Human interleukin-four induced protein

FIELD OF THE INVENTION

[0003] The invention is directed to an isolated genomic polynucleotide sequence encoding interleukin-four induced protein as well as methods for obtaining said protein by expressing said polynucleotide sequences. The invention is also directed to constructs, vectors and hosts comprising such sequences, and oligonucleotide probes which hybridize to said polynucleotide sequences and kits comprising said probes. Additionally, the invention is directed to compositions comprising said polynucleotides and methods for using said compositions to treat, prevent or ameliorate pathological disorders, e.g., immune related disorders and novel methods of using the interleukin-four induced protein. The invention is also directed to methods for obtaining an antibody that binds to an epitope on said protein. The invention is further directed to a method for measuring the activity of said protein by measuring its L-amino acid oxidase activity.

BACKGROUND OF THE INVENTION

[0004] Interleukin-4 Regulation of Differentiation, Growth and Apoptosis

[0005] Interleukin-4 (IL-4) is an important immunoregulatory cytokine with potent growth and differentiation effects on B and T lymphocytes (Paul, 1991, Blood 77:1859-1870). In mouse B cells, it acts as a comitogen with anti-IgM, lipopolysaccharide (LPS) and anti-CD40 and promotes survival in culture. IL-4 causes differentiation in mouse B cells as observed by the induction of transcription and/or expression of a series of genes including the germ line (Iγl and Iε) and mature forms of IgG1 and IgE heavy chains, MHC class II molecules, Thy-1, CD23 and as will be described below, FIG. 1 In T cells, IL-4 acts as a growth and survival factor for T cells and promotes the differentiation of CD4+ cells into TH2 cells, which are thought to promote a humoral or antibody type response.

[0006] Although IL-4 was initially characterized as an important immunoregulatory cytokine with potent effects on B and T lymphocyte growth, differentiation, and protection from apoptosis (Paul, 1991, Blood, 77:1859-1870), it has been subsequently observed to also contribute to apoptosis under certain situations. In T cells that have undergone repeated exposure to antigen, IL-4 will enhance anti-CD3 induced apoptosis (Zheng et aL, 1998, J. Immunol., 160:763-769). IL-4 has also been reported to induce apoptosis in human breast cancer cells (Gooch et al., 1998, Cancer Res, 58:4199-4205), mast cells (Oskeritzian et al., 1999, J. Immunol, 163:5105-5115), peripheral blood eosinophils (Wedi et al., 1998, J. Allergy Clin Immunol., 102:1013-1020), and gingival macrophages (Yamamoto et al., 1996, Am. J. Pathol., 148:331-339). Although IL-4 induced apoptosis in B cells has not yet been noted, a lack of IL-4 protection from Ig-induced apoptosis in WEHI-231 cells, a B cell lymphoma, has been reported (Tsubata et al., 1993, Nature, 364:645-648).

[0007]

FIG. 1

[0008] Mouse FIG. 1 (interleukin-four induced gene-1) is as an immediate-early interleukin-4 induced gene in mouse B cells (Chu, C. C., and W. E. Paul, 1997, Proc. Natl. Acad. Sci. USA 94:2507-2512 and Chu et al., 1998, Molecular Immunology 35:487-502). Other cytokines tested (IL2, IL-5, or IL-6) were not able to induce FIG. 1 RNA in B cells. The RNA expression of FIG. 1 is strikingly limited to lymphoid tissues, with expression thus far only clearly demonstrable in B cells. In addition, genetic mapping in mice places FIG. 1 in a genetic region implicated in disease susceptibility in mouse models of systemic lupus erythematosus (SLE) (Theofilopoulos et al., 1999, Proc. Assoc. Am. Physicians 111:228-40 and Vyse et al., 1998, Annu. Rev. Immunol. 16:261-292), suggesting that FIG. 1 plays a role in the development of autoimmunity.

[0009] The FIG. 1 gene maps between Klk1 and Fut1 on mouse chromosome 7 (23.1 cM, Mouse Genome Database (MGD), Mouse Genome Informatics, The Jackson Laboratory, Bar Harbor, Maine. World Wide Web (URL: http://www.informatics.jax.org/). (July, 2000)) (Blake et al., 2000, Nucleic Acids Res, 28:108-111). In mouse, chromosome 7 has been implicated in susceptibility to SLE by several studies (Theofilopoulos et al., 1999, Proc. Assoc. Am. Physicians 111:228-40 and Vyse et al., 1998, Annu. Rev. Immunol. 16:261-292). Three studies involving SLE-prone mouse models involving th e NZW strain have likely identified the same NZW locus or loci on chromosome 7 involved in SLE-susceptibility: Lbw5 (Kono et al., 1994, Proc. Natl. Acad. Sci. USA 91:10168-10172); Sle3 (Morel et al., 1994, Immunity 1:219-229), and an unnamed gene (Santiago et al., 1998, Eur. J. Immunol. 28:4257-67). FIG. 1 most closely maps to this NZW locus or loci. FIG. 1 has been genetically mapped to the smallest known chromosomal interval (˜13 cM) containing Sle3 (Chu et al., 1998, The Immunologist Suppl.1: 178.; Chu et al., 1999, Faseb J. 13, no. 5 (Part II):A955 and Chu et al., 1999, Genes Expressed in B Cells During Differentiation. In Signaling & Gene Expression in the Immune System, vol. LXIV Cold Spring Harbor Symposium on Quantitative Biology. B. Stillman, editor. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p.39).

[0010] The syntenic genetic location of human FIG. 1 is on chromosome 19q13.3-19q13.4 between the FUT1 and KLK1 markers (Deloukas et al., 1998, Science, 282:744-746). Chromosome 19q13.3-19q13.4 has been identified as a possible region of susceptibility to human SLE (Moser et al., 1998, Proc. Natl. Acad. Sci. USA 95:14869). Additionally, this area is a hot spot for autoirnmune disease susceptibility in general, including arthritis, multiple sclerosis, insulin-dependent diabetes mellitus and SLE (Vyse et al., 1996, Cell 85:311-8 and Becker et al., 1998, Proc Natl Acad. Sci USA 95:9979-84).

[0011] To investigate the relationship between FIG. 1 and Lbw5/Sle3, the NZW FIG. 1 allele was examined for any defects. No difference in RNA size, tissue expression, or induction by IL-4 was observed as compared to wild-type BALB/c (Chu et al., 1999, Faseb J. 13, no. 5 (Part II):A955). To investigate whether a defect in coding sequence of FIG. 1 could account for SLE-susceptibility, cDNA was sequenced from the NZW FIG. 1 allele. This sequence did not reveal an obvious translational defect caused by a frameshift or nonsense mutation. However, eight nucleotide polymorphisms were discovered that result in three non-conservative amino acid substitutions. The substitutions do not obviously affect any predicted functional domain. The first is an A to V substitution at residue 12 in the predicted signal peptide sequence. Although this is not a conservative substitution, both these residues are hydrophobic. Because the signal peptide sequence is defined as a continuous stretch of amino terminal hydrophobic amino acids, the signal peptide sequence is probably not perturbed by this substitution. The remaining two polymorphisms (a P to L substitution at residue 551 and an A to M substitution at residue 568) are found in the acidic C-terminal domain. Thus, these two polymorphisms may affect some as yet unknown function. The C-terminal domain is proposed to regulate FIG. 1 protein enzyme activity, in order to protect the cell from potential damaging effects of its L-amino acid oxidase (LAAO) activity (see below).

[0012] The mouse FIG. 1 cDNA sequence predicts a 630 amino acid 70 kDa protein containing a putative signal peptide sequence for secretion, a region of similarity to flavoproteins (enzymes that bind FAD (flavin adenine dinucleotide) as a cofactor) containing five binding domains, and an acidic 127 amino acid C-terminal region that has no homology to known proteins (Chu, C. C., and W. E. Paul, 1997, Proc. Natl. Acad. Sci. USA 94:2507-2512). The region of similarity to flavoproteins has the most similarity to the recently cloned snake venom L-amino acid oxidase (LAAO) (37% identical over 484 amino acids) (Raibekas et al., 1998, Biochem. Biophys. Res. Commun. 248:476-478).

[0013] LAAO, found widely in snake venom, has antibacterial activity (Stiles et al., 1991, Toxicon 29:1129-1141), has cytotoxic activity (Ahn et al., 1997, Int. J. Biochem. Cell Biol. 296:911-919), induces human platelet aggregation (Li et al., 1994, Toxicon. 32:1349-1358) and has apoptosis-inducing activity (Torii et al., 1997, J. Biol. Chem. 272:9539-9542). These lethal activities are due in part to the production of hydrogen peroxide (H2O2) during the oxidation of L-amino acids by LAAO (Suhr et al., 1999, J Biochem (Tokyo) 125:305-9). LAAO has also been found to detect peptidase activity (Sugiura et al., 1977, Clin. Chim. Acta 78:381-389) and as a biosensor for enantioselective analysis of S-enantiomers of various ACE inhibitors (Stefan et al., 1998, Prep. Biochem. Biotechnol. 28:305-315). One L-amino acid molecule plus one molecule of oxygen and water is oxidized by LAAO to one 2-oxo-acid molecule plus one molecule of hydrogen peroxide and ammonia. Preliminary data demonstrate that FIG. 1 indeed has the predicted LAAO activity (Chavan et al., 1999, Faseb J. 13, no. 5 (Part II):A318 and Naidu et al., 1999, Faseb J. 13, no. 5 (Part II):A318). Thus, FIG. 1 is an L-amino acid oxidase.

[0014] Since there appears to be a linkage between FIG. 1 and SLE, it would be useful to isolate and express the human homolog (h FIG. 1) of the mouse FIG. 1 gene. Therefore, it is an object of the invention to isolate and express such a gene. The product of such a gene is an immediate early interleukin-4 induced protein. There is a need for detecting polymorphisms in this gene and measuring RNA expression of this gene. Therefore, it is an object of the invention to detect such polymorphisms and RNA expression.

[0015] There is a further need to develop assays for measuring the activity of this protein. It is a further object of the invention to measure activity of the protein to use such assays to detect, treat and monitor the treatment of pathological disorders.

SUMMARY OF THE INVENTION

[0016] As will be discussed below, the invention is directed to a method for obtaining an isolated human immediate early interleukin-four induced protein having the following characteristics: (a) about 546 to about 567 amino acids; (b) L-amino acid oxidase activity; (c) is obtainable from interleukin-4 induced B cells; (c) contains an N-terminal signal sequence of about 21 amino acids; (d) is encoded by a genomic DNA sequence having eight exons, wherein said genomic DNA is obtainable from human chromosome 19q13.3-19q13.4; (e) has a non-conserved C-terminal sequence having about 62 amino acids; (f) has less than 80% homology to a mouse immediate early interleukin-four induced protein having the sequence depicted in SEQ ID NO: 1

1SEQ ID NO:1MAGLALRLVLAATLLGLAGSLDWKAASSLNPIEKCMEDHDYEQLLKVVTLGLNRTSKPQKVVVVGAGVAGLVAAKMLSDAGHKVTILEADNRIGGRIFTFRDEKTGWIGELGAMRMPSSHRILHKLCRTLGLNLTQFTQYDENTWTEVHNVKLRNYVVEKMPEKLGYNLNNRERGHSPEDIYQMALNKAFKDLKALGCKKAMNKFNKHTLLEYLLEEGNLSRPAVQLLGDVMSEEGFFYLSFAEALRAHACLSDRLRYSRIVGGWDLLPRALLSSLSGALLLNAPVVSITQGRNDVRVHIATSLHSEKTLTADVVLLTASGPALQRITFSPPLTRKRQEALRALHYVAASKVFLSFRRPFWHEEHIEGGHSNTDRPSRLIFYPARGEGSLLLASYTWSDAAAPFAGLSTDQTLRLVLQDVAALHGPVVFRLWDGRGVVKRWAEDPHSQGGFVVQPPLYGREAEDYDWSAPFGRIYFAGEHTALPHGWVETAVKSGLRAAVRINNNYGYGEVDPQMMEHAYAEANYLDQYPEGERPEEQQAREEVSPDEQEPSHKHLLVETSPEGQQHAFVEAIPELQGHVFVETVPQEKGHAHQNIYPSEHVQVHGEVIPEWHGHGGSGTPQMHRVGDHS

[0017] In a specific embodiment, the protein has at least 95% homology to the protein depicted in SEQ ID NO:2.

2SEQ ID NO:2MAPLALHLLVLVPILLSLVASQDWKAERSQDPFEKCMQDPDYEQLLKVVTWGLNRTLKPQRVIVVGAGVAGLVAAKVLSDAGHKVTILEADNRIGGRIFTYRDONTGWIGELGAMRMPSSHRILHKLCOGLGLNLTKFTQYDKNTWTEVHEVKLRNYVVEKVPEKLGYALRPQEKGHSPEDIYQMALNQALKDLKALGCRKAMKKFERHTLLEYLLGEGNLSRPAVQLLGDVMSEDGFFYLSFAEALRAHSCLSDRLQYSRIVGGWDLLPRALLSSLSGLVLLNAPVVAMTQGPHDVHVQIETSPPARNLKVLKADVVLLTASGPAVKRITFSPPLPRHMQEALRRLHYVPATKVFLSFRRPFWREEHIEGGHSNTDRPSRMIFYPPPREGALLLASYTWSDAAAAFAGLSREEALRLALDDVAALHGPVVRQLWDGTGVVKRWAEDQHSQGGFVVQPPALWQTEKDDWTVPYGRIYFAGEHTAYPHGWVETAVKSALRAAIKINSRKGPASDTASPEGHASDMEGQGHVHGVASSPSHDLAKEEGSHPPVQGQLSLQNTTHTRTSH

[0018] The invention is also directed to an isolated genomic polynucleotide sequence obtainable from human chromosome 19q 13.3-19q 13.4 that encodes said protein as well as nucleic acid constructs, expression vectors and host cells comprising such a sequence. In a specific embodiment, said genomic polynucleotide is selected from the group consisting of:

[0019] (a) a polynucleotide encoding human immediate early interleukin-four induced protein depicted in SEQ ID NO:2;

[0020] (b) a polynucleotide depicted in SEQ ID NO:3 which encodes said isolated human immediate early interleukin-four induced protein;

[0021] (c) a polynucleotide which is a variant of SEQ ID NO:3;

[0022] (d) a polynucleotide which is an allelic variant of SEQ ID NO:3;

[0023] (e) a polynucleotide which encodes a variant of SEQ ID NO:2;

[0024] (f) a polynucleotide which hybridizes to any one of the polynucleotides specified in (a)-(e) under stringent conditions and

[0025] (g) a polynucleotide that is a reverse complement of the polynucleotides specified in (a)-(f).

3SEQ ID NO:3CTGCAGAGAATCTGCCTCCCTCTGGTTTTCCTCCTCACGGAGCCCCTGGCTCCCTCTCCCCCACCCCAAGGAAAGTCCCTGCTATTGTTTCTCCACCCCCACCCACTCCAAGGTGCCGCCCCACTCCCTGCCCTGAGCACTGAGGTGGTTCTTAAAGCAGGGATGGGTGAAGCCTGGGCGCTTCCCTCTGCCGGGTGGTCACTCATAGAACCCCTGAGTGCCCCTGCGGCCACAGCAGCTGGCATTTCCAAAGGAGCAGCTTCCAGTACTACCCCAGGTTCCGGCCACAGAACTTAACCTGGATTCACTCACTTGATCCTAAAACACTGTGACGTCTGTAGACGTCATTTGACAGCTGTAGACGAGAGCCACAGAGGGGAGGGCGGGCTCAGAGCCCACGTCCTGACCCCGGGACCACGGCTGCCAGCCCCACTGCTACACCCATGTAACGGAGGGGGAGACTGAGGCTCTGGGTGGCAAGTGGAGAGGGAAGTGGAACCATGGGGGGCCCTGCAGGACATGGCGACACCTGACGGAGAGGGACTTGGAGGGGAAAGTTGGCCTGGCCCAGGGACCTGTAGCCCCTCTGTCCCCGTGGGAATCAAGGAGGCAGAGAGAGTGCAGGGGAGGATAGAAACAAGTGGCCTGGTGATCAGGGACGGGGGCAAACCGAAGGGGATTCCCCGTTGTTGGCACGGTTACCTGAGCATCTATTTCTGGCCTCCAAACCACCCCAGAACTATCTGGGGCAAAAGGAAGTGAATAGACTGATTAACTTCCTCCAACCCCCTTTCTCCAACCTCAACTAGACTCTGAGCATTGGACCAGAGCAGCTCCAGGAAACCAGTTTCATTTCCCTCGGCAGTTTCCCAAAAGGAGGGGTGGGCGGGGCTCTGCCAGTTTAAGGGGAGAGAAAGGGCCACAGCAGAGACAGTGGAGGGCAGTGGAGAGGACCGCGCTGTCCTGCTGTCACCAAGAGCTGGAGGTAGTGACACGGCCAGTGGTGGGGGGACAGAGAGACAGGACCCACAGAAATAGGGACTGAGAAGCAGTCAGGAGCTGGGGAGATGAGGAAGAGGGAAAAGAGGCCGGGCGCGGGGGCTCATGCCTGGAATCCCAGCACTTTGGGAGGCCGAGGTGGGTGGATCACCTAAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGCGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCAGGTTTGGTTATGGGTGTCTGTAATCCCAGTTACTTGGGAGGCTGAAGTGGGAGGATTCTTGAACCTGGGAGGCGGAGGTTGCAGTGAGCCAAGATCTTACCACTGCACTCCAGCCTGGATGGCAGAGAAAGACTGTTAAAAAAAAAAGAAAGGAAAGAAAGAGGGAAAAGAAGAGAAAGAGACAGGAACCCGAGAGAGCTGTGGCAGAGGTGGAGACCGGTGAGGAGGGAGGCAGGGACGGTCCAGGGGACTCTGAAGGAATGGGGTTAGGGGACACAGGTCCAGAGAGCAGGACAGTGACTGGGGGAGGGCCGGCTCTAGCAGCTGGGCAGCCCATGGGAGACAGAGTCAGGCTACAGTGGTCACAGGCCTCAGTCACAACAGCCCTCCCTTCTCCTCCACCCCAGACACCATCTCCCACCGAGAGTCATGGCCCCATTGGGTGAGTAAGCAGGGGAGGGGCTCTGGAACAAGTGAGTAGAACCCCTCTCCCCCGACCTGCCACAACCCCGTTGACCCTGTCCTCCTCCTCCCAGCCCTGCACCTCCTCGTCCTCGTCCCCATCCTCCTCAGCCTGGTGGCCTCCCAGGACTGGAAGGCTGAACGCAGCCAAGACCCCTTCGAGAAATGCATGCAGGATCCTGACTATGAGCAGCTGCTCAAGGTGGTGACCTGGGGGCTCAATCGGACCCTGAAGCCCCAGAGGGTGATTGTGGTTGGCGCTGGTGTGGCCGGGCTGGTGGCCGCCAAGGTGCTCAGCGATGCTGGACACAAGGTGGGAAGCACTGCTTGCAGTCTACCTGGAGTCCCTCCTGCTGCAGGCTGGAGTCCCTGGGCACAGGGACACGGCCGCGTTCCTCCTTGAGGGGGTGGAGGTGGGGATGTGGGGGGAGAGGCTATCTTTCCCTGTAGTCTGGGGGTGAGGCCCAAGCCCCCTGCTGTCACATGTCTCCACTGAGAGGCCCCAGACTTTCCTGTCCCACGTTTTTCATCCCAGAGGGAAGTCTCACTGGCTCAAAGCTCCTCTGTCCTGGGGGGGGCGCCTGTTGGTGGAGAGGGAAACTCTACCAAGGGGATGTGTGCCCTGAGGTCTAACCACTGACCTACTCACTGCAATCCGAGAATGAGAATGGGCCAGCCCAGGGGCTTCTACGCATATGGGTCATCTCCGAACTCCCCGAGCTGGGAAGTCCCTTTGCAGTCTAGCTGGCAGCCTTCCTCCTGCAGTCTGGAACTGCTCACTCAGTTCTGTTCCCCCACCTCCATACAGAAGGGGACCCTCTCCTAGTCTGGGTCTGGGCCACTGTGGGGATGGAAGAAGGCAGGGGGCTGGCACGGGGTGGTAGAGGGCAGGGGATGGTCAGTGGGTGGAGCTGAAGGGCCCTCCTCGCCCCACGGACTCCCTCAGGTCACCATCCTGGAGGCAGATAACAGGATCGGGGGCCGCATCTTCACCTACCGGGACCAGAACACGGGCTGGATTGGGGAGCTGGGAGCCATGCGCATGCCCAGCTCTCACAGGTGACCTAGCAACCCACCCACCTGTGTGCCTAGACTCAACTAGCCCCCCAGCTCCATGCCCACACCAGAGTGGGCCCGAACTTCCACTCTGACTCCCAAACCCAGCCTCCAACCCCATCCTTCGCCTAATCCTCACCTCAGTCTTGCCTCCAGCGCCAGCCCTGGGCCTCTGGCAACACTCGGGCCACCTGGTCCAACCAGACCTCCAGCTGGCCCTGCCTGGTGCAAAAGCCAGCTCCTGTGCTGGCCCCTCCAGGCCTGCAGCTCCACCATCCCCACACCCTCAGGCACCCCAGCCCCAGGCCCCTTCTTCATCTCAAACCCCCGTCCAACCCAAACTCTGTCCCCCTTCCCAATTCTAATCTGGGCTCTATCCCACTCTTAACCCAATCCTAATCCTGGTACTGCCCTGAGGCCTCCAGCCTTGTCCCCATTTCATTCCCATCCCCCAGCATTGATTACCCCAAATCTAGCCCCACCCCCAGCCTCATGCTGGTCCCCAGCTACTCTCCCCTCCCACCCCTCTTCTCCAAGTCCCACTAGGGACCAGGCCACCCCCACTGGAGCCCGGCCCTCACTCACCCACTCCCCTGATCAGGATCCTCCACAAGCTCTGCCAGGGCCTGGGGCTCAACCTGACCAAGTTCACCCAGTACGACAAGAACACGTGGACGGAGGTGCACGAAGTGAAGCTGCGCAACTATGTGGTGGAGAAGGTGCCCGAgAAgcTGGGCTACGCCTTGCGTCCCCAGGAAAAGGGCCACTCGCCCGAAGACATCTACCAGATGGCTCTCAACCAGGTGGGTACCGcCTGCACCCTCCTcCCGCCCTGACTTcTCTGTcCTcCTCCTAAGCTCGCCCCGGcCCGGCTTTTCTTCTCTcTGGCATCCCTTCAGTCCCCTCCATGGGGTGGGGCGCCTGTTGGTGGAGATGGAAATTCTACCAAGGGGATGTGTGCCATGAGGTCTAACCACTGACCTACTCACTGAAATCCGAGAATGAGAATGGGCCAGCCCAGGGGCTTCTACACATATGGGTCATCTCTCAACTCCCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTCTTACTCTGTCACCCAGGCTGGAGTGCAATGGCGCCATTTCAGCTCACTGCAACCTCCGCCTCCCAGGTTCAAGCAATTCTCTGCCTCAGCCTCCCGAGTAGCTGGGATTACAGGCGCCCGCCACCACGCCCAGCTAATTTTTGTATTTTTAATAGAGACGGGGTTTCACCATCTTGGCCAGGCTGATCTTGAACTCCTGACCTCGTGATCCACCTGCCTCAGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACTGCCCCCGGCCCCAACTCCCTTCTTTAGCTTCCCCGTAAACACTGCTGACCCCAAAGCTGCATCAGCTTTTGGCCTCTTCCCCTTTCACCCACACACTGTCCTGGATAACCTCCCCAGTCTCCACCTCTGGCTTAAACATAGATCCATGGGCTGATGGAGACATCTGCCTCCCGAGACCCTGATCTGTGTCCCTCCCCAGGACACTGGGCTCAGAAGCTTCCCCTAAACCTGTTCCTCCTCCCGGTGCCCCCATCTCACGATGGCCCCACCATCCCGCAGTCACGGGCAGACCCCTAGGAGCCATCCTCCTCCTGACCTCCTCCTCGCCCACCTCCCCCACCATCAGTGCCCACCCCTGCCGCCTGACGCTTGTGCCCTGCTCCCCTCTGCCTGCCCCAGCTCAGGCCTCTCCTCTGCCTGGACCCTCCCCCAGCCTCCTCTCAAGCTGCCCAGGCTCTAGTCTCCCCAGCCTGGTACCAGAAACGTCTTCCCACACACCCAGAGTGATGCTGCTCACAGCCCCCAGTGGCTCCCCGCTGCCTCCAGGACAAAGTCTGATCTTTTCAGGCTCAGCTTTGAGCCCTGACTGGCCTTCCCTTCTGCACCCCTCCAGCCTCATGCTCTTTTCTCCTCTAAGAAATTTTTGTCCTCATGGTGGTCCACATGCCCCCAGCTCTGTGTTCCTCACACCCACTATACCTGTGTATCACCCTCCCTTTCCAGAATGCCTTTCCCTTCCCCACACAGGAACTCCTGCTCAGCCCTCAAGAACTAGCTCCAATGTCCCCTCCTCCAGGAAGCCTTCCCTGATACCTTTAGGGAGGAACTGGCCACACCCAACATCCCCTCCCCACCCAGGTCAGAGCCCACTTGCCCATCTCCCCTTCTAGACCAGGATGGGGGCCATGTCCAGGTCATTTCTATGCCTTGACACTGACTGGTTTGGGAGTCTGAGAGATGCGAGATGCATGAGTGAGTGAAGGGGCACTGCCTTCCCTCTGAGGTCCTGGCCAGAAGGGGGAAGCCAGGTGGACCAAGAAGAAGGCTGAAATTAGATGGGGGCAGCAAGGCTTCTGGGAGGGGGTGAGATTCGAACAAAGTGCTGAAGAACACGCACTCATGTGCCACGCAAAGACAGGCAGGGAAACAATAGGTGCCAGATGTGTAGGTGGGAGAGCCCTGAGGGCAGGTGGTCTGGGCACTGTACACTGTTCACAGAACCAGAGCTGCCATAAAAGACATGGACTGAGGTAAGTGGGGATCTTGGTTCTGCTGAGATTCAGGGCAAAGAGAGTGACCATGAGGCCTTCCCCAGAGAGTCAGAAGGCGGGTACCTGGCACGCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGCAGATCACTTGAGGTCAGGAGTTCGAGACCAGCCTGACCAACATGATGAAACCCTGTCTCTACTAAAAATGCAAAAATGAGCCAGGTGTGGTGGCAGGCGCCTGTAATCCCAGCTACTTGGGAGGGTGAGGTGGGAGAATCACTTGAACCTGGGAGGCAGAGGTTTCAGTGAGCTGAGATGGCGCCACTGCACTCCAGCCTGGGCAACAGAGCGACACTCCATCTCAAAAAAAAAAAAAAAAAAAAAAGACATTGAGGGGCTAGCAGGGGCCTTGAACATGAGGCTGAGGGGCAAGAATTCTGTGGCTCCAGGCACTGGGGAGCTACAGGAGGGCTGTGGGCAGGGGACGGACGGTAGATAAACTTCTCAGCCATCATGCCCTGTCAGCACTGACCAGTGGCACAGGGGACCAGCTTCTGTCCGTGGCCACAGTGATTCAGGCAACAAATAATCACTGAGCCCCCGGGAGGACACAGCACGCCTGGGACCCTCTCCCAGTCTCCTCTCCAGCTGCCCACATCCCTGCCTcCATGGAGCTCACAGTCTGGCTGGCTGGCTGAAAAATAGGCAAAATAACACGCAACCGGGATGAGCTGTGACCAGCCGGGCCAGCCGAATGAGGGGGGATGGGCGGCGTTAACAGTTCGGTAATATCGGAGGAGCAGTGGCATCTGAGTTGCCGTGGAGGGCAAGGCGGAGGGCATGGTGTGGGCAAAAGCCCAgGGGCTTGAGAGTGGTGAATGGCCAGCCGTGGGCAGAACCACGAGGAGAAGCCGAGTGTGGGCTcCAGGCCCGGCcTcCACCCTGGCTGcCCGGGCAGcTCAGCACCATGTCTGCTTCTTCCACAACAGGCCcTcAAAGACCTcAAGGCACTGGGCTGCAGAAAGGCGATGAAGAAGTTTGAAAGGCACACGCTCTTGGTAAGTGGGGATCTTGGTTCTGCTGAGATGCAGGGCAAAGAGAGTGACCATGAGGCCTTCCCCAGAGAGTCAGAAGGCGGGTACCTGGCACCCTAGTCCTGCCACCGCCTTCCTGCCTGACCCGGGGGCTGGACACGCCCCCTGTCTGGACCTCCGTTTCCCTCATCTGTCAGAGAGTGGGGACAGGATCCTACGGTCCAAGGACATGTCATGGGACCAGGAGGCCCAGCTGCGGAGGGGCTCTCTCAgGGGTGCAGCCTGCCTGGGCCCTAACCTCGGcCTGTGCCCAACCTGCAGGAATATCTTCTCGGGGAGGGGAACCTGAgCCGGCCGGCCGTGCAgCTTCTgGGAGACGTGATGTCCGAGGATGGCTTCTTCTATCTCAgcTTCGCCGAGGcCCTcCGGGCCCACAGCTGCCTCAGCGACAGACTCCAGTAAGGGGCGGGGCTGGCGGGCAGGGGGGGGGGGCgGgGGGCAATCAGGGAAAGTAGCAGGGAGTGGGAGGAGCCTCTGGGTCTGGGGGCGGGGCTGTTGTGAAGGGCGTGGCCTAATTGATGGTCGCGGGGCTAAGGGTTCCCAGATAAAGGACACGGCCTAGGGGAGAAGTGGTGGGGTCCCTGGTGACACAATCCACTACTCCTGCGGCCTTTGGGATGTCAGGGACgGgCGGGGTCGGGAGAGGTGGATGACGGGGGAGCGGgCCGGTGCCCATAAGGGCCAGCTGCCGGGGTGGGTGGGGCAAGGCAGAGGGGCAGGGCCGCAGAGCCCAGGTCACGCCCCcACCCCGCCCCGCCCCGCCTGCAGGTACAGCCGCATCGTGGGTGGCTGGGACCTGCTGCCGCGCGCGCTGCTGAGCTCGCTGTCCGGGCTTGTGCTGTTGAACGCGCCCGTGGTGGCGATGACCCAGGGACCGCACGATGTGCACGTGCAGATCGAGACCTCTCCCCCGGCGCGGAATCTGAAGGTGCTGAAGGCCGACGTGGTGCTGCTGACGGCGAGCGGACCGGCGGTGAAGCGCATCACCTTCTCGCCGCCGCTGCCCCGCCACATGCAGGAGGCGCTGCGGAGGCTGCACTACGTGCCGGCCACCAAGGTGTTCCTAAGCTTCCGCAGGCCCTTCTGGCGCGAGGAGCACATTGAAGGCGGCCACTCAAACACCGATCGCCCGTCGCGCATGATTTTCTACCCGCCGCCGCGCGAGGGCGCGCTGCTGCTGGCCTCGTACACGTGGTCGGACGCGGCGGCAGCGTTCGCCGGCTTGAGCCGGGAAGAGGCGTTGCGCTTGGCGCTCGACGACGTGGCGGCATTGCACGGGCCTGTCGTGCGCCAGCTCTGGGACGGCACCGGCGTCGTCAAGCGTTGGGCGGAGGACCAGCACAGCCAGGGTGGCTTTGTGGTACAGCCGCCGGCGCTCTGGCAAACCGAAAAGGATGACTGGACGGTCCCTTATGGCCGCATCTACTTTGCCGGCGAGCACACCGCCTACCCGCACGGCTGGGTGGAGACGGCGGTCAAGTCGGCGCTGCGCGCCGCCATCAAGATCAACAGCCGGAAGGGGCCTGCATCGGACACGGCCAGCCCCGAGGGGCACGCATCTGACATGGAGGGGCAGGGGCATGTGCATGGGGTGGCCAGCAGCCCCTCGCATGACCTGGCAAAGGAAGAAGGCAGCCACCCTCCAGTCCAAGGCCAGTTATCTCTCCAAAACACGACCCACACGAGGACCTCGCATTAAAGTATTTTCGGAAAAAGCCGTGTGGTCCAGCCTcCCCCGTGGCTCAGTTACTTcCCCAGTTTGCCTGCATCGGAACCACTAGCCCTGCAGTTAGCAGGCGCCACGCCCATCCTGGAGCCCCCCCAAAAATCTGCCCCTCACTTCTTcCTGGACAGTGGGGCTCCAGAgGAgGGTGGGGGGTGGTGCGCACAGAGGGAACCGGAAGGACGCCCTGCACCACCGGGAAATGGCAATCACAGCGACCACCTCTGTCACGAGGCTGAAGTCCAGATGGGCCCTAATGCAAGTAACAGAAAGCCCAGCTGACAGTGGCTGAACAGGAGGTGATTTTTCTCACAAGCAGCAGCTGGGAGGGCAGGCTCCCCAGAGTGCATCTCTGCAGGCCCTTCCACCTTGGTTATGTCCGGGAAGTGTTCCCACACAGCTACCAGGTCGCCGCCACAGCCCAGAGCACCCTATCCTCTTCCAAAAACCCCAAATCAGGCCGGGCGCGGTGGCTCAGCCTGTAATACCAGCACTTTGGGAGGCCAAGGCAGGCAGATCACCTGAGGTCGGGAGTTCAAGACCAGCCTGGGCAACATGGTGGAACCCCATCTCTACCAAAATTACAAAAATCAGCCGGGTGTGGTGGCACACGCCTGTAGTCCCAGCTACTCAGGAGGCTGAGGCAGGGTAATTGCTTGAACCCGGGAGGCGGAGGCTGCAGTTAGCCGAGATCTGCCACTGCACTCCAGCCTGGGCGACAGAGCAAGACTCCATCTCATAAAACAAACAAACAAAAAAACAAATCAGGCAACTCAGGAAGCCCTGCCTCCACAAACCCCAATCATTCACCCTAGGCTCTGCCCAGCCACCATCCCCTCCTCAACCAGGTCAGAGCCCACTTGGCAGCCTTCCCCCCACCAACAAGAAGCCAACTGCACATGAGAAGTGTCAGTTTAATGAAGCCAGCTTATCAGCAGGGCGGCGGAGCACACCTGCCCCCTCGCAGGTGTGCCTGGCTCGGGCTAAAGTGCCTGTGCAGAACGAGGCTGCCTGGCGGGGTTAGGAGTCGGCGCCCTCGTCCTCCTCCTCGGGCAGGATCTCCAGGCTGCTGTCGGGCTGCGGGGCTGTGTCCGTGGAGGGCGGCGGGGTGGGCGGGGCCCGGGTGGGCGACAGAGGCAGCGGGGAGGCGGTGGGCGAGGGGCTGTGGGGCTCTGCGGGCGGGGCCAGCCCCAGGATCTCCTGCACGTTGTGGGGCAGCTCCTGGAGGCACGGGATGAGAGAAGCgAAAAGCGGAAGGTGAAGGTGAGAAGGGGGCGGGGGTCGGTCGGGGTTCGGGCGGGGCGCTGCGGGgCGGACGGGGACTCACGGGGCAGGCGGTTAGCTGgCGGTGCAGGGCCTCGGCGCGGGTCAGCACGTCCTCCACGCTCAGCTTCATAGTCAGCTCGTTGATGTGCTGCGGGAGGGGGTGGGTCAGGCGCCAGTCGGCACTCAACCACCTCCGCCCGAGCGCCCACTCTGTGCTTCGTGCTGGgAAACAATGAAGACACACCGGGCTGGCCCTCTGGGGGCTCAGAAGTGCGCTGGGCGAGACATGCTTCTCAGGCCTCCGGGAGCATTcTGAGCACGTACTCGGGGAGGgAaGgtCCCCACCCACCTGCCcGgAGgaCcTTGCTGGgAGCAGGCATGGCATGGGGCAGAAGGGAAACCTACGCGGCCCCACCCCCACTGCGCTCCCAACTAACCAGGCTTTCAATAATCCCACACTAAACACCTGCTCCCCGGAGGTACTGCGGCCCTGGGCAGCGCCCAGCTCAGGGGGgACAGGGCGGGGGCGGGGCCGGAgCCTCACCTTGAGGATCTCATTGGAGCCGAAGCCGGACAGCATGAGGGTGTCCCTCTCCATGTCCAGGATGGCGCAGGCCACCAGCAGGTGCAGATTGGGGCCAGGGAGCCCTGTCCACAGCACCTGAGGGTGCGGGACAGAGGGAGGGAGGGAGGTCATAGCCCAGCCTGGGCCTGAGGTCCCACCAGGGTCTGTCCCTCTCCGCCCAACCTCCCAGGGCAGGTGAGCCCACCCCTTCAGGCCTCTCGCCCAAACTGGCTGGCCCACCTCCCACAGCCGAAGGACATCCGGGAAGGGGAATTC

[0026] The invention is also directed to an oligonucleotide probe labeled with a detectable substance that is at least 10 nucleotides in length. In a specific embodiment, the sequence comprises an isolated polynucleotide which hybridizes to a non-coding region of SEQ ID NO:3, which non-coding region is selected from the group consisting of an intron, a splice junction, a 5′-non-coding region, a transcription factor binding region and a 3′-non-coding region. The invention is further directed to an antisense oligonucleotide or mimetic to said polynucleotide.

[0027] The invention is also directed to an isolated polynucleotide comprising a polynucleotide sequence encoding a fluorescent protein-immediate early interleukin-four induced fusion protein, said interleukin-four induced protein has the following characteristics: (i) at least about 546 amino acids; (ii) L-amino acid oxidase activity; (iii) is obtainable from interleukin-4 induced B cells; (iv) has at least about 75% homology to a mouse immediate early interleukin-four induced protein. In a specific embodiment, the interleukin-four induced protein is depicted in SEQ ID NO:1. In another embodiment, the interleukin-four induced protein is depicted in SEQ ID NO:2. In yet another embodiment, the fluorescent protein is red fluorescent protein. The invention is also directed to constructs, vectors and host cells comprising said polynucleotide. Furthermore, the invention is directed to an oligonucleotide probe comprising a fragment at least 10 nucleotides in length that hybridizes under stringent conditions to SEQ ID NO:3 or SEQ ID NO:4 linked to a sequence encoding a fluorescent protein. In a specific embodiment, the invention relates to a kit comprising such a probe.

[0028] As noted above, the invention is also directed to a method for obtaining said interleukin-four induced protein and said fusion protein, comprising (a) culturing the recombinant host cell comprising the polynucleotide of the present invention under conditions that provide for the expression of said protein and (b) recovering said expressed protein. In one embodiment, the protein may be recovered from the supernatant. Alternatively, it may be recovered from the pellet.

[0029] The invention is further directed to compositions comprising the genomic polynucleotides or antisense oligonucleotides and a pharmaceutically acceptable carrier as well as methods of using said polynucleotides or oligonucleotides to treat pathological disorders (e.g., immune related disorders or diseases) resulting from defects in human immediate early interleukin-four induced protein synthesis or defects in platelet aggregation. These compositions may also be used to kill unwanted cells such as tumor cells, bacterial cells, viruses and fungi.

[0030] The invention is also directed to a method of diagnosing a pathological condition or susceptibility to a pathological condition resulting from a defect in said protein synthesis in a subject comprising (a) determining the presence or absence of a mutation in a polynucleotide encoding said protein and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation. In a specific embodiment, the pathological condition is an immune-related disorder and said disorder is detected in a human patient. The immune-related disorder is selected from the group consisting of: systemic lupus erythematosis, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis, idiopathic inflammatory myopathies, Sjöbgren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmunethrombocytopenia thyroiditis, diabetes mellitus, immune-mediated renal disease, demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome, and chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases such as infectious, autoimmune chronic active hepatitis, primary biliary cirrhosis, gramilomatous hepatitis, and sclerosing cholangitis, inflammatory bowel disease, gluten-sensitive enteropathy, and Whipple's disease, autoimmune or immune-mediated skin diseases including bullous skin diseases,erythema multiforme and contact dermatitis, psoriasis, allergic diseases such as asthma, allergicrhinitis, atopic dermatitis, food hypersensitivity and urticaria, immunologic diseases of the lung such as eosinophilic pneumonias, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplantation associated diseases including graft rejection and graft-versus-host-disease. Furthermore, the protein or composition comprising said protein might be used to treat or prevent disorders where programmed cell death would be beneficial such as in fungal and bacterial infections and neoplasia.

[0031] An antagonist of said protein might be used to decrease cell death in mammalian cells. Therefore, it could be used in treating or preventing disorders caused by apoptosis or other types of cell death such as necrosis. These include but are not limited to HIV, neurodegenerative or neuromuscular diseases, ischemic stroke, anoxia, ischemia/reperfusion damage and intoxication septic shock.

[0032] Furthermore, an interleukin-four induced protein having the following characteristics: (a) at least about 546 amino acids; (b) L-amino acid oxidase activity; (c) is obtainable from interleukin-4 induced B cells; (c) contains an N-terminal signal sequence of about 21 amino acids; (d) has a non-conserved C-terminal sequence of about 62 amino acids; (e) has at least about 75% homology to a mouse immediate early interleukin-four induced protein having the sequence depicted in SEQ ID NO:1 or composition comprising said protein and a carrier may be used or used for the manufacture of a medicament to treat or prevent a pathological condition in a mammal by treating said mammal with a compound effective to treat or prevent said condition. As defined herein, to “treat” or “treatment” is both therapeutic treatment and prophylactic or preventative measures, specifically slow down the pathological disorder. In a specific embodiment, the interleukin-four induced protein has about 95% sequence homology to the sequence depicted in SEQ ID NO:1. In another embodiment, said protein has the following characteristics: (a) about 546 to about 567 amino acids; (b) L-amino acid oxidase activity; (c) is obtainable from interleukin-4 induced B cells; (c) contains an N-terminal signal sequence of about 21 amino acids; (d) in encoded by a genomic DNA sequence having eight exons; (e) has a non-conserved C-terrninal sequence of about 62 amino acids; (f) has less than 80% homology to a mouse immediate early interleukin-four induced protein having the sequence depicted in SEQ ID NO:1. In a specific embodiment, the protein has about 95% sequence homology to the sequence depicted in SEQ ID NO:2.

[0033] Furthermore said interleukin-four induced protein may be used to detect L-amino acids. Specifically, a sample or preparation is contacted with said interleukin-four induced protein and the presence or absence of L-amino acid oxidase activity is detected. In a specific embodiment the sample is a biological sample. In a more specific embodiment, the sample is obtained from a mammal.

[0034] As will be described in further detail infra, said protein has L-amino acid oxidase activity associated with said protein: it has phenylalanine oxidase activity and has a pH optimum of 4.0. Given that said interleukin-four induced protein does have L-amino acid oxidase activity that can be assayed, said protein can be used for therapeutic and diagnostic uses, as well as to screen potential candidate compounds. In one specific embodiment, said interleukin-four induced protein may be used for diagnosing a pathological condition in a mammal comprising (a) measuring L-amino acid oxidase activity associated with said protein (b) comparing the amount of L-amino acid oxidase activity with activity from a mammal without said disorder. In another embodiment, said interleukin-four induced protein may be used for measuring the level of activity of said immediate early interleukin-four induced protein in a sample, comprising measuring the amount of L-amino acid oxidase activity associated with said immediate early interleukin-four induced protein. Such measurements may be used to monitor a patient receiving therapy for a pathological condition, e.g., an immune related disorder.

[0035] In yet another embodiment, the invention is directed to a method for identifying a compound that acts as an antagonist or agonist to an immediate early interleukin-four induced protein having the following characteristics: (i) at least about 546 amino acids; (ii) L-amino acid oxidase activity; (iii) is obtainable from interleukin-4 induced B cells; (iv) contains an N-terminal signal sequence of about 21 amino acids; (v) has a non-conserved C-terminal sequence of about 62 amino acids; (vi) has at least about 75% homology to a mouse immediate early interleukin-four induced protein having the sequence depicted in SEQ ID NO:1 comprising contacting (a) a substrate containing said interleukin-four induced protein with a candidate compound; (b) measuring the L-amino acid oxidase activity associated with said protein, wherein a decrease in L-amino acid oxidase activity after addition of said candidate compound indicates that said candidate compound is an antagonist and wherein an increase in L-amino acid oxidase activity after addition of said candidate compound indicates that said candidate compound is an agonist. An “agonist” is any molecule that mimics a biological activity of said interleukin-induced protein disclosed herein. An “antagonist” is any molecule that partially or fully inhibits or neutralizes the biological activity of said interleukin-four induced protein. Suitable antagonist or agonist molecules include but are not limited to agonist or antagonist antibodies or antibody fragments, variants of said protein, antisense oligonucleotides and small organic molecules.

BRIEF DESCRIPTION OF THE FIGURES

[0036]
FIG. 1 shows the human FIG. 1 (hFIG. 1) gene locus diagrammed to scale as a colored line. Exons of hFIG. 1 are numbered 1 through 8 (5′ to 3′ direction) with enlarged boxes (dark blue indicating coding sequence, pink indicating 5′ untranslated (UT) sequence, light blue indicating 3′ UT sequence). Enlarged boxes that are not numbered indicate an adjacent uncharacterized gene (Unigene number Hs. 119482) that is transcribed in the opposite orientation of hFIG. 1. Alu repeats are labeled and orientation is indicated by large green arrow. Small direct repeats unique to the hFIG. 1 locus (DR1) are indicated by a labeled median size striped red box. Location of human EST sequences (IMAGE clones 1845115 and 1505169) is shown by a labeled black arrow. Region of unique human sequence (as compared to mouse FIG. 1) is shown as a labeled red overline. Extents of genomic clones are shown below as labeled black lines. The BamHI and EcoRI sites at the end of clones A4/B4 and D2/D3/D5/D7, respectively, are shown. Only the relevant portion of the high throughput genomic sequence ACO11452 is shown starting just after the first Alu repeat and arbitrarily cut off at the indicated Psti. Other BamHI, EcoRI, and PstI sites are omitted for clarity.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The invention is directed to an isolated genomic polynucleotide sequence encoding human immediate early interleukin-four induced protein having the following characteristics: (a) about 546 to about 567 amino acids; (b) L-amino acid oxidase activity; (c) is obtainable from interleukin-4 induced B cells; (d) contains an N terminal signal sequence of about 21 amino acids; (e) is encoded by a genomic DNA sequence having eight exons; (f) has a non-conserved C-terminal sequence having about 62 amino acids; (g) has less than 80% homology to a mouse immediate early interleukin-four induced protein having the sequence depicted in SEQ ID NO:1(see above).

[0038] As defined herein “isolated” refers to material removed from its original environment and is thus altered “by the hand of man” from its natural state. As defined herein, an “isolated” human immediate early interleukin-four induced protein is a protein which is essentially free of other non-human immediate early interleukin-four induced protein e.g., at least about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE.

[0039] In a specific embodiment, the protein has at least 95% identity or homology to the protein depicted in SEQ ID NO:2 (see above). A protein that has an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence is identical to the query sequence except that the subject protein sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a protein having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions interspersed either individually among residues in the referenced sequence or in one or more contiguous groups within the reference sequence.

[0040] As a practical matter, whether any particular protein is at least 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence depicted in SEQ ID NO:2 (see above) can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Com. App. Biosci. (1990) 6:237-245). In a sequence alignment, the query and subject sequence are either nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.

[0041] If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total residues of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.

[0042] The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes genomic DNA. The DNA may be double-stranded or single stranded. The polynucleotide of the present invention also includes the FIG. 1 gene. As defined herein a “FIG. 1 gene” is the segment of DNA involved in producing said interleukin-four induced protein. It includes regions preceding and following the coding region, as well as intervening sequences (introns) between exons.

[0043] The polynucleotides of the invention have at least a 95% identity and may have a 96%, 97%, 98% or 99% identity to the polynucleotides depicted in SEQ ID NO:3 (genomic DNA) (see above).

[0044] A polynucleotide having 95% “identity” to a reference nucleotide sequence of the present invention, is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the protein. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence, the ORF (open reading frame), or any fragment specified as described herein.

[0045] As a practical matter, whether any particular nucleic acid molecule or protein is at least 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.

[0046] If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence are calculated for the purposes of manually adjusting the percent identity score.

[0047] For example, a 95 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 5 bases at 5′ end. The 5 unpaired bases represent 5% of the sequence (number of bases at the 5′ and 3′ ends not matched/total numbers of bases in the query sequence) so 5% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 95 bases were perfectly matched the final percent identity would be 95%. In another example, a 95 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence, which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for purposes of the present invention.

[0048] The invention also encompasses polynucleotides that hybridize to the polynucleotides depicted in SEQ ID NO: 3 under stringent conditions. A polynucleotide “hybridizes” to another polynucleotide, when a single stranded form of the polynucleotide can anneal to the other polynucleotide under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., supra). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. “Stringent” hybridization conditions may be defined as prehybridization and hybridization at 42° C. in 40% deionized formamide, 4× SSC, 10 mM Tris (pH 7.5), 2× Denhardt's solution, 0.1 mg/ml sheared and denatured salmon sperm DNA, with or without 10% dextran sulfate. Alternatively, prehybridization and hybridization may be performed at higher temperature without deionized formamide (65° C. in 5× SSC, 0.5% SDS, 5× Denhardt's solution, with or without 0.1 mg/ml sheared and denatured salmon sperm DNA). “Stringency” is usually determined by the subsequent washes. “Low stringency” wash is usually at high salt (2× SSC, 0.1% SDS) and/or at low temperature (from room temperature to about 42° C.). “High stringency” wash is usually at low salt (0.1× SSC, 0.1% SDS) and high temperature (at 65° C.).

[0049] Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.

[0050] Polynucleotide and Protein Variants

[0051] The invention is directed to polynucleotide variants. In a specific embodiment, the polynucleotide variant encodes a protein variant. A “variant” refers to a polynucleotide or protein differing from the polynucleotide or protein of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar and in many regions, identical to the polynucleotide or protein of the present invention.

[0052] The variants may contain alterations in the coding regions, non-coding regions, or both. The variants may also be splice variants. Especially preferred are polynucleotide variants containing alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded protein. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred.

[0053] The invention also encompasses allelic variants of said polynucleotides. An allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a protein is a protein encoded by an allelic variant of a gene. In a specific embodiment, the polymorphism is an insertion of a guanine residue in intron 2.

[0054] The amino acid sequences of the variant protein may differ from the amino acid sequences depicted in SEQ ID NO:2 by an insertion or deletion of one or more amino acid residues and/or the substitution of one or more amino acid residues by different amino acid residues. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

[0055] Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions which do not generally alter the specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, LeuNal, Ala/Glu, and Asp/Gly as well as these in reverse.

[0056] Expression of Proteins

[0057] Isolated Polynucleotide Sequences

[0058] Procedures for obtaining a polynucleotide sequence of the present invention are described in the Examples. Such a polynucleotide sequence may also be referred to as the FIG. 1 gene. Generally, the mouse FIG. 1 cDNA sequence is used to probe the publicly expressed sequence tag (EST) database. Initially three human ESTs (IMAGE clones 1581345, 1845115 and 1505169) are found that are similar to the mouse FIG. 1 gene (77% amino acid sequence identity), but differed in the 3′ end (no amino acid sequence identity). The portion of the human EST that is similar to the mouse FIG. 1 gene is used to probe a human genomic DNA library. A clone is obtained and further subcloned. Positive subclones are obtained for further determination of the human FIG. 1 gene sequence. Procedures that may be used to obtain the full-length genomic DNA sequence are described in the Examples section.

[0059] cDNA may be obtained from an IL-4 induced human B cells and identified as described in the Examples section. Briefly, specific PCR primers may be used to amplify partial cDNA products. The PCR products are sequenced to verify by comparison to the genomic sequence that they are indeed the FIG. 1 cDNA sequence (SEQ ID NO: 4).

4GACAGTGGAGGGCAGTGGAGAGGACCGCGCTGTCCTGCTGTCACCAAGAGCTGGAGACACCATCTCCCACCGAGAGTCATGGCCCCATTGGCCCTGCACCTCCTCGTCCTCGTCCCCATCCTCCTCAGCCTGGTGGCCTCCCAGGACTGGAAGGCTGAACGCAGCCAAGACCCCTTCGAGAAATGCATGCAGGATCCTGACTATGAGCAGCTGCTCAAGGTGGTGACCTGGGGGCTCAATCGGACCCTGAAGCCCCAGAGGGTGATTGTGGTTGGCGCTGGTGTGGCCGGGCTGGTGGCCGCCAAGGTGCTCAGCGATGCTGGACACAAGGTCACCATCCTGGAGGCAGATAACAGGATCGGGGGCCGCATCTTCACCTACCGGGACCAGAACACGGGCTGGATTGGGGAGCTGGGAGCCATGCGCATGCCCAGCTCTCACAGGATCCTCCACAAGCTCTGCCAGGGCCTGGGGCTCAACCTGACCAAGTTCACCCAGTACGACAAGAACACGTGGACGGAGGTGCACGAAGTGAAGCTGCGCAACTATGTGGTGGAGAAGGTGCCCGAGAAGCTGGGCTACGCCTTGCGTCCCCAGGAAAAGGGCCACTCGCCCGAAGACATCTACCAGATGGCTCTCAACCAGGCCcTcAAAGACCTcAAGGCACTGGGCTGCAGAAAGGCGATGAAGAAGTTTGAAAGGCACACGCTCTTGGAATATCTTCTCGGGGAGGGGAACCTGAgCCGGCCGGCCGTGCAgCTTCTgGGAGACGTGATGTCCGAGGATGGCTTCTTCTATCTCAGCTTCGCCGAGGcCCTcCGGGCCCACAGCTGCCTCAGCGACAGACTCCAGTACAGCCGCATCGTGGGTGGCTGGGACCTGCTGCCGCGCGCGCTGCTGAGCTCGCTGTCCGGGCTTGTGCTGTTGAACGCGCCCGTGGTGGCGATGACCCAGGGACCGCACGATGTGCACGTGCAGATCGAGACCTCTCCCCCGGCGCGGAATCTGAAGGTGCTGAAGGCCGACGTGGTGCTGCTGACGGCGAGCGGACCGGCGGTGAAGCGCATCACCTTCTCGCCGCCGCTGCCCCGCCACATGCAGGAGGCGCTGCGGAGGCTGCACTACGTGCCGGCCACCAAGGTGTTCCTAAGCTTCCGCAGGCCCTTCTGGCGCGAGGAGCACATTGAAGGCGGCCACTCAAACACCGATCGCCCGTCGCGCATGATTTTCTACCCGCCGCCGCGCGAGGGCGCGCTGCTGCTGGCCTCGTACACGTGGTCGGACGCGGCGGCAGCGTTCGCCGGCTTGAGCCGGGAAGAGGCGTTGCGCTTGGCGCTCGACGACGTGGCGGCATTGCACGGGCCTGTCGTGCGCCAGCTCTGGGACGGCACCGGCGTCGTCAAGCGTTGGGCGGAGGACCAGCACAGCCAGGGTGGCTTTGTGGTACAGCCGCCGGCGCTCTGGCAAACCGAAAAGGATGACTGGACGGTCCCTTATGGCCGCATCTACTTTGCCGGCGAGCACACCGCCTACCCGCACGGCTGGGTGGAGACGGCGGTCAAGTCGGCGCTGCGCGCCGCCATCAAGATCAACAGCCGGAAGGGGCCTGCATCGGACACGGCCAGCCCCGAGGGGCACGCATCTGACATGGAGGGGCAGGGGCATGTGCATGGGGTGGCCAGCAGCCCCTCGCATGACCTGGCAAAGGAAGAAGGCAGCCACCCTCCAGTCCAAGGCCAGTTATCTCTCCAAAACACGACCCACACGAGGACCTCGCATTAAAGTATTTTCGGAAAAA

[0060] Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired genomic or cDNA may be accomplished in a number of ways. For example, if an amount of a portion of the genomic DNA or cDNA is available and can be purified and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). The present invention provides such nucleic acid probes, which can be conveniently prepared from the specific sequences disclosed herein, e.g., a hybridizable probe having a nucleotide sequence corresponding to at least a 10, and preferably a 15, nucleotide fragment of the sequences depicted in SEQ ID NOS:3 or 4. Preferably, a fragment is selected that is highly unique to the interleukin-four induced protein. Those DNA fragments with substantial homology to the probe will hybridize.

[0061] A polynucleotide encoding said protein can also be identified by MRNA selection, i.e., by nucleic acid hybridization followed by in vitro translation. In this procedure, fragments are used to isolate complementary mRNAs by hybridization. Immunoprecipitation analysis or functional assays of the in vitro translation products of the products of the isolated mRNAs identifies the MRNA and, therefore, the complementary DNA fragments, that contain the desired sequences.

[0062] Alternatively, the polynucleotide may be used to design specific PCR primers to amplify a gene-specific product. When used on genomic DNA, this product can be used as an indicator for the presence of the gene. When used on cDNA reverse transcribed from RNA, the amount of PCR product can be used as an indicator for FIG. 1 gene expression.

[0063] A polynucleotide encoding a fluorescent protein-interleukin four induced protein fusion protein may be constructed using the procedures described in the Examples. Additionally, a probe comprising a polynucleotide encoding a fluorescent protein or portion thereof that fluoresces and at least a 10 nucleotide segment that hybridizes under stringent conditions to SEQ ID NO:3 or 4.

[0064] Nucleic Acid Constructs

[0065] The present invention also relates to nucleic acid constructs comprising the polynucleotide of the present invention operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Expression will be understood to include any step involved in the production of the protein including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0066] “Nucleic acid construct” is defined herein as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention. The term “coding sequence” is defined herein as a portion of a nucleic acid sequence which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by translation initiation and stop sites (i.e. open reading frame). Proper expression may require 1) a good Kozak sequence that encompasses the translation initiation codon (required for good translation initiation), 2) 3′ untranslated region sequences (including the transcription terminator) that are important for RNA stability, and 3) 5′ untranslated region sequences are sometimes also important for RNA stability and good translation initiation. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0067] The isolated polynucleotide of the present invention may be manipulated in a variety of ways to provide for expression of the protein. Manipulation of the nucleic acid sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

[0068] The control sequence may be an appropriate promoter sequence, a nucleic acid sequence that is recognized by a host cell for expression of the nucleic acid sequence. The promoter sequence contains transcriptional control sequences that mediate the expression of the protein. The promoter may be any nucleic acid sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0069] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the E. coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothernophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

[0070] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease (WO 96/00787), NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof.

[0071] In a yeast host, useful promoters are obtained from the Saccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae galactokinase gene (GAL1), the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP), and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.

[0072] The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleic acid sequence encoding the protein. Any terminator which is functional in the host cell of choice may be used in the present invention. Preferred terminators for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Preferred terminators for yeast host cells are obtained from the genes encoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), or Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

[0073] The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the protein. Any leader sequence that is functional in the host cell of choice may be used in the present invention.

[0074] Preferred leaders for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0075] Suitable leaders for yeast host cells are obtained from the Saccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene, the Saccharomyces cerevisiae alpha-factor, and the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP).

[0076] The control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3′ terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence, which is functional in the host cell of choice, may be used in the present invention.

[0077] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, and Aspergillus niger alpha-glucosidase.

[0078] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cell Biol. 15: 5983-5990.

[0079] The control sequence may also be a heterologous signal peptide coding region, which codes for an amino acid sequence linked to the amino terminus of the protein which can direct the encoded protein into the cell's secretory pathway. The 5′ end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted protein. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not normally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to obtain enhanced secretion of the protein. However, any signal peptide coding region which directs the expressed protein into the secretory pathway of a host cell of choice may be used in the present invention.

[0080] An effective signal peptide coding region for bacterial host cells is the signal peptide coding region obtained from the maltogenic amylase gene from Bacillus NCIB 11837, the Bacillus stearothermophilus alpha-amylase gene, the Bacillus licheniformis subtilisin gene, the Bacillus licheniformis beta-lactamase gene, the Bacillus stearothennophilus neutral proteases genes (nprT, nprS, nprM), or the Bacillus subtilis prsA gene. Further signal peptides are described by Simonen and Palva, 1993, Microbiol. Rev. 57: 109-137.

[0081] An effective signal peptide coding region for filamentous fungal host cells is the signal peptide coding region obtained from the Aspergillus oryzae TAKA amylase gene, Aspergillus niger neutral amylase gene, Aspergillus niger glucoamylase gene, Rhizomucor miehei aspartic proteinase gene, Humicola lanuginosa cellulase gene, or Humicola lanuginosa lipase gene.

[0082] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are described by Romanos et al., 1992, supra.

[0083] The control sequence may also be a propeptide coding region, which codes for an amino acid sequence positioned at the amino terminus of a protein. The resultant protein is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the Bacillus subtilis alkaline protease gene (aprE), the Bacillus subtilis neutral protease gene (npr7), the Saccharomyces cerevisiae alpha-factor gene, the Rhizomucor miehei aspartic proteinase gene, or the Myceliophthora thermophila laccase gene (WO 95/33836).

[0084] Where both signal peptide and propeptide regions are present at the amino terminus of a protein, the propeptide region is positioned next to the amino terminus of a protein and the signal peptide region is positioned next to the amino terminus of the propeptide region.

[0085] It may also be desirable to add regulatory sequences which allow the regulation of the expression of the protein relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems would include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those which allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the nucleic acid sequence encoding the protein would be operably linked with the regulatory sequence.

[0086] Expression Vectors

[0087] The present invention also relates to recombinant expression vectors comprising a nucleic acid sequence or polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the protein at such sites. Alternatively, the polynucleotide of the present invention may be expressed by inserting the polynucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0088] The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

[0089] The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.

[0090] The vectors of the present invention preferably contain one or more selectable markers that permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. A selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), as well as equivalents from other species. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.

[0091] The vectors of the present invention preferably contain an element(s) that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome of the cell.

[0092] For integration into the host cell genome, the vector may rely on the polynucleotide sequence encoding the protein or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional polynucleotide sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0093] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proc. Natl. Acad. Sci. USA 75: 1433).

[0094] More than one copy of a polynucleotide sequence of the present invention may be inserted into the host cell to increase production of the gene product. An increase in the copy number of the polynucleotide sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0095] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

[0096] Host Cells

[0097] The present invention also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides. A vector comprising a nucleic acid sequence of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the protein and its source.

[0098] The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus lichenifornis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E. coli and Pseudomonas sp. In a preferred embodiment, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. In another preferred embodiment, the Bacillus cell is an alkalophilic Bacillus.

[0099] The introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. General Genetics 168: 111-115), using competent cells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5771-5278).

[0100] The host cell may be a eukaryote, such as a mammalian cell, an insect cell, a plant cell or a fungal cell.

[0101] In a preferred embodiment, the host cell is a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).

[0102] In a more preferred embodiment, the fungal host cell is a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0103] In an even more preferred embodiment, the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

[0104] In a most preferred embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. In another most preferred embodiment, the yeast host cell is a Kluyveromyces lactis cell. In another most preferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

[0105] In another more preferred embodiment, the fungal host cell is a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

[0106] In an even more preferred embodiment, the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma.

[0107] In a most preferred embodiment, the filamentous fungal host cell is an Aspergillus awamori, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most preferred embodiment, the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell. In an even most preferred embodiment, the filamentous fungal parent cell is a Fusarium venenatum (Nirenberg sp. nov.) cell. In another most preferred embodiment, the filamentous fungal host cell is a Humicola insolens or Humicola lanuginosa cell. In another most preferred embodiment, the filamentous fungal host cell is a Mucor miehei cell. In another most preferred embodiment, the filamentous fungal host cell is a Myceliophthora thermophilum cell. In another most preferred embodiment, the filamentous fungal host cell is a Neurospora crassa cell. In another most preferred embodiment, the filamentous fungal host cell is a Penicillium purpurogenum cell. In another most preferred embodiment, the filamentous fungal host cell is a Thielavia terrestris cell. In another most preferred embodiment, the Trichoderma cell is a Trichodenna harzianum, Trichodenna koningii, Trichodenna longibrachiatum, Trichodenna reesei or Trichoderma viride cell.

[0108] Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

[0109] Methods of Production

[0110] The present invention also relates to methods for producing a human immediate early interleukin-four induced protein having the following characteristics: (i) about 546 to about 567 amino acids; (ii) L-amino acid oxidase activity; (iii) is obtainable from interleukin4 induced B cells; (iv) contains an N-terminal signal sequence of about 21 amino acids; (v) in encoded by a genomic DNA sequence having eight exons; (vi) has a non-conserved C-terminal sequence of about 62 amino acids; (vi) has less than 80% homology to a mouse immediate early interleukin-four induced protein having the sequence depicted in SEQ ID NO: 1 comprising (a) cultivating a host cell under conditions conducive for production of the protein; and (b) recovering the protein.

[0111] In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the protein using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the protein to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the protein is secreted into the nutrient medium, the protein can be recovered directly from the medium. If the protein is not secreted, it can be recovered from cell lysates.

[0112] The protein may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the protein. Such assays that may be used are L-amino acid oxidase assays (as will be discussed in further detail, infra), apoptosis assays, and ammonia release assays.

[0113] The resulting protein may be recovered by methods known in the art. For example, the protein may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. Alternatively, as detailed in the Examples section, the protein may also be recovered from the pellet.

[0114] The protein may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing, differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J. -C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

[0115] Antibodies

[0116] According to the invention, the protein produced according to the method of the present invention may be used as an immunogen to generate antibodies that bind to at least one epitope on the protein of the present invention. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.

[0117] Various procedures known in the art may be used for the production of antibodies. For the production of antibody, various host animals can be immunized by injection with the polypeptide thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc. In one embodiment, the polypeptide or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

[0118] For preparation of monoclonal antibodies, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals utilizing recent technology (PCT/US90/02545). According to the invention, human antibodies may be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). In fact, according to the invention, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, J. Bacteriol. 159-870; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes from a mouse antibody molecule specific for the protein of the present invention together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention.

[0119] According to the invention, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce polypeptide-specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for the immediate early IL-4 induced protein of the present invention.

[0120] Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab′)2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab′ fragments which can be generated by reducing the disulfide bridges of the F(ab′)2, fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.

[0121] In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

[0122] Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980); Hammerling et al., “Monoclonal Antibodies And T-cell Hybridomas” (1981); Kennett et al., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890.

[0123] The antibody produced by the method of the present invention has a number of possible uses. These include the detection of FIG. 1 protein in samples by the same methods as above (Western blot, immunoprecipitation, immunohistochemistry, etc.), to aid in purification of FIG. 1 protein, to block FIG. 1 protein activity (e.g., L-amino acid oxidase, apoptosis), to detect and measure amounts of FIG. 1 protein in patients and to inactivate or remove FIG. 1 protein in patients.

[0124] Detection of Mutations

[0125] Oligonucleotides hybridizing under stringent conditions to the polynucleotide of the present invention may be used as probes for detecting mutations from samples from a mammal. Genomic DNA may be isolated from the mammal. A mammal includes but is not limited to humans, domest, form, zoo, sports or pet animals including but not limited to dogs, cats, cattle, horses, cheep, goats, pigs and rabbits. The sample may be obtained from a blood sample, a cell sample, including but not limited to lymphocytes, and antigen presenting cells such as macrophages, monocytes, Islets of Langerhans and/or tissue sample such as lung tissue, tissues from neoplasms, lymphoid tissue.

[0126] A mutation(s) may be detected by Southern blot analysis, specifically by hybridizing restriction digested genomic DNA to various probes and subjecting to agarose electrophoresis. Alternatively, these oligonucleotides may be used as PCR primers and be used to amplify the genomic DNA isolated from the patients. The sequence of the amplified genomic DNA from the patient may be determined using methods known in the art.

[0127] Alternatively, RNA may be isolated from B cells of a patient and a mutation(s) may be detected by Northern Blot analysis using the probes of the present invention. RNA isolated from a patient may also be used.

[0128] Such probes may be between 10-100 nucleotides in length and may preferably be between 20-50 or between 10-40 nucleotides in length. The probes are labeled with a detectable label selected from the group consisting of a radiolabel such as 3H, 14C, 32P, 35S, enzymes such as beta-glucuronidase, peroxidase, beta-D-glucosidase, urease, glucose oxidase plus peroxidase, and fluorescent materials such as fluorescein, rhodamine and auramine, cyanine dye Cy5, GFP (green fluorescent protein) red fluorescent protein or its variants. Sometimes people use a tag, such as biotin or dioxygenin. The tag is detected with label linked to avidin, or anti-dioxygenin antibody, respectively. Thus, the invention is directed to kits comprising these polynucleotide probes.

[0129] PCR techniques including 1) SSCP (single strand conformation polymorphism) may also be used to detect mutations. Briefly, PCR amplify a specific region of the FIG. 1 gene or cDNA. Purified PCR fragments are denatured and separated on native gels. A change in mobility of the single-stranded DNA indicates the presence of a polymorphism or mutation. 2) RFLP (Restriction fragment length polymorphism). This can be done without PCR as well (directly on genomic DNA, restriction enzyme digested, Southern blotted and probed with gene-specific probe). In brief, PCR is used to amplify a specific region of the FIG. 1 gene or cDNA. The PCR fragment is digested with a known restriction enzyme that differentially digests the normal and mutant sequence (i.e. digests the mutant and not normal, or digests the normal and not mutant). PCR fragments are separated by gel electrophoresis and visualized. Mutant exhibits a different pattern of restriction enzyme digested fragment sizes. 3) PCR techniques that utilize fluorescent dyes. These include but are not limited to the following five techniques (Wittwer et aL, 1997, Biotechniques, 22:130-131, 134-138; Tapp etal., 2000, Biotechniques, 28:732-738). i) Fluorescent dyes are used to detect specific PCR amplified double stranded DNA product (e.g. ethidium bromide, or SYBR Green I). ii) The 5′ nuclease (TaqMan) assay utilizes a specially constructed primer whose fluorescence is quenched until it is released by the nuclease activity of the Taq DNA polymerase during extension of the PCR product. iii) Assays based on Molecular Beacon technology rely on a specially constructed oligonucleotide that when self-hybridized quenches fluorescence (fluorescent dye and quencher molecule are adjacent). Upon hybridization to a specific amplified PCR product, fluorescence is increased due to separation of the quencher from the fluorescent molecule. iv) Assays based on Amplifluor (Intergen) technology utilize specially prepared primers, where again fluorescence is quenched due to self-hybridization. In this case, fluorescence is released during PCR amplification by extension through the primer sequence, which results in the separation of fluorescent and quencher molecules. v) Assays that rely on an increase in fluorescence resonance energy transfer utilize two specially designed adjacent primers, which have different fluorochromes on their ends. When these primers anneal to a specific PCR amplified product, the two fluorochromes are brought together. The excitation of one fluorochrome results in an increase in fluorescence of the other fluorochrome.

[0130] Therapeutic and Prophylactic Uses

[0131] The polynucleotides of the present invention or interleukin-four induced protein expressed by the polynucleotide of the present invention have a number of therapeutic and prophylactic uses. The interleukin-four induced protein may also have the following characteristics: (a) at least about 546 amino acids; (b) L-amino acid oxidase activity; (c) is obtainable from interleukin4 induced B cells; (c) contains an N-terminal signal sequence of about 21 amino acids; (d) has a non-conserved C-terminal sequence of about 62 amino acids; (e) has at least about 75% homology to a mouse immediate early interleukin-four induced protein having the sequence depicted in SEQ ID NO:1 or composition comprising said protein and a carrier may be used for the manufacture of a medicament to treat or prevent a pathological condition in a mammal by treating said mammal with a compound effective to treat or prevent said condition. In a specific embodiment, the interleukin-four induced protein has about 95% sequence homology to the sequence depicted in SEQ ID NO:1. In another embodiment, said protein has the following characteristics: (a) about 546 to about 567 amino acids; (b) L-amino acid oxidase activity; (c) is obtainable from interleukin-4 induced B cells; (c) contains an N-terminal signal sequence of about 21 amino acids; (d) in encoded by a genomic DNA sequence having eight exons; (e) has a non-conserved C-terminal sequence of about 62 amino acids; (f) has less than 80% homology to a mouse immediate early interleukin-four induced protein having the sequence depicted in SEQ ID NO: 1. In a specific embodiment, the protein has about 95% sequence homology to the sequence depicted in SEQ ID NO:2.

[0132] Said polynucleotide and said interleukin-four induced protein may be used to prevent, treat or ameliorate a medical condition caused by a mutation in the polynucleotide of the present invention. This may be a mutation in the coding sequence or non coding sequence. The mutation in the noncoding sequence may be in the regulatory sequence or at the exon-intron junction. The protein or polynucleotide may be used to treat immune related disorders such as systemic lupus erythematosis, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis, idiopathic inflammatory myopathies, Sjöbgren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmunethrombocytopenia thyroiditis, diabetes mellitus, immune-mediated renal disease, demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome, and chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases such as infectious, autoimmune chronic activehepatitis, primary biliary cirrhosis,gramilomatous hepatitis, and sclerosing cholangitis, inflammatory bowel disease, gluten-sensitive enteropathy, and Whipple's disease, autoimmune or immune-mediated skin diseases including bullous skin diseases,erythema multiforme and contact dermatitis, psoriasis, allergic diseases such as asthma, allergicrhinitis, atopic dermatitis, food hypersensitivity and urticaria, immunologic diseases of the lung such as eosinophilic pneumonias, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplantation associated diseases including graft rejection and graft-versus-host-disease.

[0133] Furthermore, the polynucleotide of the present invention and said immediate early interleukin-four induced protein may be used as a toxin to kill unwanted cells, such as tumor cells, bacterial cells and fungal cells. In a specific embodiment, the protein is expressed by a host cell comprising the polynucleotide of the present invention. The polynucleotides, proteins, oligonucleotides, and antagonists may be formulated into compositions. These compositions may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

[0134] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0135] Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0136] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0137] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0138] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0139] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0140] In one embodiment of the present invention, the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations including, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature, these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

[0141] The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates and can generally be estimated based on EC50 is found to be effective in in vitro and in vivo animal models.

[0142] In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the polynucleotide or protein formulation is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0143] Gene Therapy

[0144] As described herein, the polynucleotide of the present invention may be introduced into a patient's cells for therapeutic uses. As will be discussed in further detail below, cells can be transfected using any appropriate means, including viral vectors, as shown by the example, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA. See, for example, Wolff, Jon A, et al., “Direct gene transfer into mouse muscle in vivo,” Science, 247, 1465-1468, 1990; and Wolff, Jon A, “Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs,” Nature, 352, 815-818, 1991. As used herein, vectors are agents that transport the gene into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. As will be discussed in further detail below, promoters can be general promoters, yielding expression in a variety of mammalian cells, or cell specific, or even nuclear versus cytoplasmic specific. These are known to those skilled in the art and can be constructed using standard molecular biology protocols. Vectors have been divided into two classes:

[0145] a) Biological agents derived from viral, bacterial or other sources.

[0146] b) Chemical physical methods that increase the potential for gene uptake, directly introduce the gene into the nucleus or target the gene to a cell receptor.

[0147] Biological Vectors

[0148] Viral vectors have higher transaction (ability to introduce genes) abilities than do most chemical or physical methods to introduce genes into cells. Vectors that may be used in the present invention include viruses, such as adenoviruses, adeno associated virus (AAV), vaccinia, herpesviruses, baculoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression. Polynucleotides are inserted into vector genomes using methods well known in the art.

[0149] Retroviral vectors are the vectors most commonly used in clinical trials, since they carry a larger genetic payload than other viral vectors. However, they are not useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation. Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature.

[0150] Examples of promoters are SP6, T4, T7, SV40 early promoter, cytomegalovirus (CMV) promoter, mouse mammary tumor virus (MMTV) steroid-inducible promoter, Moloney murine leukemia virus (MMLV) promoter, phosphoglycerate kinase (PGK) promoter, and the like. Alternatively, the promoter may be an endogenous adenovirus promoter, for example the E1 a promoter or the Ad2 major late promoter (MLP). Similarly, those of ordinary skill in the art can construct adenoviral vectors utilizing endogenous or heterologous poly A addition signals.

[0151] Plasmids are not integrated into the genome and the vast majority of them are present only from a few weeks to several months, so they are typically very safe. However, they have lower expression levels than retroviruses and since cells have the ability to identify and eventually shut down foreign gene expression, the continuous release of DNA from the polymer to the target cells substantially increases the duration of functional expression while maintaining the benefit of the safety associated with non-viral transfections.

[0152] Chemical/Physical Vectors

[0153] Other methods to directly introduce genes into cells or exploit receptors on the surface of cells include the use of liposomes and lipids, ligands for specific cell surface receptors, cell receptors, and calcium phosphate and other chemical mediators, microinjections directly to single cells, electroporation and homologous recombination. Liposomes are commercially available from Life Technologies, Inc. (Rockville, Md.), for example, as LIPOFECTIN and LIPOFECTACE, which are formed of cationic lipids such as N-[1-(2,3 dioleyloxy)-propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Numerous methods are also published for making liposomes, known to those skilled in the art.

[0154] For example, Nucleic acid-Lipid Complexes—Lipid carriers can be associated with naked nucleic acids (e.g., plasmid DNA) to facilitate passage through cellular membranes. Cationic, anionic, or neutral lipids can be used for this purpose. However, cationic lipids are preferred because they have been shown to associate better with DNA which, generally, has a negative charge. Cationic lipids have also been shown to mediate intracellular delivery of plasmid DNA (Felgner and Ringold, Nature 337:387 (1989)). Intravenous injection of cationic lipid-plasmid complexes into mice has been shown to result in expression of the DNA in lung (Brigham et al., Am. J. Med. Sci.298:278 (1989)). See also, Osaka et al., J. Pharm. Sci. 85(6):612-618 (1996); San et al., Human Gene Therapy 4:781-788 (1993); Senior et al., Biochemica et Biophysica Acta 1070:173-179 (1991); Kabanov and Kabanov, Bioconjugate Chem. 6:7-20 (1995); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Behr, J-P., Bioconjugate Chem 5:382-389 (1994); Behr et al., Proc. Natl. Acad. Sci., USA 86:6982-6986 (1989); and Wyman et al., Biochem. 36:3008-3017 (1997).

[0155] Cationic lipids are known to those of ordinary skill in the art. Representative cationic lipids include those disclosed, for example, in U.S. Pat. No. 5,283,185; and e.g., U.S. Pat. No. 5,767,099. In a preferred embodiment, the cationic lipid is N4_spermine cholesteryl carbamate (GL-67) disclosed in U.S. Pat. No. 5,767,099. Additional preferred lipids include N4_spermidine cholestryl carbamate (GL-53) and 1-(N4-spermine)-2,3-dilaurylglycerol carbamate (GL-89).

[0156] The vectors of the invention may be targeted to specific cells by linking a targeting molecule to the vector. A targeting molecule is any agent that is specific for a cell or tissue type of interest, including for example, a ligand, antibody, sugar, receptor, or other binding molecule. Invention vectors may be delivered to the target cells in a suitable composition, either alone, or complexed, as provided above, comprising the vector and a suitably acceptable carrier. The vector may be delivered to target cells by methods known in the art, for example, intravenous, intramuscular, intranasal, subcutaneous, intubation, lavage, and the like. The vectors may be delivered via in vivo or ex vivo applications. In vivo applications involve the direct administration of an adenoviral vector of the invention formulated into a composition to the cells of an individual. Ex vivo applications involve the transfer of the adenoviral vector directly to harvested autologous cells which are maintained in vitro, followed by readministration of the transduced cells to a recipient.

[0157] In a specific embodiment, the vector is transfected into antigen-presenting cells. Suitable sources of antigen-presenting cells (APCs) include, but are not limited to, whole cells such as dendritic cells or macrophages; purified MHC class I molecule complexed to beta2-microglobulin and foster antigen-presenting cells. In a specific embodiment, the vectors of the present invention may be introduced into T cells or B cells using methods known in the art (see, for example, Tsokos and Nepom, 2000, J. Clin. Invest. 106:181-183).

[0158] L-Amino Acid Oxidase Activity (LAAO)

[0159] As described in the Examples herein, the interleukin four induced protein has associated L-amino acid oxidase activity. The activity may be assayed using, for example using the methods described in the Examples section herein. As described in further detail; infra, in a preferred embodiment, published procedures (see, for example, Raibekas et al., 1996, Proc. Natl. Acad. Sci. USA 93:7546-7551 or Torii et al., 1997, J. Biol. Chem. 272:9539-9542) are modified so that o-phenylenediamine (OPD) is used as a chromogenic substrate. Specifically, said protein described herein has phenylalanine oxidase activity. There is lesser specificity for other aromatic amino acids such as tyrosine and tryptophan. Furthermore, the activity is maximal and therefore has a pH optimum of 4.0, with lower activity at pH 5, 8 and 9.

[0160] Therefore, said protein may be used to diagnose pathological disorders by detecting the level of L-amino acid oxidase activity. In a specific embodiment, an immune related disorder in a patient may be detected by measuring the L-amino acid oxidase activity. A changed (decreased or increased) level of L-amino acid oxidase activity in a patient relative to that a control activity, e.g., activity in an individual who does not have said disorder would be indicative of an immune related disorder, such as arthritis or SLE. In another specific embodiment, a tumor may be detected in a patient by measuring the L-amino acid oxidase activity in a sample. An increased level of L-amino acid oxidase activity may be detected in tumor cells. Furthermore, the level of activity may be used as a tool to determine the treatment regimen to be undertaken.

[0161] In another embodiment, a treatment regimen may be monitored at periodic intervals by measuring the level of L-amino acid oxidase activity. In a related embodiment, measuring their effect on L-amino acid oxidase activity may be used to screen potential candidate compounds. A candidate that may act as a potential antagonist may act to decrease L-amino acid oxidase activity. A candidate compound that acts as a potential agonist may act to increase L-amino acid oxidase activity. The compound may be applied to a substrate containing said interleukin-four induced protein. Said substrate may be interleukin four induced B cells, an isolated interleukin-four induced protein described herein, or a cell extract containing said protein

[0162] Furthermore, the immediate early interleukin-four induced protein may be used for detecting an L-amino acid in a sample f by (a) contacting said sample with said immediate early interleukin-four induced protein and (b) detecting the presence or absence of L-amino acid oxidase activity. The sample may be a biological sample. In a specific embodiment, it may be obtained from a mammal.

EXAMPLES

[0163] The Examples describe the isolation of the human FIG. 1 gene after some difficulties. Specifically, the mouse FIG. 1 cDNA is used to design primers in orders to PCR the human FIG. 1 gene. This strategy failed because of the unusual GC rich sequence of the human FIG. 1 gene. The mouse FIG. 1 cDNA is used to probe a human genomic DNA library. This also failed. Again, this may be due to the unusual nature of the human FIG. 1 gene. The Examples also provide further information on the location and activity of the protein encoded by the FIG. 1 gene. Furthermore, the Examples describe an LAOO assay to measure the activity of the FIG. 1 protein.

[0164] Successful Isolation of Human FIG. 1 Gene

[0165] The mouse FIG. 1 cDNA sequence is used to probe the public expressed sequence tag (EST) database (Genbank, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.). Initially, three human ESTs (IMAGE clones 1581345, 1845115 and 1505169) were found that were similar to the mouse FIG. 1 gene (77% amino acid sequence identity), but differed in the 3′ end (no amino acid sequence identity). This sequence is verified by obtaining and completely sequencing IMAGE clones 1845115 and 1505169. The inserts of these clones are sequenced initially using primers to the ends of the cloning vector and completed using internal primers that matched the initial sequence. It appears that clones 1845115 and 1505169 are identical, containing 586 bp inserts with an additional 22 bp poly (A) tail. The sequence of these clones is contained within the 3′ end of SEQ ID NO:4 (1213-1798). The first 380 bp is similar to mouse FIG. 1, whereas the last 206 bp did not resemble mouse FIG. 1. A 357 bp EcoRI-PshAI fragment from clone 1845115, containing vector sequence plus the first 342 bp of the human EST that is similar to the mouse FIG. 1 gene, is used to probe two human genomic DNA libraries (22585-265p15-PAC and 22586-31p20-PAC) (Genome Systems, St. Louis, MO). Two PAC clones (265(P15) and 31 (P20)) hybridized to this probe. DNA prepared from one PAC clone (31 (P20) is digested with either BamHI, EcoRI, or KpnI and subcloned into pZeroI (InVitrogen, Carlsbad, Calif.). The two identical BamHI subclones (A4, B4) contain sequences similar to mouse FIG. 1. The plasmid pZeroI containing subclone A4 and designated plasmid A4 is deposited under the terms of the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110, and is given accession number ______. Probing a Southern blot containing subclone plasmid DNA digested to separate the insert and vector sequences, revealed four EcoRI subclones (D2, D3, D5, D7) that contained sequence that hybridized to the same human EST probe as above. The pZeroI plasmid containing subclone D2 is deposited deposited under the terms of the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110, and is given accession number ______. These six genomic subclones are completely sequenced by primer walking and found to contain portions of the human FIG. 1 gene that spanned exon 5 to exon 8 (the 3′ end of the FIG. 1 gene). A genomic map of exons, introns, and clone locations is shown in FIG. 1. Clones A4 and B4 (3257 bp) contained exons 5 and 6. Clones D2, D3, D5, and D7 (4493 bp) contained exons 6 to 8, including the part of the human EST used as a probe. Exons 5, 6 and 7 could be predicted by similarity to mouse FIG. 1 protein sequence (82%, 78%, 91% identity, respectively). Exon 8 is similar to mouse FIG. 1 (77% identity), except for the 3′ end which corresponded to the human ESTs. Clones A4/B4 and D2/D3/D5/D7 overlap and can be combined to form a contiguous stretch of genornic DNA (6933 bp).

[0166] The remaining genomic sequence that includes exons 1 through 4 is obtained from the public preliminary high throughput genomic sequence (htgs) database (Genbank, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.). One sequence (AC011452) had some similarity to the mouse FIG. 1 gene. By comparison of AC011452 to the genomic sequence that we determined above, we discovered that AC011452 contained hFIG. 1 exon 5 and a long region of 5′ genomic sequence (>45 kb) that should contain hFIG. 1 exons 1 through 4. The entire combined genomic sequence of human FIG. 1 containing clones A4/B4 and D2/D3/D5/D7 plus 3350 bp from ACO 1452 is shown in SEQ ID NO:3 and illustrated in FIG. 1. SEQ ID NO:3 (10283 bp) contains all of the human FIG. 1 exons. Since exon 3 and exon 4 are entirely coding sequence, we could provisionally determine hFIG. 1 exon 3 and 4 based on similarity to mouse FIG. 1 protein sequence (74% and 92% identity, respectively). However, exons 1 and 2 could not be predicted, because exon 1 is noncoding sequence and exon 2 is only partially coding sequence. In order to determine these remaining exons, as well as confirm the exon predictions, the human FIG. 1 cDNA (representing the expressed mRNA) sequence is determined.

[0167] The genetic location of human FIG. 1 on chromosome 19q13.3-19q13.4 between the FUT1 and KLK1 is based on the following two pieces of evidence. First, some of the additional PAC subclones, which did not contain hFIG. 1, could be identified by matching the initial end sequences to the public sequence database (Genbank, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.). Two genes are identified that have been genetically mapped in human (GeneMap′ 98, World Wide Web (URL: http://www.ncbi.nlm.nih.gov/genemap98/)) (Deloukas et al., 1998, Science, 282:744-746). The NUP62 gene (Genbank No. XX58521), which encodes p62 nucleoporin (Carmo-Fonseca et al., 1991, Eur J Cell Biol, 55:17-30), has been localized to chromosome 19 between FUT1 and KLK1. An EST (Genbank No. AI097295), which represents an unidentified transcribed gene (Unigene number Hs.16552) has also been mapped to human chromosome 19 between FUT1 and KLK1. Since, hFIG. 1 is obtained from a PAC clone containing these two genes, this suggests that hFIG. 1 also resides on human chromosome 19 between FUT1 and KLK1. Second, part of the hFIG. 1 genomic sequence comes from Genbank No. AC011452. AC011452 sequence comes from a single BAC clone, CIT-HSPC—326K19, which is one of a series of BAC clones containing insert DNA from human chromosome 19 (DOE Joint Genome Institute, Walnut Creek, Calif.). Furthermore, the preliminary sequence of AC011452 indicates that the NUP62 gene is adjacent to the 5′ end of hFIG. 1 (11233 bp upstream of exon 1). Because the hFIG. 1 gene is physically linked to NUP62, this indicates that hFIG. 1 is also localized to 19q13.3-19q13.4.

[0168] Determination of Human FIG. 1 cDNA Sequence

[0169] In order to confirm and deterrmine the remaining hFIG. 1 cDNA sequence, the genomic hFIG. 1 sequence is used to design specific PCR primers to amplify hFIG. 1 cDNA by RT-PCR (reverse transcription-polymerase chain reaction) or RACE (rapid amplification of cDNA ends) techniques described below. The source of human cDNA is B cells purified from mononuclear cells prepared from peripheral blood of human volunteers by Ficoll-Hypaque (Amersham Pharmacia Biotech) density gradient centrifugation. B cells are purified by positive selection using Dynabeads M-450 PanB (anti-CD19 monoclonal antibody) (Dynal, Lake Success, N.Y.) or by negative selection using MACS B cell isolation kit (Miltenyi Biotec, Auburn, Calif.) using a cocktail of hapten-conjugated antibodies to non-B cell markers (anti-CD2, CD4, CD11b, CD16, CD36, IgE). Cells are cultured at 2×105 cells/mil in AMV medium (Life Technologies) supplemented with antibiotics and 10 nglml recombinant human IL-4 (R&D Systems) for 18-20 hr. Total RNA is prepared from purified B cells using a guanidine thiocyanate procedure (Chomczynski and Sacchi, 1987, Anal Biochem, 162:156-159). Briefly, cultured cells are lysed in guanidine thiocyanate solution, extracted with phenol/chloroform and then precipitated with isopropanol. The resulting RNA is resuspended in diethyl-pyrocarbonate-treated water, quantitated by UV adsorption at 260 nm, and used in the RT-PCR and RACE procedures.

[0170] In order to confirm the predicted exons from the genomic DNA sequence, RT-PCR is performed on total RNA obtained from IL-4 induced human B cells. First strand cDNA is synthesized from 1 μg of total RNA with 20 Units Avian Myeloblastosis Virus reverse transcriptase (Boehringer Mannheim, Indianapolis, Ind.), 30 Units PRIME RNase inhibitor (5 Prime →3 Prime, Boulder, Colo.), 0.25 mM of each deoxynucleotide (dATP, dCTP, dGTP, dTTP), and 1.875 μM oligo d(T)-anchor (5′ GAC CAC GCG TAT CGA TGT CGA CTT TTT TTT TTT TTT TTV (SEQ ID NO:5), Boehringer Mannheim) or 1.0 μM CCC48 primer (5′ GAC TCG AGT CGA CAT CGA TIT TTT TTT TTT TT (SEQ ID NO:6)) in cDNA synthesis buffer (50 mM Tris-HCl, 8 mM MgCl2, 30 mM KCl, 1 mM dithiotreitol, pH 8.5) and incubated at 55° C. for 1 hour. After heat inactivation at 65° C., this cDNA served as template for PCR using four primer pairs designed to generate overlapping PCR products that spanned the predicted exons.

[0171] Primer pair CCC268 (5′ CCA AGA CCC CTT CGA GAA ATG (SEQ ID NO:7)/CCC293 (5′ GCC TCG GCG AAG CTG AGA TAG (SEQ ID NO:8) is designed to traverse exons 3 to 7 to produce a 651 bp PCR product. Primer pair CCC262 (5′ ATC CTC CAC AAG CTC TGC C) (SEQ ID NO:9)/CCC295 (5′ AAC AGC ACA AGC CCG GAC AG) (SEQ ID NO:10) produces a 482 bp PCR product that traverses exons 5 to 8. Primer pairs CCC294 (5′ GTG GGT GGC TGG GAC CTG) (SEQ ID NO:11)/CCC288 (5′ GCG GGT AGA AAA TCA TGC G) (SEQ ID NO:12) and CCC151 (5′ CCT TCT GGC GCG AGG AGC AC) (SEQ ID NO:13)/CCC152 (5′ GCC GAC TTG ACC GCC GTC TC) (SEQ ID NO:14) both amplify within exon 8 to produce 373 and 406 bp products, respectively. PCR is performed in a 20 ll reaction containing 1 μM cDNA, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM of each deoxynucleotide, 1 μM of each primer, and 1.5 Units AmpliTaq Gold (Roche Molecular Systems, Branchburg, N.J.). Mixtures containing primer pairs CCC268/CCC293 and CCC262/CCC295 are thermocycled under the following conditions: 9 min at 95 C, 37×(5 sec at 94 C, 15 sec at 65 C, 30 sec at 72 C), 30 sec at 72° C., and soak at 4° C. 1M betaine is added to mixtures containing primer pair CCC294/CCC288 and thermocycled under the following conditions: 9 min at 95° C., 40× (5 sec at 95° C., 15 sec at 65° C., 30 sec at 72° C.), 30 sec at 72° C., and soak at 4° C. Primer pair CCC151/CCC152 is also amplified in 1 M betaine under the following conditions: 9 min at 95° C., 40× (5 sec at 94 C, 15 sec at 70° C., 30 sec at 72° C.), 30 sec at 72° C., and soak at 4 C.

[0172] The PCR products are directly cloned into pCR2.1 using the Original TA Cloning Kit (InVitrogen, Carlsbad, Calif.). After transformation of TOP10F′ E. coli (InVitrogen) to ampicillin resistance, individual bacterial colonies are boiled and tested by PCR for inserts in the cloning plasmid (Chu and Paul, 1998, Mol. Immunol., 35:487-502). RT-PCR with CCC268/CCC293, CCC262/CCC295, CCC294/CCC288, or CCC151/CCC152 primers resulted in four plasmid clones (pSC268/293#9, 10, 11, 12), seven plasmid clones (pSC262/295#5, 8, 9, 13, 14, 15, 16), six plasmid clones (pSC294/288#5, 6, 12, 14, 18, 19), and three plasmid clones (pSC151/152#25, 26, 28), respectively.

[0173] Inserts in these clones are PCR amplified using primers (CCC35 (T7) (5′ TAA TAC GAC TCA CTA TAG GG) (SEQ ID NO:15), M13R2 (5′ CAG GAA ACA GCT ATG AC) (SEQ ID NO:16) to the ends of the plasmid vector, purified by Chromaspin-100 columns (Clontech Laboratories, Palo Alto, Calif.), and sequenced using the same primers. In addition, pSC151/152#25, 26, and 28 clones are sequenced with CCC151, CCC152, and CCC156 (5′ CGT CGC GCA TGA TTT TCT ACC) (SEQ ID NO:17) primers. DNA sequencing is performed on an ABI 373 DNA Sequencer using the ABI PRISM® BigDye™ Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, FS (PE Applied Biosystems, Foster City, Calif.). Sequences are assembled and compared to hFIG. 1 genomic sequence using AssemblyLIGN (Oxford Molecular, Campbell, Calif.) on Macintosh computers (Apple, Cupertino, Calif.). The resulting assembled sequence confirmed the predicted exons 3, 4, 5, 6, and 7. The 5′ end of exon 8 that is conserved between mouse and human is also confirmed.

[0174] In order to determine if the 3′ end of the human FIG. 1 cDNA is indeed different from mouse FIG. 1 and not due to some splicing difference, 3′ RACE is performed on the 3′ end of exon 8 using the 5′/3′ RACE kit (Boehringer Mannheim). Briefly, first strand cDNA is synthesized as before using 1 μg of total RNA from IL-4 stimulated B cells and oligo d(T)-anchor primer. This cDNA is used as a template for PCR amplification with oligo d(T)-anchor and CCC287 (5′ TGG AGA CGG CGG TCA AGT C) (SEQ ID NO:18) primers. PCR is performed in a 20 μl reaction containing 1 μl cDNA, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM of each deoxynucleotide, 1 μM of each primer, 1.5 Units AmpliTaq Gold, and 2.5% DMSO. This mixture is thermocycled for: 9 min at 95 C, 37×(5 sec at 94 C, 15 sec at 65 C, 30 sec at 72 C), 30 sec at 72 C, and soak at 4 C. The PCR product is ligated into pCR2.1 and used to transform bacteria to ampicillin resistance. The resulting transformants are checked for insert by PCR using vector end primers CCC35 and M13R2. The resulting four clones (pSC3RACE8, 12, 14, 16) contained an identical 285 bp insert (including 33 bp oligo d(T)-anchor tail) that is sequenced using CCC35 and M13R2 primers as before. This sequence corroborated the 3′ end of the human FIG. 1 cDNA that is predicted from the genomic hFIG. 1 DNA sequence and found in the human EST sequences. Thus, human FIG. 1 differs from mouse FIG. 1 at the 3′ end.

[0175] In order to determine exons 1 and 2, as well as confirm exon 3, 5′ RACE is performed on total RNA obtained from IL-4 induced human B cells as before. 5′ RACE is performed using the 5′/3′ RACE Kit (Boehringer Mannheim). Briefly, first strand cDNA is synthesized as before, except with gene specific primer CCC265 (5′ TTC TTC ATC GCC TTT CTG C) (SEQ ID NO:19). This cDNA is purified and tailed at the 3′ end with homopolymeric dATP using 10 Units terminal transferase in 25 μl according to the manufacturer's directions. This dA-tailed cDNA is amplified by PCR in a 50 μl reaction containing 5 μl dA-tailed cDNA, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM of each deoxynucleotide, 0.4 PM CCC269 (5′ ACC AGC GCC AAC CAC AAT C) (SEQ ID NO:20) primer, 0.75 μM oligo d(T)-anchor primer, and 1 Unit ELONGASE Enzyme Mix (Life Technologies) and thermocycled under the following conditions: 2 min at 94 C, 10×(15 sec at 94 C, 30 sec at 64 C, 40 sec at 72 C), 20×(15 sec at 94 C, 30 sec at 64 C, 40 sec (plus 20 sec for each cycle) at 72 C), 7 min at 72 C, and soak at 4 C. This first PCR product (2.0 μl) is reamplified by a second PCR using 0.4 μM of a nested, specific primer CCC335 (5′ GGA CGA GGA CGA GGA GGT G) (SEQ ID NO:21) and 0.25 μM of the PCR anchor primer (5′ GAC CAC GCG TAT CGA TGT CGA C) (SEQ ID NO:22) in a 50 μl reaction containing 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM of each deoxynucleotide, and 1 Unit ELONGASE Enzyme Mix and thermocycled under the following conditions: 2 min at 94 C, 40×(15 sec at 94 C, 30 sec at 60 C, 30 sec at 72 C), 2 min at 72 C, and soak at 4 C. The first and second PCR products are cloned respectively into pCR2.1 vector using Original TA Cloning Kit resulting in four (pB2, pB13, pB15, pB19) and three clones (pD105, pD107, pD109), respectively. The insert sizes for clones pB2, pB13, pB15, pBl9, pD105, pD107, and pD109 are 227, 169, 281, 269, 102, 115, and 102 base pairs (bp), respectively. To finish the 5′ end of the hFIG. 1 gene, another cDNA synthesis is performed as before except with primer CCC269. cDNA is purified and dA-tailed as before. This material (5 μl) is PCR amplified in a 50 μl reaction containing 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM of each deoxynucleotide, 0.4 μM CCC335 primer, 0.75 μM oligo d(T)-anchor primer, and 1 Unit ELONGASE Enzyme Mix (Life Technologies) and therrnocycled under the following conditions: 2 min at 94 C, 10×(15 sec at 94 C, 30 sec at 63 C, 40 sec at 72 C), 20×(15 sec at 94 C, 30 sec at 63 C, 40 sec (plus 20 sec for each cycle) at 72 C), 7 min at 72 C, and soak at 4 C. The PCR product is cloned as before resulting in two clones (pC115 and pC122) with inserts of 115 bp. The insert sequence of all nine clones is determined as before. Briefly, inserts in these clones are PCR amplified using primers (CCC35, M13R2) to the ends of the plasmid vector, purified by Chromaspin-100 columns, and sequenced using the same primers. DNA sequencing is performed on an ABI 373 DNA Sequencer using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS. Sequences are assembled and compared to hFIG. 1 genomic sequence using AssemblyLIGN on Macintosh computers. The aligned sequences confirmed the exon 3 designation determined previously and defined exons 1 and 2 that compose the 5′ end of hFIG. 1.

[0176] Construction of Complete hFIG. 1 cDNA

[0177] Mononuclear cells are prepared from human peripheral blood by Ficoll-Hypaque (Amersham Pharmacia Biotech) density gradient centrifugation. From this cell population, B cells are purified by negative selection using MACS B cell isolation kit (Miltenyi Biotec, Auburn, Calif.) using a cocktail of hapten-conjugated antibodies to non-B cell markers (anti-CD2, CD4, CD11b, CD16, CD36, IgE). B cells (2×1o5 /ml) are cultured for 18 to 20 hours at 37° C. and 5% CO2 in RPMI Medium 1640 plus 100 Units/ml penicillin G sodium, 100 μg/rnl streptomycin sulfate, 2 mM glutamine (Life Technologies, Rockville, Md.), 10% fetal bovine serum (HyClone Laboratories, Logan Utah) and 10 ng/ml recombinant human IL-4 (R&D Systems).

[0178] Total RNA is prepared using a guanidine thiocyanate procedure. Briefly, cultured cells are lysed in guanidine thiocyanate solution, extracted with phenol/chloroform and then precipitated with isopropanol. The resulting RNA is resuspended in diethyl pyrocarbonate-treated water and quantitated by UV adsorption at 260 nm.

[0179] For RT-PCR, first strand cDNA is synthesized from 1 μg of total RNA obtained from IL-4 induced human B cells with 200 Units Moloney Murine Leukemia Virus Reverse Transcriptase (Life Technologies), 30 Units PRIME RNase inhibitor (5 Prime→3 Prime), 1.0 mM of each deoxynucleotide (dATP, dCTP, dGTP, dTTP), and 1.0 μM oligo dT15 in cDNA synthesis buffer (50 mM Tris-HCl, 3 mM MgCl2, 75 mM KCl, 5 mM dithiotreitol, pH 8.3) and incubated at 42° C. for 1 hour. After heat inactivation at 99° C., this cDNA served as template for PCR.

[0180] The full length hFIG. 1 cDNA is amplified using two primer pairs. Primer pair ccc372 (5′ TTC GAA TTC GCC GCC GCC ATG GCC CCA TTG GCC CTG CAC)/ccc 373 (5′ CCG CGG TAC CGT ATG GGA GGT CCT CGT GTG GGT) produces a 1737 bp hFIG. 1 cDNA with restriction enzyme sites engineered at either end (5′ EcoRI site and 3′ KpnI site) and without a translational stop codon (suitable for fusion of the hFIG. 1 protein with another protein). PCR with ccc372/ccc374 (5′ CCG CGG TAC CGT TTA ATG GGA GGT CCT CGT GTG GGT) produces a 1740 bp full length hFIG. 1 cDNA with the same restriction enzyme sites engineered at either end and with the normal translational stop codon (suitable for the expression of hFIG. 1 protein alone). PCR is performed in a 20 μl reaction containing 1 μl cDNA, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM of each deoxynucleotide, 1 μM of each primer, 1 M betaine (Sigma, St. Louis, Mo.) and 2 μl Elongase (Life Technologies). PCR is carried out under the following conditions: 35×(5 sec at 94° C., 30 sec annealing at 68° C., 2 min at 72° C.), 4 min at 72° C. and soak at 4° C. The expected size PCR product is eluted from the gel and purified by phenol/chloroform extraction.

[0181] The purified PCR product is mixed with plasmid vector pDsRed1-N1 (Clontech Laboratories, Inc., Palo Alto, Calif.) at a 10:1 molar ratio, sequentially digested with EcoRI and KpnI restriction enzymes, purified by ethanol precipitation, followed by ligation overnight. Ligation of pDsRed1-N1 with ccc372/ccc373 PCR product produces a hFIG. 1-dsRED (red fluorescence protein) fusion cDNA.

[0182]
FIG. 1 Expression Plasmids

[0183] A 1985 bp BamHI fragment containing the full length mouse FIG. 1 cDNA is inserted into the BamHI site of the pREP9 vector (InVitrogen, Carlsbad, Calif.), resulting in pMN10-9, a plasmid that expresses FIG. 1 mRNA from the vector's RSV 3′ LTR promoter. A 1930 bp PvuII/EcoRI fragment containing the full length mouse FIG. 1 cDNA is inserted into the Ec1126II/EcoRI sites of pIRES2-EGFP (Clontech), resulting in plasmid mFIG. 1-iGFP (also known as pAFFP), a plasmid that expresses FIG. 1 mRNA from the vector's CMV promoter.

[0184] Both mouse and human FIG. 1 have been constructed as fusions with the dsRED fluorescent protein.

[0185] For mouse FIG. 1, the full length cDNA is PCR amplified from the previously constructed plasmid containing full length mouse FIG. 1 cDNA (LXSN-FIG. 1) using primer pair NS217 (5′ AAA CCC GGG GCC GCC ACC ATG GCT GGG CTG GCC CTG CGT)/NS218 (5′ GGC GAC CGG TGA GTG GTC CCC CAC TCG GTG CAT). The resulting PCR product (1917 bp) is designed to generate mouse FIG. 1 cDNA with restriction enzyme sites engineered at either end (5′ SmaI and 3′ Agel), with an optimized Kozak sequence for translation initiation, and without a translational stop codon (suitable for fusion of the mFIG. 1 protein with another protein). The Smal and Agel restriction enzyme digested PCR product (1907 bp) is inserted into the SmaI and AgeI sites of plasmid vector pDsRed1-N1 (Clontech). The resulting plasmid pmFIG. 1-dsRED expresses a fusion protein consisting of the full length mouse FIG. 1 protein followed by a 4 amino acid linker followed by the full length dsRED fluorescent protein. This mFIG. 1-dsRED fusion construct is verified by DNA sequencing to be as expected.

[0186] For human FIG.1, the full length cDNA is PCR amplified from interleukin-4 stimulated human B cell cDNA using primer pair ccc372 (5′ TTC GAA TTC GCC GCC GCC ATG GCC CCA TTG GCC CTG CAC)/ccc 373 (5′ CCG CGG TAC CGT ATG GGA GGT CCT CGT GTG GGT). The ccc372/ccc373 PCR product (1731 bp) is designed to generate human FIG.1 cDNA with restriction enzyme sites engineered at either end (5′ EcoRI site and 3′ KpnI site) and without a translational stop codon (suitable for fusion of the hFIG. 1 protein with another protein). The EcoRI and KpnI restriction enzyme digested PCR product (1724 bp) is inserted into the EcoRI and KpnI sites of plasmid vector pDsRed1-N1 (Clontech Laboratories, Inc., Palo Alto, Calif.). The resulting plasmid phFIG. 1-dsRED expresses a fusion protein consisting of the full length human FIG. 1 protein followed by a 12 amino acid linker followed by the full length dsRED fluorescent protein. This hFIG. 1-dsRED fusion construct is verified by DNA sequencing to be as expected.

[0187] L-Amino Acid Oxidase (LAOO) Activity

[0188] The following procedure is used to measure FIG. 1 LAAO activity. The LAAO enzyme reaction produces hydrogen peroxide (H2O2) as follows:

[0189] The generated hydrogen peroxide can be conveniently measured by a chromogenic substrate (X) in a second coupled peroxidase reaction:

[0190] The color generated during the reaction can be measured by a spectrophotometer. Knowing the time of the reaction, the enzyme activity in Units can be calculated. For example, Worthington Biochemical Corporation (Lakewood, N.J.) measures the activity of snake venom LAAO using o-dianisidine as a chromogenic substrate, which is measured by absorbance (Abs) at 436 nm. Add 0.1 ml of enzyme sample to 2.9 ml reaction buffer (0.2 M triethanolamine (pH 7.6), 0.1% L-leucine, 0.0065% o-dianisidine, 34 μg/ml peroxidase). Incubate 4 to 5 minutes at 25° C. and measure the change in Abs 436 nm over time. Units can be calculated as follows:

Units (μmol / \min) = \frac{(ΔAbs436) / \min \times V}{E \times 1}

[0191] Where:

[0192] (ΔAbs436)/min=the change in Abs 436 nm per minute,

[0193] E=the Extinction coefficient of o-dianisidine at 436 nm

[0194] V=assay volume=3.0 ml (in this example)

[0195] 1=path length of light through sample volume

[0196] E×I=8.1 mM−1 for o-dianisidine in this example

[0197] One unit oxidizes one micromole of L-leucine per minute at 25° C. and pH 7.6.

[0198] Specific activity (Units/mg protein) can be calculated as follows:

Units / mg protein = \frac{Units}{(vol / dilution) \times [protein]}

[0199] Where:

[0200] vol=volume of sample=0.1 ml (in this example)

[0201] dilution=the dilution of the sample used in measurement

[0202] [protein]=protein concentration of sample (mg/ml)

[0203] This protocol (Raibekas et al., 1996, Proc. Natl. Acad. Sci. USA 93:7546-7551) or close variation (Torii et al., 1997, J. Biol. Chem. 272:9539-9542) has been used to purify and characterize snake venom LAAO.

[0204] 3,3′,5,5′-tetramethylbenzidine (TMB) or o-phenylenediamine (OPD) is used as a chromogenic substrate in the FIG. 1 assay. TMB is advantageous because it is not mutagenic and therefore not likely to be carcinogenic, whereas OPD and o-dianisidine are mutagenic. OPD is advantageous because its chromogenic property is not significantly altered by pH, especially acidic pH.

[0205]
FIG. 1 Enzyme Assay Ising TMB

[0206] For the FIG. 1 enzyme assay using TMB, a reaction mixture (pH 6.0) is prepared consisting of TMB (3 drops/5 ml distilled water) and its buffer (2 drops/5 ml distilled water) made according to manufacturer's specifications (Vector Labs. Inc., Burlingame, Calif.), 1 mg/ml L-leucine (Sigma), and 15-30 μg/ml horseradish peroxidase (Sigma). After incubation at room temperature (RT, ˜25° C.) for 5 minutes, 190 μl of the reaction mixture is added to 10 μl of sample and incubated at RT for 10-15 minutes. The Abs at 630 nm is measured in a 96-well plate reader. The reaction is terminated at 12-15 minutes by adding 50p of 1N H2SO4. The Abs at 450 nm is measured in the terminated sample. FIG. 1 enzyme units can be calculated as follows:

Units (μmol / \min) = \frac{(Abs) / \min \times V}{E \times 1}

[0207] Where:

[0208] (ΔAbs)/min=the change in Abs (630 nm or 450 nm) per minute,

[0209] E=the Extinction coefficient (mM−1cm−1) of TMB at 630 nm or 450 nm

[0210] V=assay volume=0.2 ml

[0211] 1=path length of light through sample volume (0.51 cm in 96 well plate)

[0212] The extinction coefficient for TMB at 630 nm and 450 nm is determined to be 35 and 72 mM−1cm−1, respectively. One unit oxidizes one micromole of L-leucine per minute at 25° C. and pH 6.

[0213] Specific activity (Units/mg protein) can be calculated as follows:

Units / mg protein = \frac{Units}{(vol / dilution) \times [protein]}

[0214] Where:

[0215] vol=volume of sample=0.01 ml (in this example)

[0216] dilution=the dilution of the sample used in measurement

[0217] [protein]=protein concentration of sample (mg/ml)

[0218] This assay works well with snake venom LAAO, but is less useful for measurement of FIG. 1, because we determined that 1) FIG. 1 protein prefers aromatic L-amino acids for substrates, particularly L-phenylalanine, and 2) FIG. 1 protein works best at acidic pH (pH 4). To perform these measurements, we developed a FIG. 1 enzyme assay using OPD as a substrate.

[0219]
FIG. 1 Enzyme Assay Using OPD

[0220] For the FIG. 1 enzyme assay using OPD, 10 μl of sample is added to a 190 μl assay mixture resulting in a 200 μl reaction with final concentration of 5 to 50 mM L-phenylalanine (Sigma, St. Louis, Mo.), 10 U/ml peroxidase (Sigma), 0.5 mg/ml OPD (Sigma), and 52.5 mM sodium acetate (pH 4). The reaction is incubated for 2 hours at 37° C., after which the reaction is stopped by the addition of 11 μl 36N sulfuric acid (H2SO4). The quantity of H2O2 generated is determined by measuring absorbance at 490 nm. FIG. 1 enzyme activity in Units is determined by comparison with a standard curve generated using known concentrations of H2O2. The amount of H2O2 (μM) corresponding to the observed absorbance at 490 nm is the amount of L-phenylalanine oxidized, since one H2O2 molecule is produced per L-amino acid oxidized. Therefore, FIG. 1 enzyme units can be calculated as follows:

[0221] Units (μmol/min)=(μM)/min×V

[0222] Where:

[0223] (μM)/min=the μM peroxide concentration corresponding to the observed change in Abs (490 nm) per minute,

[0224] V=assay volume=0.2 ml

[0225] One unit oxidizes one micromole of L-phenylalanine per minute at 37° C. and pH 4.

[0226] Specific activity (Units/mg protein) can be calculated as follows:

Units / mg protein = \frac{Units}{(vol / dilution) \times [protein]}

[0227] Where:

[0228] vol=volume of sample=0.01 ml (in this example)

[0229] dilution=the dilution of the sample used in measurement

[0230] [protein]=protein concentration of sample (mg/ml) Ps Activity of FIG. 1

[0231] A murine B cell line (M.12.4.1) and fibroblast line (NIH3T3) is transfected with vectors expressing mouse FIG. 1. Specifically, a 1985 bp BamHI fragment containing the full length mouse FIG. 1 cDNA is inserted into the BamHI site of the pREP9 vector (InVitrogen, Carlsbad, Calif.), resulting in pMN10-9, a plasmid that expresses FIG. 1 mRNA from the vector's RSV 3′ LTR promoter. A 1930 bp PvuII/EcoRI fragment containing the full length mouse FIG. 1 cDNA is inserted into the Ecl126II/EcoRI sites of pIRES2-EGFP (Clontech), resulting in plasmid mFIG. 1-iGFP (also known as pAFFP), a plasmid that expresses FIG. 1 mRNA from the vector's CMV promoter.

[0232] NIH 3T3 cells are transfected with plasmid DNA using the calcium phosphate method. In particular, a Calcium Phosphate Mammalian Cell Transfection Kit (5 Prime→3 Prime, Inc., Boulder, Colo.) or Calcium Phosphate Transfection System (Life Technologies, Inc., Rockville, Md.) is used. A 40 μg plasmid DNA/calcium phosphate precipitate is prepared according to manufacturer's protocol and used to transfect 2×106 cells. M12.4. 1 cells are transfected by electorporation or with an Effectene Transfection Reagent (Qiagen, Inc., Valencia, Calif.). For electroporation, 20 μg plasmid DNA is added to 5×106 cells in 1 ml CRPMI (RPMI Medium 1640 plus 100 Units/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, 2 mM glutamine (Life Technologies, Rockville, Md.), 10% fetal bovine serum (HyClone Laboratories, Logan UT)). This DNA/cell mixture is electroporated at 960 μF and 200 V in a Gene Pulser II Electorporator using 0.4 cm Gene Pulser cuvettes (Bio-Rad Laboratories, Hercules, Calif.). For Effectene, a 1 μg plasmid DNA/Effectene complex is prepared according to manufacturer's protocol and used to transfect 5×105 cells. Effectene is a lipid carrier of nucleic acids. Both pMN10-9 and mFIG. 1-iGFP are used to transfect M12.4.1 cells. Only mFIG. 1-iGFP is used to transfect NIH3T3 cells.

[0233] Stable transfectants, could not be obtained, suggesting that FIG. 1 does cause cell toxicity. During the selection of M.12.1 bulk transfectants, LAAO activity is detected after 15 days, peaking at 17 days, declining after 20 days to undetectable using procedures described above. Since stable clones are not obtained from this bulk transfection, individual clones are selected in the subsequent experiment. Nine clones showed LAAO activity after 24 days, peaking at 31 days and disappearing by 46 days. Six clones secreted FIG. 1 and three retained it in the insoluble cell fraction. Although no stable cell lines are obtained, only cells expressing FIG. 1 in the insoluble fraction showed significant cell death, with some cells showing condensed cytoplasmic staining resembling apoptosis. Thus, FIG. 1 is able to cause cell death by a mechanism that may involve apoptosis or other mechanisms, such as necrosis or autophagy.

[0234] In addition to the toxicity of hydrogen peroxide, another mechanism that FIG. 1 may kill cells is by the removal of essential amino acids resulting in the starvation of cells. FIG. 1 prefers aromatic amino acids as a substrate, with phenylalanine (Phe) being the optimal substrate. Phe is an essential amino acid, whose removal from the cell and/or its surrounding media would result in death. Additionally, it appears that the pH optimum is 4.0.

[0235] Localization of FIG. 1

[0236] Surprisingly, FIG. 1 protein is found in the water insoluble fraction of the cell pellet in some cases, whereas initially and for the most part subsequently, FIG. 1 protein is been found to be secreted into the supernatants. FIG. 1 has a signal peptide sequence that suggests targeting to the endoplasmic reticulum (ER) prior to being secreted. However, some proteins in the ER are not secreted but move to other subcellular locations, such as the lysosome. In these cells, FIG. 1 is localized to an intracellular organelle, such as a lysosome, in order to prevent inadvertent cell death caused by the generation of hydrogen peroxide by oxidation of the pool of amino acids found inside the cell. To test this hypothesis, a FIG. 1-red fluorescent protein (dsRED) fusion expression plasmid is constructed. By fluorescent light microscopy, FIG. 1-dsRED transfectants show a distinct cytoplasmic punctate pattern, which excluded the nucleus. In contrast, dsRED alone transfectants expressed dsRED protein diffusely throughout the cell. To determine to which subcellular compartment FIG. 1-dsRED is localized, co-transfections of FIG. 1-dsRED are performed with vectors expressing green fluorescence targeted to specific cellular locations. The overlap of red and green fluorescence visualized by confocal microscopy determined co-localization. As predicted, FIG. 1 is found in the endoplasmic reticulum (with the pEYFP-ER vector) and also in lysosomes (using the pHYAL2-EGFP vector). The latter result is confirmed by co-localization of FIG. 1-dsRED with acriflavine dye that is taken up by cellular lysosomes. Furthermore, FIG. 1 has LAAO activity with an optimum at pH 4 (lysosomal enzymes have acidic optimums, whereas cytoplasmic enzymes have neutral optimums), as well as potential glycosylation sites for lysosomal targeting. Thus, FIG. 1 is targeted to lysosomes, which may protect immune cells from its toxicity. Possible roles for FIG. 1 in lysosomes include involvement in autophagy or antigen presentation.

[0237] The lysosomal localization may explain the observation of some cell lines expressing FIG. 1 in the insoluble cell pellet. These cell lines are less frequent and show some obvious signs of distress. The majority of cell lines we detected seem to secrete FIG. 1. This may be a mechanism to avoid cell death from overexpression of FIG. 1 inside the cell. However, this may only avoid death for a short time, since the FIG. 1 secreting cell lines also succumb.

[0238] Polymorphisms

[0239] As noted above, human FIG. 1 maps to the predicted mouse FIG. 1 syntenic region on human chromosome 19q13.3-19q13.4. This region is a hot spot for susceptibility for several autoimmune diseases, including SLE. We have been searching for relevant FIG. 1 polymorphisms in humans that may associate with SLE. Too date, no polymorphisms that affect the protein coding sequence of FIG. 1 have been found in the public database or from the sequence data of SLE patients that we have generated. We have identified one polymorphism in intron 2 of FIG. 1 that results in an insertion or a deletion of guanine (G) nucleotide. This polymorphism does not affect any obvious conserved splicing sequences. Thus, at present this intron 2 polymorphism does not have a known effect on FIG. 1 protein levels. In any case, this polymorphism shows a trend towards the presence of the G insertion in SLE patients (p=0.13, n=25 SLE patients, n=182 normals).

[0240] The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

[0241] Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

Human interleukin-four induced protein

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