Novel human hydrolase family members and uses thereof (28-Oct-2004)

RELATED APPLICATIONS

[0001] This application is a continuation-in-part and claims priority to U.S. application Ser. No. 09/816,664, filed Mar. 23, 2001, which claims the benefit of U.S. Provisional Application Ser. No. 60/191,973, filed Mar. 24, 2000; and U.S. application Ser. No. 09/841,880, filed Apr. 24, 2001, which claims the benefit of U.S. Provisional Application Ser. No. 60/199,559, filed Apr. 25, 2000; and U.S. application Ser. No. 09/862,556, filed May 22, 2001, and International Application Serial No. PCT/US01/16424, filed May 22, 2001, which claim the benefit of U.S. Provisional Application Ser. No. 60/206,036, filed May 22, 2000; and U.S. application Ser. No. 09/861,165, filed May 18, 2001, and International Application Serial No. PCT/US01/16014, filed May 18, 2001, which claim the benefit of U.S. Provisional Application Ser. No. 60/205,442, filed May 19, 2000; and U.S. application Ser. No. 09/875,353, filed Jun. 6, 2001, and International Application Serial No. PCT/US01/18335, filed Jun. 6, 2001, which claim the benefit of U.S. Provisional Application Ser. No. 60/209,949, filed Jun. 6, 2000; and U.S. application Ser. No. 09/896,578, filed Jun. 29, 2001, and International Application Serial No. PCT/US01/20880, filed Jun. 29, 2001, which claim the benefit of U.S. Provisional Application Ser. No. 60/214,948, filed Jun. 29, 2000; and U.S. application Ser. No. 09/911,150, filed Jul. 23, 2001, and International Application Serial No. PCT/US01/23153, filed Jul. 23, 2001, which claim the benefit of U.S. Provisional Application Ser. No. 60/220,008, filed Jul. 21, 2000; and U.S. application Ser. No. 09/911,317, filed Jul. 23, 2001, and International Application Serial No. PCT/US01/23160, filed Jul. 23, 2001, which claim the benefit of U.S. Provisional Application Ser. No. 60/220,040, filed Jul. 21, 2000; and U.S. application Ser. No. 09/934,323, filed Aug. 21, 2001, and International Application Serial No. PCT/US01/26091, filed Aug. 21, 2001, which claim the benefit of U.S. Provisional Application Ser. No. 60/226,774, filed Aug. 21, 2000; and U.S. application Ser. No. 09/963,959, filed Sep. 25, 2001, and International Application Serial No. PCT/US01/29962, filed Sep. 25, 2001, which claim the benefit of U.S. Provisional Application Ser. No. 60/235,033, filed Sep. 25, 2000; and U.S. Application Serial No. 09/971,490, filed Oct. 5, 2001, and International Application Serial No. PCT/US01/31674, filed Oct. 5, 2001, which claim the benefit of U.S. Provisional Application Ser. No. 60/238,170, filed Oct. 5, 2000; and U.S. application Ser. No. 10/071,275, filed Feb. 7, 2002, and International Application Serial No. PCT/US02/03793, filed Feb. 7, 2002, which claim the benefit of U.S. Provisional Application Ser. No. 60/267,054, filed Feb. 7, 2001; and U.S. application Ser. No. 09/888,911, filed Jun. 25, 2001, and International Application Serial No. PCT/US01/19967, filed Jun. 25, 2001, which claim the benefit of U.S. Provisional Application Ser. No. 60/213,688, filed Jun. 23, 2000, the contents of which are incorporated herein by reference.

BACKGROUND OF THE 26443 AND 46873 INVENTION

[0002] Asparaginase is an enzyme that catalyzes the hydrolysis of asparagine to aspartic acid and ammonia. Saccharomyces cerevisiae expresses two forms of asparaginase: L-asparaginase I, a cytoplasmic enzyme that is synthesized constitutively, and asparaginase II, a cell wall mannan protein localized external to the cell membrane which plays a role in hydrolysis of exogenous asparagines and uptake of aspartic acid. The two enzymes are biochemically and genetically distinct.

[0003] Because some lymphoid tumor cells are deficient in L-asparagine synthetase and cannot synthesize sufficient L-asparagine, asparagine is, for these cells, an essential amino acid. Therefore, asparagine depletion by administration of asparaginase rapidly results in decreased protein synthesis, followed by a decrease in DNA and RNA synthesis, and ultimately cell death.

SUMMARY OF THE 26443 And 46873 INVENTION

[0004] The present invention is based, in part, on the discovery of novel asparaginases, referred to herein as “26443” and “46873” nucleic acid and protein molecules. The nucleotide sequence of a cDNA encoding 26443 and 46873 is shown in SEQ ID NO: 1 and SEQ ID NO:4, respectively, and the amino acid sequence of a 26443 and 46873 polypeptide is shown in SEQ ID NO:2 and SEQ ID NO:5, respectively. In addition, the nucleotide sequence of the coding regions of 26443 and 46873 are depicted in SEQ ID NO:3 and SEQ ID NO:6, respectively.

[0005] Accordingly, in one aspect, the invention features a nucleic acid molecule that encodes a 26443 or 46873 protein or polypeptide, e.g., a biologically active portion of the 26443 or 46873 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5. In other embodiments, the invention provides isolated 26443 or 46873 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______ or Accession Number ______. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______ or Accession Number ______. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______ or Accession Number ______, wherein the nucleic acid encodes a full length 26443 or 46873 protein or a biologically active fragment thereof.

[0006] In a related aspect, the invention further provides nucleic acid constructs, which include a 26443 or 46873 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 26443 or 46873 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 26443 or 46873 nucleic acid molecules and polypeptides.

[0007] In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 26443 or 46873-encoding nucleic acids.

[0008] In still another related aspect, isolated nucleic acid molecules that are antisense to a 26443 or 46873 encoding nucleic acid molecule are provided.

[0009] In another aspect, the invention features, 26443 or 46873 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 26443- or 46873-mediated or related disorders. In another embodiment, the invention provides 26443 or 46873 polypeptides having a 26443 or 46873 activity. Preferred polypeptides are 26443 or 46873 proteins including at least one asparaginase domain, and, preferably, having a 26443 or 46873 activity, e.g., a 26443 or 46873 activity as described herein.

[0010] In other embodiments, the invention provides 26443 or 46873 polypeptides, e.g., a 26443 or 46873 polypeptide having the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:5, respectively; the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______ or Accession Number ______; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:5; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______ or Accession Number ______, wherein the nucleic acid encodes a full length 26443 or 46873 protein or an active fragment thereof.

[0011] In a related aspect, the invention further provides nucleic acid constructs that include a 26443 or 46873 nucleic acid molecule described herein.

[0012] In a related aspect, the invention provides 26443 or 46873 polypeptides or fragments operatively linked to non-26443 or -46873 polypeptides to form fusion proteins.

[0013] In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably, specifically bind 26443 or 46873 polypeptides.

[0014] In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 26443 or 46873 polypeptides or nucleic acids.

[0015] In still another aspect, the invention provides a process for modulating 26443 or 46873 polypeptide or nucleic acid expression or activity, e.g., using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the 26443 or 46873 polypeptides or nucleic acids, such as metabolic diseases and conditions involving aberrant or deficient oxidation of long- and medium-chain fatty acids.

[0016] The invention also provides assays for determining the activity of, or the presence or absence of, 26443 or 46873 polypeptides or nucleic acid molecules in a biological sample, including for the purpose of disease diagnosis.

[0017] In a further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 26443 or 46873 polypeptide or nucleic acid molecule, including for the purpose of disease diagnosis.

[0018] In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 26443 or 46873 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 26443 or 46873 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 26443 or 46873 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.

[0019] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIGS. 1A-1B depicts a cDNA sequence (SEQ ID NO:1) and predicted amino acid sequence (SEQ ID NO:2) of human 26443. The methionine-initiated open reading frame of human 26443 (without the 5′ and 3′untranslated regions) starts at nucleotide 91 and continues through to nucleotide 1344 of SEQ ID NO: 1 (coding sequence also shown in SEQ ID NO:3).

[0021] FIG. 2 depicts a hydropathy plot of human 26443. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (Cys) are indicated by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human 26443 are indicated. Polypeptides of the invention include 26443 fragments that include: all or part of a hydrophobic sequence (a sequence above the dashed line; all or part of a hydrophilic fragment (e.g., a fragment below the dashed line). Other fragments include a cysteine or a glycosylation site.

[0022] FIG. 3 depicts a series of plots summarizing an analysis of the primary and secondary protein structure of a human asparaginase. The particular algorithm used for each plot is indicated at the right hand side of each plot. The following plots are depicted: Gamier-Robson plots providing the predicted location of alpha-, beta-, turn and coil regions (Gamier et al. (1978) J. Mol. Biol. 120:97); Chou-Fasman plots providing the predicted location of alpha-, beta-, turn and coil regions (Chou and Fasman (1978) Adv. In Enzymol. Mol. 47:45-148); Kyte-Doolittle hydrophilicity/hydrophobicity plots (Kyte and Doolittle (1982) J. Mol. Biol. 157:105-132); Eisenberg plots providing the predicted location of alpha- and beta-amphipathic regions (Eisenberg et al. (1982) Nature 299:371-374); a Karplus-Schultz plot providing the predicted location of flexible regions (Karplus and Schulz (1985) Naturwissens-Chafen 72:212-213); a plot of the antigenic index (Jameson-Wolf) (Jameson and Wolf (1988) CABIOS 4:121-136); and a surface probability plot (Emini algorithm) (Emini et al. (1985) J. Virol. 55:836-839). The numbers corresponding to the amino acid sequence of human 26443 are indicated.

[0023] FIG. 4 depicts an alignment of the asparaginase domain of human 26443 with a consensus amino acid sequence derived from a hidden Markov model. The upper sequence is the consensus amino acid sequence (SEQ ID NO:7), while the lower amino acid sequence corresponds to amino acids 38 to 345 of SEQ ID NO:2.

[0024] FIG. 5 depicts a cDNA sequence (SEQ ID NO:4) and predicted amino acid sequence (SEQ ID NO:5) of human 46873. The methionine-initiated open reading frame of human 46873 (without the 5′ and 3′untranslated regions) starts at nucleotide 134 and continues through to nucleotide 1057 of SEQ ID NO:4 (coding sequence also shown in SEQ ID NO:6).

[0025] FIG. 6 depicts a hydropathy plot of human 46873. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (Cys) are indicated by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human 46873 are indicated. Polypeptides of the invention include 46873 fragments that include: all or part of a hydrophobic sequence (a sequence above the dashed line; all or part of a hydrophilic fragment (e.g., a fragment below the dashed line).

[0026] FIG. 7 depicts a series of plots summarizing an analysis of the primary and secondary protein structure of a human asparaginase. The particular algorithm used for each plot is indicated at the right hand side of each plot. The following plots are depicted: Gamier-Robson plots providing the predicted location of alpha-, beta-, turn and coil regions (Gamier et al. (1978) J. Mol. Biol. 120:97); Chou-Fasman plots providing the predicted location of alpha-, beta-, turn and coil regions (Chou and Fasman (1978) Adv. In Enzymol. Mol. 47:45-148); Kyte-Doolittle hydrophilicity/hydrophobicity plots (Kyte and Doolittle (1982) J. Mol. Biol. 157:105-132); Eisenberg plots providing the predicted location of alpha- and beta-amphipathic regions (Eisenberg et al. (1982) Nature 299:371-374); a Karplus-Schultz plot providing the predicted location of flexible regions (Karplus and Schulz (1985) Naturwissens-Chafen 72:212-213); a plot of the antigenic index (Jameson-Wolf) (Jameson and Wolf (1988) CABIOS 4:121-136); and a surface probability plot (Emini algorithm) (Emini et al. (1985) J. Virol. 55:836-839). The numbers corresponding to the amino acid sequence of human 46873 are indicated.

[0027] FIG. 8 depicts an alignment of the asparaginase domain of human 46873 with a consensus amino acid sequence derived from a hidden Markov model. The upper sequence is the consensus amino acid sequence (SEQ ID NO:7), while the lower amino acid sequence corresponds to amino acids 1 to 302 of SEQ ID NO:5.

[0028] FIG. 9 depicts a hydropathy plot of human 61833. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 61833 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 30 to 50, from about 276 to 293, and from about 323 to 341 of SEQ ID NO:11; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 300 to 310 of SEQ ID NO:11.

[0029] FIGS. 10A-10B depicts an alignment of the pyridoxyl-dependent decarboxylase domain of human 61833 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:13), while the lower amino acid sequence corresponds to amino acids 41 to 401 of SEQ ID NO:11.

[0030] FIG. 11 depicts a hydropathy plot of human 26493. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human 26493 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence of from about amino acid residue 85 to 101, and from about 350 to 360 of SEQ ID NO:17; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence from about amino acid residue 360 to 370 of SEQ ID NO:17; or a sequence which includes a Cys, or an N-glycosylation site.

[0031] FIG. 12 depicts an alignment of the mutT domain of human 26493 with consensus amino acid sequences derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence for a mutT domain (SEQ ID NO:19), while the lower amino acid sequence corresponds to amino acids 122 to 251 of SEQ ID NO: 17.

[0032] FIG. 13 depicts a hydropathy plot of human 58224. The SNF2 domain and the C-terminal helicase domain are indicated. The numbers corresponding to the amino acid sequence of human 58224 (SEQ ID NO:23) are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence of 255-265 of SEQ ID NO:23; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of 150-160 of SEQ ID NO:23; a sequence which includes a Cys, or a glycosylation site.

[0033] FIGS. 14A-14B depicts an alignment of the SN2 N-terminal domain of human 58224 with a consensus amino acid sequence derived from a hidden Markov model. The upper sequence is the consensus amino acid sequence (SEQ ID NO:25), while the lower amino acid sequence corresponds to amino acids 226 to 577 of SEQ ID NO:23.

[0034] FIG. 14C depicts an alignment of the helicase conserved C-terminal domain of human 58224 with a consensus amino acid sequence derived from a hidden Markov model. The upper sequence is the consensus amino acid sequence (SEQ ID NO:26), while the lower amino acid sequence corresponds to amino acids 629 to 712 of SEQ ID NO:23.

[0035] FIG. 15 depicts a hydropathy plot of human 46980. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 46980 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 1 to 29, from about 187 to 211, and from about 675 to 696 of SEQ ID NO:28; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 80 to 100, or 440 to 450 of SEQ ID NO:28. Also indicated are an extracellular domain from about amino acid 43 to 674 of SEQ ID NO:28, a transmembrane domain from about amino acid 675 to 696 of SEQ ID NO:28, an intracellular domain from about amino acid 697 to 816 of SEQ ID NO:28, and a carboxylesterase domain from about amino acid 25 to 590 of SEQ ID NO:28.

[0036] FIGS. 16A-16C depicts an alignment of the carboxylesterase domain of human 46980 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:30), while the lower amino acid sequence corresponds to amino acids 25 to 590 of SEQ ID NO:28.

[0037] FIGS. 17A-17B depicts a BLAST alignment of a human 46980 polypeptide with a rat neuroligin 3 (SEQ ID NO:31).

[0038] FIG. 18 depicts a hydropathy plot of human 32225. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 32225 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 71 to 88, from about 135 to 157, and from about 186 to 199 of SEQ ID NO:34; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 106 to 123, from about 220 to 236, and from about 299 to 316 of SEQ ID NO:34.

[0039] FIG. 19 depicts an alignment of the hydrolase domain of human 32225 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:36), while the lower amino acid sequence corresponds to amino acids 95 to 338 of SEQ ID NO:34.

[0040] FIGS. 20A-20C depicts BLAST alignments of the α/β hydrolase domain of human 32225 with consensus amino acid sequences derived from ProDom families PD349163, PD021903, and PD034252, (see ProDomain Release 2001.1; http://www.toulouse.inra.fr/prodom.html). The BLAST algorithm identifies multiple local alignments between the consensus amino acid sequence and human 32225. (A) The lower sequence is the consensus amino acid sequence for ProDom PD349163 (SEQ ID NO:37), while the upper amino acid sequence corresponds to a first fragment of the α/β hydrolase domain of human 32225, about amino acids 8 to 69 of SEQ ID NO:34. (B) The lower sequence is the consensus amino acid sequence for ProDom PD021903 (SEQ ID NO:38), while the upper amino acid sequence corresponds to a second fragment of the α/β hydrolase domain of human 32225, about amino acids 187 to 277 of SEQ ID NO:34. (C) The lower sequence is the consensus amino acid sequence for ProDom PD034252 (SEQ ID NO:39), while the upper amino acid sequence corresponds to a third fragment of the α/β hydrolase domain of human 32225, about amino acids 280 to 342 of SEQ ID NO:34.

[0041] FIG. 21 depicts a hydropathy plot of human 47508. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 47508 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 151 to 172, from about 215 to 232, and from about 377 to 393 of SEQ ID NO:42; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 19 to 35, from about 245 to 263, and from about 286 to 310 of SEQ ID NO:42.

[0042] FIG. 22 depicts an alignment of the histone deacetylase domain of human 47508 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:44), while the lower amino acid sequence corresponds to amino acids 83 to 392 of SEQ ID NO:42.

[0043] FIGS. 23A-23C depict BLAST alignments of portions of the histone deacetylase domain of human 47508 with representative amino acid sequences derived from ProDomains No. 345193, 001400, and 021448 (ProDomain Release 2000.1; http://www.toulouse.inra.fr/prodom.html). The BLAST algorithm identifies multiple local alignments between the consensus amino acid sequence and human 47508. In FIG. 23A, the lower sequence is the representative amino acid sequence of ProDomain 345193 (SEQ ID NO:45), while the upper amino acid sequence corresponds to an N-terminal portion of the histone deacetylase domain of human 47508, about amino acid residues 71 to 115 of SEQ ID NO:42. In FIG. 23B, the lower sequence is the representative amino acid sequence of ProDomain 001400 (SEQ ID NO:46), while the upper amino acid sequence corresponds to a central portion of the histone deacetylase domain of human 47508, about amino acid residues 120 to 258. In FIG. 23C, the lower sequence is the representative amino acid sequence of ProDomain 021448 (SEQ ID NO:47), while the upper amino acid sequence corresponds to a C-terminal portion of the histone deacetylase domain of human 47508, about amino acid residues 251 to 372 of SEQ ID NO:42.

[0044] FIG. 24 depicts a hydropathy plot of human 56939. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The numbers corresponding to the amino acid sequence of human 56939 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence of from about amino acid 78 to 83, from about 100 to 104, and from about 223 to 231 of SEQ ID NO:49; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 110 to 115, from about 323 to 330, and from about 339 to 349 of SEQ ID NO:49.

[0045] FIG. 25 depicts an alignment of the acyl-CoA thioesterase domain of human 56939 with a consensus amino acid sequence derived from the ProDomain family PD0006914. The lower sequence is the consensus amino acid sequence (SEQ ID NO:51), while the upper sequence corresponds to amino acids 1 to 415 of SEQ ID NO:49 (SEQ ID NO:52).

[0046] FIG. 26 depicts a hydropathy plot of human 33410. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human 33410 are indicated. Polypeptides of the invention include 33410 fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 60 to 72, from about 260 to 277, and from about 780 to 793; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 330 to 350, from about 480 to 505, and from about 695 to 720; and/or a sequence which includes a cysteine, or a glycosylation site.

[0047] FIG. 27 depicts an alignment of the carboxylesterase domain of human 33410 with a consensus amino acid sequence derived from a hidden Markov model (HMM). The upper sequence is the consensus amino acid sequence (SEQ ID NO:56), while the lower amino acid sequence corresponds to amino acids 42 to 601 of SEQ ID NO:54.

[0048] FIGS. 28A-28B depicts alignment of the rat neuroligin-2 amino acid sequence and the human 33410 (SEQ ID NO:54) amino acid sequences. The location of the transmembrane domain in the rat neuroligin-2 (SEQ ID NO:57) and 33410 amino acid sequences is indicated as “TM1”.

[0049] FIGS. 29A-29B depicts alignment of the partial human KIAA1366 (Genbank Accession Number AB037787; SEQ ID NO:58) and the human 33410 amino acid sequences (SEQ ID NO:54).

[0050] FIG. 30 depicts a hydropathy plot of human 33521. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human 33521 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 722 to 730, from about 883 to 891, and from about 966 to 975, of SEQ ID NO:62; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 741 to 750, from about 756 to 762, and from about 1363 to 1372, of SEQ ID NO:62; a sequence which includes a Cys, or a glycosylation site.

[0051] FIG. 31A depicts an alignment of the first PH domain of human 33521 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:64), while the lower amino acid sequence corresponds to amino acids 507 to 620 of SEQ ID NO:62.

[0052] FIG. 31B depicts an alignment of the RBD domain of human 33521 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:65), while the lower amino acid sequence corresponds to amino acids 810 to 853 of SEQ ID NO:62.

[0053] FIG. 31C depicts an alignment of the PDZ domain of human 33521 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:66), while the lower amino acid sequence corresponds to amino acids 890 to 975 of SEQ ID NO:62.

[0054] FIG. 31D depicts an alignment of the Rho GEF domain of human 33521 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:67), while the lower amino acid sequence corresponds to amino acids 1103 to 1292 of SEQ ID NO:62.

[0055] FIG. 31E depicts an alignment of the second PH domain of human 33521 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:68), while the lower amino acid sequence corresponds to amino acids 1353 to 1455 of SEQ ID NO:62.

[0056] FIG. 32A depicts an alignment of the first PH domain of human 33521 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from SMART. The upper sequence is the consensus amino acid sequence (SEQ ID NO:69), while the lower amino acid sequence corresponds to amino acids 507 to 622 of SEQ ID NO:62.

[0057] FIG. 32B depicts an alignment of the RBD domain of human 33521 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from SMART. The upper sequence is the consensus amino acid sequence (SEQ ID NO:70), while the lower amino acid sequence corresponds to amino acids 810 to 881 of SEQ ID NO:62.

[0058] FIG. 32C depicts an alignment of the PDZ domain of human 33521 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from SMART. The upper sequence is the consensus amino acid sequence (SEQ ID NO:71), while the lower amino acid sequence corresponds to amino acids 900 to 976 of SEQ ID NO:62.

[0059] FIG. 32D depicts an alignment of the Rho GEF domain of human 33521 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from SMART. The upper sequence is the consensus amino acid sequence (SEQ ID NO:72), while the lower amino acid sequence corresponds to amino acids 1103 to 1292 of SEQ ID NO:62.

[0060] FIG. 32E depicts an alignment of the second PH domain of human 33521 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from SMART. The upper sequence is the consensus amino acid sequence (SEQ ID NO:73), while the lower amino acid sequence corresponds to amino acids 1326 to 1457 of SEQ ID NO:62.

[0061] FIG. 33 depicts a hydropathy plot of human 23479 polypeptide. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 23479 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 100 to 110, from about amino acid 295 to 310, and from about amino acid 920 to 930, of SEQ ID NO:75; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence from about amino acid 275 to 290, from about amino acid 530 to 550, and from about amino acid 640 to 650, of SEQ ID NO:75.

[0062] FIG. 34A depicts an alignment of the first ubiquitin carboxyl-terminal hydrolase domain (UCH-1) of human 23479 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:83), while the lower amino acid sequence corresponds to amino acids 296-327 of SEQ ID NO:75.

[0063] FIG. 34B depicts an alignment of the second ubiquitin carboxyl-terminal hydrolase domain (UCH-2) of human 23479 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:84), while the lower amino acid sequence corresponds to amino acids 546-640 of SEQ ID NO:75.

[0064] FIG. 35 depicts a hydropathy plot of human 48120 polypeptide. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 48120 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 1040 to 1055 of SEQ ID NO:78; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence from about amino acid 120 to 155, from about amino acid 680 to 700, and from about amino acid 770 to 800, of SEQ ID NO:78.

[0065] FIG. 36A depicts an alignment of the first ubiquitin carboxyl-terminal hydrolase domain (UCH-1) of human 48120 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:83), while the lower amino acid sequence corresponds to amino acids 162 to 193 of SEQ ID NO:78.

[0066] FIG. 36B depicts an alignment of the second ubiquitin carboxyl-terminal hydrolase domain (UCH-2) of human 48120 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:84), while the lower amino acid sequence corresponds to amino acids 580 to 649 of SEQ ID NO:78.

[0067] FIG. 36C depicts an alignment of the ubiquitin associated (UBA) domain of human 48120 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:85), while the lower amino acid sequence corresponds to amino acids 20 to 61 of SEQ ID NO:78.

[0068] FIG. 36D depicts an alignment of the ubiquitin interaction motif (UIM) domain of human 48120 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:86), while the lower amino acid sequence corresponds to amino acids 96 to 113 of SEQ ID NO:78.

[0069] FIG. 37 depicts a hydropathy plot of human 46689 polypeptide. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 46689 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 1 to 23, from about 133 to 145, and from about 150 to 168 of SEQ ID NO:81; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 34 to 51, from about 333 to 347, and from about 438 to 449 of SEQ ID NO:81.

[0070] FIG. 38 depicts an alignment of the α/β hydrolase domain of human 46689 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:87), while the lower amino acid sequence corresponds to about amino acid residues 186 to 419 of SEQ ID NO:81.

[0071] FIG. 39 depicts a BLAST alignment of the α/β hydrolase domain of human 46689 with a consensus amino acid sequence derived from a ProDom family PD007763 (Release 2001.1; http://www.toulouse.inra.fr/prodom.html). The lower sequence is the consensus amino acid sequence (SEQ ID NO:88), while the upper amino acid sequence corresponds to the a/b hydrolase domain of human 46689 along with some flanking sequence, about amino acid residues 97 to 424 of SEQ ID NO:81.

[0072] FIG. 40 depicts a hydropathy plot of human 80091. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (Cys) are indicated by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human 80091 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequences of about amino acids 188 to 205, about 540 to 550, about 700 to 725, about 817 to 825, and about 980 to 995 of SEQ ID NO:95; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequences of about amino acids 315 to 339, about 530 to 539, about 680 to 695, and about 1185 to 1220 of SEQ ID NO:95; a sequence which includes a Cys, or a glycosylation site.

[0073] FIG. 41A depicts an alignment of the ubiquitin carboxy-terminal hydrolase-1 (UCH-1) domain of human 80091 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:96), while the lower amino acid sequence corresponds to amino acids 447 to about 478 of SEQ ID NO:95.

[0074] FIG. 41B depicts an alignment of the ubiquitin carboxy-terminal hydrolase-2 (UCH-2) domain of human 80091 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:97), while the lower amino acid sequence corresponds to amino acids 1219 to 1279 of SEQ ID NO:95.

[0075] FIGS. 42A-42C depicts a cDNA sequence (SEQ ID NO:101) and predicted amino acid sequence (SEQ ID NO:102) of human 46508. The coding sequence (without the 5′ and 3′untranslated regions), which starts at the initiator methionine of the open reading frame of human 46508 until the termination codon of SEQ ID NO:101 are also indicated (shown as SEQ ID NO:103).

[0076] FIG. 43 depicts a hydropathy plot of human 46508. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 46508 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 60 to 70, from about 86 to 102, and from about 189 to 195 of SEQ ID NO: 102; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 77 to 85, from about 217 to 224 of SEQ ID NO: 102, a sequence which includes a Cys, or a glycosylation site, of SEQ ID NO: 102.

[0077] FIG. 44 depicts an alignment of the peptidyl-tRNA hydrolase domain of human 46508 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 104), while the lower amino acid sequence corresponds to amino acids 44 to 221 of SEQ ID NO:102.

DETAILED DESCRIPTION OF 26443 AND 46873

[0078] The human 26443 sequence (FIG. 1; SEQ ID NO: 1), which is approximately 1888 nucleotides long, including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1254 nucleotides (SEQ ID NO:3, and nucleotides 91-1344 of SEQ ID NO:1). The coding sequence encodes an 418 amino acid protein (SEQ ID NO:2).

[0079] Human 26443 contains a predicted asparaginase domain from about amino acids 38 to 345 of SEQ ID NO:2.

[0080] The 26443 protein also includes the following domains: a predicted N-glycosylation site (PFAM Accession PS0001) located at about amino acid residues 225-228 of SEQ ID NO:2; two predicted glycosaminoglycan attachment sites (PFAM Accession PS0002) located at about amino acid residues 7-10 and 289-292 of SEQ ID NO:2; a predicted cAMP- and cGMP-dependent protein kinase phosphorylation site (PFAM Accession PS0004) located at about amino acid residues 217-220 of SEQ ID NO:2; five predicted Protein Kinase C phosphorylation sites (PS00005) at about amino acids 24-26, 33-35, 186-188, 221-223 and 346-348 of SEQ ID NO:2; six predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino acids 6-9, 24-27, 33-36, 116-119, 221-224 and 381-384 of SEQ ID NO:2; and eight predicted N-myristoylation sites (PS00008) from about amino acids 4-9, 77-82, 100-105, 126-131, 228-233, 242-247, 336-341 and 397-402 of SEQ ID NO:2.

[0081] The human 46873 sequence (FIG. 4; SEQ ID NO:4), which is approximately 1358 nucleotides long, including untranslated regions, contains a predicted methionine-initiated coding sequence of about 924 nucleotides (SEQ ID NO:6, and nucleotides 134-1057 of SEQ ID NO:4). The coding sequence encodes a 308 amino acid protein (SEQ ID NO:5).

[0082] Human 46873 contains a predicted asparaginase domain from about amino acids 1 to 302 of SEQ ID NO:5.

[0083] The 46873 protein also includes the following domains: one predicted Protein Kinase C phosphorylation site (PS00005) at about amino acids 141-143 of SEQ ID NO:5; five predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino acids 43-46, 71-74, 80-83, 243-246 and 303-306 of SEQ ID NO:5; and eight predicted N-myristoylation sites (PS00008) from about amino acids 26-31, 50-55, 66-71, 90-05, 156-161, 167-172, 187-192 and 214-219 of SEQ ID NO:5.

[0084] For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc.edu/general/software/packages/pfam/pfam.html.

[0085] Plasmids containing the nucleotide sequence encoding human 26443 and 46873 (clones “Fbh26443FL” and “Fbh46873FL,” respectively) were deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______ and assigned Accession Numbers ______ or ______. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112. Table 1 contains a summary of sequence information for 26443 and 46873. 1

TABLE 1
(Summary of Sequence Information
for Asparaginase Polypeptides

					ATCC
			Poly-		Accession
GENE	cDNA	ORF	peptide	FIG	No.
26443	SEQ ID	SEQ ID	SEQ ID	1	—
	NO: 1	NO: 3	NO: 2
46873	SEQ ID	SEQ ID	SEQ ID	5	—
	NO: 4	NO: 6	NO: 5

[0086] The 26443 and 46873 proteins contain a significant number of structural characteristics in common with members of the asparaginase family. The term “family” when referring to the protein and nucleic acid molecules of the invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.

[0087] 26443 and 46873 polypeptides or 26443 and 46873 family members can include an “asparaginase domain” or regions homologous with an “asparaginase domain”.

[0088] As used herein, the term “asparaginase domain” refers to a protein domain having an amino acid sequence of about 50 to 600 amino acids, preferably about 150 to 450 amino acid residues, more preferably about 300 to 310 amino acids. An asparaginase domain typically includes two conserved threonine residues that play a role in the catalytic properties of asparaginases. The first is typically located in the N-terminal extremity of the protein, while the second is located at the end of the first third of the amino acid sequence. Consensus patterns for asparaginases are as follows: [LIVM]-x(2)-T-G-G-T-[IV]-[AGS], SEQ ID NO:8, the second T is an active site residue, and G-x-[LIVM]-x(2)-H-G-T-D-T-[LIVM], SEQ ID NO:9, wherein the first T is an active site residue. Preferably, an “asparaginase domain” includes an amino acid sequence of about 250 to 400 amino acid residues in length and having a bit score for the alignment of the sequence to the asparaginase domain (HMM) of at least 75. More preferably, an asparaginase domain includes at least about 50 to 600 amino acids, even more preferably about 150 to 400 amino acids, or even most preferably, 300-310 amino acids, and has a bit score for the alignment of the sequence to the asparaginase domain (HMM) of at least 75, 100, 200, 300, 400 or greater. Asparaginase domains (HMM) have been assigned PFAM Accession PF00710 and PFAM Accession PF01112 (http://genome.wustl.edu/Pfam/html). An alignment of the asparaginase domain (SEQ ID NO:7, corresponding to amino acids 38 to 345 of SEQ ID NO:2) of human 26443 with a consensus amino acid sequence derived from a hidden Markov model is depicted in FIG. 4. An alignment of the asparaginase domain (SEQ ID NO:7, corresponding to amino acids 1 to 302 of SEQ ID NO:5) of human 46873 with a consensus amino acid sequence derived from a hidden Markov model is depicted in FIG. 8.

[0089] In a preferred embodiment, A 26443 or 46873 polypeptide or protein has an “asparaginase domain” or a region which includes at least about 50-600, more preferably about 150-450 or 300-310 amino acid residues, and having at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “asparaginase domain,” e.g., the asparaginase domain of human 26443 or 46873 (e.g., residues 38-345 of SEQ ID NO:2 or residues 1-302 of SEQ ID NO:5, respectively).

[0090] To identify the presence of a “asparaginase domain” in a 26443 or 46873 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of an “asparaginase domain” in the amino acid sequence of human 26443 and 46873 at about residues 38-345 of SEQ ID NO:2 (see FIG. 4) and 1-302 of SEQ ID NO:5 (see FIG. 8), respectively.

[0091] As the 26443 or 46873 polypeptides of the invention may modulate 26443- or 46873-mediated activities, they may be useful as, or for, developing novel diagnostic and therapeutic agents for 26443- or 46873-mediated or related disorders, as described below.

[0092] As used herein, a “26443 or 46873 activity”, “biological activity of 26443 or 46873” or “functional activity of 26443 or 46873”, refers to an activity exerted by a 26443 or 46873 protein, polypeptide or nucleic acid molecule on, e.g., a 26443- or 46873-responsive cell or on a 26443 or 46873 substrate, e.g., a protein substrate, as determined in vivo or in vitro. In one embodiment, a 26443 or 46873 activity is a direct activity, such as an association with a 26443 or 46873 target molecule. A “target molecule” or “binding partner” is a molecule with which a 26443 or 46873 protein binds or interacts in nature. In an exemplary embodiment, a “target molecule” is, e.g., an asparagine. A 26443 or 46873 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the 26443 or 46873 protein with a 26443 or 46873 ligand. For example, the 26443 or 46873 proteins of the present invention can have one or more of the following activities: (1) catalyzes the hydrolysis of asparagine to aspartic acid and ammonia; (2) regulates cellular amounts of asparagine; (3) regulates the cellular amounts of aspartic acid; (4) regulates cellular amounts of ammonia; and (5) antagonizes or inhibits, e.g., competitively or noncompetitively, any of activities 1-4.

[0093] Based on the above-described sequence similarities, the 26443 or 46873 molecules of the present invention are predicted to have similar biological activities as asparaginase family members. Asparaginase enzymes assist in the hydrolysis of asparagine to aspartic acid and ammonia. Thus, the 26443 or 46873 molecules can act as novel diagnostic targets and therapeutic agents for controlling, e.g., the amount of asparagine (and likewise, aspartic acid) in a cell.

[0094] The 26443 or 46873 protein may be involved in disorders characterized by aberrant activity of the cells in which it is expressed. Since asparaginase enzymes are typically found in most cells in bacterial fungi, plants and mammals, e.g., cells that contain or metabolize asparagine, it is likely that 26443 or 46873 proteins may also be expressed in such cells. Therefore, altered expression and/or activity of a 26443 or 46873 molecule can lead to defects in the metabolism of asparagine and/or aspartic acid.

[0095] The 26443 or 46873 molecules can also act as novel diagnostic targets and therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders, disorders associated with bone metabolism, immune disorders, hematopoietic disorders, cardiovascular disorders, liver disorders, viral diseases, pain or metabolic disorders.

[0096] Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

[0097] 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. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. 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.

[0098] The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[0099] The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0100] The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[0101] The 26443 or 46873 nucleic acid and protein of the invention can be used to treat and/or diagnose a variety of 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.

[0102] Asparaginases are generally more effective in treating acute lymphoblastic leukemia and lymphosarcomas, than other forms of leukemia or solid tumors, since remissions of these types of cancers are invariably of short duration. Whereas most normal tissues synthesize L-asparagine in amounts sufficient for their metabolic needs, certain neoplastic tissues, primarily acute lymphoblastic leukemia (ALL) and lymphosarcoma cells, require an exogenous source of asparagines (i.e., from nearby host tissues). Administration of L-asparaginase enzymatically catalyzes the hydrolysis of asparagine to aspartic acid and ammonia, which deprives the malignant cells of the asparagine from extracellular fluid and eventually results in cell death. Clinical use of asparaginase from, e.g., Escherichia coli or Erwinia chrysanthemi, oftentimes results in hypersensitive immune responses after multiple administrations. Since the two asparaginase enzymes from E. coli and E. chrysanthemi do not exhibit any cross-reactivity, the two enzymes can be used in a treatment regimen to reduce or avoid the hypersensitivity response.

[0103] Additionally, asparaginases can be administered in combination with other traditional or experimental cancer treatments. Asparaginases can be combined with a treatment modality which inhibits cell proliferation, e.g., cytotoxic agents, e.g., agents with diverse structures and mechanisms of action, including but not limited to, antimicrotubule agents, topoisomerase I inhibitors, topoisomerase II inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, intercalating agents, agents capable of interfering with a signal transduction pathway (e.g., protein kinase C inhibitors, e.g., anti-hormones, e.g., antibodies against growth factor receptors), agents that promote apoptosis and/or necrosis, biological response modifiers (e.g., interferons, interleukins, tumor necrosis factors), and radiation.

[0104] The 26443 or 46873 protein, fragments thereof, and derivatives and other variants of the sequence in SEQ ID NO:2 or SEQ ID NO:5, respectively, are collectively referred to as “polypeptides or proteins of the invention” or “26443 or 46873 polypeptides or proteins”. Nucleic acid molecules encoding such polypeptides or proteins are collectively referred to as “nucleic acids of the invention” or “26443 or 46873 nucleic acids”. 26443 or 46873 molecules refer to 26443 or 46873 nucleic acids, polypeptides, and antibodies.

[0105] As used herein, the term “nucleic acid molecule” includes DNA molecules (e.g., a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0106] The term “isolated or purified nucleic acid molecule” includes nucleic acid molecules that are separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term “isolated” includes nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′ and/or 3′ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0107] 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 non-aqueous 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× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2× SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6× SSC at about 45° C., followed by one or more washes in 0.2× SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6× SSC at about 45° C., followed by one or more washes in 0.2× SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2× SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 or 3, corresponds to a naturally-occurring nucleic acid molecule.

[0108] As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

[0109] As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a 26443 or 46873 protein, preferably a mammalian 26443 or 46873 protein, and can further include non-coding regulatory sequences and introns.

[0110] 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. In one embodiment, the language “substantially free” means preparation of 26443 or 46873 protein having less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-26443 or -46873 protein (also referred to herein as a “contaminating protein”), or of chemical precursors or non-26443 or -46873 chemicals. When the 26443 or 46873 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

[0111] A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of 26443 or 46873 (e.g., the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ or Accession Number ______) without abolishing or more preferably, without substantially altering a biological activity, whereas an “essential” amino acid residue results in such a change. For example, amino acid residues that are conserved among the polypeptides of the present invention, e.g., those present in the asparaginase domain, are predicted to be particularly unamenable to alteration.

[0112] 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 26443 or 46873 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a 26443 or 46873 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 26443 or 46873 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ or as Accession Number ______, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[0113] As used herein, a “biologically active portion” of a 26443 or 46873 protein includes a fragment of a 26443 or 46873 protein that participates in an interaction between a 26443 or 46873 molecule and a non-26443 or -46873 molecule. Biologically active portions of a 26443 or 46873 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the 26443 or 46873 protein, e.g., the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:5, respectively, which include less amino acids than the full length 26443 or 46873 proteins, and exhibit at least one activity of a 26443 or 46873 protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the 26443 or 46873 protein, e.g., asparaginase. A biologically active portion of a 26443 or 46873 protein can be a polypeptide that is, for example, 50, 100, 200 or more amino acids in length. Biologically active portions of a 26443 or 46873 protein can be used as targets for developing agents, which modulate a 26443- or 46873-mediated activity, e.g., asparaginase.

[0114] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

[0115] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence (e.g., when aligning a second sequence to the 26443 amino acid sequence of SEQ ID NO:2 having 125 amino acid residues, at least 167, preferably at least 209, more preferably at least 251, and even more preferably at least 293, 334, 376 or 418 amino acid residues are aligned; when aligning a second sequence to the 46873 amino acid sequence of SEQ ID NO:5 having 92 amino acid residues, at least 123, preferably at least 154, more preferably at least 185, and even more preferably at least 216, 246, 277 or 308 amino acid residues are aligned). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0116] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if the molecule is within the sequence identity limits of a claim) is using a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0117] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0118] The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to 26443 or 46873 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to 26443 or 46873 protein molecules of the invention. To obtain-gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0119] “Misexpression or aberrant expression”, as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over- or under-expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.

[0120] “Subject”, as used herein, can refer to a mammal, e.g., a human, or to an experimental or animal or disease model. The subject can also be a non-human animal, e.g., a horse, cow, goat, or other domestic animal.

[0121] A “purified preparation of cells”, as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.

[0122] Various aspects of the invention are described in further detail below.

[0123] Isolated Nucleic Acid Molecules of 26443 and 46873

[0124] In one aspect, the invention provides, an isolated or purified, nucleic acid molecule that encodes a 26443 or 46873 polypeptide described herein, e.g., a full-length 26443 or 46873 protein or a fragment thereof, e.g., a biologically active portion of a 26443 or 46873 protein. Also included is a nucleic acid fragment suitable for use as a hybridization probe, which can be used, e.g., to a identify a nucleic acid molecule encoding a polypeptide of the invention, 26443 or 46873 mRNA, and fragments suitable for use as primers, e.g., PCR primers for the amplification or mutation of nucleic acid molecules.

[0125] In one embodiment, an isolated nucleic acid molecule of the invention includes the nucleotide sequence shown in SEQ ID NO: 1, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ or Accession Number ______, or a portion of any of these nucleotide sequences. In one embodiment, the nucleic acid molecule includes sequences encoding the human 26443 protein (i.e., “the coding region”, from nucleotides 91-1344 of SEQ ID NO:1), as well as 5′untranslated sequences (nucleotides 1-90 of SEQ ID NO:1) and 3′untranslated sequences (nucleotides 1345-1888 of SEQ ID NO:1). Alternatively, the nucleic acid molecule can include only the coding region of SEQ ID NO:1 (e.g., nucleotides 91-1344, corresponding to SEQ ID NO:3) and, e.g., no flanking sequences which normally accompany the subject sequence. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to the mature protein from about amino acid 1 to amino acid 418 of SEQ ID NO:2.

[0126] In another embodiment, an isolated nucleic acid molecule of the invention includes the nucleotide sequence shown in SEQ ID NO:4, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ or Accession Number ______, or a portion of any of these nucleotide sequences. In one embodiment, the nucleic acid molecule includes sequences encoding the human 46873 protein (i.e., “the coding region”, from nucleotides 134-1057 of SEQ ID NO:4), as well as 5′untranslated sequences (nucleotides 1-133 of SEQ ID NO:4) and 3′untranslated sequences (nucleotides 1058-1358 of SEQ ID NO:4). Alternatively, the nucleic acid molecule can include only the coding region of SEQ ID NO:4 (e.g., nucleotides 134-1057, corresponding to SEQ ID NO:6) and, e.g., no flanking sequences which normally accompany the subject sequence. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to the mature protein from about amino acid 1 to amino acid 308 of SEQ ID NO:5.

[0127] 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: 1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ or as Accession Number ______, 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: 1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ or as Accession Number ______ such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ or as Accession Number ______, thereby forming a stable duplex.

[0128] In one embodiment, an isolated nucleic acid molecule of the present invention includes a nucleotide sequence which is at least about 60%, 65%, 70%, 75%, 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:1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the entire length of the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ or as Accession Number ______, or a portion, preferably of the same length, of any of these nucleotide sequences.

[0129] 26443 or 46873 Nucleic Acid Fragments

[0130] A nucleic acid molecule of the invention can include only a portion of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ or as Accession Number ______. For example, such a nucleic acid molecule can include a fragment that can be used as a probe or primer or a fragment encoding a portion of a 26443 or 46873 protein, e.g., an immunogenic or biologically active portion of a 26443 or 46873 protein. A fragment can comprise nucleotides 202 to 1125 of SEQ ID NO:1, which encodes an asparaginase domain of human 26443, or nucleotides 134 to 1039 of SEQ ID NO:4, which also encodes an asparaginase domain of human 46873. The nucleotide sequence determined from the cloning of the 26443 or 46873 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 26443 or 46873 family members, or fragments thereof, as well as 26443 or 46873 homologues, or fragments thereof, from other species.

[0131] In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, of the coding region and extends into either (or both) the 5′ or 3′noncoding region. Other embodiments include a fragment that includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof that are at least 200, preferably 300 amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.

[0132] A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domain, region, or functional site described herein. Thus, for example, a nucleic acid fragment can include a sequence corresponding to an asparaginase domain.

[0133] In a preferred embodiment, the fragment is at least 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides in length.

[0134] 26443 or 46873 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under stringent conditions to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ or as Accession Number ______, or of a naturally occurring allelic variant or mutant of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ or as Accession Number ______.

[0135] In a preferred embodiment the nucleic acid is a probe which is at least 5 or 10, and less than 200, more preferably less than 100, or less than 50, base pairs in length. It should be identical, or differ by 1, or less than in 5 or 10 bases, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[0136] A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes an asparaginase domain (corresponding to residues 38-345 of SEQ ID NO:2 or residues 1-302 of SEQ ID NO:5.

[0137] In another embodiment, a set of primers is provided, e.g., primers suitable for use in a PCR, which can be used to amplify a selected region of a 26443 or 46873 sequence. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. E.g., primers suitable for amplifying all or a portion of a domain or region described herein, e.g., any of the following regions, are provided an asparaginase domain corresponding to residues 38-345 of SEQ ID NO:2 or residues 1-302 of SEQ ID NO:5.

[0138] A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.

[0139] A nucleic acid fragment encoding a “biologically active portion of a 26443 or 46873 polypeptide” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ or Accession Number ______, which encodes a polypeptide having a 26443 or 46873 biological activity (e.g., the biological activities of the 26443 or 46873 proteins described herein), expressing the encoded portion of the 26443 or 46873 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the 26443 or 46873 protein. For example, a nucleic acid fragment encoding a biologically active portion of 26443 or 46873 includes an asparaginase domain, e.g., amino acid residues 38 to 345 of SEQ ID NO:2 or amino acid residues 1 to 302 of SEQ ID NO:5. A nucleic acid fragment encoding a biologically active portion of a 26443 or 46873 polypeptide may comprise a nucleotide sequence that is greater than 300 or more nucleotides in length (e.g., greater than about 400 nucleotides in length).

[0140] In preferred embodiments, a nucleic acid fragment of 26443 includes a nucleotide sequence which is at least about 300, at least about 353 (e.g., 355, 375, 400), at least about 400 (e.g., 500, 600, 700, 800), at least about 457 (e.g., 460, 500, 600, 700), or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO: 1, or SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______.

[0141] In a preferred embodiment, a nucleic acid fragment of 26443 includes a nucleotide sequence comprising nucleotides 183-842, 459-842, 1195-1244, or 1644-1888 of SEQ ID NO: 1, or a portion thereof, wherein each fragment hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO: 1, or SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______. In another preferred embodiment, a nucleic acid fragment of 26443 includes a nucleotide sequence comprising nucleotides 1-842 of SEQ ID NO: 1, or a portion thereof, wherein each portion is about 183 or longer nucleotides and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO: 1, or SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______.

[0142] In a preferred embodiment, a nucleic acid fragment has a nucleotide sequence other than AI793006, AA262517, R89654, or C07777.

[0143] In preferred embodiments, a nucleic acid fragment of 46873 includes a nucleotide sequence which is at least about 300, 400, 500, 560 (e.g., 570, 580, 590, 600), at least about 662 (e.g., 665, 666, 667, 668, 670, 680, 690, 700), or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO:4, or SEQ ID NO:6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______.

[0144] In a preferred embodiment, a nucleic acid fragment of 46873 includes a nucleotide sequence of SEQ ID NO:4 or 6, or a portion thereof; or a portion of the 46873 sequence comprising nucleotides 1-680, 1-686, 1-692 or 1-785 of SEQ ID NO:4, or a portion thereof, wherein each fragment hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO:4, or SEQ ID NO:6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______.

[0145] In a preferred embodiment, a nucleic acid fragment has a nucleotide sequence other than AI879995, AI928914, AW131805, or AI978667.

[0146] 26443 or 46873 Nucleic Acid Variants

[0147] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ or Accession Number ______. Such differences can be due to degeneracy of the genetic code (and result in a nucleic acid that encodes the same 26443 or 46873 proteins as those encoded by the nucleotide sequence disclosed herein. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence which differs, by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues that shown in SEQ ID NO:2 or SEQ ID NO:5. If alignment is needed for this comparison the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[0148] Nucleic acids of the inventor can be chosen for having codons, which are preferred, or non-preferred, for a particular expression system. E.g., the nucleic acid can be one in which at least one colon, at preferably at least 10%, or 20% of the codons has been altered such that the sequence is optimized for expression in E. Coli, yeast, human, insect, or CHO cells.

[0149] Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non-naturally occurring. Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).

[0150] In a preferred embodiment, the nucleic acid differs from that of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the sequence in ATCC Accession Number ______ or Accession Number ______, e.g., as follows: by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 2%, 5%, 10% or 20% of the subject nucleic acid. If necessary for this analysis the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[0151] Orthologs, homologs, and allelic variants can be identified using methods known in the art. These variants comprise a nucleotide sequence encoding a polypeptide that is 50%, at least about 55%, typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-95% or more identical to the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:5 or a fragment of those sequences. Nucleic acid molecules encoding such polypeptides can readily be identified as being able to hybridize under stringent conditions, to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or a fragment of the sequence. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of the 26443 or 46873 cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the 26443 or 46873 gene.

[0152] Preferred variants include those that are correlated with asparaginase activity.

[0153] Allelic variants of 26443 or 46873, e.g., human 26443 or 46873, include both functional and non-functional proteins. Functional allelic variants are naturally occurring amino acid sequence variants of the 26443 or 46873 protein within a population that maintain the ability to function as an asparaginase. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:2 or SEQ ID NO:5, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein. Non-functional allelic variants are naturally-occurring amino acid sequence variants of the 26443 or 46873, e.g., human 26443 or 46873, protein within a population that do not have the ability to function as an asparaginase. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5, or a substitution, insertion, or deletion in critical residues or critical regions of the protein.

[0154] Moreover, nucleic acid molecules encoding other 26443 or 46873 family members and, thus, which have a nucleotide sequence which differs from the 26443 or 46873 sequences of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ or Accession Number ______ are intended to be within the scope of the invention.

[0155] Antisense Nucleic Acid Molecules, Ribozymes and Modified 26443 or 46873 Nucleic Acid Molecules

[0156] In another aspect, the invention features, an isolated nucleic acid molecule that is antisense to 26443 or 46873. An “antisense” nucleic acid can include a nucleotide sequence that 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 26443 or 46873 coding strand, or to only a portion thereof (e.g., the coding region of human 26443 or 46873 corresponding to SEQ ID NO:3 or SEQ ID NO:6, respectively). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding 26443 or 46873 (e.g., the 5′ or 3′untranslated regions).

[0157] An antisense nucleic acid can be designed such that it is complementary to the entire coding region of 26443 or 46873 mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of 26443 or 46873 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of 26443 or 46873 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.

What is claimed is:

1. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 10, 12, 16, 18, 22, 24, 27, 29, 33, 35, 41, 43, 48, 50, 53, 55, 61, 63, 74, 76, 77, 79, 80, 82, 94, 101 or 103; and b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 5, 11, 17, 23, 28, 34, 42, 49, 54, 62, 75, 78, 81, 95 or 102.

2. The nucleic acid molecule of claim 1, further comprising vector nucleic acid sequences.

3. The nucleic acid molecule of claim 1, further comprising nucleic acid sequences encoding a heterologous polypeptide.

4. A host cell which contains the nucleic acid molecule of claim 1.

5. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2, 5, 11, 17, 23, 28, 34, 42, 49, 54, 62, 75, 78, 81, 95 or 102.

6. The polypeptide of claim 5 further comprising heterologous amino acid sequences.

7. An antibody or antigen-binding fragment thereof that selectively binds to a polypeptide of claim 5.

8. A method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 5, 11, 17, 23, 28, 34, 42, 49, 54, 62, 75, 78, 81, 95 or 102, the method comprising culturing the host cell of claim 4 under conditions in which the nucleic acid molecule is expressed.

9. A method for detecting the presence of a polypeptide of claim 5 in a sample, comprising: a) contacting the sample with a compound which selectively binds to the polypeptide; and b) determining whether the compound binds to the polypeptide in the sample.

10. The method of claim 9, wherein the compound which binds to the polypeptide is an antibody.

11. A kit comprising a compound which selectively binds to a polypeptide of claim 5 and instructions for use.

12. A method for detecting the presence of a nucleic acid molecule of claim 1 in a sample, comprising the steps of: a) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule; and b) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample.

13. The method of claim 12, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.

14. A kit comprising a compound which selectively hybridizes to a nucleic acid molecule of claim 1 and instructions for use.

15. A method for identifying a compound which binds to a polypeptide of claim 5 comprising the steps of: a) contacting a polypeptide, or a cell expressing a polypeptide of claim 5 with a test compound; and b) determining whether the polypeptide binds to the test compound.

16. A method for modulating the activity of a polypeptide of claim 5, comprising contacting a polypeptide or a cell expressing a polypeptide of claim 5 with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.

17. A method of inhibiting aberrant activity of a 26443, 46873, 61833, 26493, 58224, 46980, 32225, 47508, 56939, 33410, 33521, 23479, 48120, 46689, 80091, or 46508-expressing cell, comprising contacting a 26443, 46873, 61833, 26493, 58224, 46980, 32225, 47508, 56939, 33410, 33521, 23479, 48120, 46689, 80091, or 46508-expressing cell with a compound that modulates the activity or expression of a polypeptide of claim 5, in an amount which is effective to reduce or inhibit the aberrant activity of the cell.

18. The method of claim 17, wherein the compound is selected from the group consisting of a peptide, a phosphopeptide, a small organic molecule, and an antibody.

19. A method of treating or preventing a disorder characterized by aberrant activity of a 26443, 46873, 61833, 26493, 58224, 46980, 32225, 47508, 56939, 33410, 33521, 23479, 48120, 46689, 80091, or 46508-expressing cell, in a subject, comprising: administering to the subject an effective amount of a compound that modulates the activity or expression of a nucleic acid molecule of claim 1, such that the aberrant activity of the 26443, 46873, 61833, 26493, 58224, 46980, 32225, 47508, 56939, 33410, 33521, 23479, 48120, 46689, 80091, or 46508-expressing cell is reduced or inhibited.