Inhibitors of Taspase1 protease activity

TECHNICAL FIELD

This invention relates to a novel protease.

BACKGROUND

MLL/HRX/ALL1 encodes a 3,969 amino acid nuclear protein bearing multiple conserved domains with assigned activities including: an N terminus with three AT-hook motifs that bind AT rich DNA segments (Zeleznik-Le et al., (1994) Proc. Natl. Acad. Sci. USA, 91:10610-10614), a DNA methyl transferase homology domain that represses transcription (Xia et al., (2003) Proc. Natl. Acad. Sci. USA), four PHD fingers that mediate protein-protein interactions (Fair et al., (2001) Mol. Cell. Biol., 21:3589-3597), a transactivation domain that interacts with CBP (Ernst et al., (2001) Mol. Cell. Biol., 21:2249-2258), and a C-terminal SET domain with histone H3 lysine 4 methyl transferase activity (Milne et al., (2002) Mol. Cell., Vol. 10:1107-1117; Nakamura et al., (2002) Mol. Cell., 10:1119-1128) (FIG. 1A). MLL and its Drosophila homologue trithorax are required for maintaining proper Hox and homeotic gene expression patterns, respectively (Breen and Harte, (1993) Development, 117:119-134; Yu et al., (1998) Proc. Natl. Acad. Sci. USA, 95:10632-10636).

Chromosome translocations characteristically found in human infant leukemia disrupt MLL (11q23), generating chimeric proteins between the MLL N-terminus and multiple translocation partners that vary substantially (Ayton and Cleary, Oncogene, (2001) 20:5695-5707; Domer et al., (1993) Proc. Natl. Acad. Sci. USA, 90:7884-7888; Downing and Look, (1996) Cancer Treat. Res., 84:79-92; Gu et al., (1992) Cell, 71:701-708; Thirman et al., (1993) New England Journal of Medicine, 329:909-914; Tkachuk et al., (1992) Cell, 71:691-700). Mice carrying engineered Mll translocations develop leukemia (Corral et al., (1996) Cell, 85:853-851; Forster et al., (2003) New England Journal of Medicine, 326:800-806). Gene expression profiles of infant leukemias bearing MLL translocations identified a characteristic gene expression profile that distinguishes this poor prognosis leukemia from other leukemias (Armstrong et al., (2002) Nat. Genet., 30:41-47; Yeoh et al., (2002) Cancer Cell, 1: 133-143). Among the upregulated genes were some recognized targets of MLL including select HOX genes. Deregulated expression of HOX genes typifies certain malignancies (Buske and Humphries, (2002) Int. J. of Hematol., 71:391-398; Cillo et al., (2001) Int. J. Hematol., 71:161-169; Dash and Gilliland, (2001) Best Pract. Res. Clin. Haematol., 14:49-64).

Recently, we and others demonstrated that MLL is normally processed at two cleavage sites, CS1 (D/GADD) and CS2 (D/GVDD), and that mutation of both sites abolishes the proteolysis (Hsieh et al., (2003) Mol. Cell. Biol., 23:186-194; Yokoyama et al., (2002) Blood, 100:3710-3718) (FIG. 1B). The sequence of the cleavage site is highly conserved in MLL homologues from flies to mammals. MLL cleavage generates N-terminal p320 (N320) and C-terminal p180 (C180) fragments, which heterodimerize to form a stable complex that localizes to a subnuclear compartment. Within this complex, the FYRN domain of N320 directly interacts with the FYRC and SET domains of C180. This dynamic post-cleavage association confers stability to N320 and correct nuclear sublocalization of the MLL complex for proper target gene expression (Hsieh et al., (2003) Mol. Cell. Biol., 23:186-194).

Site-specific proteolysis is essential in many important biological pathways including the sequential activation of blood coagulation factors (Furie and Furie, (1992) New England Journal of Medicine, 326:800-806), cholesterol-gauged liberation of SREBP from the ER (Brown et al., (2000) Cell, 100:391-398), ligand-activated cleavage and subsequent release of the intracellular domain of Notch (Brown et al., (2000) Cell, 100:391-398), maturation of the hedgehog signaling molecule (Ye and Fortini, Semin. (2000) Cell Dev. Biol., 11:211-221), separation of HCF-1 for proper cell cycle regulation (Wilson et al., (1995) Genes. Dev., 9:2445-2458), and activation of caspases and their subsequent cleavage of death substrates during apoptosis (Thornberry and Lazebnik, (1998) Science, 281:1312-1316). Identification and characterization of the responsible proteases has not only proven critical to understanding such biologic processes but also for developing targeted therapeutics for diseases involving specific pathways.

SUMMARY

The present invention is based, in part, on the discovery of a novel protease, referred to herein as "Taspase1". The amino acid sequence of a human Taspase1 polypeptide is shown in SEQ ID NO:1 (See e.g., FIG. 12), and the nucleotide sequence of a cDNA encoding human Taspase1 is shown in SEQ ID NO:2 (See e.g., FIG. 13).

Accordingly, in one aspect, the invention features, Taspase1 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of Taspase1-mediated or Taspase1-related disorders or as antigens for eliciting antibodies directed against Taspase1. In another embodiment, the invention provides Taspase1 polypeptides having a Taspase1 activity. Preferred polypeptides are Taspase1 proteins including at least one Taspase1 domain, e.g., an Asparaginase.sub.--2 homology domain, and, preferably, having MLL CS1 and/or CS2 cleavage activity.

In other embodiments, the invention provides Taspase1 polypeptides, e.g., a Taspase1 polypeptide having the amino acid sequence shown in SEQ ID NO: 1; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO: 1; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2, wherein the nucleic acid encodes a full length Taspase1 protein or an active fragment thereof.

In a related aspect, the invention provides Taspase1 polypeptides or fragments operatively linked to non-Taspase1 polypeptides to form fusion proteins.

In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind Taspase1 polypeptides or fragments thereof.

In another aspect, the invention provides methods of screening for compounds that modulate (e.g., inhibit) the expression or activity of a Taspase1 polypeptide or nucleic acid.

In still another aspect, the invention provides a process for modulating (e.g., inhibiting) Taspase1 polypeptide or nucleic acid expression or activity, e.g. using a peptide, derived peptide, or small molecule that inhibits the ability of Taspase1 to cleave a Taspase1 substrate, e.g., MLL (which is shown in SEQ ID NO: 59 (See e.g., FIG. 14)). Thus a suitable inhibitor might have a K.sub.i for inhibition of MLL cleavage of about 10.sup.-4 M, 10.sup.-5 M, 10.sup.-6 M, 10.sup.-7 M, 10.sup.-8 M, 10.sup.-9 M or less. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the Taspase1 polypeptides or nucleic acids, such as conditions involving aberrant or deficient HOX gene expression and cellular proliferation or differentiation. Thus, such inhibitors can be used to reduce undesirable Taspase1 proteolytic activity. The inhibitors can also inhibit other proteases having the same mechanism as Taspase1. In some instances it is desirable to inhibit normal Taspase1 expression or activity, wherein the inhibition of such Taspase1 activity will reduce the expression or activity of other gene products such as MLL or HOX gene products, which are expressed, for example, in malignant cells.

In yet another aspect, the invention provides methods for reducing Taspase1 expression. The method includes contacting the cell with a compound or agent (e.g., a compound identified using the methods described herein) that modulates (e.g., inhibits) the expression, of the Taspase1 polypeptide or nucleic acid. In a preferred embodiment, the contacting step is effective in vitro or ex vivo. In other embodiments, the contacting step is effected in vivo, e.g., in a subject (e.g., a mammal, e.g., a human), as part of a therapeutic or prophylactic protocol.

In a preferred embodiment, the compound is an inhibitor of a Taspase1 polypeptide. Preferably, the inhibitor is chosen from a peptide (e.g., a polypeptide including naturally occurring as well as non-naturally occurring amino acids), a peptidomimetic, a phosphopeptide, a small organic molecule, a small inorganic molecule and an antibody. In another preferred embodiment, the compound is an inhibitor of a Taspase1 nucleic acid, e.g., an antisense, a ribozyme, or an RNAi or a triple helix molecule.

The compound can be administered in combination with a cytotoxic agent. Examples of cytotoxic agents include anti-microtubule agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, an anti-metabolite, a mitotic inhibitor, an alkylating agent, an intercalating agent, an agent capable of interfering with a signal transduction pathway, an agent that promotes apoptosis or necrosis, and radiation.

In another aspect, the invention features methods for treating or preventing a disorder characterized by aberrant cellular proliferation or differentiation of a Taspase1-expressing cell in a subject. Preferably, the method includes administering to the subject (e.g., a mammal, e.g., a human) an effective amount of a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the Taspase1 polypeptide or nucleic acid (e.g., inhibits proteolytic cleavage of MLL). In a preferred embodiment, the disorder is a cancerous or pre-cancerous condition (e.g., leukemia).

In another aspect, the invention provides methods for evaluating the efficacy of a therapeutic or prophylactic agent (e.g., an anti-neoplastic agent). The method includes: contacting a sample with an agent (e.g., a polypeptide inhibitor or a compound identified using the methods described herein) and, evaluating the expression or function of Taspase1 nucleic acid or polypeptide in the sample before and after the contacting step. A change, e.g., a decrease or increase, in the level of Taspase1 nucleic acid (e.g., mRNA) or polypeptide function (e.g., proteolysis of MLL) in the sample obtained after the contacting step, relative to the level of expression in the sample before the contacting step, is indicative of the efficacy of the agent. The level of Taspase1 nucleic acid or polypeptide expression or function can be detected by any method described herein (e.g., measuring the cleavage of MLL using a labeled MLL substrate and SDS-PAGE).

The invention also features a nucleic acid molecule that encodes a Taspase1 protein or polypeptide, e.g., a biologically active portion of the Taspase1 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:1. In other embodiments, the invention provides isolated Taspase1 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO:2. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2, wherein the nucleic acid encodes a full length Taspase1 protein or an active fragment thereof.

In a related aspect, the invention further provides nucleic acid constructs that include a Taspase1 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included are vectors and host cells containing the Taspase1 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing Taspase1 nucleic acid molecules and polypeptides.

In still another related aspect, isolated nucleic acid molecules that are antisense to or interfere with (e.g., an RNAi) a Taspase1 encoding nucleic acid molecule are provided.

In one aspect, the invention features an isolated polypeptide having an amino acid sequence, which is at least about 80% identical to the amino acid sequence of SEQ ID NO:1.

The peptide can have one or more of the following features. The polypeptide can include a heterologous polypeptide. The polypeptide can cleave a polypeptide comprising an Asp-Gly-Ala-Asp-Asp (SEQ ID NO:3) or Asp-Gly-Val-Asp-Asp (SEQ ID NO:4) sequence between the Asp and the Gly amino acids of the Asp-Gly-Ala-Asp-Asp (SEQ ID NO:3) or Asp-Gly-Val-Asp-Asp (SEQ ID NO:4) sequence. The polypeptide can be intramolecularly proteolyzed into a first peptide fragment and a second peptide fragment. One of the first or second peptide fragments can include a threonine at the N-terminus. The polypeptide can have a conserved Leu-Asp-Thr-Val-Gly (SEQ ID NO:5) motif. Amino acids 232-236 of the peptide can be Leu-Asp-Thr-Val-Gly (SEQ ID NO:5). The polypeptide can include the amino acid sequence of SEQ ID NO: 1, wherein up to 20 amino acids are substituted.

In another embodiment, the invention features an isolated polypeptide encoded by a nucleic acid that hybridizes under high stringency conditions to the nucleic acid of SEQ ID NO:2.

In one embodiment, the invention features a host cell including the any one of the polypeptides described herein.

In one embodiment, the invention features a method for producing a polypeptide described herein including culturing a host cell under conditions in which the nucleic acid encoding the polypeptide is expressed.

The invention also features an antibody that selectively binds to a polypeptide described herein.

In one embodiment, the invention features an inhibitor of a polypeptide described herein. The inhibitor can be a polypeptide. In some instances, the polypeptide includes one of the following amino acid sequences, Ser-Gln-Leu-Ala (SEQ ID NO:6), Ile-Ser-Gln-Leu-Ala (SEQ ID NO:7) or Lys-Ile-Ser-Gln-Leu-Ala (SEQ ID NO:8), Ser-Gln-Leu-Asp-aldehyde (SEQ ID NO:9), Ile-Ser-Gln-Leu-Asp-aldehyde (SEQ ID NO:10), or Lys-Ile-Ser-Gln-Leu-Asp-aldehyde (SEQ ID NO:11), Ser-Gln-Leu-Asp-chloromethylketone (SEQ ID NO:12), Ile-Ser-Gln-Leu-Asp-chloromethylketone (SEQ ID NO:13), or Lys-Ile-Ser-Gln-Leu-Asp-chloromethylketone (SEQ ID NO:14). The inhibitor can also include a peptidomimetic of one of amino acid sequences Ser-Gln-Leu-Ala (SEQ ID NO:6), Ile-Ser-Gln-Leu-Ala (SEQ ID NO:7) or Lys-Ile-Ser-Gln-Leu-Ala (SEQ ID NO:8), Ser-Gln-Leu-Asp-aldehyde (SEQ ID NO:9), Ile-Ser-Gln-Leu-Asp-aldehyde (SEQ ID NO:10), or Lys-Ile-Ser-Gln-Leu-Asp-aldehyde (SEQ ID NO:11), Ser-Gln-Leu-Asp-chloromethylketone (SEQ ID NO:12), Ile-Ser-Gln-Leu-Asp-chloromethylketone (SEQ ID NO:13), or Lys-Ile-Ser-Gln-Leu-Asp-chloromethylketone (SEQ ID NO:14).

In one embodiment, the invention features a method of treating a subject including administering to the subject an inhibitor of any of the polypeptides described herein. In some instances, the method includes administering an additional therapeutic agent.

In another embodiment, the invention features a method of treating cancer in a subject including administering to the subject an inhibitor of any of the polypeptides described herein. In some instances, the method includes administering an additional therapeutic agent (e.g., an anti-cancer agent). In some instances, the cancer is a hematopoietic neoplasm or a solid cancer. In other instances, the cancer is a leukemia.

In one embodiment, the invention features a method of identifying an inhibitor of a polypeptide described herein, the method including: (a) providing a polypeptide described herein; (b) contacting the polypeptide with a candidate inhibitor and a proteolytic substrate; (c) measuring proteolysis of the substrate in the presence of the candidate inhibitor, and (d) comparing the proteolysis of the substrate in the presence of the candidate inhibitor to the proteolysis of the substrate in the absence of the candidate inhibitor, wherein a decrease in proteolytic activity identifies the candidate inhibitor as an inhibitor.

The method can include one or more of the following features: The polypeptide can be provided in vivo or in vitro. The substrate can include a Taspase1 substrate. Alternatively, the substrate can include a CS1-like or CS2-like motif. The substrate can include a fragment of an MLL family protein including one or more of a CS1, CS2, Ile-Ser-Gln-Leu-Asp (SEQ ID NO:15), or Glu-Gly-Gln-Val-Asp (SEQ ID NO:16) motif. The method can be performed in an array format. The method can also include generating dataset correlating a value for the measured function with the determination of whether the agent is an inhibitor of the polypeptide.

In another aspect, the invention features a method of treating a subject including administering to the subject an inhibitor identified by a method described herein.

In yet another aspect, the invention features a method of treating cancer in a subject including administering to the subject an inhibitor identified by a method described herein. In some instances, the cancer can be a solid tumor or leukemia.

In still another aspect, the invention features a method of treating a heomatopoetic proliferative disorder in a subject including administering to the subject an inhibitor identified by a method described herein.

In still another aspect, the invention features an inhibitor identified by a method described herein.

The Taspase1 polypeptide, fragments thereof, and derivatives and other variants of the sequence in SEQ ID NO:1 thereof are collectively referred to as "polypeptides or proteins of the invention" or "Taspase1 polypeptides or proteins".

As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6.times. sodium chloride/sodium citrate (SSC) at about 45.degree. C., followed by two washes in 0.2.times.SSC, 0.1% SDS at least at 50.degree. C. (the temperature of the washes can be increased to 55.degree. C. for low stringency conditions); 2) medium stringency hybridization conditions in 6.times.SSC at about 45.degree. C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at 60.degree. C.; 3) high stringency hybridization conditions in 6.times.SSC at about 45.degree. C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at 65.degree. C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65.degree. C., followed by one or more washes at 0.2.times.SSC, 1% SDS at 65.degree. C.

An "isolated" or "purified" polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.

A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of Taspase1 without abolishing or substantially altering a Taspase1 activity. An "essential" amino acid residue is a residue that, when altered from the wild-type sequence of Taspase1, results in abolishing or substantially abolishing a Taspase1 activity. For example, conserved amino acid residues in Taspase1 are predicted to be particularly unamenable to alteration.

A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a Taspase1 protein is preferably replaced with another amino acid residue from the same side chain family.

The term "peptidomimetic" refers to a chemical compound that mimics the ability of a peptide to recognize certain physiological molecules, such as proteins (e.g., Taspase1) and DNA.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A depicts conserved domain structures of human MLL with cleavage sites (CS1 (D/GADD (SEQ ID NO:3)) and CS2 (D/GVDD (SEQ ID NO:4))) positioned thereon.

FIG. 1B depicts the conservation of CS1 (D/GADD) (SEQ ID NO:3) and CS2 (D/GVDD) (SEQ ID NO:4) among MLL family members.

FIG. 1C depicts the results of a study showing that Taspase1 has a preference for the CS2 over the CS1 cleavage site.

FIG. 2A depicts the results of a study demonstrating in vitro reconstitution of MLL cleavage using subcellular fractions.

FIG. 2B depicts the results of a study showing that Taspase1 proteolysis of MLL is not affected by various protease inhibitors.

FIG. 3A schematically depicts the methods used for purification of the MLL cleaving protease, Taspase1.

FIG. 3B depicts a silver stained SDS-PAGE of the mono S fraction with maximum enzymatic activity. The amino acid sequence SLNFGAVGALSGIK corresponds to amino acids 124-137 of SEQ ID NO:1. The amino acid sequence NPVSVANR corresponds to amino acids 138-145 of SEQ ID NO:1.

FIG. 3C depicts alignment of active sites among Asparaginase.sub.--2 family proteins.

FIG. 3D depicts the results of a study demonstrating the self-proteolysis of Taspase1.

FIG. 3E depicts alignment of the amino acid sequences of putative Taspase1 proteins in various species.

FIG. 4A depicts the results of a study comparing cleavage of MLL wild type substrate and mutant substrate by recombinant Taspase1 versus Mono S fraction.

FIG. 4B depicts the results of a study demonstrating that Taspase1 sequentially cleaves CS1 and CS2.

FIG. 5A depicts the results of coomassie blue staining of purified recombinant Taspase1, demonstrating that Taspase1 proenzyme undergoes intramolecular processing to an active 29 kDa .alpha./22 kDa .beta. heterodimer.

FIG. 5B, depicts the results of coomassie blue staining of purified recombinant Taspase1, demonstrating Threonine 234 of Taspase1 is essential for its enzymatic activity.

FIG. 6A depicts the results of an assay demonstrating that Taspase1 cleaves the MLL reporter but not the CS1/2 mutant and that wild type Taspase1 but not the T234A Taspase1 mutant underwent intramolecular processing.

FIG. 6B depicts the results of a study demonstrating that RNAi against Taspase1 resulted in the decrease of Taspase1 expression as well as MLL cleavage.

FIG. 6C depicts the results of a study indicating that knockdown of Taspase1 diminished the expression of the earlier expressed HOX genes, but not the later expressed HOX genes.

FIG. 7 is a schematic model depicting the intramolecular proteolysis of Taspase1 followed by MLL processing required for proper HOX gene expression.

FIG. 8 depicts the results of a study demonstrating the effects of various point mutations in the CS2 cleavage site.

FIG. 9 depicts the results of a study demonstrating the effect of a change in length of peptide inhibitor on substrate cleavage and demonstrating the effect of a change in amino acid P1 from D to A on substrate cleavage.

FIG. 10 depicts the results of a study demonstrating the difference in effectiveness of a five amino acid Taspase1 inhibitor and a six amino acid Taspase1 inhibitor.

FIGS. 11A-C depicts the results of a study demonstrating the dose dependent inhibitory effects of three different amino acid aldehyde Taspase1 inhibitors, SQLA-aldehyde (SEQ ID NO: 17), SQLD-aldehyde (SEQ ID NO:9), and KISQLD-aldehyde (SEQ ID NO:11).

FIG. 12 depicts the amino acid sequence of human Taspase1 (SEQ ID NO:1).

FIG. 13 depicts the cDNA sequence of human Taspase1 (SEQ ID NO:2).

FIG. 14 depicts the amino acid sequence of human MLL (SEQ ID NO:59).

DETAILED DESCRIPTION

The invention is based, in part, on the discovery of a novel protease, Taspase1. Taspase1 cleaves MLL at two conserved sites (D/GADD (referred to herein as CS1) and D/GVDD (referred to herein as CS2)) generating N-terminal 320 kDa and C-terminal 180 kDa fragments, which heterodimerize to both stabilize the complex and confer it subnuclear destination.

Taspase1 was purified and cloned using highly conserved cleavage motifs within MLL. Upon the purification and cloning of Taspase1, it was discovered that Taspase1 (threonine aspartase 1) is the first endopeptidase within a family of enzymes possessing an Asparaginase.sub.--2 homology domain. Other members present in both prokaryotes and eukaryotes include the amidohydrolases, L-Asparaginase in intermediary amino acid metabolism and Glycosylasparaginase. Glycosylasparaginase participates in the ordered degradation of N-linked glycoproteins by cleaving Asn-GlcNAc linkages that join oligosaccharides to proteins.

Site Specific MLL Cleavage Substrate

To facilitate the purification and characterization of the MLL protease, we generated a tractable cleavage substrate. We found that aa 2,400 to 2,900 of MLL containing CS1 (aa 2,666) and CS2 (aa 2,718) are sufficient to recapitulate endogenous MLL cleavage (FIG. 1C). Proteolysis of this p75 MLL test substrate at CS 1 or CS2 would generate N-terminal Myc-tagged p42 or p47 fragments and C-terminal Flag-tagged p33 or p28 respectively (FIG. 1A). The p47 and p28 fragments were most abundant, indicating processing at CS2 is more efficient than at CS1. This is consistent with our prior observations of the proteolysis of full-length MLL protein (Hsieh et al., Mol. Cell. Biol., Vol. 23, pp. 186-194; 2003)). The transfected p75 MLL substrate with mutant CS1/CS2 sites (CS1/2 mt) was not cleaved, indicating the specificity of this substrate (FIG. 1C). Subcellular fractions derived from 293T cells (a human embryonic kidney cell line) were incubated with in vitro transcribed/translated .sup.35S-methionine labeled p75 MLL substrates. The light membrane (LM) fraction displayed the most enzymatic activity (FIG. 2A, left panel) and did not cleave the mutant fragment (CS1/2 mt) (FIG. 2A, right panel). Inhibitors of multiple classes of proteases including serine proteases, cysteine proteases, metalloproteases, acid proteases, and the 26S proteosome, were examined but again showed no substantial inhibition of MLL proteolysis in this fraction enriched for endoplasmic reticulum (FIG. 2B). Only heat incubation at 70.degree. C. for 30 minutes inactivated the proteolytic activity in light membranes.

Purification

The LM fraction possessing the protease activity was subjected to serial column chromatography and the activity followed by an in vitro cleavage assay. Seven chromatographic steps achieved an approximately 200,000-fold enrichment of the proteolytic activity (FIG. 3A). The ultimate mono S fractions displaying the highest enzymatic activity were subjected to SDS-PAGE followed by a silver stain (FIG. 3B). Polypeptide bands whose presence best correlated with the proteolytic activity were digested with trypsin and subjected to liquid chromatography and tandem mass spectrometry (LC-MS/MS) sequence analysis. Two peptide sequences corresponding to aa 124 to 137 and aa 138 to 145 of an uncharacterized open reading frame (orf) present on chromosome 20 (c20orf13) were identified from a gel slice that migrated at .about.28 kDa (FIG. 3B). The orf predicts a 420 aa protein possessing an Asparaginase.sub.--2 homology domain (pfam 01112, interpro 000246) from aa 41 to 391 (FIG. 3C). Typical proteins that contain this signature motif include L-Asparaginase and Glycosylasparaginase (FIG. 3C). Three distinct conserved motifs distinguish the Taspase1, Glycosylasparaginase and L-Asparaginase subfamilies. L-Asparaginase catalyzes an amide bond hydrolysis to convert L-asparagine to L-aspartate. Glycosylasparaginase is an amidohydrolase which catalyzes the N-acetylglucosamine-asparagine bond that links oligosaccharides to asparagine. However, no endopeptidase activity had been demonstrated to date among the Asparaginase.sub.--2 family enzymes. The characteristics of this MLL cleaving protease (c20orf13) as subsequently detailed here prompts its designation as Taspase1 (Threonine aspartase1). Sequence alignment searches of the public databases identified highly conserved Taspase1 orthologues in fly, mosquito, pufferfish, zebrafish, rat, mouse, and human (FIG. 3C and FIG. 3E), but not in the nematode, C. elegans. The conserved LDTVG motif that surrounds a putative threonine active site is distinct from L-Asparaginase and Glycosylasparaginase (FIG. 3C) suggesting it may have a unique specificity.

Recombinant Taspase1 Activity

To assess the potential activity of this candidate protease, we expressed and purified recombinant N-terminal His-tagged Taspase1 from E. coli. Recombinant Taspase1 (rTaspase1) cleaved the wt p75 MLL but not the CS1/2 mt substrate (FIG. 4A). rTaspase1 more efficiently processed p75 MLL to completion based on the predominance of the p42 fragment when compared to the activity in the original mono S fraction (FIG. 4A). The p42 fragment results from cleavage at the CS1 (D/GADD) (SEQ ID NO:3) site, which is less conserved than CS2 (D/GVDD) (SEQ ID NO:4) and is also less efficiently processed in vivo (Hsieh et al., (2003) Mol. Cell. Biol., 23:186-194). We next examined the differential sensitivity of CS1 and CS2 sites for cleavage by rTaspase1. The p47 fragment appears first, while higher enzyme concentration or more time is needed for the appearance of the p42 fragment indicating a preference of rTaspase1 for CS2 over CS1 (FIG. 4B). Thus the proteolytic characteristics of rTaspase1 match the pattern of endogenous MLL proteolysis.

Characterization of Taspase1

Purification of recombinant N-terminal His-tagged Taspase1 yielded an expected 50 kDa product, a His-tagged 28 kDa a subunit, and a co-purified 22 kDa polypeptide (FIG. 5A). This 22 kDa polypeptide was subjected to N-terminal Edman degradation analysis, which identified threonine 234 of Taspase1 as the N-terminal amino acid of the apparent 22 kDa .beta. subunit (FIG. 5A). This represents proteolysis between aspartate 233 and threonine 234 of the 50 kDa putative proenzyme. Similarly when an N-terminal and C-terminal epitope tagged human Taspase1 cDNA was expressed in the human 293 T cell line, the 50 kDa product also appeared to be processed to a 28 kDa N-terminal .alpha. subunit and a 22 kDa C-terminal .beta. subunit (FIG. 3D). This suggested that Taspase1 may be intramolecularly proteolyzed and processed subunits reassembled through a non-covalent association. Amino acid substitution of either aspartate 233 (D233A) or threonine 234 (T234A) to alanine abolished the intramolecular processing of Taspase1 expressed in E. coli (FIG. 5A) or in mammalian cells (FIG. 3D). However, the D233A mutant retained some residual enzymatic activity, although it was .about.1000 fold less efficient than the wt enzyme (FIG. 5B, middle panel). Conversely, threonine 234 which became the N-terminus of the .beta. subunit is absolutely essential for cleavage activity (FIG. 5B, lower panel). These enzymatic characteristics are similar to properties shared by L-Asparaginase and Glycosylasparaginase which also demonstrate autoproteolysis of a proenzyme into an active .alpha./.beta. heterodimeric enzyme in which the N-terminal threonine of the .beta. subunit is the active site nucleophile for catalysis (Guan et al., (1996) J. Biol. Chem., 27:1732-1737; Liu et al., (1998) J. Biol. Chem., 273:9688-9694; Tikkanen et al., (1996) Embo. J., 15:2954-2960; Xu et al., (1999) Cell, 98:651-661). Thus, this MLL cleaving protease is the first endopeptidase within the Asparaginase.sub.--2 family with the novel characteristic of being a threonine aspartase, Taspase1.

Proteolysis of MLL In Vivo Requires Taspase1

We next asked whether Taspase1 was required to cleave MLL within mammalian cells. As a model system to test specificity, we co-expressed the p75 MLL substrate reporter together with Taspase1 in 293T cells. Taspase1 resulted in cleavage of wt p75 MLL but not the p75 CS1/2 mt reporter (FIG. 6A). Wt Taspase1, but not the T234A mutant Taspase1, enhanced the processing of p75 MLL to the final p42 product (FIGS. 1A and 6A). Only wt Taspase1, but not the T234A mutant of the nucleophile site demonstrated intramolecular processing into .alpha./.beta. fragments (FIGS. 3D and 6A). To assess the role of endogenous Taspase1, we designed duplex RNAi against Taspase1 which knocked down the expression of endogenous Taspase1 by .about.80% (FIG. 6B). Taspase1 RNAi resulted in a .about.50% decrease in the endogenous, processed C180 MLL fragment and in the appearance of full-length p500 MLL (FIG. 6B). In contrast, MLL RNAi resulted in the marked reduction of the C180 MLL fragment, but did not increase p500 MLL. In total, both the in vitro and in vivo cleavage assays confirm the role of Taspase1 in the proper processing of MLL.

Taspase1 is Required for Proper HOX Gene Expression

Genetic studies in both mice and flies establish that Mll and trithorax regulate Hox and homeotic gene expression, respectively (Mazo et al., (1990) Proc. Natl. Acad. Sci. USA, 87:2112-2116; Yu et al., (1995) Nature, 378:505-508). Mice heterozygous for an Mll.sup.-exon3LacZ disruption demonstrated haploinsufficiency with bi-directional homeotic transformations and shifted anterior boundaries of several Hox genes (Yu et al., (1995) Nature, 378:505-508). Mll-/-deficient embryos and mouse embryonic fibroblasts (MEFs) demonstrated Mll is required for the maintenance of selected Hox gene expression (Hanson et al., (1999) Proc. Natl. Acad. Sci. USA, 96:14372-14377; Yu et al., (1998) Proc. Natl. Acad. Sci. USA, 95:10632-10636; Yu et al., (1995) Nature, 378:505-508). As a first assessment of whether reduced Taspase1 activity would alter gene expression, we examined the gene expression profile of HeLa cells treated with the Taspase1 RNAi versus a control RNAi (FIG. 6B). Initial analysis of Affymetrix (HG-U133A) oligonucleotide array based RNA profiles indicated diminished expression of selected HOX genes (data not shown). Consequently, we used a quantitative RT-PCR approach to determine the relative expression of genes across the HOX A cluster. Of note, the knockdown of Taspase1 diminished the expression of the 3' located and "earlier" expressed genes in the HOX A cluster (A1, A3, and A4), but not those genes located more 5' and expressed "later" during embryonic development (A5, A9, and A10) (FIG. 6C). This selected attenuation contrasts with the global decrease in expression of most HOX A genes (A1 to A10) in cells with MLL knocked down (FIG. 6C). These data suggest the importance of Taspase1 in the correct expression of the early HOX A gene cluster (equivalent to the ANT-C cluster of Drosophila) (FIG. 7).

Inhibitors of Taspase1 Activity

Point Mutations of CS2 Cleavage Site

In order to identify essential amino acids relating to the CS2 cleavage site, mutant MLL substrates were prepared and labeled with .sup.35S-methionine. The mutant MLL substrates included p45, a 300 amino acid portion of the MLL protein including the amino acids from 2500-2800. In order to prevent proteolytic cleavage at the CS1 cleavage site, the CS1 cleavage site was mutated at amino acids 2666-2670 from amino acid sequence DGADD to amino acid sequence AAADD. Individual mutant substrates of the CS1 mutated MLL substrate were then generated to provide mutants having point mutations at each of P7 through P5' (i.e., amino acids 2712-2723). In each mutant, the naturally occurring amino acid was substituted with an alanine as depicted in FIG. 8. The labeled mutant substrates were incubated with rTaspase1 and the results analyzed by SDS-PAGE followed by autoradiography. As seen in FIG. 8, mutation at P1 or P1' virtually eliminates any detectable proteolytic cleavage of the MLL substrate. Mutations at P2, P3 and P5 significantly reduce detectable proteolytic cleavage of the MLL substrate.

Polypeptide Inhibitors of Taspase1

Polypeptides of varying length (i.e., 4 to 7 amino acids in length) were prepared to identify preferred lengths and sequences of polypeptide inhibitors of Taspase1. The polypeptide inhibitors were based on the amino acid sequence that includes a upstream portion of the CS2 cleavage site of MLL as well as a portion of the CS2 cleavage site (See FIG. 1B and FIG. 9). The peptide inhibitors were purchased from Tufts University Peptide Core Facility and AnaSpec Inc. of San Jose, Calif. 1 mM of each of the inhibitors was incubated with 5 ng of rTaspase1 for 20 minutes before adding labeled substrate for another 60 minutes at 37.degree. C. The results were analyzed by SDS-PAGE followed by autoradiography. As depicted in FIG. 9, polypeptides SQLD (SEQ ID NO:52), ISQLD (SEQ ID NO:15), and KISQLD (SEQ ID NO:53) had little inhibitory effect on the rTaspase1 enzyme. A likely reason for this result is due to the efficiency of the enzyme, where it cleaves the polypeptide inhibitor quickly, allowing the active site of Taspase1 to become available for another MLL substrate. On the other hand, in polypeptides where the P1 aspartate residue was substituted for an alanine residue (i.e., KISQLA (SEQ ID NO:8), KISdQLA (SEQ ID NO:54), and KISQA (SEQ ID NO:55)), inhibitory effect was observed. This suggests that the P1 is an essential amino acid for MLL cleavage activity. As seen in FIG. 9, polypeptides having greater than four amino acids have improved inhibitory effect. The results of the study also demonstrate the importance of the P2 amino acid, as mutation of P2 (i.e., omission of the leucine residue at P2) resulted in significantly decreased inhibition of rTaspase1 relative to the Taspase1 inhibition of the corresponding five amino acid alanine containing polypeptide having a P2 leucine (ISQLA) (SEQ ID NO:7).

Dose Response of Taspase1 with Two Polypeptide Inhibitors

In order to determine a preferred length of polypeptide inhibitors of Taspase1, dose responses were tested for a six amino acid polypeptide inhibitor (KISQLA) (SEQ ID NO:8) and a five amino acid polypeptide inhibitor (ISQLA) (SEQ ID NO:7). The polypeptides were incubated at the concentrations depicted in FIG. 10 with 5 ng of rTaspase1 for 20 minutes before adding labeled MLL substrate for another 60 minutes at 37.degree. C. The results were analyzed by SDS-PAGE followed by autoradiography. As can be seen in FIG. 10, the five amino acid polypeptide was a more effective inhibitor at lower concentrations than the corresponding six amino acid polypeptide. Thus, based on the results of this experiment, a five amino acid polypeptide inhibitor is likely to be more effective than a six amino acid polypeptide inhibitor.

Dose Responses of Polypeptide-Aldehyde Taspase1 Inhibitors

Three modified polypeptide inhibitors were prepared wherein the carboxy terminals of the polypeptides were replaced with an aldehyde (SQLA-aldehyde (SEQ ID NO:17), SQLD-aldehyde (SEQ ID NO:9), and KISQLD-aldehyde (SEQ ID NO:11)). Chemical syntheses of these modified polypeptides is well known to one of skill in the art, and the modified polypeptides are also are available commercially at AnaSpec Inc. of San Jose, Calif. The polypeptides were incubated in the concentrations depicted in FIGS. 11A-11C with 5 ng of rTaspase1 for 20 minutes before adding labeled MLL substrate for another 60 minutes at 37.degree. C. The results were analyzed by SDS-PAGE followed by autoradiography. As depicted in FIGS. 11A and 11B, the SQLD-aldehyde (SEQ ID NO:9) was a more effective inhibitor than the SQLA-aldehyde (SEQ ID NO:17). Although the prior experiments showed that polypeptide sequences including SQLA (SEQ ID NO:6) were more effective Taspase1 inhibitors than polypeptide sequences including SQLD (SEQ ID NO:52), the modification of the C-terminal portion of the peptide significantly reduces the cleavage efficiency of the enzyme by causing a reversible (but very inefficiently reversible) bond between the polypeptide inhibitor and the enzyme. Accordingly, the modified polypeptides can keep the active site of the enzyme occupied for a greater length of time, blocking entrance of the MLL substrate from entering the active site as required for cleavage. Moreover, experimental studies showed that the six amino acid aldehyde (KISQLD) (SEQ ID NO:53) was more effective than both of the four amino acid aldehyde. (See FIG. 11C.).

Methods and Materials

Plasmid Construction and Antibody Production

PCR fragments consisting of MLL aa 2,400 to 2,900 derived from either wild type or noncleavable MLL mt (CS1/CS2 mt) were inserted into a Myc/Flag doubly-tagged eukaryotic expression vector for transient transfection assays. These constructs also contain a 5' T7 promoter for generating in vitro transcription/translation product of .sup.35S-methionine labeled p75 MLL substrates. Full-length Taspase1 was cloned from 293T cell cDNA and inserted into the Myc/Flag doubly-tagged expression vector, a Protein C tagged vector, and a His-tagged bacteria expression vector, ET15b (Novagen). Taspase1 mutants were generated using QuikChange site-directed mutagenesis kit (Stratagene). Rabbit anti-Taspase1 polyclonal antibody was raised against aa 7 to 212 of purified recombinant human Taspase1. Transient transfection, in vitro transcription/translation, .sup.35S-methionine labeling, and immunoblot assays were performed as previously described (Hsieh et al., (2003) Mol. Cell. Biol., 23:186-194).

In Vitro Cleavage Assays

.sup.35S-methionine labeled MLL substrate was incubated with 2 .mu.l of indicated subcellular fractions or specified amounts of rTaspase1 in cleavage buffer (100 mM HEPES [pH 7.9], 5 mM MgCl.sub.2, 20 mM KCl, 5 mM DTT, and 10% sucrose) for 1 hour at 37.degree. C. or indicated periods of time. Protease inhibitors utilized include 8.5 .mu.M Phosphoramidon, 100 .mu.M TLCK, 100 .mu.M TPCK, 5 mM Iodoacetamide, 5 mM N-Ethylmaleimide, 0.3 .mu.M Aprotinin, 100 .mu.M Leupeptin, 1 .mu.M Pepstatin, 1.times. Complete protease inhibitor cocktail (Roche), 100 .mu.M Antipain, 100 .mu.M APMSF, 10 .mu.M Bestatin, 25 .mu.M ALLN, 100 .mu.M Chymostatin, 10M E-64, 5 mM EDTA, 1 mM PMSF, 1 mM EGTA, 50 .mu.M BAF (Boc-Aspartyl-FMK), and 50 .mu.M z-VAD (z-VAD-FMK).

Purification and LC-MS/MS

Human 293T cells from one hundred 15-cm dishes were collected and incubated in hypotonic buffer (10 mM HEPES [pH 7.9], 1.5 mM MgCl.sub.2, 10 mM KCl, 0.2 mM PMSF, 1 mM EDTA, 1 mM EDTA, 1 mM EGTA, and protease inhibitor cocktail [Roche]) for 15 min on ice. Supplemented protease inhibitors were omitted during the initial characterization of MLL cleaving protease. Cells were homogenized using a glass dounce and the homogenized cellular extract was subjected to centrifugal fractionation. Nuclei (Nuc) and unbroken cells were twice separated at 700 g for 10 min. The heavy membrane (HM) fraction pellet was collected after two centrifugations of the supernatant at 7,000 g for 10 minutes. The resulting supernatant was centrifuged at 100,000 g for 30 minutes to yield the light membrane pellet (LM) and final soluble fraction (S100). Proteins were solubilized in buffer A (20 mM HEPES [pH 7.9], 100 mM KCl, 1.5 mM MgCl.sub.2, 0.2 mM PMSF, 1 mM DTT, 1 mM EDTA, 1 mM EGTA, 0.1% Tween 20, and 10% glycerol) with additional 0.5% of Tween 20. Solubilized LM fraction was applied to a P11 column and the bound protease was eluted with gradients of KCl. Positive fractions were collected and dialyzed against buffer B (10 mM HEPES [pH 7.9], 100 mM KCl, 1 mM MgCl.sub.2, 10 uM CaCl.sub.2, 0.2 mM PMSF, 1 mM DTT, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 0.1% Tween 20, and 10 mM potassium phosphate [pH 7.9]) and applied to a hydroxyapatite column. Elution was performed with a phosphate gradient and the protease positive fractions were dialyzed against buffer A before loading onto indicated chromatographic columns. LC-MS/MS was performed by the Taplin Biological Mass Spectrometry Facility at the Harvard Medical School.

Recombinant Enzyme and Edman Degradation Analysis

His-tagged Taspase1 was expressed in BL21(DE3) cells and purified with a TALON column (Clontech). N-terminal protein sequencing was performed by Molecular Biology Core Facility at the Dana-Farber Cancer Institute.

RNAi, Reverse Transcription and Quantitative PCR

HeLa cells were transfected with indicated duplex RNAi (Dharmacon) using oligofectamine (Invitrogen). Double-stranded ribo-oligonucleotides with overhanging 3' deoxy TT were prepared to target mRNAs of either hTaspase1 (GACUCACAUUUCAAGACUU) (SEQ ID NO:56) or hMLL (GAAGUCAGAGUGCGAAGUC) (SEQ ID NO:57). Cells harvested 72 hours after transfection were either lysed in RIPA buffer for immunoblots or with Trizol (Invitrogen) for RNA purification using RNeasy (Qiagen). Reverse transcription were performed with oligo-dT primers using Superscript II (Invitrogen). Quantitative PCR was performed in triplicate using indicated gene specific primers (supplementary methods) with SYBR green (PE biosystems) on the ABI Prism 7700 sequence detection system.

Determination of Sequence Homology or Identity

The "percent identity" of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLASTN and BLASTX programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used (available on the Internet at ncbi.nlm.nih.gov).

Particularly preferred Taspase1 polypeptides have an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO:1. In the context of an amino acid sequence, the term "substantially identical" is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1 are termed substantially identical.

Isolated Taspase1 Polypeptides

In another aspect, the invention features, an isolated Taspase1 protein or fragment, e.g., a biologically active portion. Taspase1 protein can be isolated from cells or tissue sources using standard protein purification techniques. Taspase1 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.

The polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g., glycosylation or cleavage, present when expressed in a native cell.

The Taspase1 proteins, or fragment thereof, can differ from the corresponding sequence in SEQ ID: 1, for example, by at least one but by less than 20, 15, 10 or 5 amino acid residues. Alternatively, it can differ from the corresponding sequence in SEQ ID NO:1 by at least one residue but less than 20%, 15%, 10% or 5% of the residues in it differ from the corresponding sequence in SEQ ID NO:1. The differences can be conservative, non-conservative or both.

In one embodiment, the protein includes an amino acid sequence at least about 80%, 85%, 90%, 95%, 98%, 99% or more homologous to SEQ ID NO:1.

The peptides of this invention can be made by chemical synthesis methods, which are well known to the ordinarily skilled artisan. See, for example, Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77. Hence, peptides can be synthesized using the automated Merrifield techniques of solid phase synthesis with the .alpha.-NH2 protected by either t-Boc or F-moc chemistry using side chain protected amino acids on, for example, an Applied Biosystems Peptide Synthesizer Model 430A or 431.

One manner of making of the peptides described herein is using solid phase peptide synthesis (SPPS). The C-terminal amino acid is attached to a cross-linked polystyrene resin via an acid labile bond with a linker molecule. This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products. The N-terminus is protected with the Fmoc group, which is stable in acid, but removable by base. Any side chain functional groups are protected with base stable, acid labile groups.

Alternatively, the longer synthetic peptides can be synthesized by well known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct a gene encoding a peptide of this invention, the amino acid sequence is reverse translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably with codons that are optimum for the organism in which the gene is to be expressed. Next, a synthetic gene is made, typically by synthesizing oligonucleotides which encode the peptide and any regulatory elements, if necessary. The synthetic gene is inserted in a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods.

Screening Assays

The invention provides methods (also referred to herein as "screening assays") for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which have a stimulatory or inhibitory effect on, for example, Taspase1 expression or Taspase1 activity (e.g., proteolytic cleavage of an MLL substrate), have a stimulatory or inhibitory effect on, for example, the expression or activity of a Taspase1 substrate (e.g., MLL), or which bind to Taspase1. Desirable inhibitors of Taspase1 activity reduce the proteolytic activity of Taspase1 and include those which reduce cleavage of MLL at CS1 or CS2 or both CS1 and CS2. Inhibitors can be identified by their ability to reduce cleavage of MLL family proteins or some other substrate, e.g., a fragment of MLL containing CS1 and/or CS2 such as the p75 fragment of MLL (amino acids 2400-2900 of SEQ ID NO: 3) or the p45 fragment of MLL (amino acids 2500-2800 of SEQ ID NO: 3).

In one embodiment, the invention includes assays to determine the ability of a candidate compound to modulate the proteolytic cleavage of a Taspase1 substrate such as MLL or a fragment of MLL (e.g., an MLL fragment containing CS1 and/or CS2, such as the p75 fragment or the p45 fragment). Taspase1 is exposed to a candidate compound in the presence of MLL or a fragment thereof under conditions sufficient to allow cleavage of the MLL or MLL fragment (e.g., 37.degree. C. for about 60 minutes). The reaction mixture is then analyzed (for example, using labeled MLL or a labeled MLL fragment and SDS-PAGE followed by autoradiography) to determine whether a candidate compound modulates (e.g., stimulates or inhibits) the activity of Taspase1 (e.g., the proteolytic cleavage of MLL or an MLL fragment). In some instances, it is desirable for the candidate compound to inhibit the activity of Taspase1 (e.g., decrease the level of MLL proteolytic cleavage). In other instances, it is desirable for the compound to enhance or stimulate the activity of Taspase1 (e.g., increase the level of MLL proteolytic cleavage).

The K.sub.i of candidate compounds can be determined using, for example, a titration assay. Taspase1 can be exposed to varying concentrations of candidate compound (e.g., 1 nM, 10 nM, 100 nM, 1 .mu.M, 10 .mu.M, 100 .mu.M, 1 mM, and 10 mM) in the presence of a substrate such as MLL or a fragment thereof (e.g., a CS1 and/or CS2 containing MLL fragment). The effect of each concentration of candidate compound is then analyzed (e.g., using labeled MLL and SDS-PAGE followed by autoradiography) to determine the effect of the candidate compound on Taspase1 activity (e.g., inhibition of MLL cleavage) at varying concentrations, which can be used to calculate the K.sub.i of the candidate compound. The candidate compound can modulate Taspase1 activity in a competitive or non-competitive manner.

The assays described herein can be performed with individual candidate compounds or can be performed with a plurality of candidate compounds. Where the assays are performed with a plurality of candidate compounds, the assays can be performed using mixtures of candidate compounds or can be run in parallel reactions with each reaction having a single candidate compound. The test compounds or agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art.

In one embodiment, an assay is a cell-based assay in which a cell that expresses a Taspase1 protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate Taspase1 activity is determined. Determining the ability of the test compound to modulate Taspase1 activity can be accomplished by monitoring, for example, MLL cleavage.

In yet another embodiment, a cell-free assay is provided in which a Taspase1 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate Taspase1 activity is evaluated. Preferred biologically active portions of the Taspase1 proteins to be used in assays of the present invention include fragments which have the ability to proteolytically cleave a Taspase1 substrate, e.g., MLL or a CS1 and/or CS1 containing fragment thereof such as the p75 fragment or the p45 fragment. Preferred biologically active portions of the Taspase1 proteins used in the assays described herein include fragments that have the ability to proteolytically cleave a Taspase1 substrate, e.g., MLL. For example a cell-free assay can involve preparing a reaction mixture of a Taspase1 polypeptide or a fragment thereof and the candidate compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected. Candidate compounds that have the ability to form a covalent bond with Taspase1 can be detected, for example, by preparing a labeled candidate compound (e.g., a modified polypeptide including a peptide aldehyde and a chloromethylketone or a fluoromethylketone peptide derivative), exposing Taspase1 to the candidate compound, and subsequently measuring the amount of radioactive Taspase1. Alternatively, cell free assays can be used to determine the ability of the compound to modulate (i.e., enhance or inhibit) Taspase1 activity (e.g., MLL cleavage), for example, using labeled MLL or a CS1 and/or CS2 containing fragment thereof.

In one embodiment, a cell free assay can measure the ability of Taspase1 to proteolytically cleave a substrate using a peptide-based fluorescence resonance energy transfer (FRET) assay. FRET assays are known to one of skill in the art. (see, for example, Cummings G. et al., (2002) PNAS 99:6603-6) For example, Taspase1 or a fragment thereof can be incubated in the presence of a doubly labeled peptide substrate (e.g., MLL, a p75 fragment of MLL, a p45 fragment of MLL, or a fragment of MLL containing a CS1 and/or a CS2) and a candidate compound, wherein the peptide substrate is doubly labeled with suitable fluorophore/quencher pair (e.g., a coumarin fluorophore paired with either DABCYL or QSY-35 as the quencher). After incubation under conditions to allow proteolytic cleavage of the substrate, the peptide is then removed from the mixture and the peptide substrates and products separated (e.g., using HPLC). The degree of inhibition of the candidate compound is then measured by a change in fluorescence relative to a control sample, wherein a decrease in Taspase1 activity (e.g., MLL cleavage) corresponds to a relative decrease in fluorescence and an increase Taspase1 activity corresponds to a relative increase in fluorescence.

In another embodiment, Taspase1 or a fragment thereof is incubated in the presence of a candidate compound and a substrate (e.g., MLL, a p75 fragment of MLL, a p45 fragment of MLL, or a fragment of MLL containing a CS1 and/or a CS2). After incubation under conditions sufficient to allow proteolytic cleavage of the substrate, the reaction mixture is analyzed using MS (e.g., LC/MS, ESI-LC/MS, FAB-MS). (see, for example, Zhu et al., (2003) J. Biol. Chem., 278:22418-23) Quantitative measurements of substrate conversion can be made using ratiometric analysis of the substrate (e.g., MLL or a fragment thereof) and product (e.g., the cleaved MLL or fragment thereof) peak areas in the extracted ion chromatograms of each species. The ratios of substrate and product in the presence of a candidate compound can be compared to a control to determine whether the candidate compound had an inhibitory effect or an enhancing effect on the Taspase1 activity (e.g., proteolytic cleavage of substrate).

In one embodiment, Taspase1, Taspase1 fragment, or test compound is anchored onto a solid phase. The Taspase1/test compound complexes anchored on the solid phase can be detected at the end of the reaction. Preferably, the Taspase1 or a fragment thereof is anchored onto a solid surface, and the test compound (which is not anchored) can be labeled, either directly or indirectly, with detectable labels discussed herein.

Cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A. P., (1993) Trends Biochem. Sci. 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel, F. et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel, F. et al., eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, N. H., (1998) J Mol Recognit 11: 141-8; Hage, D. S., and Tweed, S. A. (1997) J Chromatogr. B. Biomed. Sci. Appl. 699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, candidate compounds that interfere with the interaction between Taspase1 and an MLL polypeptide, e.g., by competition, can be identified by conducting an MLL proteolytic cleavage reaction in the presence of the candidate compound.

In a heterogeneous assay system, either the Taspase1 or the MLL polypeptide, is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.

In an alternate embodiment of the invention, a homogeneous assay can be used. For example, a preformed complex of Taspase1 or a fragment thereof and MLL or a fragment thereof can be prepared in that either the Taspase1 or MLL are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 that utilizes this approach for immunoassays). The addition of candidate compound that competes with and displaces one of the species from the preformed Taspase1/MLL substrate complex will result in the generation of a signal above background. In this way, test substances that disrupt Taspase1/MLL interaction can be identified. In some instances it is desirable to modify the MLL substrate in order to prevent proteolytic cleavage of the substrate upon interaction with Taspase1, thus maintaining the MLL substrate in the Taspase1/MLL substrate complex for a greater length of time.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a Taspase1 modulating agent such as a Taspase1 inhibitor, an antisense Taspase1 nucleic acid molecule, a Taspase1-specific antibody, or a Taspase1-binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein.

Inhibitors of Taspase1 Activity

In one embodiment, the invention includes an inhibitor of Taspase1 activity. The inhibitor can be, for example a polypeptide, a modified polypeptide, or a peptidomimetic. Preferred peptides are of between about 5 and 7 amino acids in length. However, longer and shorter polypeptides are also envisioned. Preferred polypeptides include amino acid sequences of Ile-Xaa-Gln-Leu-Xaa (SEQ ID NO:58) (e.g., Ile-Ser-Gln-Leu-Asp (SEQ ID NO:16) or Ile-Ser-Gln-Leu-Ala (SEQ ID NO:7)).

In some instances, the polypeptides are modified at the C-terminal end or the N-terminal end. Modification of the C-terminal end can provide a chemically reactive group that will form a covalent bond between Taspase1 and the polypeptide, wherein the bond is either irreversible or inefficiently reversible, thus allowing the polypeptide to occupy the active site of Taspase1, for a longer period of time. Some examples of C-terminal modifications include replacing the carboxy end of the polypeptide with an aldehyde, a chloromethylketone or a fluoromethylketone. Other C-terminal modifications are envisioned for the polypeptide inhibitors described herein. Methods of modification of polypeptides are well known to one of skill in the art.

In some instances, it is desirable to modify a backbone of a polypeptide in order to improve the bioavailability of the polypeptide, improve the potency of the polypeptide, or prevent (e.g., slow) the metabolism of the polypeptide in the body. Preferably, one or more hydrolyzable amide bonds of the polypeptide are replaced with a non-hydrolyzable isosteric group of the amide or the transition state of the amide during hydrolysis. Some examples of peptide backbone modifications include replacing the amide bond with a hydroxyethylamine, hydroxyelthylene, hydroxyethylurea, urea, norstatine, a C2 symmetric monoalcohol, or diol(dihydroxyethylene). (See e.g., Abdel-Rahman et al., (2002) Cur. Med. Chem. 9:1905-1922.) Other peptide modifications are also envisioned. For example, the use of a terminal (e.g., N-terminal or C-terminal) thiazole group can increase the chemical stability towards metabolic oxidation while maintaining water solubility, or the addition of a pyridyl group to the polypeptide (e.g., a terminal portion of the polypeptide) can improve the water solubility of the polypeptide.

Small molecule inhibitors are also envisioned. For example, the small molecule inhibitors can include heterocyclic compounds having motifs that mimic the CS1 or CS2 binding sites of MLL.

Isolated Nucleic Acid Molecules

In one aspect the invention provides an isolated or purified, nucleic acid molecule that encodes a Taspase1 polypeptide described herein, e.g., a full-length Taspase1 protein or a fragment thereof, e.g., a biologically active portion of Taspase1 protein.

In one embodiment, an isolated nucleic acid molecule of the invention includes the nucleotide sequence shown in SEQ ID NO:2, or a portion of any of these nucleotide sequences. In one embodiment, the nucleic acid molecule includes sequences encoding the human Taspase1 protein, as well as 5' untranslated sequences.

In another embodiment, an isolated nucleic acid molecule of the invention includes a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:2, or a portion of any of these nucleotide sequences. In other embodiments, the nucleic acid molecule of the invention is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:2, such that it can hybridize (e.g., under a stringency condition described herein) to the nucleotide sequence shown in SEQ ID NO:2, thereby forming a stable duplex.

In one embodiment, an isolated nucleic acid molecule of the present invention includes a nucleotide sequence which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to the entire length of the nucleotide sequence shown in SEQ ID NO:2, or a portion, preferably of the same length, of any of these nucleotide sequences.

Antisense Nucleic Acid Molecules, Ribozymes, RNAi, and Modified Taspase1 Nucleic Acid Molecules

To inhibit the expression of Taspase1, one can administer one or more nucleic acid inhibitory agents, such as antisense RNA, a small inhibitory RNA (i.e., RNAi), or a ribozyme, any of which can be designed to target a sequence within Taspase1 or a fragment thereof.

In another aspect, the invention features, an isolated nucleic acid molecule which is antisense to Taspase1. An "antisense" nucleic acid can include a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire Taspase1 coding strand, or to only a portion thereof. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of Taspase1 mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

The antisense nucleic acid molecules of the invention are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a Taspase1 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.

Taspase1 Chimeric or Fusion Proteins

In another aspect, the invention provides Taspase1 chimeric or fusion proteins. As used herein, a Taspase1 "chimeric protein" or "fusion protein" includes a Taspase1 polypeptide linked to a non-Taspase1 polypeptide. A "non-Taspase1 polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the Taspase1 protein, e.g., a protein which is different from the Taspase1 protein and which is derived from the same or a different organism. The Taspase1 polypeptide of the fusion protein can correspond to all or a portion e.g., a fragment described herein of a Taspase1 amino acid sequence. In a preferred embodiment, a Taspase1 fusion protein includes at least one (or two) biologically active portion of a Taspase1 protein. The non-Taspase1 polypeptide can be fused to the N-terminus or C-terminus of the Taspase1 polypeptide.

The Taspase1 fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo and can be used to affect the bioavailability of a Taspase1 substrate. Additionally, Taspase1 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a Taspase1 protein; (ii) mis-regulation of the Taspase1 gene; and (iii) aberrant post-translational modification of a Taspase1 protein.

Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A Taspase1-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in frame to the Taspase1 protein.

Anti-Taspase1 Antibodies

The Taspase1 polypeptide can be used to produce an anti-Taspase1 antibody, or a fragment thereof (e.g., an antigen-binding fragment thereof). The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. As used herein, the term "antibody" refers to a protein comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL).

The term "antigen-binding fragment" of an antibody (or simply "antibody portion," or "fragment"), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to the antigen, e.g., Taspase1 polypeptide or fragment thereof. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

The anti-Taspase1 antibody can be a polyclonal or a monoclonal antibody. In other embodiments, the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods.

Phage display and combinatorial methods for generating anti-Taspase1 antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated by reference herein).

Recombinant Expression Vectors, Host Cells and Genetically Engineered Cells

In another aspect, the invention includes, vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.

Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.

In another embodiment, the promoter is an inducible promoter, e.g., a promoter regulated by a steroid hormone, by a polypeptide hormone (e.g., by means of a signal transduction pathway), or by a heterologous polypeptide (e.g., the tetracycline-inducible systems, "Tet-On" and "Tet-Off"; see, e.g., Clontech Inc., CA, Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983).

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus.

Another aspect the invention provides a host cell which includes a nucleic acid molecule described herein, e.g., a Taspase1 nucleic acid molecule within a recombinant expression vector or a Taspase1 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms "host cell" and "recombinant host cell" are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell.

A host cell can be any prokaryotic or eukaryotic cell. For example, a Taspase1 protein can be expressed in bacterial cells (such as E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells (African green monkey kidney cells CV-1 origin SV40 cells; Gluzman (1981) Cell I23:175-182)). Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.

Pharmaceutical Compositions

The nucleic acids and polypeptides, fragments thereof, anti-Taspase1 antibodies, inhibitors of Taspase1 activity, or enhancers of Taspase1 activity (also referred to herein as "active compounds") of the invention can be incorporated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecule, protein, antibody, or inhibitor and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The active compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD.sub.50 (the dose lethal to 50% of the population) and the ED.sub.50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED.sub.50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC.sub.50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

For antibodies, the preferred dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. (1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

The present invention encompasses agents which modulate expression or activity (e.g., inhibit Taspase1 activity or enhance Taspase1 activity). An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

Methods of Treatment

The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted Taspase1 expression or activity (e.g., MLL proteolytic cleavage). As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

It is possible that some Taspase1 disorders can be caused, at least in part, by an abnormal level of Taspase1, or by the presence of Taspase1 exhibiting abnormal activity. As such, the reduction in the level and/or activity of Taspase1 would bring about the amelioration of disorder symptoms. Moreover, even normal levels of activity of Taspase1 may lead to expression of HOX genes that are present in malignant cells, thus consequently inhibiting Taspase1 activity could reduce the level of target proteins such as the HOX products and ameliorate the disorder (e.g., a cancer).

The Taspase1 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders, for example cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias.

As used herein, the terms "cancer", "hyperproliferative" and "neoplastic" refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. "Pathologic hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.

Examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term "hematopoietic neoplastic disorders" includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

As discussed, successful treatment of Taspase1 dependent disorders can be brought about by techniques that serve to inhibit the expression or activity of Taspase1. For example, compounds, e.g., an agent identified using an assays described above, that proves to exhibit negative modulatory activity (e.g., inhibits MLL proteolysis), can be used in accordance with the invention to ameliorate symptoms of Taspase1 dependent disorders, such as cancer. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab')2 and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof).

Further, antisense, siRNA (small interfering RNA), and ribozyme molecules that inhibit expression of Taspase1 can also be used in accordance with the invention to reduce the level of Taspase1 expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of Taspase1 activity. Antisense, ribozyme, siRNA, and triple helix molecules are discussed above.

It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances in that the target gene encodes an extracellular protein, it can be preferable to co-administer normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

The identified compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate Taspase1 disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disorders. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures as described above.

Another aspect of the invention pertains to methods of modulating Taspase1 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell with a Taspase1 or agent that modulates one or more of the activities of Taspase1 protein activity associated with the cell. An agent that modulates Taspase1 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a Taspase1 protein (e.g., a Taspase1 substrate or receptor), a Taspase1 antibody, a Taspase1 agonist or antagonist, a peptidomimetic of a Taspase1 agonist or antagonist, or other small molecule.

In one embodiment, the agent stimulates Taspase1 activity. Examples of such stimulatory agents include active Taspase1 protein and a nucleic acid molecule encoding Taspase1. In another embodiment, the agent inhibits one or more Taspase1 activities. Examples of such inhibitory agents include antisense Taspase1 nucleic acid molecules, anti-Taspase1 antibodies, and Taspase1 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a Taspase1 protein or nucleic acid molecule.

Stimulation of Taspase1 activity is desirable in situations in which Taspase1 is abnormally downregulated and/or in which increased Taspase1 activity is likely to have a beneficial effect. Likewise, inhibition of Taspase1 activity is desirable in situations in which Taspase1 is abnormally upregulated and/or in which decreased Taspase1 activity is likely to have a beneficial effect.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

SEQUENCE LISTINGS

1

591420PRTHomo sapiens 1Met Thr Met Glu Lys Gly Met Ser Ser Gly Glu Gly Leu Pro Ser Arg 1 5 10 15Ser Ser Gln Val Ser Ala Gly Lys Ile Thr Ala Lys Glu Leu Glu Thr 20 25 30Lys Gln Ser Tyr Lys Glu Lys Arg Gly Gly Phe Val Leu Val His Ala 35 40 45Gly Ala Gly Tyr His Ser Glu Ser Lys Ala Lys Glu Tyr Lys His Val 50 55 60Cys Lys Arg Ala Cys Gln Lys Ala Ile Glu Lys Leu Gln Ala Gly Ala65 70 75 80Leu Ala Thr Asp Ala Val Thr Ala Ala Leu Val Glu Leu Glu Asp Ser 85 90 95Pro Phe Thr Asn Ala Gly Met Gly Ser Asn Leu Asn Leu Leu Gly Glu 100 105 110Ile Glu Cys Asp Ala Ser Ile Met Asp Gly Lys Ser Leu Asn Phe Gly 115 120 125Ala Val Gly Ala Leu Ser Gly Ile Lys Asn Pro Val Ser Val Ala Asn 130 135 140Arg Leu Leu Cys Glu Gly Gln Lys Gly Lys Leu Ser Ala Gly Arg Ile145 150 155 160Pro Pro Cys Phe Leu Val Gly Glu Gly Ala Tyr Arg Trp Ala Val Asp 165 170 175His Gly Ile Pro Ser Cys Pro Pro Asn Ile Met Thr Thr Arg Phe Ser 180 185 190Leu Ala Ala Phe Lys Arg Asn Lys Arg Lys Leu Glu Leu Ala Glu Arg 195 200 205Val Asp Thr Asp Phe Met Gln Leu Lys Lys Arg Arg Gln Ser Ser Glu 210 215 220Lys Glu Asn Asp Ser Gly Thr Leu Asp Thr Val Gly Ala Val Val Val225 230 235 240Asp His Glu Gly Asn Val Ala Ala Ala Val Ser Ser Gly Gly Leu Ala 245 250 255Leu Lys His Pro Gly Arg Val Gly Gln Ala Ala Leu Tyr Gly Cys Gly 260 265 270Cys Trp Ala Glu Asn Thr Gly Ala His Asn Pro Tyr Ser Thr Ala Val 275 280 285Ser Thr Ser Gly Cys Gly Glu His Leu Val Arg Thr Ile Leu Ala Arg 290 295 300Glu Cys Ser His Ala Leu Gln Ala Glu Asp Ala His Gln Ala Leu Leu305 310 315 320Glu Thr Met Gln Asn Lys Phe Ile Ser Ser Pro Phe Leu Ala Ser Glu 325 330 335Asp Gly Val Leu Gly Gly Val Ile Val Leu Arg Ser Cys Arg Cys Ser 340 345 350Ala Glu Pro Asp Ser Ser Gln Asn Lys Gln Thr Leu Leu Val Glu Phe 355 360 365Leu Trp Ser His Thr Thr Glu Ser Met Cys Val Gly Tyr Met Ser Ala 370 375 380Gln Asp Gly Lys Ala Lys Thr His Ile Ser Arg Leu Pro Pro Gly Ala385 390 395 400Val Ala Gly Gln Ser Val Ala Ile Glu Gly Gly Val Cys Arg Leu Glu 405 410 415Ser Pro Val Asn 42021263DNAHomo sapiens 2atgaccatgg agaaggggat gagttctgga gaagggctgc cttccagatc atctcaggtt 60tcggctggta aaataacagc caaagagttg gaaacaaagc agtcctataa agagaaacga 120ggaggctttg tgttggtgca tgcaggtgca ggttatcatt ctgaatccaa agccaaggag 180tataaacatg tatgcaaacg agcttgtcag aaggcaattg aaaagctgca ggccggtgct 240cttgcaactg acgcagtcac tgcagcactg gtggaacttg aggattctcc ttttacaaat 300gcaggaatgg gatctaatct aaatctgtta ggtgaaattg agtgtgatgc cagcataatg 360gatggaaaat ccttaaattt tggagcagtt ggagcactga gtggaatcaa gaacccagtc 420tcggttgcca acagactctt atgtgaaggg cagaagggca agctctcggc tggcagaatt 480cctccctgct ttttagttgg agaaggagcc tacagatggg cagtagatca tggaataccc 540tcttgccctc ctaacatcat gaccacaaga ttcagtttag ctgcatttaa aagaaacaag 600aggaaactag agctggcaga aagggtggac acagatttta tgcaactaaa gaaaagaaga 660caatcaagtg agaaggaaaa tgactcaggc actttggaca cggtaggcgc tgtggttgtg 720gaccacgaag ggaatgttgc tgctgctgtc tccagtggag gcttggcctt gaaacatccg 780gggagagttg ggcaggctgc tctttatgga tgtggctgct gggctgaaaa tactggagct 840cataacccct actccacagc tgtgagtacc tcaggatgtg gagagcatct tgtgcgcacc 900atactggcta gagaatgttc acatgcttta caagctgagg atgctcacca agccctgttg 960gagactatgc aaaacaagtt tatcagttca cctttccttg ccagtgaaga tggcgtgctt 1020ggcggagtga ttgtcctccg ttcatgcaga tgttctgccg agcctgactc ctcccaaaat 1080aagcagacac ttctagtgga atttctgtgg agccacacga cggagagcat gtgtgtcgga 1140tatatgtcag cccaggatgg gaaagccaag actcacattt caagacttcc tcctggtgcg 1200gtggcaggac agtctgtggc aatcgaaggt ggggtgtgcc gcctggagag cccagtgaac 1260tga 126335PRTHomo sapiens 3Asp Gly Ala Asp Asp 1 545PRTHomo sapiens 4Asp Gly Val Asp Asp 1 555PRTHomo sapiens 5Leu Asp Thr Val Gly 1 564PRTHomo sapiens 6Ser Gln Leu Ala 175PRTHomo sapiens 7Ile Ser Gln Leu Ala 1 586PRTHomo sapiens 8Lys Ile Ser Gln Leu Ala 1 594PRTArtificial SequenceSynthetically generated peptide 9Ser Gln Leu Asp 1105PRTArtificial SequenceSynthetically generated peptide 10Ile Ser Gln Leu Asp 1 5116PRTArtificial SequenceSynthetically generated peptide 11Lys Ile Ser Gln Leu Asp 1 5124PRTArtificial SequenceSynthetically generated peptide 12Ser Gln Leu Asp 1135PRTArtificial SequenceSynthetically generated peptide 13Ile Ser Gln Leu Asp 1 5146PRTArtificial SequenceSynthetically generated peptide 14Lys Ile Ser Gln Leu Asp 1 5155PRTHomo sapiens 15Ile Ser Gln Leu Asp 1 5165PRTHomo sapiens 16Glu Gly Gln Val Asp 1 5174PRTArtificial SequenceSynthetically generated peptide 17Ser Gln Leu Ala 1185PRTHomo sapiens 18Ala Ala Ala Asp Asp 1 51920PRTHomo sapiens 19Gly Lys Arg Ser Ala Glu Gly Gln Val Asp Gly Ala Asp Asp Leu Ser 1 5 10 15Thr Ser Asp Glu 202020PRTMus musculus 20Gly Lys Arg Ser Ala Glu Gly Gln Val Asp Gly Ala Asp Asp Leu Ser 1 5 10 15Thr Ser Asp Glu 202120PRTDanio Rerio 21Arg Lys Lys Ser Ala Glu Gly Gln Val Asp Gly Ala Asp Asp Ile Ser 1 5 10 15Ser Thr Ser Ser 202220PRTHomo sapiens 22Cys Asp Leu Pro Lys Ile Ser Gln Leu Asp Gly Val Asp Asp Gly Thr 1 5 10 15Glu Ser Asp Thr 202320PRTMus musculus 23Cys Asp Leu Pro Lys Ile Ser Gln Leu Asp Gly Val Asp Asp Gly Thr 1 5 10 15Glu Ser Asp Thr 202420PRTDanio rerio 24Leu Gly Lys Pro Gln Ile Gly Gln Leu Asp Gly Val Asp Asp Gly Ser 1 5 10 15Glu Ser Asp Ala 202520PRTAnopheles trathorax 25Phe Gln Lys Leu Lys Ile Ser Gln Leu Asp Gly Val Asp Asp Ile Cys 1 5 10 15Leu Asp Gly Thr 202620PRTDrosophilia trithorax 26Ala Ala Lys Met Arg Ile Met Gln Met Asp Gly Val Asp Asp Ser Ile 1 5 10 15Thr Glu Phe Arg 202720PRTHomo sapiens 27Pro Gly Ala Pro Arg Ile Glu Gln Leu Asp Gly Val Asp Asp Gly Thr 1 5 10 15Asp Ser Glu Ala 202810PRTHomo sapiens 28Thr Leu Asp Thr Val Gly Ala Val Val Val 1 5 102910PRTMus musculus 29Thr Leu Asp Thr Val Gly Ala Val Val Val 1 5 103010PRTRattus norvegicus 30Ala Leu Asp Thr Val Gly Ala Val Val Val 1 5 103110PRTFugu rubripes 31Cys Leu Asp Thr Val Gly Ala Val Val Val 1 5 103210PRTDanio rerio 32Cys Leu Asp Thr Val Gly Ala Val Val Val 1 5 103310PRTAnopheles gambiae 33Pro Leu Asp Thr Val Gly Ala Val Cys Val 1 5 103410PRTDrosophilia melanogaster 34Ala Leu Asp Thr Val Gly Ala Val Cys Val 1 5 103510PRTHomo sapiens 35Gly His Asp Thr Ile Gly Met Val Val Ile 1 5 103610PRTMus musculus 36Ser His Asp Thr Ile Gly Met Val Val Ile 1 5 103710PRTFugu rubripes 37Ala His Asp Thr Ile Gly Met Ile Ala Ile 1 5 103810PRTSpodotera frugiperda 38Asn His Asp Thr Ile Gly Met Val Ala Ile 1 5 103910PRTFlavobacterium meningosepticum 39Asn His Asp Thr Ile Gly Met Ile Ala Ile 1 5 104010PRTHomo sapiens 40Asn Leu Gly Thr Val Gly Ala Val Ala Leu 1 5 104110PRTMus musculus 41Asn Ser Gly Thr Val Gly Ala Val Ala Leu 1 5 104210PRTFugu rubripes 42Lys Met Gly Thr Val Gly Ala Val Ala Val 1 5 104310PRTArabidopsis thaliana 43Gln Ile Gly Thr Val Gly Cys Val Ala Val 1 5 104410PRTLupinus arboreus 44Gln Ile Gly Thr Val Gly Cys Val Ala Val 1 5 1045420PRTMus musculus 45Met Ile Met Glu Lys Gly Met Asn Ser Gly Glu Gly Leu Pro Ser Arg 1 5 10 15Ser Ser Gln Ala Ser Ala Ala Lys Val Thr Val Lys Glu Leu Glu Thr 20 25 30Gln Gln Pro Cys Lys Glu Lys Arg Gly Gly Phe Val Leu Val His Ala 35 40 45Gly Ala Gly Tyr His Ser Glu Ser Lys Ala Lys Glu Tyr Lys His Val 50 55 60Cys Lys Arg Ala Cys Gln Lys Ala Ile Glu Lys Leu Gln Ala Gly Ala65 70 75 80Leu Ala Thr Asp Ala Val Ala Ala Ala Leu Val Glu Leu Glu Asp Ser 85 90 95Pro Phe Thr Asn Ala Gly Ile Gly Ser Asn Leu Asn Leu Leu Gly Glu 100 105 110Ile Glu Cys Asp Ala Ser Ile Met Asp Gly Lys Ser Leu Asn Phe Gly 115 120 125Ala Val Gly Ala Leu Ser Gly Ile Lys Asn Pro Val Ser Val Ala His 130 135 140Arg Leu Leu Cys Glu Gly Gln Lys Gly Lys Leu Ser Ala Gly Arg Ile145 150 155 160Pro Pro Cys Phe Leu Val Gly Glu Gly Ala Tyr Arg Trp Ala Val Asp 165 170 175His Gly Ile Pro Ser Cys Pro Pro Ser Thr Met Thr Thr Arg Phe Ser 180 185 190Leu Ala Ala Phe Lys Arg Asn Lys Arg Lys Leu Glu Leu Ala Glu Arg 195 200 205Val Glu Thr Asp Phe Ile Gln Leu Lys Arg Arg Arg Gln Ser Ser Ala 210 215 220Lys Glu Asn Asp Ser Gly Thr Leu Asp Thr Val Gly Ala Val Val Val225 230 235 240Asp His Glu Gly Asn Val Ala Ala Ala Val Ser Ser Gly Gly Leu Ala 245 250 255Leu Lys His Pro Gly Arg Val Gly Gln Ala Ala Leu Tyr Gly Cys Gly 260 265 270Cys Trp Ala Glu Asn Thr Gly Ala Gln Asn Pro Tyr Ser Thr Ala Val 275 280 285Ser Thr Ser Gly Cys Gly Glu His Leu Val Arg Thr Ile Leu Ala Arg 290 295 300Glu Cys Ser His Ala Leu Gln Ala Glu Asp Ala His Gln Ala Leu Leu305 310 315 320Glu Thr Met Gln Asn Lys Phe Ile Ser Ser Pro Phe Leu Ala Cys Glu 325 330 335Asp Gly Val Leu Gly Gly Val Ile Val Leu Arg Ser Cys Arg Cys Ser 340 345 350Ser Glu Ser Asp Ser Ser Gln Asp Lys Gln Thr Leu Leu Val Glu Phe 355 360 365Leu Trp Ser His Thr Thr Glu Ser Met Cys Val Gly Tyr Met Ser Ala 370 375 380Gln Asp Gly Lys Ala Lys Thr His Ile Ser Arg Leu Pro Pro Gly Ala385 390 395 400Val Ala Gly Gln Ser Val Ala Ile Glu Gly Gly Val Cys Arg Leu Glu 405 410 415Ser Pro Val Asn 42046386PRTRattus norvegicus 46Met Leu Lys Phe Val Asp Asp Ser Glu Tyr Arg Asp Ser Pro Phe Thr 1 5 10 15Asn Ala Gly Val Gly Ser Asn Leu Asn Leu Leu Gly Glu Ile Glu Cys 20 25 30Asp Ala Ser Ile Met Asp Gly Lys Ser Leu Ser Phe Gly Ala Val Gly 35 40 45Ala Leu Ser Gly Ile Lys Asn Pro Val Ser Val Ala His Arg Leu Leu 50 55 60Cys Glu Gly Gln Lys Gly Lys Leu Ser Ala Gly Arg Ile Pro Pro Cys65 70 75 80Phe Leu Val Gly Glu Gly Ala Tyr Arg Trp Ala Val Asp His Gly Ile 85 90 95Pro Ser Cys Pro Pro Ser Thr Met Thr Thr Arg Phe Ser Leu Ala Ala 100 105 110Phe Lys Arg Asn Lys Arg Lys Leu Glu Leu Ala Glu Arg Val Glu Thr 115 120 125Asp Phe Ile Gln Leu Lys Arg Arg Arg Gln Ser Ser Ala Lys Glu Asn 130 135 140Asp Ser Gly Ala Leu Asp Thr Val Gly Ala Val Val Val Asp His Glu145 150 155 160Gly Asn Val Ala Ala Ala Val Ser Ser Gly Gly Leu Ala Leu Lys His 165 170 175Pro Gly Arg Val Gly Gln Asp Val Glu Ser Ile Leu Cys Ala Pro Tyr 180 185 190Trp Leu Glu Asn Val His Thr Leu Tyr Lys Leu Lys Thr Leu Thr Lys 195 200 205Leu Cys Trp Arg Leu Cys Lys Thr Ser Leu Ser Asp Thr Glu Arg Ile 210 215 220Pro Gly Leu Asp Ser Ala Tyr Gly Thr Gly Lys Lys Arg Glu Gly Ile225 230 235 240Pro Ser Leu Gly Ser Glu Lys Glu Phe His Asn Gly Gln Pro His Asp 245 250 255Ala Trp Asn Ser Asp Thr His Leu His Ser Thr Ala Arg Thr Leu His 260 265 270His Gly Ser Pro Phe Leu Ala Ser Glu Asp Gly Val Leu Gly Gly Val 275 280 285Ile Val Leu Arg Ser Cys Arg Cys Pro Ser Glu Ser Asp Pro Ser Gln 290 295 300Asp Lys Gln Thr Leu Leu Val Glu Phe Leu Trp Ser His Ser Thr Glu305 310 315 320Ser Met Cys Val Gly Tyr Met Ser Ala Gln Asp Gly Lys Ala Lys Val 325 330 335Ile Val Arg Asn Met Thr Leu Glu Ser Leu Leu His Phe Ser Lys Val 340 345 350Phe Asn Ala Val Ile Ile Tyr Leu Trp Pro Leu Phe Leu Asn Val Ile 355 360 365Asp Asp Val Ile Cys Glu Val Ser Leu Asn Asp Asp Leu Ile Leu Pro 370 375 380Cys Tyr38547360PRTDanio rerio 47Lys Glu Glu Ser Ser Lys Asn Gln Lys Ala Lys Pro Val Gly Gly Phe 1 5 10 15Val Leu Val His Ala Gly Ala Gly Tyr His Ser Glu Ser Lys Ala Lys 20 25 30Glu Tyr Lys His Val Cys Lys Arg Ala Cys Gln Arg Ala Val Asp Gln 35 40 45Leu Asn Ala Gly Ala Leu Ala Val Glu Ala Val Ala Ala Ala Leu Val 50 55 60Glu Leu Glu Asp Ser Pro Phe Thr Asn Ala Gly Met Gly Ser Asn Leu65 70 75 80Asn Leu Leu Gly Glu Ile Glu Cys Asp Ala Ser Ile Met Asp Gly Lys 85 90 95Ser Leu Gln Tyr Gly Ala Val Gly Ser Ile Ser Gly Val Lys Asn Pro 100 105 110Val Leu Val Ala Asn Arg Leu Leu Ser Glu Ala Gln Arg Gly Lys Leu 115 120 125Ser Ala Gly Arg Ile Pro Pro Cys Phe Leu Val Gly Arg Gly Ala Leu 130 135 140Glu Trp Ala Val Ser His Gly Ile Ala Pro Cys Pro Ser Glu Lys Met145 150 155 160Ala Thr Lys Phe Ser Leu Ser Ala Tyr Lys Arg Asn Lys Arg Lys Met 165 170 175Glu Leu Ala Glu Leu Ala Glu Lys Met Glu Ser Gly His Asn Gln Ile 180 185 190Lys Lys Arg Arg Gln Ser Thr Glu Thr Ala Asn Pro Thr Glu Asp Gly 195 200 205Ser Gly Cys Leu Asp Thr Val Gly Ala Val Val Val Asp Leu Glu Gly 210 215 220Asn Val Ala Ala Ala Val Ser Ser Gly Gly Leu Ala Met Lys His Pro225 230 235 240Gly Arg Val Gly Gln Val Gly Leu Ser Gln Tyr Asn Asn Ala His Phe 245 250 255Cys Val Thr Ala Ser Phe Cys Cys Gly Glu His Leu Ile Arg Thr Met 260 265 270Leu Ala Arg Glu Cys Ser Ala Ala Met Arg Ser Glu Asp Ala His Gln 275 280 285Ala Leu Leu Glu Ala Met Gln Asn Lys Phe Ile Ser Ser Pro Phe Leu 290 295 300Ala Gly Glu Asp Arg Val Leu Gly Gly Val Ile Val Leu Arg Gly Cys305 310 315 320Arg Cys Val Glu Ala Pro Ser Ser Gln Asn Ile Gln Gly Ile Leu Val 325 330 335Glu Phe Leu Trp Ser His Thr Thr Glu Ser Met Cys Val Gly Tyr Met 340 345 350Ser Ala Gln Asp Ser Lys Ala Lys 355 36048328PRTDanio rerio 48Lys Leu Asn Ser Thr Ile Ser His Arg Asp Gly Lys Asn Met Gly Phe 1 5 10 15Val Leu Val His Ala Gly Ala Gly Tyr His Ser Glu Ser Lys Ala Lys 20 25 30Glu Tyr Lys His Val Cys Lys Arg Ala Cys Gln Lys Leu Gln Asp Ser 35

40 45Pro Phe Thr Asn Ala Gly Thr Gly Ser Asn Leu Asn Leu Ser Gly Glu 50 55 60Val Glu Cys Asp Ala Ser Ile Met Asp Gly Lys Ser Leu Asn Tyr Gly65 70 75 80Ala Val Gly Ala Leu Ser Gly Ile Lys Asn Pro Val Leu Val Ser Arg 85 90 95Arg Leu Leu Ser Glu Thr Gln Lys Gly Lys Leu Ser Ala Gly Arg Ile 100 105 110Pro Pro Phe Leu Val Gly Lys Gly Ala Glu Gln Trp Ala Ile Ser His 115 120 125Gly Ile Pro Ala Cys Pro Thr Glu Lys Met Thr Thr Glu Glu Asn Asn 130 135 140Ser Ala Cys Leu Asp Thr Val Gly Ala Val Val Val Asp Gly Glu Gly145 150 155 160Asn Val Ala Thr Ala Val Ser Ser Gly Gly Leu Ala Met Lys His Pro 165 170 175Gly Arg Val Gly Gln Ala Ala His Tyr Gly Cys Gly Cys Trp Ala Glu 180 185 190Asn Ala Arg Asp Val Ser Leu Tyr Ser Thr Ala Val Ser Gly Ser Gly 195 200 205Glu His Leu Ile Pro Pro Met Leu Ala Arg Glu Cys Ser Thr Ala Met 210 215 220His Ala Glu Asn Pro Pro Pro Ala Pro Leu Glu Pro Met Gln Asn Lys225 230 235 240Phe Ile Ser Ser Pro Phe Leu Ala Gly Glu Asp Cys Val Leu Gly Gly 245 250 255Val Ile Val Leu Arg Cys Cys Thr Cys Gly Glu Ala Gln Arg Ser Glu 260 265 270Asp Ile Gln Ala Leu Leu Val Glu Phe Leu Trp Ser His Thr Thr Glu 275 280 285Ser Met Cys Val Gly Tyr Met Ser Ala Gln Asp Ser Lys Ala Arg Thr 290 295 300His Ile Ser Arg Leu Pro Pro Gly Ala Val Ala Gly Gln Ser Leu Ala305 310 315 320Ile Glu Gly Gly Val Cys Arg Leu 32549388PRTAnopheles gambiae str 49Asn Ile Leu Pro Ser Gln Cys Ala Val Leu Lys Ser Ile Gln Leu Pro 1 5 10 15Ser Thr Ser Cys Leu Cys Gln Cys Ser Cys Cys Pro Asn Val Ile Val 20 25 30Arg Lys Lys Ala Arg Ala Leu Val Thr Met Thr Gly Phe Val Ala Val 35 40 45His Thr Gly Ala Gly Asn Phe Leu Asp Glu Thr Leu Tyr Glu His Val 50 55 60Cys Arg Glu Ala Cys Asn Gln Ala Val Asn Val Leu Tyr Ala Gly Gly65 70 75 80Thr Ala Leu Asp Ala Cys Glu Arg Ala Ile Val Leu Leu Glu Asn Ser 85 90 95Thr Ala Thr Asn Ala Gly Ile Gly Ser Asn Leu Asn Trp Asp Arg Arg 100 105 110Val Glu Cys Asp Ala Cys Ile Met Asp Gly Ala Ser Leu Gln Phe Gly 115 120 125Ala Cys Thr Asn Val Thr Asp Val Lys Asn Pro Ile Ser Leu Ala Arg 130 135 140His Leu Cys Glu Arg Gln Ser Lys Leu Leu Ser Phe Gly Arg Ile Phe145 150 155 160Phe Met Val Leu Val Gly Gln Gly Ala Ser Ala Tyr Ala Arg Glu Val 165 170 175Gly Leu Gln Leu Val Pro Ala Glu His Met Ile Ser Val Asn Ala Ala 180 185 190Lys Lys Tyr Asp His Tyr Arg Ser Gln Ile Met Gln Tyr Glu Glu Met 195 200 205Asn Arg Ala Lys Leu Ser Pro Leu Asp Thr Val Gly Ala Val Cys Val 210 215 220Asp Ala Glu Gly Ser Ile Val Ala Gly Cys Ser Ser Gly Gly Met Leu225 230 235 240Lys Leu Ser Gly Arg Val Gly Gln Ala Ala Thr Tyr Gly Ala Gly Cys 245 250 255Trp Ala Leu Met Asp Glu Ser Thr Ser Met Ser Ala Ala Ser Cys Thr 260 265 270Thr Gly Asn Gly Glu Tyr Leu Met Lys Thr Leu Phe Ala Lys Glu Leu 275 280 285Val Asp Asp Leu Ile Ser Cys Asn Cys Pro Ile Thr Ser Gln His Leu 290 295 300Thr Tyr Lys Lys Lys Leu Leu Glu Ser Pro Phe Leu Ser Lys Gln Lys305 310 315 320Ala Ile His Ala Gly Ser Leu Ser Ile Ile Tyr Asn Thr Ala Ser Gly 325 330 335Asp Gly Asp Leu Leu Trp Ala His Thr Thr Asn Ser Met Cys Ile Gly 340 345 350Phe Met Ser Thr Lys Gln Lys Lys Pro Lys Phe Val Leu Ser Lys Leu 355 360 365Pro Gln Asn Leu Thr Cys Gly Thr Lys Pro Val Ile Asn Gly His His 370 375 380Phe Lys Leu Ile38550360PRTDrosophila melanogaster 50Met Ala Gly Phe Val Ala Val His Thr Gly Ala Gly Asn Cys Ile Asp 1 5 10 15Glu Thr Lys Tyr Gln Arg Val Ile Lys Glu Ala Cys Leu Arg Ala Thr 20 25 30Glu Ile Leu Arg Asn Gly Gly Ser Ala Val Asp Ala Cys Glu Ala Ala 35 40 45Ile Val Arg Leu Glu Asn Cys Gly Tyr Thr Asn Ala Gly Tyr Gly Ser 50 55 60Asn Leu Cys Met Asp Gly Ser Val Gln Cys Asp Ala Ala Ile Met Asp65 70 75 80Gly Ser Thr Leu Asn Phe Gly Ala Cys Thr Asn Val Ser Arg Val Lys 85 90 95Asn Pro Ile Gln Leu Ala Arg Arg Ile Cys Asp Ala Gln Ser Ser Pro 100 105 110Gln Leu Leu Glu Arg Ile Pro Pro Met Ile Leu Ala Gly Thr Gly Ala 115 120 125Glu His Tyr Ala Asp Glu Val Gly Cys Ser Met Val Glu Pro Gly Val 130 135 140Leu Ile Ser Ser Lys Ala Lys Phe Gln Phe Asn His Tyr Lys Ser Lys145 150 155 160Tyr Asp Leu Val Val Asn Ser Arg Leu Gly Lys Ala Thr Ser Glu Glu 165 170 175Ser Val Gln Val Pro Glu Pro Gly Asn Glu Val Glu Leu Ala Ala Ala 180 185 190Leu Asp Thr Val Gly Ala Val Cys Val Asp Gly Ala Gly Asn Thr Ala 195 200 205Ala Gly Cys Ser Ser Gly Gly Ile Leu Leu Lys Val Pro Gly Arg Val 210 215 220Gly Gln Ala Ala Thr Tyr Gly Ala Gly Cys Trp Ala Thr Asp Thr Asp225 230 235 240Glu Leu Ala Ile Ala Thr Cys Thr Thr Gly Asn Gly Glu Tyr Leu Met 245 250 255Lys Thr Leu Leu Ala Arg Glu Ile Cys His Gly Ala Phe Ser Ser Asp 260 265 270Cys Ala Val Thr Ser Leu His Lys Thr Phe Lys Gln Lys Phe Leu Asp 275 280 285Ser Pro Leu Leu Pro Arg Gln Gln Asp Leu Tyr Ala Gly Ala Leu Thr 290 295 300Leu Leu Tyr Tyr Pro Gly Gln Ser Ser Gly Glu Val Met Trp Ser His305 310 315 320Thr Thr Gln Ser Phe Cys Val Gly Tyr Met Ala Thr Asn Gln Arg Val 325 330 335Pro Lys Phe Val His Ser Pro Leu Pro Thr Tyr Ser Val Pro Gly Arg 340 345 350Ser Cys Val Val Asn Gly His Asn 355 3605111PRTHomo sapiensVARIANT7Xaa = Asp or Gly 51Pro Lys Ile Ser Gln Leu Xaa Val Asp Asp Gly 1 5 10524PRTHomo sapiens 52Ser Gln Leu Asp 1536PRTHomo sapiens 53Lys Ile Ser Gln Leu Asp 1 5547PRTHomo sapiens 54Lys Ile Ser Asp Gln Leu Ala 1 5555PRTHomo sapiens 55Lys Ile Ser Gln Ala 1 55619RNAArtificial SequencePrimer 56gacucacauu ucaagacuu 195719RNAArtificial SequencePrimer 57gaagucagag ugcgaaguc 19585PRTArtificial SequenceSynthetically generated peptide 58Ile Xaa Gln Leu Xaa 1 5593969PRTHomo sapiensVARIANT30Xaa = any amino acid 59Met Ala His Ser Cys Arg Trp Arg Phe Pro Ala Arg Pro Gly Thr Thr 1 5 10 15Gly Gly Gly Gly Gly Gly Gly Arg Arg Gly Leu Gly Gly Xaa Pro Arg 20 25 30Gln Arg Val Pro Ala Leu Leu Leu Pro Pro Gly Pro Pro Val Gly Gly 35 40 45Gly Gly Pro Gly Ala Pro Pro Ser Pro Pro Ala Val Ala Ala Ala Ala 50 55 60Ala Ala Ala Gly Ser Ser Gly Ala Gly Val Pro Gly Gly Ala Ala Ala65 70 75 80Ala Ser Ala Ala Ser Ser Ser Ser Ala Ser Ser Ser Ser Ser Ser Ser 85 90 95Ser Ser Ala Ser Ser Gly Pro Ala Leu Leu Arg Val Gly Pro Gly Phe 100 105 110Asp Ala Ala Leu Gln Val Ser Ala Ala Ile Gly Thr Asn Leu Arg Arg 115 120 125Phe Arg Ala Val Phe Gly Glu Ser Gly Gly Gly Gly Gly Ser Gly Glu 130 135 140Asp Glu Gln Phe Leu Gly Phe Gly Ser Asp Glu Glu Val Arg Val Arg145 150 155 160Ser Pro Thr Arg Ser Pro Ser Val Lys Thr Ser Pro Arg Lys Pro Arg 165 170 175Gly Arg Pro Arg Ser Gly Ser Asp Arg Asn Ser Ala Ile Leu Ser Asp 180 185 190Pro Ser Val Phe Ser Pro Leu Asn Lys Ser Glu Thr Lys Ser Gly Asp 195 200 205Lys Ile Lys Lys Lys Asp Ser Lys Ser Ile Glu Lys Lys Arg Gly Arg 210 215 220Pro Pro Thr Phe Pro Gly Val Lys Ile Lys Ile Thr His Gly Lys Asp225 230 235 240Ile Ser Glu Leu Pro Lys Gly Asn Lys Glu Asp Ser Leu Lys Lys Ile 245 250 255Lys Arg Thr Pro Ser Ala Thr Phe Gln Gln Ala Thr Lys Ile Lys Lys 260 265 270Leu Arg Ala Gly Lys Leu Ser Pro Leu Lys Ser Lys Phe Lys Thr Gly 275 280 285Lys Leu Gln Ile Gly Arg Lys Gly Val Gln Ile Val Arg Arg Arg Gly 290 295 300Arg Pro Pro Ser Thr Glu Arg Ile Lys Thr Pro Ser Val Ser Ser Leu305 310 315 320Ile Leu Asn Trp Lys Ser Pro Arg Lys Ser Gly Lys Thr Arg Lys Glu 325 330 335His Leu His Leu Gln Lys Lys Ile Arg Gln Leu Ser Asp Lys Ala Leu 340 345 350Glu Gly Leu Ser Gln Leu Gly Leu Phe Leu Leu Gln Lys Gly Gln Met 355 360 365Gln Pro Leu Leu Ser Asn Ser Tyr Arg Gly Gln Lys Lys Gly Ala Gln 370 375 380Lys Lys Ile Glu Lys Glu Ala Ala Gln Leu Gln Gly Arg Lys Val Lys385 390 395 400Thr Gln Val Lys Asn Ile Arg Gln Phe Ile Met Pro Val Val Ser Ala 405 410 415Ile Ser Ser Arg Ile Ile Lys Thr Pro Arg Arg Phe Ile Glu Asp Glu 420 425 430Asp Tyr Asp Pro Pro Ile Lys Ile Ala Arg Leu Glu Ser Thr Pro Asn 435 440 445Ser Arg Phe Ser Ala Pro Ser Cys Gly Ser Ser Glu Lys Ser Ser Ala 450 455 460Ala Ser Gln His Ser Ser Gln Met Ser Ser Asp Ser Ser Arg Ser Ser465 470 475 480Ser Pro Ser Val Asp Thr Ser Thr Asp Ser Gln Ala Ser Glu Glu Ile 485 490 495Gln Val Leu Pro Glu Glu Arg Ser Asp Thr Pro Glu Val His Pro Pro 500 505 510Leu Pro Ile Ser Gln Ser Pro Glu Asn Glu Ser Asn Asp Arg Arg Ser 515 520 525Arg Arg Tyr Ser Val Ser Glu Arg Ser Phe Gly Ser Arg Thr Thr Lys 530 535 540Lys Leu Ser Thr Leu Gln Ser Ala Pro Gln Gln Gln Thr Ser Ser Ser545 550 555 560Pro Pro Pro Pro Leu Leu Thr Pro Pro Pro Pro Leu Gln Pro Ala Ser 565 570 575Ser Ile Ser Asp His Thr Pro Trp Leu Met Pro Pro Thr Ile Pro Leu 580 585 590Ala Ser Pro Phe Leu Pro Ala Ser Thr Ala Pro Met Gln Gly Lys Arg 595 600 605Lys Ser Ile Leu Arg Glu Pro Thr Phe Arg Trp Thr Ser Leu Lys His 610 615 620Ser Arg Ser Glu Pro Gln Tyr Phe Ser Ser Ala Lys Tyr Ala Lys Glu625 630 635 640Gly Leu Ile Arg Lys Pro Ile Phe Asp Asn Phe Arg Pro Pro Pro Leu 645 650 655Thr Pro Glu Asp Val Gly Phe Ala Ser Gly Phe Ser Ala Ser Gly Thr 660 665 670Ala Ala Ser Ala Arg Leu Phe Ser Pro Leu His Ser Gly Thr Arg Phe 675 680 685Asp Met His Lys Arg Ser Pro Leu Leu Arg Ala Pro Arg Phe Thr Pro 690 695 700Ser Glu Ala His Ser Arg Ile Phe Glu Ser Val Thr Leu Pro Ser Asn705 710 715 720Arg Thr Ser Ala Gly Thr Ser Ser Ser Gly Val Ser Asn Arg Lys Arg 725 730 735Lys Arg Lys Val Phe Ser Pro Ile Arg Ser Glu Pro Arg Ser Pro Ser 740 745 750His Ser Met Arg Thr Arg Ser Gly Arg Leu Ser Ser Ser Glu Leu Ser 755 760 765Pro Leu Thr Pro Pro Ser Ser Val Ser Ser Ser Leu Ser Ile Ser Val 770 775 780Ser Pro Leu Ala Thr Ser Ala Leu Asn Pro Thr Phe Thr Phe Pro Ser785 790 795 800His Ser Leu Thr Gln Ser Gly Glu Ser Ala Glu Lys Asn Gln Arg Pro 805 810 815Arg Lys Gln Thr Ser Ala Pro Ala Glu Pro Phe Ser Ser Ser Ser Pro 820 825 830Thr Pro Leu Phe Pro Trp Phe Thr Pro Gly Ser Gln Thr Glu Arg Gly 835 840 845Arg Asn Lys Asp Lys Ala Pro Glu Glu Leu Ser Lys Asp Arg Asp Ala 850 855 860Asp Lys Ser Val Glu Lys Asp Lys Ser Arg Glu Arg Asp Arg Glu Arg865 870 875 880Glu Lys Glu Asn Lys Arg Glu Ser Arg Lys Glu Lys Arg Lys Lys Gly 885 890 895Ser Glu Ile Gln Ser Ser Ser Ala Leu Tyr Pro Val Gly Arg Val Ser 900 905 910Lys Glu Lys Val Val Gly Glu Asp Val Ala Thr Ser Ser Ser Ala Lys 915 920 925Lys Ala Thr Gly Arg Lys Lys Ser Ser Ser His Asp Ser Gly Thr Asp 930 935 940Ile Thr Ser Val Thr Leu Gly Asp Thr Thr Ala Val Lys Thr Lys Ile945 950 955 960Leu Ile Lys Lys Gly Arg Gly Asn Leu Glu Lys Thr Asn Leu Asp Leu 965 970 975Gly Pro Thr Ala Pro Ser Leu Glu Lys Glu Lys Thr Leu Cys Leu Ser 980 985 990Thr Pro Ser Ser Ser Thr Val Lys His Ser Thr Ser Ser Ile Gly Ser 995 1000 1005Met Leu Ala Gln Ala Asp Lys Leu Pro Met Thr Asp Lys Arg Val Ala 1010 1015 1020Ser Leu Leu Lys Lys Ala Lys Ala Gln Leu Cys Lys Ile Glu Lys Ser1025 1030 1035 1040Lys Ser Leu Lys Gln Thr Asp Gln Pro Lys Ala Gln Gly Gln Glu Ser 1045 1050 1055Asp Ser Ser Glu Thr Ser Val Arg Gly Pro Arg Ile Lys His Val Cys 1060 1065 1070Arg Arg Ala Ala Val Ala Leu Gly Arg Lys Arg Ala Val Phe Pro Asp 1075 1080 1085Asp Met Pro Thr Leu Ser Ala Leu Pro Trp Glu Glu Arg Glu Lys Ile 1090 1095 1100Leu Ser Ser Met Gly Asn Asp Asp Lys Ser Ser Ile Ala Gly Ser Glu1105 1110 1115 1120Asp Ala Glu Pro Leu Ala Pro Pro Ile Lys Pro Ile Lys Pro Val Thr 1125 1130 1135Arg Asn Lys Ala Pro Gln Glu Pro Pro Val Lys Lys Gly Arg Arg Ser 1140 1145 1150Arg Arg Cys Gly Gln Cys Pro Gly Cys Gln Val Pro Glu Asp Cys Gly 1155 1160 1165Val Cys Thr Asn Cys Leu Asp Lys Pro Lys Phe Gly Gly Arg Asn Ile 1170 1175 1180Lys Lys Gln Cys Cys Lys Met Arg Lys Cys Gln Asn Leu Gln Trp Met1185 1190 1195 1200Pro Ser Lys Ala Tyr Leu Gln Lys Gln Ala Lys Ala Val Lys Lys Lys 1205 1210 1215Glu Lys Lys Ser Lys Thr Ser Glu Lys Lys Asp Ser Lys Glu Ser Ser 1220 1225 1230Val Val Lys Asn Val Val Asp Ser Ser Gln Lys Pro Thr Pro Ser Ala 1235 1240 1245Arg Glu Asp Pro Ala Pro Lys Lys Ser Ser Ser Glu Pro Pro Pro Arg 1250 1255 1260Lys Pro Val Glu Glu Lys Ser Glu Glu Gly Asn Val Ser Ala Pro Gly1265 1270 1275 1280Pro Glu Ser Lys Gln Ala Thr Thr Pro Ala Ser Arg Lys Ser Ser Lys 1285 1290 1295Gln Val Ser Gln Pro Ala Leu Val Ile Pro Pro Gln Pro Pro Thr Thr 1300 1305 1310Gly Pro Pro Arg Lys Glu Val Pro Lys Thr Thr Pro Ser Glu Pro Lys 1315 1320 1325Lys Lys Gln Pro Pro Pro Pro Glu Ser Gly Pro Glu Gln Ser Lys Gln 1330

1335 1340Lys Lys Val Ala Pro Arg Pro Ser Ile Pro Val Lys Gln Lys Pro Lys1345 1350 1355 1360Glu Lys Glu Lys Pro Pro Pro Val Asn Lys Gln Glu Asn Ala Gly Thr 1365 1370 1375Leu Asn Ile Leu Ser Thr Leu Ser Asn Gly Asn Ser Ser Lys Gln Lys 1380 1385 1390Ile Pro Ala Asp Gly Val His Arg Ile Arg Val Asp Phe Lys Glu Asp 1395 1400 1405Cys Glu Ala Glu Asn Val Trp Glu Met Gly Gly Leu Gly Ile Leu Thr 1410 1415 1420Ser Val Pro Ile Thr Pro Arg Val Val Cys Phe Leu Cys Ala Ser Ser1425 1430 1435 1440Gly His Val Glu Phe Val Tyr Cys Gln Val Cys Cys Glu Pro Phe His 1445 1450 1455Lys Phe Cys Leu Glu Glu Asn Glu Arg Pro Leu Glu Asp Gln Leu Glu 1460 1465 1470Asn Trp Cys Cys Arg Arg Cys Lys Phe Cys His Val Cys Gly Arg Gln 1475 1480 1485His Gln Ala Thr Lys Gln Leu Leu Glu Cys Asn Lys Cys Arg Asn Ser 1490 1495 1500Tyr His Pro Glu Cys Leu Gly Pro Asn Tyr Pro Thr Lys Pro Thr Lys1505 1510 1515 1520Lys Lys Lys Val Trp Ile Cys Thr Lys Cys Val Arg Cys Lys Ser Cys 1525 1530 1535Gly Ser Thr Thr Pro Gly Lys Gly Trp Asp Ala Gln Trp Ser His Asp 1540 1545 1550Phe Ser Leu Cys His Asp Cys Ala Lys Leu Phe Ala Lys Gly Asn Phe 1555 1560 1565Cys Pro Leu Cys Asp Lys Cys Tyr Asp Asp Asp Asp Tyr Glu Ser Lys 1570 1575 1580Met Met Gln Cys Gly Lys Cys Asp Arg Trp Val His Ser Lys Cys Glu1585 1590 1595 1600Asn Leu Ser Asp Glu Met Tyr Glu Ile Leu Ser Asn Leu Pro Glu Ser 1605 1610 1615Val Ala Tyr Thr Cys Val Asn Cys Thr Glu Arg His Pro Ala Glu Trp 1620 1625 1630Arg Leu Ala Leu Glu Lys Glu Leu Gln Ile Ser Leu Lys Gln Val Leu 1635 1640 1645Thr Ala Leu Leu Asn Ser Arg Thr Thr Ser His Leu Leu Arg Tyr Arg 1650 1655 1660Gln Ala Ala Lys Pro Pro Asp Leu Asn Pro Glu Thr Glu Glu Ser Ile1665 1670 1675 1680Pro Ser Arg Ser Ser Pro Glu Gly Pro Asp Pro Pro Val Leu Thr Glu 1685 1690 1695Val Ser Lys Gln Asp Asp Gln Gln Pro Leu Asp Leu Glu Gly Val Lys 1700 1705 1710Arg Lys Met Asp Gln Gly Asn Tyr Thr Ser Val Leu Glu Phe Ser Asp 1715 1720 1725Asp Ile Val Lys Ile Ile Gln Ala Ala Ile Asn Ser Asp Gly Gly Gln 1730 1735 1740Pro Glu Ile Lys Lys Ala Asn Ser Met Val Lys Ser Phe Phe Ile Arg1745 1750 1755 1760Gln Met Glu Arg Val Phe Pro Trp Phe Ser Val Lys Lys Ser Arg Phe 1765 1770 1775Trp Glu Pro Asn Lys Val Ser Ser Asn Ser Gly Met Leu Pro Asn Ala 1780 1785 1790Val Leu Pro Pro Ser Leu Asp His Asn Tyr Ala Gln Trp Gln Glu Arg 1795 1800 1805Glu Glu Asn Ser His Thr Glu Gln Pro Pro Leu Met Lys Lys Ile Ile 1810 1815 1820Pro Ala Pro Lys Pro Lys Gly Pro Gly Glu Pro Asp Ser Pro Thr Pro1825 1830 1835 1840Leu His Pro Pro Thr Pro Pro Ile Leu Ser Thr Asp Arg Ser Arg Glu 1845 1850 1855Asp Ser Pro Glu Leu Asn Pro Pro Pro Gly Ile Glu Asp Asn Arg Gln 1860 1865 1870Cys Ala Leu Cys Leu Thr Tyr Gly Asp Asp Ser Ala Asn Asp Ala Gly 1875 1880 1885Arg Leu Leu Tyr Ile Gly Gln Asn Glu Trp Thr His Val Asn Cys Ala 1890 1895 1900Leu Trp Ser Ala Glu Val Phe Glu Asp Asp Asp Gly Ser Leu Lys Asn1905 1910 1915 1920Val His Met Ala Val Ile Arg Gly Lys Gln Leu Arg Cys Glu Phe Cys 1925 1930 1935Gln Lys Pro Gly Ala Thr Val Gly Cys Cys Leu Thr Ser Cys Thr Ser 1940 1945 1950Asn Tyr His Phe Met Cys Ser Arg Ala Lys Asn Cys Val Phe Leu Asp 1955 1960 1965Asp Lys Lys Val Tyr Cys Gln Arg His Arg Asp Leu Ile Lys Gly Glu 1970 1975 1980Val Val Pro Glu Asn Gly Phe Glu Val Phe Arg Arg Val Phe Val Asp1985 1990 1995 2000Phe Glu Gly Ile Ser Leu Arg Arg Lys Phe Leu Asn Gly Leu Glu Pro 2005 2010 2015Glu Asn Ile His Met Met Ile Gly Ser Met Thr Ile Asp Cys Leu Gly 2020 2025 2030Ile Leu Asn Asp Leu Ser Asp Cys Glu Asp Lys Leu Phe Pro Ile Gly 2035 2040 2045Tyr Gln Cys Ser Arg Val Tyr Trp Ser Thr Thr Asp Ala Arg Lys Arg 2050 2055 2060Cys Val Tyr Thr Cys Lys Ile Val Glu Cys Arg Pro Pro Val Val Glu2065 2070 2075 2080Pro Asp Ile Asn Ser Thr Val Glu His Asp Glu Asn Arg Thr Ile Ala 2085 2090 2095His Ser Pro Thr Ser Phe Thr Glu Ser Ser Ser Lys Glu Ser Gln Asn 2100 2105 2110Thr Ala Glu Ile Ile Ser Pro Pro Ser Pro Asp Arg Pro Pro His Ser 2115 2120 2125Gln Thr Ser Gly Ser Cys Tyr Tyr His Val Ile Ser Lys Val Pro Arg 2130 2135 2140Ile Arg Thr Pro Ser Tyr Ser Pro Thr Gln Arg Ser Pro Gly Cys Arg2145 2150 2155 2160Pro Leu Pro Ser Ala Gly Ser Pro Thr Pro Thr Thr His Glu Ile Val 2165 2170 2175Thr Val Gly Asp Pro Leu Leu Ser Ser Gly Leu Arg Ser Ile Gly Ser 2180 2185 2190Arg Arg His Ser Thr Ser Ser Leu Ser Pro Gln Arg Ser Lys Leu Arg 2195 2200 2205Ile Met Ser Pro Met Arg Thr Gly Asn Thr Tyr Ser Arg Asn Asn Val 2210 2215 2220Ser Ser Val Ser Thr Thr Gly Thr Ala Thr Asp Leu Glu Ser Ser Ala2225 2230 2235 2240Lys Val Val Asp His Val Leu Gly Pro Leu Asn Ser Ser Thr Ser Leu 2245 2250 2255Gly Gln Asn Thr Ser Thr Ser Ser Asn Leu Gln Arg Thr Val Val Thr 2260 2265 2270Val Gly Asn Lys Asn Ser His Leu Asp Gly Ser Ser Ser Ser Glu Met 2275 2280 2285Lys Gln Ser Ser Ala Ser Asp Leu Val Ser Lys Ser Ser Ser Leu Lys 2290 2295 2300Gly Glu Lys Thr Lys Val Leu Ser Ser Lys Ser Ser Glu Gly Ser Ala2305 2310 2315 2320His Asn Val Ala Tyr Pro Gly Ile Pro Lys Leu Ala Pro Gln Val His 2325 2330 2335Asn Thr Thr Ser Arg Glu Leu Asn Val Ser Lys Ile Gly Ser Phe Ala 2340 2345 2350Glu Pro Ser Ser Val Ser Phe Ser Ser Lys Glu Ala Leu Ser Phe Pro 2355 2360 2365His Leu His Leu Arg Gly Gln Arg Asn Asp Arg Asp Gln His Thr Asp 2370 2375 2380Ser Thr Gln Ser Ala Asn Ser Ser Pro Asp Glu Asp Thr Glu Val Lys2385 2390 2395 2400Thr Leu Lys Leu Ser Gly Met Ser Asn Arg Ser Ser Ile Ile Asn Glu 2405 2410 2415His Met Gly Ser Ser Ser Arg Asp Arg Arg Gln Lys Gly Lys Lys Ser 2420 2425 2430Cys Lys Glu Thr Phe Lys Glu Lys His Ser Ser Lys Ser Phe Leu Glu 2435 2440 2445Pro Gly Gln Val Thr Thr Gly Glu Glu Gly Asn Leu Lys Pro Glu Phe 2450 2455 2460Met Asp Glu Val Leu Thr Pro Glu Tyr Met Gly Gln Arg Pro Cys Asn2465 2470 2475 2480Asn Val Ser Ser Asp Lys Ile Gly Asp Lys Gly Leu Ser Met Pro Gly 2485 2490 2495Val Pro Lys Ala Pro Pro Met Gln Val Glu Gly Ser Ala Lys Glu Leu 2500 2505 2510Gln Ala Pro Arg Lys Arg Thr Val Lys Val Thr Leu Thr Pro Leu Lys 2515 2520 2525Met Glu Asn Glu Ser Gln Ser Lys Asn Ala Leu Lys Glu Ser Ser Pro 2530 2535 2540Ala Ser Pro Leu Gln Ile Glu Ser Thr Ser Pro Thr Glu Pro Ile Ser2545 2550 2555 2560Ala Ser Glu Asn Pro Gly Asp Gly Pro Val Ala Gln Pro Ser Pro Asn 2565 2570 2575Asn Thr Ser Cys Gln Asp Ser Gln Ser Asn Asn Tyr Gln Asn Leu Pro 2580 2585 2590Val Gln Asp Arg Asn Leu Met Leu Pro Asp Gly Pro Lys Pro Gln Glu 2595 2600 2605Asp Gly Ser Phe Lys Arg Arg Tyr Pro Arg Arg Ser Ala Arg Ala Arg 2610 2615 2620Ser Asn Met Phe Phe Gly Leu Thr Pro Leu Tyr Gly Val Arg Ser Tyr2625 2630 2635 2640Gly Glu Glu Asp Ile Pro Phe Tyr Ser Ser Ser Thr Gly Lys Lys Arg 2645 2650 2655Gly Lys Arg Ser Ala Glu Gly Gln Val Asp Gly Ala Asp Asp Leu Ser 2660 2665 2670Thr Ser Asp Glu Asp Asp Leu Tyr Tyr Tyr Asn Phe Thr Arg Thr Val 2675 2680 2685Ile Ser Ser Gly Gly Glu Glu Arg Leu Ala Ser His Asn Leu Phe Arg 2690 2695 2700Glu Glu Glu Gln Cys Asp Leu Pro Lys Ile Ser Gln Leu Asp Gly Val2705 2710 2715 2720Asp Asp Gly Thr Glu Ser Asp Thr Ser Val Thr Ala Thr Thr Arg Lys 2725 2730 2735Ser Ser Gln Ile Pro Lys Arg Asn Gly Lys Glu Asn Gly Thr Glu Asn 2740 2745 2750Leu Lys Ile Asp Arg Pro Glu Asp Ala Gly Glu Lys Glu His Val Thr 2755 2760 2765Lys Ser Ser Val Gly His Lys Asn Glu Pro Lys Met Asp Asn Cys His 2770 2775 2780Ser Val Ser Arg Val Lys Thr Gln Gly Gln Asp Ser Leu Glu Ala Gln2785 2790 2795 2800Leu Ser Ser Leu Glu Ser Ser Arg Arg Val His Thr Ser Thr Pro Ser 2805 2810 2815Asp Lys Asn Leu Leu Asp Thr Tyr Asn Thr Glu Leu Leu Lys Ser Asp 2820 2825 2830Ser Asp Asn Asn Asn Ser Asp Asp Cys Gly Asn Ile Leu Pro Ser Asp 2835 2840 2845Ile Met Asp Phe Val Leu Lys Asn Thr Pro Ser Met Gln Ala Leu Gly 2850 2855 2860Glu Ser Pro Glu Ser Ser Ser Ser Glu Leu Leu Asn Leu Gly Glu Gly2865 2870 2875 2880Leu Gly Leu Asp Ser Asn Arg Glu Lys Asp Met Gly Leu Phe Glu Val 2885 2890 2895Phe Ser Gln Gln Leu Pro Thr Thr Glu Pro Val Asp Ser Ser Val Ser 2900 2905 2910Ser Ser Ile Ser Ala Glu Glu Gln Phe Glu Leu Pro Leu Glu Leu Pro 2915 2920 2925Ser Asp Leu Ser Val Leu Thr Thr Arg Ser Pro Thr Val Pro Ser Gln 2930 2935 2940Asn Pro Ser Arg Leu Ala Val Ile Ser Asp Ser Gly Glu Lys Arg Val2945 2950 2955 2960Thr Ile Thr Glu Lys Ser Val Ala Ser Ser Glu Ser Asp Pro Ala Leu 2965 2970 2975Leu Ser Pro Gly Val Asp Pro Thr Pro Glu Gly His Met Thr Pro Asp 2980 2985 2990His Phe Ile Gln Gly His Met Asp Ala Asp His Ile Ser Ser Pro Pro 2995 3000 3005Cys Gly Ser Val Glu Gln Gly His Gly Asn Asn Gln Asp Leu Thr Arg 3010 3015 3020Asn Ser Ser Thr Pro Gly Leu Gln Val Pro Val Ser Pro Thr Val Pro3025 3030 3035 3040Ile Gln Asn Gln Lys Tyr Val Pro Asn Ser Thr Asp Ser Pro Gly Pro 3045 3050 3055Ser Gln Ile Ser Asn Ala Ala Val Gln Thr Thr Pro Pro His Leu Lys 3060 3065 3070Pro Ala Thr Glu Lys Leu Ile Val Val Asn Gln Asn Met Gln Pro Leu 3075 3080 3085Tyr Val Leu Gln Thr Leu Pro Asn Gly Val Thr Gln Lys Ile Gln Leu 3090 3095 3100Thr Ser Ser Val Ser Ser Thr Pro Ser Val Met Glu Thr Asn Thr Ser3105 3110 3115 3120Val Leu Gly Pro Met Gly Gly Gly Leu Thr Leu Thr Thr Gly Leu Asn 3125 3130 3135Pro Ser Leu Pro Thr Ser Gln Ser Leu Phe Pro Ser Ala Ser Lys Gly 3140 3145 3150Leu Leu Pro Met Ser His His Gln His Leu His Ser Phe Pro Ala Ala 3155 3160 3165Thr Gln Ser Ser Phe Pro Pro Asn Ile Ser Asn Pro Pro Ser Gly Leu 3170 3175 3180Leu Ile Gly Val Gln Pro Pro Pro Asp Pro Gln Leu Leu Val Ser Glu3185 3190 3195 3200Ser Ser Gln Arg Thr Asp Leu Ser Thr Thr Val Ala Thr Pro Ser Ser 3205 3210 3215Gly Leu Lys Lys Arg Pro Ile Ser Arg Leu Gln Thr Arg Lys Asn Lys 3220 3225 3230Lys Leu Ala Pro Ser Ser Thr Pro Ser Asn Ile Ala Pro Ser Asp Val 3235 3240 3245Val Ser Asn Met Thr Leu Ile Asn Phe Thr Pro Ser Gln Leu Pro Asn 3250 3255 3260His Pro Ser Leu Leu Asp Leu Gly Ser Leu Asn Thr Ser Ser His Arg3265 3270 3275 3280Thr Val Pro Asn Ile Ile Lys Arg Ser Lys Ser Ser Ile Met Tyr Phe 3285 3290 3295Glu Pro Ala Pro Leu Leu Pro Gln Ser Val Gly Gly Thr Ala Ala Thr 3300 3305 3310Ala Ala Gly Thr Ser Thr Ile Ser Gln Asp Thr Ser His Leu Thr Ser 3315 3320 3325Gly Ser Val Ser Gly Leu Ala Ser Ser Ser Ser Val Leu Asn Val Val 3330 3335 3340Ser Met Gln Thr Thr Thr Thr Pro Thr Ser Ser Ala Ser Val Pro Gly3345 3350 3355 3360His Val Thr Leu Thr Asn Pro Arg Leu Leu Gly Thr Pro Asp Ile Gly 3365 3370 3375Ser Ile Ser Asn Leu Leu Ile Lys Ala Ser Gln Gln Ser Leu Gly Ile 3380 3385 3390Gln Asp Gln Pro Val Ala Leu Pro Pro Ser Ser Gly Met Phe Pro Gln 3395 3400 3405Leu Gly Thr Ser Gln Thr Pro Ser Thr Ala Ala Ile Thr Ala Ala Ser 3410 3415 3420Ser Ile Cys Val Leu Pro Ser Thr Gln Thr Thr Gly Ile Thr Ala Ala3425 3430 3435 3440Ser Pro Ser Gly Glu Ala Asp Glu His Tyr Gln Leu Gln His Val Asn 3445 3450 3455Gln Leu Leu Ala Ser Lys Thr Gly Ile His Ser Ser Gln Arg Asp Leu 3460 3465 3470Asp Ser Ala Ser Gly Pro Gln Val Ser Asn Phe Thr Gln Thr Val Asp 3475 3480 3485Ala Pro Asn Ser Met Gly Leu Glu Gln Asn Lys Ala Leu Ser Ser Ala 3490 3495 3500Val Gln Ala Ser Pro Thr Ser Pro Gly Gly Ser Pro Ser Ser Pro Ser3505 3510 3515 3520Ser Gly Gln Arg Ser Ala Ser Pro Ser Val Pro Gly Pro Thr Lys Pro 3525 3530 3535Lys Pro Lys Thr Lys Arg Phe Gln Leu Pro Leu Asp Lys Gly Asn Gly 3540 3545 3550Lys Lys His Lys Val Ser His Leu Arg Thr Ser Ser Ser Glu Ala His 3555 3560 3565Ile Pro Asp Gln Glu Thr Thr Ser Leu Thr Ser Gly Thr Gly Thr Pro 3570 3575 3580Gly Ala Glu Ala Glu Gln Gln Asp Thr Ala Ser Val Glu Gln Ser Ser3585 3590 3595 3600Gln Lys Glu Cys Gly Gln Pro Ala Gly Gln Val Ala Val Leu Pro Glu 3605 3610 3615Val Gln Val Thr Gln Asn Pro Ala Asn Glu Gln Glu Ser Ala Glu Pro 3620 3625 3630Lys Thr Val Glu Glu Glu Glu Ser Asn Phe Ser Ser Pro Leu Met Leu 3635 3640 3645Trp Leu Gln Gln Glu Gln Lys Arg Lys Glu Ser Ile Thr Glu Lys Lys 3650 3655 3660Pro Lys Lys Gly Leu Val Phe Glu Ile Ser Ser Asp Asp Gly Phe Gln3665 3670 3675 3680Ile Cys Ala Glu Ser Ile Glu Asp Ala Trp Lys Ser Leu Thr Asp Lys 3685 3690 3695Val Gln Glu Ala Arg Ser Asn Ala Arg Leu Lys Gln Leu Ser Phe Ala 3700 3705 3710Gly Val Asn Gly Leu Arg Met Leu Gly Ile Leu His Asp Ala Val Val 3715 3720 3725Phe Leu Ile Glu Gln Leu Ser Gly Ala Lys His Cys Arg Asn Tyr Lys 3730 3735 3740Phe Arg Phe His Lys Pro Glu Glu Ala Asn Glu Pro Pro Leu Asn Pro3745 3750 3755 3760His Gly Ser Ala Arg Ala Glu Val His Leu Arg Lys Ser Ala Phe Asp 3765 3770 3775Met Phe Asn Phe Leu Ala Ser Lys His Arg Gln Pro Pro Glu Tyr Asn 3780 3785 3790Pro Asn Asp Glu Glu Glu Glu Glu Val

Gln Leu Lys Ser Ala Arg Arg 3795 3800 3805Ala Thr Ser Met Asp Leu Pro Met Pro Met Arg Phe Arg His Leu Lys 3810 3815 3820Lys Thr Ser Lys Glu Ala Val Gly Val Tyr Arg Ser Pro Ile His Gly3825 3830 3835 3840Arg Gly Leu Phe Cys Lys Arg Asn Ile Asp Ala Gly Glu Met Val Ile 3845 3850 3855Glu Tyr Ala Gly Asn Val Ile Arg Ser Ile Gln Thr Asp Lys Arg Glu 3860 3865 3870Lys Tyr Tyr Asp Ser Lys Gly Ile Gly Cys Tyr Met Phe Arg Ile Asp 3875 3880 3885Asp Ser Glu Val Val Asp Ala Thr Met His Gly Asn Arg Ala Arg Phe 3890 3895 3900Ile Asn His Ser Cys Glu Pro Asn Cys Tyr Ser Arg Val Ile Asn Ile3905 3910 3915 3920Asp Gly Gln Lys His Ile Val Ile Phe Ala Met Arg Lys Ile Tyr Arg 3925 3930 3935Gly Glu Glu Leu Thr Tyr Asp Tyr Lys Phe Pro Ile Glu Asp Ala Ser 3940 3945 3950Asn Lys Leu Pro Cys Asn Cys Gly Ala Lys Lys Cys Arg Lys Phe Leu 3955 3960 3965Asn