His Glu Ala Thr Cys Tyr Asp Asp 2075 2080 2085 Gly Lys Thr Tyr His Val Gly Glu Gln Trp Gln Lys Glu Tyr Leu 2090 2095 2100 Gly Ala Ile Cys Ser Cys Thr Cys Phe Gly Gly Gln Arg Gly Trp 2105 2110 2115 Arg Cys Asp Asn Cys Arg Arg Pro Gly Gly Glu Pro Ser Pro Glu 2120 2125 2130 Gly Thr Thr Gly Gln Ser Tyr Asn Gln Tyr Ser Gln Arg Tyr His 2135 2140 2145 Gln Arg Thr Asn Thr Asn Val Asn Cys Pro Ile Glu Cys Phe Met 2150 2155 2160 Pro Leu Asp Val Gln Ala Asp Arg Glu Asp Ser Arg Glu 2165 2170 2175 231657PRTHomo sapiensFibronectin 1 isoform 7 231Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys 1 5 10 15 Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln 20 25 30 Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser 35 40 45 Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 50 55 60 Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly 65 70 75 80 Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr 85 90 95 Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr 100 105 110 Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala 115 120 125 Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly 130 135 140 Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr 145 150 155 160 Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu 165 170 175 Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly 180 185 190 Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp 195 200 205 Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 210 215 220 Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr 225 230 235 240 Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu 245 250 255 Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg 260 265 270 His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp 275 280 285 Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro 290 295 300 Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met 305 310 315 320 Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu 325 330 335 Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly 340 345 350 Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly 355 360 365 Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu 370 375 380 Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe 385 390 395 400 Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn 405 410 415 Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr 420 425 430 Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr 435 440 445 Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 450 455 460 Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile 465 470 475 480 Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys 485 490 495 Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser 500 505 510 Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn 515 520 525 Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr 530 535 540 Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln 545 550 555 560 Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp 565 570 575 Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg 580 585 590 Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser 595 600 605 Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 610 615 620 Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys 625 630 635 640 Tyr Ile Leu Arg Trp Arg Pro Val Ser Ile Pro Pro Arg Asn Leu Gly 645 650 655 Tyr 232278PRTHomo sapiensinsulin-like growth factor-binding protein-like 1 232Met Pro Arg Leu Ser Leu Leu Leu Pro Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 Pro Leu Leu Pro Pro Leu Ser Pro Ser Leu Gly Ile Arg Asp Val Gly 20 25 30 Gly Arg Arg Pro Lys Cys Gly Pro Cys Arg Pro Glu Gly Cys Pro Ala 35 40 45 Pro Ala Pro Cys Pro Ala Pro Gly Ile Ser Ala Leu Asp Glu Cys Gly 50 55 60 Cys Cys Ala Arg Cys Leu Gly Ala Glu Gly Ala Ser Cys Gly Gly Arg 65 70 75 80 Ala Gly Gly Arg Cys Gly Pro Gly Leu Val Cys Ala Ser Gln Ala Ala 85 90 95 Gly Ala Ala Pro Glu Gly Thr Gly Leu Cys Val Cys Ala Gln Arg Gly 100 105 110 Thr Val Cys Gly Ser Asp Gly Arg Ser Tyr Pro Ser Val Cys Ala Leu 115 120 125 Arg Leu Arg Ala Arg His Thr Pro Arg Ala His Pro Gly His Leu His 130 135 140 Lys Ala Arg Asp Gly Pro Cys Glu Phe Ala Pro Val Val Val Val Pro 145 150 155 160 Pro Arg Ser Val His Asn Val Thr Gly Ala Gln Val Gly Leu Ser Cys 165 170 175 Glu Val Arg Ala Val Pro Thr Pro Val Ile Thr Trp Arg Lys Val Thr 180 185 190 Lys Ser Pro Glu Gly Thr Gln Ala Leu Glu Glu Leu Pro Gly Asp His 195 200 205 Val Asn Ile Ala Val Gln Val Arg Gly Gly Pro Ser Asp His Glu Ala 210 215 220 Thr Ala Trp Ile Leu Ile Asn Pro Leu Arg Lys Glu Asp Glu Gly Val 225 230 235 240 Tyr Gln Cys His Ala Ala Asn Met Val Gly Glu Ala Glu Ser His Ser 245 250

255 Thr Val Thr Val Leu Asp Leu Ser Lys Tyr Arg Ser Phe His Phe Pro 260 265 270 Ala Pro Asp Asp Arg Met 275 233848PRTHomo sapiensNCAM1 isoform 1 233Met Leu Gln Thr Lys Asp Leu Ile Trp Thr Leu Phe Phe Leu Gly Thr 1 5 10 15 Ala Val Ser Leu Gln Val Asp Ile Val Pro Ser Gln Gly Glu Ile Ser 20 25 30 Val Gly Glu Ser Lys Phe Phe Leu Cys Gln Val Ala Gly Asp Ala Lys 35 40 45 Asp Lys Asp Ile Ser Trp Phe Ser Pro Asn Gly Glu Lys Leu Thr Pro 50 55 60 Asn Gln Gln Arg Ile Ser Val Val Trp Asn Asp Asp Ser Ser Ser Thr 65 70 75 80 Leu Thr Ile Tyr Asn Ala Asn Ile Asp Asp Ala Gly Ile Tyr Lys Cys 85 90 95 Val Val Thr Gly Glu Asp Gly Ser Glu Ser Glu Ala Thr Val Asn Val 100 105 110 Lys Ile Phe Gln Lys Leu Met Phe Lys Asn Ala Pro Thr Pro Gln Glu 115 120 125 Phe Arg Glu Gly Glu Asp Ala Val Ile Val Cys Asp Val Val Ser Ser 130 135 140 Leu Pro Pro Thr Ile Ile Trp Lys His Lys Gly Arg Asp Val Ile Leu 145 150 155 160 Lys Lys Asp Val Arg Phe Ile Val Leu Ser Asn Asn Tyr Leu Gln Ile 165 170 175 Arg Gly Ile Lys Lys Thr Asp Glu Gly Thr Tyr Arg Cys Glu Gly Arg 180 185 190 Ile Leu Ala Arg Gly Glu Ile Asn Phe Lys Asp Ile Gln Val Ile Val 195 200 205 Asn Val Pro Pro Thr Ile Gln Ala Arg Gln Asn Ile Val Asn Ala Thr 210 215 220 Ala Asn Leu Gly Gln Ser Val Thr Leu Val Cys Asp Ala Glu Gly Phe 225 230 235 240 Pro Glu Pro Thr Met Ser Trp Thr Lys Asp Gly Glu Gln Ile Glu Gln 245 250 255 Glu Glu Asp Asp Glu Lys Tyr Ile Phe Ser Asp Asp Ser Ser Gln Leu 260 265 270 Thr Ile Lys Lys Val Asp Lys Asn Asp Glu Ala Glu Tyr Ile Cys Ile 275 280 285 Ala Glu Asn Lys Ala Gly Glu Gln Asp Ala Thr Ile His Leu Lys Val 290 295 300 Phe Ala Lys Pro Lys Ile Thr Tyr Val Glu Asn Gln Thr Ala Met Glu 305 310 315 320 Leu Glu Glu Gln Val Thr Leu Thr Cys Glu Ala Ser Gly Asp Pro Ile 325 330 335 Pro Ser Ile Thr Trp Arg Thr Ser Thr Arg Asn Ile Ser Ser Glu Glu 340 345 350 Lys Thr Leu Asp Gly His Met Val Val Arg Ser His Ala Arg Val Ser 355 360 365 Ser Leu Thr Leu Lys Ser Ile Gln Tyr Thr Asp Ala Gly Glu Tyr Ile 370 375 380 Cys Thr Ala Ser Asn Thr Ile Gly Gln Asp Ser Gln Ser Met Tyr Leu 385 390 395 400 Glu Val Gln Tyr Ala Pro Lys Leu Gln Gly Pro Val Ala Val Tyr Thr 405 410 415 Trp Glu Gly Asn Gln Val Asn Ile Thr Cys Glu Val Phe Ala Tyr Pro 420 425 430 Ser Ala Thr Ile Ser Trp Phe Arg Asp Gly Gln Leu Leu Pro Ser Ser 435 440 445 Asn Tyr Ser Asn Ile Lys Ile Tyr Asn Thr Pro Ser Ala Ser Tyr Leu 450 455 460 Glu Val Thr Pro Asp Ser Glu Asn Asp Phe Gly Asn Tyr Asn Cys Thr 465 470 475 480 Ala Val Asn Arg Ile Gly Gln Glu Ser Leu Glu Phe Ile Leu Val Gln 485 490 495 Ala Asp Thr Pro Ser Ser Pro Ser Ile Asp Gln Val Glu Pro Tyr Ser 500 505 510 Ser Thr Ala Gln Val Gln Phe Asp Glu Pro Glu Ala Thr Gly Gly Val 515 520 525 Pro Ile Leu Lys Tyr Lys Ala Glu Trp Arg Ala Val Gly Glu Glu Val 530 535 540 Trp His Ser Lys Trp Tyr Asp Ala Lys Glu Ala Ser Met Glu Gly Ile 545 550 555 560 Val Thr Ile Val Gly Leu Lys Pro Glu Thr Thr Tyr Ala Val Arg Leu 565 570 575 Ala Ala Leu Asn Gly Lys Gly Leu Gly Glu Ile Ser Ala Ala Ser Glu 580 585 590 Phe Lys Thr Gln Pro Val Gln Gly Glu Pro Ser Ala Pro Lys Leu Glu 595 600 605 Gly Gln Met Gly Glu Asp Gly Asn Ser Ile Lys Val Asn Leu Ile Lys 610 615 620 Gln Asp Asp Gly Gly Ser Pro Ile Arg His Tyr Leu Val Arg Tyr Arg 625 630 635 640 Ala Leu Ser Ser Glu Trp Lys Pro Glu Ile Arg Leu Pro Ser Gly Ser 645 650 655 Asp His Val Met Leu Lys Ser Leu Asp Trp Asn Ala Glu Tyr Glu Val 660 665 670 Tyr Val Val Ala Glu Asn Gln Gln Gly Lys Ser Lys Ala Ala His Phe 675 680 685 Val Phe Arg Thr Ser Ala Gln Pro Thr Ala Ile Pro Ala Asn Gly Ser 690 695 700 Pro Thr Ser Gly Leu Ser Thr Gly Ala Ile Val Gly Ile Leu Ile Val 705 710 715 720 Ile Phe Val Leu Leu Leu Val Val Val Asp Ile Thr Cys Tyr Phe Leu 725 730 735 Asn Lys Cys Gly Leu Phe Met Cys Ile Ala Val Asn Leu Cys Gly Lys 740 745 750 Ala Gly Pro Gly Ala Lys Gly Lys Asp Met Glu Glu Gly Lys Ala Ala 755 760 765 Phe Ser Lys Asp Glu Ser Lys Glu Pro Ile Val Glu Val Arg Thr Glu 770 775 780 Glu Glu Arg Thr Pro Asn His Asp Gly Gly Lys His Thr Glu Pro Asn 785 790 795 800 Glu Thr Thr Pro Leu Thr Glu Pro Glu Lys Gly Pro Val Glu Ala Lys 805 810 815 Pro Glu Cys Gln Glu Thr Glu Thr Lys Pro Ala Pro Ala Glu Val Lys 820 825 830 Thr Val Pro Asn Asp Ala Thr Gln Thr Lys Glu Asn Glu Ser Lys Ala 835 840 845 234858PRTHomo sapiensNCAM1 isoform 2 234Met Leu Gln Thr Lys Asp Leu Ile Trp Thr Leu Phe Phe Leu Gly Thr 1 5 10 15 Ala Val Ser Leu Gln Val Asp Ile Val Pro Ser Gln Gly Glu Ile Ser 20 25 30 Val Gly Glu Ser Lys Phe Phe Leu Cys Gln Val Ala Gly Asp Ala Lys 35 40 45 Asp Lys Asp Ile Ser Trp Phe Ser Pro Asn Gly Glu Lys Leu Thr Pro 50 55 60 Asn Gln Gln Arg Ile Ser Val Val Trp Asn Asp Asp Ser Ser Ser Thr 65 70 75 80 Leu Thr Ile Tyr Asn Ala Asn Ile Asp Asp Ala Gly Ile Tyr Lys Cys 85 90 95 Val Val Thr Gly Glu Asp Gly Ser Glu Ser Glu Ala Thr Val Asn Val 100 105 110 Lys Ile Phe Gln Lys Leu Met Phe Lys Asn Ala Pro Thr Pro Gln Glu 115 120 125 Phe Arg Glu Gly Glu Asp Ala Val Ile Val Cys Asp Val Val Ser Ser 130 135 140 Leu Pro Pro Thr Ile Ile Trp Lys His Lys Gly Arg Asp Val Ile Leu 145 150 155 160 Lys Lys Asp Val Arg Phe Ile Val Leu Ser Asn Asn Tyr Leu Gln Ile 165 170 175 Arg Gly Ile Lys Lys Thr Asp Glu Gly Thr Tyr Arg Cys Glu Gly Arg 180 185 190 Ile Leu Ala Arg Gly Glu Ile Asn Phe Lys Asp Ile Gln Val Ile Val 195 200 205 Asn Val Pro Pro Thr Ile Gln Ala Arg Gln Asn Ile Val Asn Ala Thr 210 215 220 Ala Asn Leu Gly Gln Ser Val Thr Leu Val Cys Asp Ala Glu Gly Phe 225 230 235 240 Pro Glu Pro Thr Met Ser Trp Thr Lys Asp Gly Glu Gln Ile Glu Gln 245 250 255 Glu Glu Asp Asp Glu Lys Tyr Ile Phe Ser Asp Asp Ser Ser Gln Leu 260 265 270 Thr Ile Lys Lys Val Asp Lys Asn Asp Glu Ala Glu Tyr Ile Cys Ile 275 280 285 Ala Glu Asn Lys Ala Gly Glu Gln Asp Ala Thr Ile His Leu Lys Val 290 295 300 Phe Ala Lys Pro Lys Ile Thr Tyr Val Glu Asn Gln Thr Ala Met Glu 305 310 315 320 Leu Glu Glu Gln Val Thr Leu Thr Cys Glu Ala Ser Gly Asp Pro Ile 325 330 335 Pro Ser Ile Thr Trp Arg Thr Ser Thr Arg Asn Ile Ser Ser Glu Glu 340 345 350 Lys Ala Ser Trp Thr Arg Pro Glu Lys Gln Glu Thr Leu Asp Gly His 355 360 365 Met Val Val Arg Ser His Ala Arg Val Ser Ser Leu Thr Leu Lys Ser 370 375 380 Ile Gln Tyr Thr Asp Ala Gly Glu Tyr Ile Cys Thr Ala Ser Asn Thr 385 390 395 400 Ile Gly Gln Asp Ser Gln Ser Met Tyr Leu Glu Val Gln Tyr Ala Pro 405 410 415 Lys Leu Gln Gly Pro Val Ala Val Tyr Thr Trp Glu Gly Asn Gln Val 420 425 430 Asn Ile Thr Cys Glu Val Phe Ala Tyr Pro Ser Ala Thr Ile Ser Trp 435 440 445 Phe Arg Asp Gly Gln Leu Leu Pro Ser Ser Asn Tyr Ser Asn Ile Lys 450 455 460 Ile Tyr Asn Thr Pro Ser Ala Ser Tyr Leu Glu Val Thr Pro Asp Ser 465 470 475 480 Glu Asn Asp Phe Gly Asn Tyr Asn Cys Thr Ala Val Asn Arg Ile Gly 485 490 495 Gln Glu Ser Leu Glu Phe Ile Leu Val Gln Ala Asp Thr Pro Ser Ser 500 505 510 Pro Ser Ile Asp Gln Val Glu Pro Tyr Ser Ser Thr Ala Gln Val Gln 515 520 525 Phe Asp Glu Pro Glu Ala Thr Gly Gly Val Pro Ile Leu Lys Tyr Lys 530 535 540 Ala Glu Trp Arg Ala Val Gly Glu Glu Val Trp His Ser Lys Trp Tyr 545 550 555 560 Asp Ala Lys Glu Ala Ser Met Glu Gly Ile Val Thr Ile Val Gly Leu 565 570 575 Lys Pro Glu Thr Thr Tyr Ala Val Arg Leu Ala Ala Leu Asn Gly Lys 580 585 590 Gly Leu Gly Glu Ile Ser Ala Ala Ser Glu Phe Lys Thr Gln Pro Val 595 600 605 Gln Gly Glu Pro Ser Ala Pro Lys Leu Glu Gly Gln Met Gly Glu Asp 610 615 620 Gly Asn Ser Ile Lys Val Asn Leu Ile Lys Gln Asp Asp Gly Gly Ser 625 630 635 640 Pro Ile Arg His Tyr Leu Val Arg Tyr Arg Ala Leu Ser Ser Glu Trp 645 650 655 Lys Pro Glu Ile Arg Leu Pro Ser Gly Ser Asp His Val Met Leu Lys 660 665 670 Ser Leu Asp Trp Asn Ala Glu Tyr Glu Val Tyr Val Val Ala Glu Asn 675 680 685 Gln Gln Gly Lys Ser Lys Ala Ala His Phe Val Phe Arg Thr Ser Ala 690 695 700 Gln Pro Thr Ala Ile Pro Ala Asn Gly Ser Pro Thr Ser Gly Leu Ser 705 710 715 720 Thr Gly Ala Ile Val Gly Ile Leu Ile Val Ile Phe Val Leu Leu Leu 725 730 735 Val Val Val Asp Ile Thr Cys Tyr Phe Leu Asn Lys Cys Gly Leu Phe 740 745 750 Met Cys Ile Ala Val Asn Leu Cys Gly Lys Ala Gly Pro Gly Ala Lys 755 760 765 Gly Lys Asp Met Glu Glu Gly Lys Ala Ala Phe Ser Lys Asp Glu Ser 770 775 780 Lys Glu Pro Ile Val Glu Val Arg Thr Glu Glu Glu Arg Thr Pro Asn 785 790 795 800 His Asp Gly Gly Lys His Thr Glu Pro Asn Glu Thr Thr Pro Leu Thr 805 810 815 Glu Pro Glu Lys Gly Pro Val Glu Ala Lys Pro Glu Cys Gln Glu Thr 820 825 830 Glu Thr Lys Pro Ala Pro Ala Glu Val Lys Thr Val Pro Asn Asp Ala 835 840 845 Thr Gln Thr Lys Glu Asn Glu Ser Lys Ala 850 855 235761PRTHomo sapiensNCAM1 isoform 3 235Met Leu Gln Thr Lys Asp Leu Ile Trp Thr Leu Phe Phe Leu Gly Thr 1 5 10 15 Ala Val Ser Leu Gln Val Asp Ile Val Pro Ser Gln Gly Glu Ile Ser 20 25 30 Val Gly Glu Ser Lys Phe Phe Leu Cys Gln Val Ala Gly Asp Ala Lys 35 40 45 Asp Lys Asp Ile Ser Trp Phe Ser Pro Asn Gly Glu Lys Leu Thr Pro 50 55 60 Asn Gln Gln Arg Ile Ser Val Val Trp Asn Asp Asp Ser Ser Ser Thr 65 70 75 80 Leu Thr Ile Tyr Asn Ala Asn Ile Asp Asp Ala Gly Ile Tyr Lys Cys 85 90 95 Val Val Thr Gly Glu Asp Gly Ser Glu Ser Glu Ala Thr Val Asn Val 100 105 110 Lys Ile Phe Gln Lys Leu Met Phe Lys Asn Ala Pro Thr Pro Gln Glu 115 120 125 Phe Arg Glu Gly Glu Asp Ala Val Ile Val Cys Asp Val Val Ser Ser 130 135 140 Leu Pro Pro Thr Ile Ile Trp Lys His Lys Gly Arg Asp Val Ile Leu 145 150 155 160 Lys Lys Asp Val Arg Phe Ile Val Leu Ser Asn Asn Tyr Leu Gln Ile 165 170 175 Arg Gly Ile Lys Lys Thr Asp Glu Gly Thr Tyr Arg Cys Glu Gly Arg 180 185 190 Ile Leu Ala Arg Gly Glu Ile Asn Phe Lys Asp Ile Gln Val Ile Val 195 200 205 Asn Val Pro Pro Thr Ile Gln Ala Arg Gln Asn Ile Val Asn Ala Thr 210 215 220 Ala Asn Leu Gly Gln Ser Val Thr Leu Val Cys Asp Ala Glu Gly Phe 225 230 235 240 Pro Glu Pro Thr Met Ser Trp Thr Lys Asp Gly Glu Gln Ile Glu Gln 245 250 255 Glu Glu Asp Asp Glu Lys Tyr Ile Phe Ser Asp Asp Ser Ser Gln Leu 260 265 270 Thr Ile Lys Lys Val Asp Lys Asn Asp Glu Ala Glu Tyr Ile Cys Ile 275 280 285 Ala Glu Asn Lys Ala Gly Glu Gln Asp Ala Thr Ile His Leu Lys Val 290 295 300 Phe Ala Lys Pro Lys Ile Thr Tyr Val Glu Asn Gln Thr Ala Met Glu 305 310 315 320 Leu Glu Glu Gln Val Thr Leu Thr Cys Glu Ala Ser Gly Asp Pro Ile 325 330 335 Pro Ser Ile Thr Trp Arg Thr Ser Thr Arg Asn Ile Ser Ser Glu Glu 340 345 350 Lys Thr Leu Asp Gly His Met Val Val Arg Ser His Ala Arg Val Ser 355 360 365 Ser Leu Thr Leu Lys Ser Ile Gln Tyr Thr Asp Ala Gly Glu Tyr Ile 370 375 380 Cys Thr Ala Ser Asn Thr Ile Gly Gln Asp Ser Gln Ser Met Tyr Leu 385 390 395 400 Glu Val Gln Tyr Ala Pro Lys Leu Gln Gly Pro Val Ala Val Tyr Thr 405 410 415 Trp Glu Gly Asn Gln Val Asn Ile Thr Cys Glu Val Phe Ala Tyr Pro 420 425 430 Ser Ala Thr Ile Ser Trp Phe Arg Asp Gly Gln Leu Leu Pro Ser Ser 435 440 445 Asn Tyr Ser Asn Ile Lys Ile Tyr Asn Thr Pro Ser Ala Ser Tyr Leu 450 455 460 Glu Val Thr Pro Asp Ser Glu Asn Asp Phe Gly Asn Tyr Asn Cys Thr 465 470 475 480 Ala Val Asn Arg Ile Gly Gln Glu Ser Leu Glu Phe Ile Leu Val Gln 485 490 495 Ala Asp Thr Pro Ser Ser Pro Ser Ile Asp Gln Val Glu Pro Tyr Ser 500 505 510 Ser Thr Ala Gln Val Gln Phe Asp Glu Pro Glu Ala Thr Gly Gly Val 515 520 525 Pro Ile Leu Lys Tyr Lys Ala Glu Trp Arg Ala Val Gly Glu Glu Val 530 535 540 Trp His Ser Lys Trp Tyr Asp Ala Lys Glu Ala Ser Met Glu Gly Ile 545 550 555

560 Val Thr Ile Val Gly Leu Lys Pro Glu Thr Thr Tyr Ala Val Arg Leu 565 570 575 Ala Ala Leu Asn Gly Lys Gly Leu Gly Glu Ile Ser Ala Ala Ser Glu 580 585 590 Phe Lys Thr Gln Pro Val His Ser Pro Pro Pro Pro Ala Ser Ala Ser 595 600 605 Ser Ser Thr Pro Val Pro Leu Ser Pro Pro Asp Thr Thr Trp Pro Leu 610 615 620 Pro Ala Leu Ala Thr Thr Glu Pro Ala Lys Gly Glu Pro Ser Ala Pro 625 630 635 640 Lys Leu Glu Gly Gln Met Gly Glu Asp Gly Asn Ser Ile Lys Val Asn 645 650 655 Leu Ile Lys Gln Asp Asp Gly Gly Ser Pro Ile Arg His Tyr Leu Val 660 665 670 Arg Tyr Arg Ala Leu Ser Ser Glu Trp Lys Pro Glu Ile Arg Leu Pro 675 680 685 Ser Gly Ser Asp His Val Met Leu Lys Ser Leu Asp Trp Asn Ala Glu 690 695 700 Tyr Glu Val Tyr Val Val Ala Glu Asn Gln Gln Gly Lys Ser Lys Ala 705 710 715 720 Ala His Phe Val Phe Arg Thr Ser Ala Gln Pro Thr Ala Ile Pro Ala 725 730 735 Thr Leu Gly Gly Asn Ser Ala Ser Tyr Thr Phe Val Ser Leu Leu Phe 740 745 750 Ser Ala Val Thr Leu Leu Leu Leu Cys 755 760