WO2008001045A1

WO2008001045A1 - Members of the glycoside hydrolase family 31 family of proteins

Info

Publication number: WO2008001045A1
Application number: PCT/GB2007/002274
Authority: WO
Inventors: Richard Joseph Fagan; Ellie Louise James; Melanie Yorke-Smith; Christine Power
Original assignee: Ares Trading S.A.
Priority date: 2006-06-26
Filing date: 2007-06-18
Publication date: 2008-01-03
Also published as: GB0612623D0; WO2008001045A8

Abstract

This invention relates to novel proteins, termed INTP041, INTP042 and INTP043, herein identified as members of the glycoside hydrolase family 31 family of proteins and to the use of these proteins and nucleic acid sequences from the encoding genes in the diagnosis, prevention and treatment of disease.

Description

Members of the glycoside hydrolase family 31 family of proteins

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

Background

The process of drug discovery is presently undergoing a fundamental revolution as the era of functional genomics comes of age. The term "functional genomics" applies to an approach utilising bioinformatics tools to ascribe function to protein sequences of interest. Such tools are becoming increasingly necessary as the speed of generation of sequence data is rapidly outpacing the ability of research laboratories to assign functions to these protein sequences.

As bioinformatics tools increase in potency and in accuracy, these tools are rapidly replacing the conventional techniques of biochemical characterisation. Indeed, the advanced bioinfoπnatics tools used in identifying the present invention are now capable of outputting results in which a high degree of confidence can be placed.

Various institutions and commercial organisations are examining sequence data as they become available and significant discoveries are being made on an on-going basis. However, there remains a continuing need to identify and characterise further genes and the polypeptides that they encode, as targets for research and for drug discovery.

Introduction

Glycoside hydrolase family 31

O-Glycosyl hydrolases are a widespread group of enzymes that hydrolyse the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non- carbohydrate moiety. In particular, glycoside hydrolase family 31 comprises enzymes with several known activities including alpha-glucosidase (maltase glucoamylase) and sucrase- isomaltase. In mammals, these are intestinal brush border-anchored enzymes, which play key roles in the final steps of starch digestion. They are heavily N- and O-glycosylated proteins, which are comprised of two homologous domains preceded by a Ser/Thr-rich domain in immediate proximity to the membrane and at least one P-type trefoil domain. The cysteine-rich "trefoil" or "P" domain of approximately forty five amino-acid residues contains six cysteine residues linked by three disulfide bonds with a connectivity pattern of 1-5, 2-4, 3-6. An aspartic acid residue has been implicated (Hermans et al. 1991. J. Biol. Chem. 266; 13507-13512) in the catalytic activity of sucrase, isomaltase, and lysosomal alpha-glucosidase and a WIDMNE motif is conserved at this site.

Human sucrase-isomaltase (SIM) is an 1826 amino acid long protein responsible for the hydrolysis of sucrose and maltose by an alpha-D-glucosidase-type action. It is comprised of a sucrase and an isomaltase domain, which share 35% homology to each other. The two subunits reside on the same polypeptide chain and the enzyme is synthesised as a single chain that is cleaved extracellularly in the intestinal lumen by pancreatic trypsin, generating two active subunits, which remain closely associated with each other via strong noncovalent interactions (Jacob et al, 2002. J. Biol. Chem. 277; 35. pρ32l41-32148).

Human maltase-glucoamylase (MGA) is an 1857 amino acid long protein responsible for the hydrolysis of terminal, non-reducing 1 ,4-linked D-glucose residues with release of D- glucose. It is thought that this enzyme may serve as an alternative pathway for starch digestion when luminal alpha-amylase activity is reduced because of immaturity or malnutrition and may also play a unique role in the digestion of malted dietary oligosaccharides used in food manufacturing. Like SIM, MGA is synthesised as a single polypeptide chain containing two homologous domains, maltase and glucoamylase, however, MGA does not undergo any intracellular or extracellular proteolytic cleavage event. Enzyme substrate specificities of MGA overlap with those of SIM (Nichols et al., 1998. J. Biol. Chem. 273;5. pp3076-3081).

In terms of drug therapy, the use of alpha-glucosidase inhibitors (acarbose or miglitol), either as mono-therapy or in combination with other oral agents or insulin is a current approach to the treatment of type 2 diabetes. These agents act by competitively inhibiting these carbohydrate-digesting enzymes (sucrase, glucoamylase, maltase, isomaltase), effectively delaying glucose absorption and decreasing post-prandial hyperglycemia and hyperinsulinemia. Further, Sucraid™ (sacrosidase) is an enzyme replacement therapy used in the treatment of congenital sucrase-isomaltase deficiency (CSID). CSID is a condition where the body lacks this enzyme, required for the proper break down and absorption of sucrose and isomaltase from the intestines. A clinical deficiency of intestinal glucoamylase has also been reported that consisted of chronic diarrhoea responding to a starch elimination diet in children with normal mucosal morphology and low starch hydrolyzing activity (Lebenthal, E., U, K. M., Zheng, B. Y., Lu, R. B., Lerner, A. (1994) J Pediatr. 124, 541-546 ). A genetic form of glucoamylase deficiency has also been reported in mice (Quezada-Calvillo, R, Senchyna, M., and Underdown, B. J. (1993) Am. J. Physiol. 265, G1150-G1157).

Increasing knowledge of these domains is therefore of extreme importance in increasing the understanding of the underlying pathways that lead to the disease states and associated disease states mentioned above, and in developing more effective gene and/or drug therapies to treat these disorders.

THE INVENTION

The invention is based on the discovery that the INTP041, INTP042 and INTP043 polypeptides are members of the glycoside hydrolase family 31 family of proteins .

In one embodiment of the first aspect of the invention, there is provided a polypeptide which:

(i) comprises the amino acid sequence as recited in SEQ ID NO:2, SEQ ID NO.4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166; SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO: 178, SEQ ID NO: 180 and/or SEQ ID NO: 182;

(ii) is a fragment thereof which functions as a member of the glycoside hydrolase family 31 family of proteins, or has an antigenic determinant in common with the polypeptides of (i); or (iii) is a functional equivalent of (i) or (ii).

Preferably, the polypeptide according to this first aspect of the invention:

(i) comprises the amino acid sequence as recited in SEQ ID NO:96, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180 or SEQ ID NO: 182;

(ii) is a fragment thereof which functions as a member of the glycoside hydrolase family 31 family of proteins, or has an antigenic determinant in common with the polypeptides of (i); or

(iii) is a functional equivalent of (i) or (ii).

According to a second embodiment of this first aspect of the invention, there is provided a polypeptide which:

(i) consists of the amino acid sequence as recited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO.12, SEQ ID NO:14, SEQ ED NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166; SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:178, SEQ ID NO:180 and/or SEQ ID NO:182;

(ii) is a fragment thereof which functions as a member of the glycoside hydrolase family 31 family of proteins, or having an antigenic determinant in common with the polypeptides of (i); or

(iii) is a functional equivalent of (i) or (ii). Preferably, the polypeptide

(i) consists of the amino acid sequence as recited in SEQ ID NO:96, SEQ ID NO:158, SEQ ID NO: 162, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180 or SEQ ID NO: 182;

(iii) is a functional equivalent of (i) or (ii).

The polypeptide having the sequence recited in SEQ ID NO:2 is referred to hereafter as "INTP041 exon 1 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:4 is referred to hereafter as "INTP041 exon 2 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:6 is referred to hereafter as "INTP041 exon 3 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 8 is referred to hereafter as "INTP041 exon 4 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 10 is referred to hereafter as "INTP041 exon 5 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 12 is referred to hereafter as "INTP041 exon 6 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:14 is referred to hereafter as "INTP041 exon 7 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 16 is referred to hereafter as "INTP041 exon 8 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 18 is referred to hereafter as "INTP041 exon 9 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:20 is referred to hereafter as "INTP041 exon 10 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:22 is referred to hereafter as "INTP041 exon 11 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:24 is referred to hereafter as "INTP041 exon 12 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:26 is referred to hereafter as "INTP041 exon 13 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:28 is referred to hereafter as "INTP041 exon 14 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:30 is referred to hereafter as "INTP041 exon 15 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:32 is referred to hereafter as "INTP041 exon 16 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:34 is referred to hereafter as "INTP041 exon 17 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:36 is referred to hereafter as "INTP041 exon 18 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:38 is referred to hereafter as "INTP041 exon 19 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:40 is referred to hereafter as "INTP041 exon 20 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:42 is referred to hereafter as "INTP041 exon 21 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:44 is referred to hereafter as "INTP041 exon 22 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:46 is referred to hereafter as "INTP041 exon 23 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:48 is referred to hereafter as "INTP041 exon 24 polypeptide". The polypeptide having the sequence recited in SEQ ID NO.50 is referred to hereafter as "INTP041 exon 25 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 52 is referred to hereafter as "INTP041 exon 26 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 54 is referred to hereafter as "INTP041 exon 27 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:56 is referred to hereafter as "INTP041 exon 28 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:58 is referred to hereafter as "INTP041 exon 29 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 60 is referred to hereafter as "INTP041 exon 30 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:62 is referred to hereafter as "INTP041 exon 31 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:64 is referred to hereafter as "INTP041 exon 32 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:66 is referred to hereafter as "INTP041 exon 33 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:68 is referred to hereafter as "INTP041 exon 34 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:70 is referred to hereafter as "INTP041 exon 35 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:72 is referred to hereafter as "INTP041 exon 36 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:74 is referred to hereafter as "INTP041 exon 37 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:76 is referred to hereafter as "INTP041 exon 38 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:78 is referred to hereafter as "INTP041 exon 39 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:80 is referred to hereafter as "INTP041 exon 40 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:82 is referred to hereafter as "INTP041 exon 41 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:84 is referred to hereafter as "INTP041 exon 42 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:86 is referred to hereafter as "INTP041 exon 43 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:88 is referred to hereafter as "INTP041 exon 44 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:90 is referred to hereafter as "INTP041 exon 45 polypeptide". The polypeptide having the sequence recited in SEQ ID NO.92 is referred to hereafter as "INTP041 exon 46 polypeptide". The polypeptide having the sequence recited in SEQ ID NO :94 is referred to hereafter as 'TNTP041 exon 47 polypeptide".

The polypeptide having the sequence recited in SEQ ID NO:96 is referred to hereafter as the "INTP041 partial polypeptide". The coding sequence for the FNTP041 partial polypeptide does not have a stop codon at its 3' end, suggesting that this polypeptide is a partial polypeptide. When the INTP041 coding sequence was cloned, three potential 3' ends downstream of the 3' end of the coding sequence of the INTP041 partial polypeptide were identified (INTP041-V1, INTP041-V2 and INTP041-V3), as shown in Figure 12. Only the INTP041-V1 end contains a potential polyadenylation signal sequence downstream of the 5'-most stop codon and upstream of the polyA tail. The INTP041-V1 end is therefore believed to be the true end of the INTP041 polypeptide. The polypeptide having the sequence recited in SEQ ID NO: 158 is referred to hereafter as the "INTP041 full-length polypeptide". The INTP041 full-length polypeptide comprises the INTP041 partial polypeptide and the seven additional amino acids encoded by the INTP041-V1 end.

The invention also includes alternative INTP041 full-length polypeptides comprising the INTP041 partial polypeptide and the additional amino acids encoded by the INTP041-V2 or INTP041-V3 ends shown in Figure 12. The sequences for these alternative full-length polypeptides are not provided in the list of sequences but the creation of these polypeptides is within the ability of one of skill in the art.

In addition to encoding seven more amino acids at the 3' end compared to the INTP041 partial polypeptide sequence, the INTP041-V1 end identified by RACE contains a base change which results in a Q (GIn) to D (Asp) change at position 1754 of the INTP041 full- length polypeptide sequence. This change may be a polymorphism. The invention includes INTP041 polypeptides that include an asparagine residue in place of the glutamine residue found at position 1733 of the INTP041 full-length or partial polypeptide sequences. The sequences of polypeptides containing this postulated polymorphism are not provided in the lsit of sequences found in this application but the creation of such polypeptides is within the ability of one of skill in the art.

The polypeptide having the sequence recited in SEQ ID NO: 162 is referred to hereafter as the "INTP041 cloned polypeptide". The INTP041 cloned polypeptide is a fragment of the INTP041 foil-length polypeptide, the cloning of which is described in the Examples.

The INTP041 cloned polypeptide contains amino acid differences in exons 19 and 20 compared to the INTP041 foil-length polypeptide indicative of a splice variant. The polypeptide having the sequence recited in SEQ ID NO: 164 is referred to hereafter as "INTP041 polypeptide sequence exon 19 Splice Variant". The polypeptide having the sequence recited in SEQ ID NO: 166 is referred to hereafter as "INTP041 polypeptide sequence exon 20 Splice Variant". The polypeptide having the sequence recited in SEQ ID NO: 168 is the foil-length INTP041 polypeptide containing the splice variation in exons 19 and 20, and is hereafter referred to as "INTP041 foil-length polypeptide sequence Splice Variant".

The INTP041 cloned polypeptide also contains an amino acid polymorphism H678R in exon 17 compared to the INTP041 foil-length polypeptide. The polypeptide having the sequence recited in SEQ ID NO: 170 is referred to hereafter as "INTP041 polypeptide sequence exon 17 H678R polymorphism". The polypeptide having the sequence recited in SEQ ID NO: 172 is the foil-length INTP041 polypeptide containing the H678R polymorphism and is referred to hereafter as "INTP041 foil-length polypeptide sequence H678R polymorphism".

The polypeptide having the sequence recited in SEQ ID NO: 174 is the foil-length polypeptide containing both the H678R polymorphism in exon 17 and the splice variation in exons 19 amd 20, and is referred to hereafter as "INTP041 foil-length polypeptide sequence Splice Variant and H678R polymorphism". The cloned INTP041 polypeptide is thus a fragment of the INTP041 foil-length polypeptide sequence Splice Variant and H678R polymorphism.

The INTP041 polypeptide is predicted to be a membrane anchored protein. The membrane anchor is predicted to be located at the N-terminal of the INTP041 polypeptide. The extracellular region of the INTP041 polypeptide is predicted to start at around amino acid 30 of the foil-length INTP041 polypeptide sequences. The invention includes extracellular fragments of the INTP041 foil-length polypeptide, the INTP041 foil-length polypeptide Splice Variant, the INTP041 foil-length polypeptide H678R polymorphism and the INTP041 foil-length polypeptide Splice Variant and H678R polymorphism which do not contain the membrane anchor. The polypeptide having the sequence recited in SEQ ID NO: 176 is the INTP041 extracellular fragment polypeptide sequence. The polypeptide having the sequence recited in SEQ ID NO: 178 is the INTP041 extracellular fragment polypeptide sequence Splice Variant. The polypeptide having the sequence recited in SEQ ID NO: 180 is the INTP041 extracellular fragment polypeptide sequence polymorphism. The polypeptide having the sequence recited in SEQ ID NO: 182 is the INTP041 extracellular fragment polypeptide sequence Splice Variant and polymorphism. Although these extracellular fragment sequences start at amino acid 30 in the full-length sequences, it will be appreciated that the invention also includes extracellular fragments that start at positions within the full-length sequences a few amino acids either side of amino acid 30. Specifically, the invention includes fragments that start at amino acid 28, 29, 31, 32, 33, 34, 35, 36, 37 or 38 of the full-length INTP041 polypeptide sequences. These extracellular fragments are particularly useful in assays to identify ligands, agonists and antagonists of the INTP041 polypeptides, as described herein.

The term "INTP041 polypeptides" as used herein includes polypeptides comprising the INTP041 exon 1 polypeptide, the INTP041 exon 2 polypeptide, the INTP041 exon 3 polypeptide, the INTP041 exon 4 polypeptide, the INTP041 exon 5 polypeptide, the INTP041 exon 6 polypeptide, the INTP041 exon 7 polypeptide, the INTP041 exon 8 polypeptide, the INTP041 exon 9 polypeptide, the INTP041 exon 10 polypeptide, the INTP041 exon 11 polypeptide, the INTP041 exon 12 polypeptide, the INTP041 exon 13 polypeptide, the INTP041 exon 14 polypeptide, the INTP041 exon 15 polypeptide, the INTP041 exon 16 polypeptide, the INTP041 exon 17 polypeptide, the INTP041 exon 18 polypeptide, the INTP041 exon 19 polypeptide, the INTP041 exon 20 polypeptide, the INTP041 exon 21 polypeptide, the INTP041 exon 22 polypeptide, the INTP041 exon 23 polypeptide, the INTP041 exon 24 polypeptide, the INTP041 exon 25 polypeptide, the INTP041 exon 26 polypeptide, the INTP041 exon 27 polypeptide, the INTP041 exon 28 polypeptide, the INTP041 exon 29 polypeptide, the INTP041 exon 30 polypeptide, the INTP041 exon 31 polypeptide, the INTP041 exon 32 polypeptide, the INTP041 exon 33 polypeptide, the INTP041 exon 34 polypeptide, the INTP041 exon 35 polypeptide, the INTP041 exon 36 polypeptide, the INTP041 exon 37 polypeptide, the INTP041 exon 38 polypeptide, the INTP041 exon 39 polypeptide, the INTP041 exon 40 polypeptide, the INTP041 exon 41 polypeptide, the INTP041 exon 42 polypeptide, the INTP041 exon 43 polypeptide, the INTP041 exon 44 polypeptide, the INTP041 exon 45 polypeptide, the INTP041 exon 46 polypeptide, the INTP041 exon 47 polypeptide, the INTP041 full-length polypeptide, the INTP041 cloned polypeptide, the INTP041 exon 19 polypeptide splice variant, the INTP041 exon 20 polypeptide splice variant, the INTP041 full-length polypeptide sequence Splice Variant, the INTP041 full-length polypeptide sequence H678R polymorphism, the INTP041 full-length polypeptide sequence Splice Variant and H678R polymorphism, the INTP041 extracellular fragment polypeptide sequence, the INTP041 extracellular fragment polypeptide sequence Splice Variant, the INTP041 extracellular fragment polypeptide sequence polymorphism, and the INTP041 extracellular fragment polypeptide sequence Splice Variant and polymorphism. The term "INTP041 polypeptides" also includes variants of any one of these sequences having a Q to D polymorphism at position 1733 of the full-length sequences (position 1703 of extracellular fragments) discussed above and variants having the INTP041-V2 end or INTP041-V3 end in place of the INTP041-V1 end.

In a third embodiment of the first aspect of the invention, there is provided a polypeptide which:

(i) comprises the amino acid sequence as recited in SEQ ID NO:98, SEQ ID NO.100 and/or SEQ ID NO: 160;

(iii) is a functional equivalent of (i) or (ii).

Preferably, the polypeptide according to this first aspect of the invention:

(i) comprises the amino acid sequence as recited in SEQ ID NO: 100 or SEQ ID NO: 160;

(iii) is a functional equivalent of (i) or (ii).

According to an fourth embodiment of this first aspect of the invention, there is provided a polypeptide which:

(i) consists of the amino acid sequence as recited in SEQ ID NO:98, SEQ ID NO: 100 and/or SEQ ID NO: 160; (ii) is a fragment thereof which functions as a member of the glycoside hydrolase family 31 family of proteins, or having an antigenic determinant in common with the polypeptides of (i); or

(iii) is a functional equivalent of (i) or (ii).

The polypeptide having the sequence recited in SEQ ID NO: 100 is referred to hereafter as the "INTP042 partial polypeptide". The INTP042 partial polypeptide is a further splice variant of the INTP041 partial polypeptide. The only difference between the INTP041 partial polypeptide and the INTP042 partial polypeptide is that the INTP041 partial polypeptide does not contain the INTP042 exon 2 polypeptide (SEQ ID NO:98).

The polypeptide having the sequence recited in SEQ ID NO: 160 is referred to hereafter as "the INTP042 full-length polypeptide". The INTP042 full-length polypeptide comprises the INTP042 partial polypeptide and the additional seven amino acids encoded by the INTP041-V1 3' end. The only difference between the INTP041 full-length polypeptide and the INTP042 full-length polypeptide is that the INTP041 full-length polypeptide does not contain the INTP042 exon 2 polypeptide (SEQ ID NO:98).

The term "INTP042 polypeptides" as used herein includes the INTP042 exon 2 polypeptide, the INTP042 partial polypeptide, the INTP042 full-length polypeptide and variants of the INTP042 full-length polypeptide.

Variants of the INTP042 full-length polypeptide included in the term ^uINTP042 polypeptides" are variants of the INTP042 full-length polypeptide which contain the exon 19-exon 20 splice variant or H678R polymorphism described above in connection with the INTP041 full-length polypeptide. The splice variant found in exons 19 and 20 of the INTP041 full-length polypeptide will occur in exons 20 and 21 of the full-length INTP042 polypeptide in view of the additional exon in the INTP042 full-length polypeptide. The H to R polymorphism found at position 678 of the INTP041 full-length polypeptide will occur at position 699 of the INTP042 full-length polypeptide.

Further variants included in the term "INTP042 polypeptides" are variants of the INTP042 full-length polypeptide having a Q to D polymorphism at position 1754 discussed above in connection with INTP041 and variants having the INTP041-V2 end or INTP041-V3 end in place of the INTP041-V1 end. Extracellular fragments of the INTP042 full-length polypeptide and variants thereof which start at positions within the full-length sequences a few amino acids either side of amino acid 30 are also included within "INTP042 polypeptides". Specifically, the invention includes fragments that start at amino acid 28, 29, 31, 32, 33, 34, 35, 36, 37 or 38 of the full-length INTP042 polypeptide sequences.

In a fifth embodiment of this first aspect of the invention, there is provided a polypeptide which:

(i) comprises the amino acid sequence as recited in SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO.-114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO: 152, SEQ ID NO: 154 and/or SEQ ID NO: 156;

(iii) is a functional equivalent of (i) or (ii).

Preferably, the polypeptide according to this first aspect of the invention:

(i) comprises the amino acid sequence as recited in SEQ ID NO: 156;

(iii) is a functional equivalent of (i) or (ii).

According to a sixth embodiment of this first aspect of the invention, there is provided a polypeptide which:

(i) consists of the amino acid sequence as recited in SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154 and/or SEQ ID NO: 156; (ii) is a fragment thereof which functions as a member of the glycoside hydrolase family 31 family of proteins, or having an antigenic determinant in common with the polypeptides of (i); or

(iii) is a functional equivalent of (i) or (ii).

The polypeptide having the sequence recited in SEQ ID NO: 102 is referred to hereafter as "INTP043 exon 1 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 104 is referred to hereafter as "INTP043 exon 2 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 106 is referred to hereafter as "INTP043 exon 3 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 108 is referred to hereafter as "INTP043 exon 4 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 110 is referred to hereafter as "INTP043 exon 5 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 112 is referred to hereafter as "INTP043 exon 6 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:114 is referred to hereafter as "INTP043 exon 7 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:116 is referred to hereafter as "INTP043 exon 8 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:118 is referred to hereafter as "INTP043 exon 9 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 120 is referred to hereafter as "INTP043 exon 10 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 122 is referred to hereafter as "INTP043 exon 11 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 124 is referred to hereafter as "INTP043 exon 12 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 126 is referred to hereafter as "INTP043 exon 13 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:128 is referred to hereafter as "INTP043 exon 14 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 130 is referred to hereafter as "INTP043 exon 15 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 132 is referred to hereafter as "INTP043 exon 16 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 134 is referred to hereafter as "INTP043 exon 17 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 136 is referred to hereafter as "INTP043 exon 18 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:138 is referred to hereafter as "INTP043 exon 19 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:140 is referred to hereafter as "INTP043 exon 20 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 142 is referred to hereafter as "INTP043 exon 21 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 144 is referred to hereafter as "INTP043 exon 22 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 146 is referred to hereafter as "INTP043 exon 23 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 148 is referred to hereafter as "INTP043 exon 24 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 150 is referred to hereafter as "INTP043 exon 25 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 152 is referred to hereafter as "INTP043 exon 26 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 154 is referred to hereafter as "INTP043 exon 27 polypeptide". The polypeptide having the sequence recited in SEQ ID NO.T56 is referred to hereafter as the "INTP043 partial polypeptide". As there is no methionine start codon at the start of INTP043 exon 1 (SEQ ID NO: 101), it is considered very likely that there are further exons 5' to SEQ ID NO: 101 and SEQ ID NO: 156 in the genome.

The term "INTP043 polypeptides" as used herein includes polypeptides comprising the INTP043 exon 1 polypeptide, the INTP043 exon 2 polypeptide, the INTP043 exon 3 polypeptide, the INTP043 exon 4 polypeptide, the INTP043 exon 5 polypeptide, the INTP043 exon 6 polypeptide, the INTP043 exon 7 polypeptide, the INTP043 exon 8 polypeptide, the INTP043 exon 9 polypeptide, the INTP043 exon 10 polypeptide, the INTP043 exon 11 polypeptide, the INTP043 exon 12 polypeptide, the INTP043 exon 13 polypeptide, the INTP043 exon 14 polypeptide, the INTP043 exon 15 polypeptide, the INTP043 exon 16 polypeptide, the INTP043 exon 17 polypeptide, the INTP043 exon 18 polypeptide, the INTP043 exon 19 polypeptide, the INTP043 exon 20 polypeptide, the INTP043 exon 21 polypeptide, the INTP043 exon 22 polypeptide, the INTP043 exon 23 polypeptide, the INTP043 exon 24 polypeptide, the INTP043 exon 25 polypeptide, the INTP043 exon 26 polypeptide, the INTP043 exon 27 polypeptide and the INTP043 partial polypeptide.

The term "member of the glycoside hydrolase family 31 family of proteins" refers to a molecule containing at least one domain from the glycosylase family 31 family of proteins.

Preferably, the member of the glycoside hydrolase family 31 family of proteins may be a molecule containing a domain from the glycoside hydrolase family 31 family of proteins detected with an e-value lower than 0.1, 0.01, 0.001, 0.0001, 0.0002, 0.00001, 0.000001 or 0.0000001. Preferably, the term "member of the glycoside hydrolase family 31 of proteins" refers to a molecule matching the HMM build of the Pfam entry detected with an e-value lower than 0.1, 0.01, 0.001, 0.0001, 0.0002, 0.00001, 0.000001 or 0.0000001.

Preferably the polypeptide of according to any one of the above described aspects of the invention functions as a member of the glycoside hydrolase family 31 family of proteins.

By "functions as a member of the glycoside hydrolase family 31 family of proteins" we refer to polypeptides that comprise amino acid sequence or structural features that can be identified as conserved features within the polypeptides of the glycoside hydrolase family 31 family of proteins, such that the polypeptide's interaction with ligand is not substantially affected detrimentally in comparison to the function of the full length wild type polypeptide. In particular, we refer to the presence of cysteine residues in specific positions within the polypeptide that allow the formation of intra-domain disulphide bonds. The polypeptides of the invention may also be identified as functioning as a member of the glycoside hydrolase family 31 family of proteins using standard assays known in the art. For example, the disaccharidase activity of a candidate protein may be measured by the TGO (Tris-Glucose Oxidase) assay described in R. Quezada-Calvillo, AJ. Markowitz, P. G. Traber and BJ. Underdo wn (Murine intestinal disaccharidases: identification of structural variants of sucrase-isomaltase complex. Am. J. Physiol. 265 (1993), pp. GI l 41-Gl 149); A. Dahlqvist (Method for assay of intestinal disaccharidases. Anal. Biochem. 7 (1964), pp. 18-25); and R. Quezada-Calvillo, F. Rodriguez-Zuniga and BJ. Underdown, (Partial characterization of murine intestinal maltase-glucoamylase, Biochemical and Biophysical Research Communications, 295(2), p 394-400).

The polypeptides of the present invention may modulate a variety of physiological and pathological processes or disorders. Thus, the biological activity or function of these polypeptides can be examined in ystems that allow the study of such modulatory activities, using a variety of suitable assays.

Thus, preferably, a member of the glycoside hydrolase 31 family of proteins shows biological activity in at least one of the assays described in R. Quezada-Calvillo, AJ. Markowitz, P. G. Traber and BJ. Underdown (Murine intestinal disaccharidases: identification of structural variants of sucrase— isomaltase complex. Am. J. Physiol. 265 (1993), pp. G1141-G1149); A. Dahlqvist (Method for assay of intestinal disaccharidases. Anal Biochem. 7 (1964), pp. 18-25); and R. Quezada-Calvillo, F. Rodriguez-Zuniga and B.J. Underdown, (Partial characterization of murine intestinal maltase-glucoamylase, Biochemical and Biophysical Research Communications, 295(2), p 394-400).

The polypeptides of the first aspect of the invention may further comprise a histidine tag. Preferably, the histidine tag is found at the C-terminus of the polypeptides. Preferably the histidine tag comprises 1-10 histidine residues (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues). More preferably, the histidine tag comprises 6 histidine residues. Preferred polypeptides of the invention comprising a histindine tag are the polypeptides comprising the sequence recited in SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, or SEQ ID NO: 198. The polypeptide comprising the sequence recited in SEQ ID NO: 184 is the INTP041 full-length polypeptide with a histidine tag. The polypeptide comprising the sequence recited in SEQ ID NO: 186 is the INTP041 full-length polypeptide sequence Splice Variant with a histidine tag. The polypeptide comprising the sequence recited in SEQ ID NO: 188 is the INTP041 full-length polypeptide sequence H678R polymorphism with a histidine tag. The polypeptide comprising the sequence recited in SEQ ID NO: 190 is the INTP041 full-length polypeptide sequence Splice Variant and H678R polymorphism with a histidine tag. The polypeptide comprising the sequence recited in SEQ ID NO: 192 is the INTP041 extracellular fragment polypeptide sequence with a histidine tag. The polypeptide comprising the sequence recited in SEQ ID NO: 194 is the INTP041 extracellular fragment polypeptide sequence Splice Variant with a histidine tag. The polypeptide comprising the sequence recited in SEQ ID NO: 196 is the INTP041 extracellular fragment polypeptide sequence polymorphism with a histidine tag. The polypeptide comprising the sequence recited in SEQ ID NO: 198 is the INTP041 extracellular fragment polypeptide sequence Splice Variant and polymorphism with a histidine tag. Preferably, the polypeptides consist of the the sequence recited in SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, or SEQ ID NO: 198. The invention also includes equivalent INTP042 and INTP043 polypeptides comprising a histidine tag.

An "antigenic determinant" of the present invention may be a part of a polypeptide of the present invention, which binds to an antibody-combining site or to a T-cell receptor (TCR). Alternatively, an "antigenic determinant" may be a site on the surface of a polypeptide of the present invention to which a single antibody molecule binds. Generally an antigen has several or many different antigenic determinants and reacts with antibodies of many different specificities. Preferably, the antibody is immunospecific to a polypeptide of the invention. Preferably, the antibody is imrnunospecific to a polypeptide of the invention, which is not part of a fusion protein. Preferably, the antibody is immunospecific to INTP041, INTP042, INTP043, or a fragment thereof. Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics. Preferably, the "antigenic determinant" refers to a particular chemical group on a polypeptide of the present invention that is antigenic, i.e. that elicit a specific immune response.

In a second aspect, the invention provides a purified nucleic acid molecule which encodes a polypeptide of the first aspect of the invention.

The term "purified nucleic acid molecule" preferably refers to a nucleic acid molecule of the invention that (1) has been separated from at least about 50 percent of proteins, lipids, carbohydrates, or other materials with which it is naturally found when total nucleic acid is isolated from the source cells, (2) is not linked to all or a portion of a polynucleotide to which the "purified nucleic acid molecule" is linked in nature, (3) is operably linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature as part of a larger polynucleotide sequence. Preferably, the isolated nucleic acid molecule of the present invention is substantially free from any other contaminating nucleic acid molecule(s) or other contaminants that are found in its natural environment that would interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use. In a preferred embodiment, genomic DNA are specifically excluded from the scope of the invention. Preferably, genomic DNA larger than 10 kbp (kilo base pairs), 50 kbp, 100 kbp, 150 kbp, 200 kbp, 250 kbp or 300 kbp are specifically excluded from the scope of the invention. Preferably, the "purified nucleic acid molecule" consists of cDNA only.

Preferably, the purified nucleic acid molecule comprises the nucleic acid sequence as recited in SEQ ID NO:1 (encoding the INTP041 exon 1 polypeptide), SEQ ID NO:3 (encoding the INTP041 exon 2 polypeptide), SEQ ID NO:5 (encoding the INTP041 exon 3 polypeptide), SEQ ID NO:7 (encoding the INTP041 exon 4 polypeptide), SEQ ID NO:9 (encoding the INTP041 exon 5 polypeptide), SEQ ID NO: 11 (encoding the INTP041 exon 6 polypeptide), SEQ ID NO: 13 (encoding the INTP041 exon 7 polypeptide), SEQ ID NO: 15 (encoding the INTP041 exon 8 polypeptide), SEQ ID NO: 17 (encoding the INTP041 exon 9 polypeptide), SEQ ID NO: 19 (encoding the INTP041 exon 10 polypeptide), SEQ ID NO:21 (encoding the INTP041 exon 11 polypeptide), SEQ ID NO:23 (encoding the INTP041 exon 12 polypeptide), SEQ ID NO.25 (encoding the INTP041 exon 13 polypeptide), SEQ ID NO:27 (encoding the INTP041 exon 14 polypeptide), SEQ ID NO:29 (encoding the INTP041 exon 15 polypeptide), SEQ ID NO:31 (encoding the INTP041 exon 16 polypeptide), SEQ ID NO.33 (encoding the INTP041 exon 17 polypeptide), SEQ ID NO:35 (encoding the INTP041 exon 18 polypeptide), SEQ ID NO:37 (encoding the INTP041 exon 19 polypeptide), SEQ ID NO:39 (encoding the INTP041 exon 20 polypeptide), SEQ ID NO.41 (encoding the INTP041 exon 21 polypeptide), SEQ ID NO:43 (encoding the INTP041 exon 22 polypeptide), SEQ ID NO:45 (encoding the INTP041 exon 23 polypeptide), SEQ ID NO:47 (encoding the INTP041 exon 24 polypeptide), SEQ ID NO.49 (encoding the INTP041 exon 25 polypeptide), SEQ ID NO:51 (encoding the INTP041 exon 26 polypeptide), SEQ ID NO.53 (encoding the INTP041 exon 27 polypeptide), SEQ ID NO:55 (encoding the INTP041 exon 28 polypeptide), SEQ ID NO.57 (encoding the INTP041 exon 29 polypeptide), SEQ ID NO:59 (encoding the INTP041 exon 30 polypeptide), SEQ ID NO.61 (encoding the INTP041 exon 31 polypeptide), SEQ ID NO:63 (encoding the INTP041 exon 32 polypeptide), SEQ ID NO:65 (encoding the INTP041 exon 33 polypeptide), SEQ ID NO:67 (encoding the INTP041 exon 34 polypeptide), SEQ ID NO.69 (encoding the INTP041 exon 35 polypeptide), SEQ ID NO:71 (encoding the INTP041 exon 36 polypeptide), SEQ ID NO:73 (encoding the INTP041 exon 37 polypeptide), SEQ ID NO:75 (encoding the INTP041 exon 38 polypeptide), SEQ ID NO:77 (encoding the INTP041 exon 39 polypeptide), SEQ ID NO:79 (encoding the INTP041 exon 40 polypeptide), SEQ ID NO:81 (encoding the INTP041 exon 41 polypeptide), SEQ ID NO:83 (encoding the INTP041 exon 42 polypeptide), SEQ ID NO:85 (encoding the INTP041 exon 43 polypeptide), SEQ ID NO:87 (encoding the INTP041 exon 44 polypeptide), SEQ ID NO:89 (encoding the INTP041 exon 45 polypeptide), SEQ ID NO:91 (encoding the INTP041 exon 46 polypeptide), SEQ ID NO:93 (encoding the INTP041 exon 47 polypeptide), SEQ ID NO:95 (encoding the INTP041 partial polypeptide), SEQ ID NO:97 (encoding the INTP042 exon 2 polypeptide), SEQ ID NO.99 (encoding the INTP042 partial polypeptide), SEQ ID NO: 101 (encoding the INTP043 exon 1 polypeptide), SEQ ID NO: 103 (encoding the INTP043 exon 2 polypeptide), SEQ ID NO: 105 (encoding the INTP043 exon 3 polypeptide), SEQ ID NO: 107 (encoding the INTP043 exon 4 polypeptide), SEQ ID NO: 109 (encoding the INTP043 exon 5 polypeptide), SEQ ID NO:111 (encoding the INTP043 exon 6 polypeptide), SEQ ID NO: 113 (encoding the INTP043 exon 7 polypeptide), SEQ ID NO:115 (encoding the INTP043 exon 8 polypeptide), SEQ ID NO:117 (encoding the INTP043 exon 9 polypeptide), SEQ ID NO: 119 (encoding the INTP043 exon 10 polypeptide), SEQ ID NO: 121 (encoding the INTP043 exon 11 polypeptide), SEQ ID NO:123 (encoding the INTP043 exon 12 polypeptide), SEQ ID NO:125 (encoding the INTP043 exon 13 polypeptide), SEQ ID NO:127 (encoding the INTP043 exon 14 polypeptide), SEQ ID NO: 129 (encoding the INTP043 exon 15 polypeptide), SEQ ID NO:131 (encoding the INTP043 exon 16 polypeptide), SEQ ID NO: 133 (encoding the INTP043 exon 17 polypeptide), SEQ ID NO: 135 (encoding the INTP043 exon 18 polypeptide), SEQ ID NO: 137 (encoding the INTP043 exon 19 polypeptide), SEQ ID NO: 139 (encoding the INTP043 exon 20 polypeptide), SEQ ID NO:141 (encoding the INTP043 exon 21 polypeptide), SEQ ID NO:143 (encoding the INTP043 exon 22 polypeptide), SEQ ID NO: 145 (encoding the INTP043 exon 23 polypeptide), SEQ ID NO: 147 (encoding the INTP043 exon 24 polypeptide), SEQ ID NO: 149 (encoding the INTP043 exon 25 polypeptide), SEQ ID NO:151 (encoding the INTP043 exon 26 polypeptide), SEQ ID NO: 153 (encoding the INTP043 exon 27 polypeptide) SEQ ID NO: 155 (encoding the INTP043 partial polypeptide), SEQ ID NO: 157 (encoding the INTP041 full-length polypeptide), SEQ ID NO: 159 (encoding the INTP042 full-length polypeptide), SEQ ID NO: 161 (encoding the INTP041 cloned polypeptide), SEQ ID NO:163 (encoding the INTP041 exon 19 splice variant), SEQ ID NO: 165 (encoding the INTP041 exon 20 splice variant), SEQ ID NO: 167 (encoding the INTP041 foil-length polypeptide Splice Variant), SEQ ID NO:169 (encoding the INTP041 exon 17 H678R polymorphism), SEQ ID NO:171 (encoding the INTP041 foil-length polypeptide H678R polymorphism), SEQ ID NO: 173 (encoding the INTP041 foil-length polypeptide Splice Variant and H678R polymorphism), SEQ ID NO: 175 (encoding the INTP041 extracellular fragment polypeptide), SEQ ID NO:177 (encoding the INTP041 extracellular fragment polypeptide Splice Variant), SEQ ID NO: 179 (encoding the INTP041 extracellular fragment polypeptide polymorphism), SEQ ID NO:181 (encoding the INTP041 extracellular fragment polypeptide Splice Variant and polymorphism), SEQ ID NO: 183 (encoding INTP041 foil-length polypeptide sequence HIS-TAG), SEQ ID NO: 185 (encoding INTP041 foil-length polypeptide sequence Splice Variant HIS-TAG), SEQ ID NO: 187 (encoding the INTP041 foil-length polypeptide sequence H678R polymorphism HIS-TAG), SEQ ID NO: 189 (encoding the INTP041 foil-length polypeptide Splice Variant and H678R polymorphism HIS-TAG), SEQ ID NO:191 (encoding the INTP041 extracellular fragment polypeptide HIS-TAG), SEQ ID NO: 193 (encoding the INTP041 extracellular fragment polypeptide Splice Variant HIS-TAG), SEQ ID NO:195 (encoding the INTP041 extracellular fragment polypeptide polymorphism HIS- TAG), SEQ ID NO: 197 (encoding the INTP041 extracellular fragment polypeptide Splice Variant and polymorphism HIS-TAG), or is a redundant equivalent or fragment of any one of these sequences.

The invention further provides that the purified nucleic acid molecule consists of the nucleic acid sequence as recited SEQ ID NO:1 (encoding the INTP041 exon 1 polypeptide), SEQ ID NO:3 (encoding the INTP041 exon 2 polypeptide), SEQ ID NO:5 (encoding the INTP041 exon 3 polypeptide), SEQ ID NO:7 (encoding the INTP041 exon 4 polypeptide), SEQ ID NO:9 (encoding the INTP041 exon 5 polypeptide), SEQ ID NO: 11 (encoding the INTP041 exon 6 polypeptide), SEQ ID NO: 13 (encoding the INTP041 exon 7 polypeptide), SEQ ID NO: 15 (encoding the INTP041 exon 8 polypeptide), SEQ ID NO: 17 (encoding the INTP041 exon 9 polypeptide), SEQ ID NO: 19 (encoding the INTP041 exon 10 polypeptide), SEQ ID NO:21 (encoding the INTP041 exon 11 polypeptide), SEQ ID NO:23 (encoding the INTP041 exon 12 polypeptide), SEQ ID NO:25 (encoding the INTP041 exon 13 polypeptide), SEQ ID NO:27 (encoding the INTP041 exon 14 polypeptide), SEQ ID NO:29 (encoding the INTP041 exon 15 polypeptide), SEQ ID NO:31 (encoding the INTP041 exon 16 polypeptide), SEQ ID NO:33 (encoding the INTP041 exon 17 polypeptide), SEQ ID NO:35 (encoding the INTP041 exon 18 polypeptide), SEQ ID NO:37 (encoding the INTP041 exon 19 polypeptide), SEQ ID NO:39 (encoding the INTP041 exon 20 polypeptide), SEQ ID NO:41 (encoding the INTP041 exon 21 polypeptide), SEQ ID NO:43 (encoding the INTP041 exon 22 polypeptide), SEQ ID NO:45 (encoding the INTP041 exon 23 polypeptide), SEQ ID NO:47 (encoding the INTP041 exon 24 polypeptide), SEQ ID NO:49 (encoding the INTP041 exon 25 polypeptide), SEQ ID NO:51 (encoding the INTP041 exon 26 polypeptide), SEQ ID NO:53 (encoding the INTP041 exon 27 polypeptide), SEQ ID NO:55 (encoding the INTP041 exon 28 polypeptide), SEQ ID NO:57 (encoding the INTP041 exon 29 polypeptide), SEQ ID NO:59 (encoding the INTP041 exon 30 polypeptide), SEQ ID NO:61 (encoding the INTP041 exon 31 polypeptide), SEQ ID NO:63 (encoding the INTP041 exon 32 polypeptide), SEQ ID NO:65 (encoding the INTP041 exon 33 polypeptide), SEQ ID NO:67 (encoding the INTP041 exon 34 polypeptide), SEQ ID NO:69 (encoding the INTP041 exon 35 polypeptide), SEQ ID NO:71 (encoding the INTP041 exon 36 polypeptide), SEQ ID NO:73 (encoding the INTP041 exon 37 polypeptide), SEQ ID NO:75 (encoding the INTP041 exon 38 polypeptide), SEQ ID NO:77 (encoding the INTP041 exon 39 polypeptide), SEQ ID NO:79 (encoding the INTP041 exon 40 polypeptide), SEQ ID NO:81 (encoding the INTP041 exon 41 polypeptide), SEQ ID NO:83 (encoding the INTP041 exon 42 polypeptide), SEQ ID NO:85 (encoding the INTP041 exon 43 polypeptide), SEQ ID NO: 87 (encoding the INTP041 exon 44 polypeptide), SEQ ID NO: 89 (encoding the INTP041 exon 45 polypeptide), SEQ ID NO:91 (encoding the INTP041 exon 46 polypeptide), SEQ ID NO:93 (encoding the INTP041 exon 47 polypeptide), SEQ ID NO:95 (encoding the INTP041 partial polypeptide), SEQ ID NO:97 (encoding the INTP042 exon 2 polypeptide), SEQ ID NO:99 (encoding the INTP042 partial polypeptide), SEQ ID NO: 101 (encoding the INTP043 exon 1 polypeptide), SEQ ID NO: 103 (encoding the INTP043 exon 2 polypeptide), SEQ ID NO: 105 (encoding the INTP043 exon 3 polypeptide), SEQ ID NO: 107 (encoding the INTP043 exon 4 polypeptide), SEQ ID NO: 109 (encoding the INTP043 exon 5 polypeptide), SEQ ID NO-.l l l (encoding the INTP043 exon 6 polypeptide), SEQ ID NO:113 (encoding the INTP043 exon 7 polypeptide), SEQ ID NO:115 (encoding the INTP043 exon 8 polypeptide), SEQ ID NO:1 17 (encoding the INTP043 exon 9 polypeptide), SEQ ID NO: 119 (encoding the INTP043 exon 10 polypeptide), SEQ ID NO: 121 (encoding the INTP043 exon 11 polypeptide), SEQ ID NO:123 (encoding the INTP043 exon 12 polypeptide), SEQ ID NO: 125 (encoding the INTP043 exon 13 polypeptide), SEQ ID NO:127 (encoding the INTP043 exon 14 polypeptide), SEQ ID NO:129 (encoding the INTP043 exon 15 polypeptide), SEQ ID NO:131 (encoding the INTP043 exon 16 polypeptide), SEQ ID NO: 133 (encoding the INTP043 exon 17 polypeptide), SEQ ID NO:135 (encoding the INTP043 exon 18 polypeptide), SEQ ID NO:137 (encoding the INTP043 exon 19 polypeptide), SEQ ID NO:139 (encoding the INTP043 exon 20 polypeptide), SEQ ID NO:141 (encoding the INTP043 exon 21 polypeptide), SEQ ID NO:143 (encoding the INTP043 exon 22 polypeptide), SEQ ID NO:145 (encoding the INTP043 exon 23 polypeptide), SEQ ID NO: 147 (encoding the INTP043 exon 24 polypeptide), SEQ ID NO: 149 (encoding the INTP043 exon 25 polypeptide), SEQ ID NO:151 (encoding the INTP043 exon 26 polypeptide), SEQ ID NO: 153 (encoding the INTP043 exon 27 polypeptide) SEQ ID NO: 155 (encoding the INTP043 partial polypeptide), SEQ ID NO:157 (encoding the INTP041 full-length polypeptide), SEQ ID NO: 159 (encoding the INTP042 foil-length polypeptide), SEQ ID NO: 161 (encoding the INTP041 cloned polypeptide), SEQ ID NO: 163 (encoding the INTP041 exon 19 splice variant), SEQ ID NO: 165 (encoding the INTP041 exon 20 splice variant), SEQ ID NO: 167 (encoding the INTP041 full-length polypeptide Splice Variant), SEQ ID NO:169 (encoding the INTP041 exon 17 H678R polymorphism), SEQ ID NO:171 (encoding the INTP041 full-length polypeptide H678R polymorphism), SEQ ID NO:173 (encoding the INTP041 full-length polypeptide Splice Variant and H678R polymorphism), SEQ ID NO: 175 (encoding the INTP041 extracellular fragment polypeptide), SEQ ID NO: 177 (encoding the INTP041 extracellular fragment polypeptide Splice Variant), SEQ ID NO:179 (encoding the INTP041 extracellular fragment polypeptide polymorphism), SEQ ID NO:181 (encoding the INTP041 extracellular fragment polypeptide Splice Variant and polymorphism), SEQ ID NO: 183 (encoding INTP041 full-length polypeptide sequence HIS-TAG), SEQ ID NO:185 (encoding INTP041 full-length polypeptide sequence Splice Variant HIS-TAG), SEQ ID NO: 187 (encoding the INTP041 full-length polypeptide sequence H678R polymorphism HIS-TAG), SEQ ID NO: 189 (encoding the INTP041 full- length polypeptide Splice Variant and H678R polymorphism HIS-TAG), SEQ ID NO: 191 (encoding the INTP041 extracellular fragment polypeptide HIS-TAG), SEQ ID NO: 193 (encoding the INTP041 extracellular fragment polypeptide Splice Variant HIS-TAG), SEQ ID NO: 195 (encoding the INTP041 extracellular fragment polypeptide polymorphism HIS- TAG), SEQ ID NO: 197 (encoding the INTP041 extracellular fragment polypeptide Splice Variant and polymorphism HIS-TAG), or is a redundant equivalent or fragment of any one of these sequences.

In a third aspect, the invention provides a purified nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule of the second aspect of the invention. High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (15OmM NaCl, 15mM trisodium citrate), 5OmM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C.

In a fourth aspect, the invention provides a vector, such as an expression vector, that contains a nucleic acid molecule of the second or third aspect of the invention.

In a fifth aspect, the invention provides a host cell transformed with a vector of the fourth aspect of the invention. In a sixth aspect, the invention provides a ligand which binds specifically to members of the glycoside hydrolase family 31 family of proteins of the first aspect of the invention. Preferably, the ligand inhibits the function of a polypeptide of the first aspect of the invention which is a member of the glycoside hydrolase family 31 family of proteins. Ligands to a polypeptide according to the invention may come in various forms, including natural or modified substrates, enzymes, receptors, small organic molecules such as small natural or synthetic organic molecules of up to 2000Da, preferably 800Da or less, peptidomimetics, inorganic molecules, peptides, polypeptides, antibodies, structural or functional mimetics of the aforementioned.

In a seventh aspect, the invention provides a compound that is effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention. Such compounds may be identified using the assays and screening methods disclosed herein.

A compound of the seventh aspect of the invention may either increase (agonise) or decrease (antagonise) the level of expression of the gene or the activity of the polypeptide.

Importantly, the identification of the function of the INTP041, INTP042 and INTP043 polypeptides allows for the design of screening methods capable of identifying compounds that are effective in the treatment and/or diagnosis of disease. Ligands and compounds according to the sixth and seventh aspects of the invention may be identified using such methods. These methods are included as aspects of the present invention.

Another aspect of this invention resides in the use of an INTP041, INTP042 or INTP043 gene or polypeptide as a target for the screening of candidate drug modulators, particularly candidate drugs active against disorders in which members of the glycoside hydrolase family 31 family of proteins are implicated.

A further aspect of this invention resides in methods of screening of compounds for therapy of glycoside hydrolase family 31 related disorders, comprising determining the ability of a compound to bind to an INTP041, INTP042 or INTP043 gene or polypeptide, or a fragment thereof.

In an eighth aspect, the invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in therapy or diagnosis of a disease or disorder in which members of the glycoside hydrolase family 31 family of proteins are implicated. Such diseases and disorders may include reproductive disorders, cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis¹ sarcoma; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, multiple sclerosis, peripheral nervous system disease and pain; developmental disorders; metabolic disorders including diabetes mellitus, hypoglycemia, osteoporosis, and obesity, AIDS and renal disease; infections including viral infection, bacterial infection, fungal infection and parasitic infection and other pathological conditions. Preferably, the disease is one in which glycoside hydrolase family 31 proteins are implicated, particularly diabetes and sugar metabolism related disorders.

Thus, embodiments of the invention provide for the use of the polypeptides in the treatment of sugar metabolism related disorders (in particular diabetes) or cancer.

In one embodiment of the invention, the cancer is selected from carcinoma, including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma or leukemia. In one embodiment of the invention, the cancer is selected from lung, colorectal, breast, pancreas and head and neck carcinomas.

The moieties of the present invention (i.e. the polypeptides of the first aspect of the invention, a nucleic acid molecule of the second or third aspect of the invention, a vector of the fourth aspect of the invention, a host cell of the fifth aspect of the invention, a ligand of the sixth aspect of the invention, a compound of the seventh aspect of the invention) may have particular utility in the therapy or diagnosis of disorders/diseases (the two terms are used interchangeably herein) of sugar metabolism disorders and diabetes.

In a ninth aspect, the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide of the first aspect of the invention or the activity of a polypeptide of the first aspect of the invention in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease. Such a method will preferably be carried out in vitro. Similar methods may be used for monitoring the therapeutic treatment of disease in a patient, wherein altering the level of expression or activity of a polypeptide or nucleic acid molecule over the period of time towards a control level is indicative of regression of disease.

A preferred method for detecting polypeptides of the first aspect of the invention comprises the steps of: (a) contacting a ligand, such as an antibody, of the sixth aspect of the invention with a biological sample under conditions suitable for the formation of a ligand- polypeptide complex; and (b) detecting said complex.

A number of different such methods according to the ninth aspect of the invention exist, as the skilled reader will be aware, such as methods of nucleic acid hybridization with short probes, point mutation analysis, polymerase chain reaction (PCR) amplification and methods using antibodies to detect aberrant protein levels. Similar methods may be used on a short or long term basis to allow therapeutic treatment of a disease to be monitored in a patient. The invention also provides kits that are useful in these methods for diagnosing disease.

In a tenth aspect, the invention provides for the use of a polypeptide of the first aspect of the invention as a glycoside hydrolase family 31 protein. Suitable uses of the polypeptides of the invention as glycoside hydrolase family 31 proteins include use as a regulator of cellular growth, metabolism or differentiation, use as part of a receptor/ligand pair and use as a diagnostic marker for a physiological or pathological condition selected from the list given above.

In an eleventh aspect, the invention provides a pharmaceutical composition comprising a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, in conjunction with a pharmaceutically- acceptable earner.

In a twelfth aspect, the present invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in the manufacture of a medicament for the diagnosis or treatment of a disease. Such diseases and disorders may include reproductive disorders, cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis' sarcoma; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, multiple sclerosis, peripheral nervous system disease and pain; developmental disorders; metabolic disorders including diabetes mellitus, hypoglycemia, osteoporosis, and obesity, AIDS and renal disease; infections including viral infection, bacterial infection, fungal infection and parasitic infection and other pathological conditions. Preferably, the disease is one in which glycoside hydrolase family 31 proteins are implicated, particularly diabetes and sugar metabolism related disorders.

Thus, embodiments of the invention provide for the use of the polypeptides of the invention in the manufacture of a medicament for the treatment of sugar metabolism related disorders (in particular diabetes) or cancer.

In a thirteenth aspect, the invention provides a method of treating a disease in a patient comprising administering to the patient a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention. For diseases in which the expression of a natural gene encoding a polypeptide of the first aspect of the invention, or in which the activity of a polypeptide of the first aspect of the invention, is lower in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an agonist. Conversely, for diseases in which the expression of the natural gene or activity of the polypeptide is higher in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an antagonist. Examples of such antagonists include antisense nucleic acid molecules, ribozymes and ligands, such as antibodies.

The INTP041, INTP042 and INTP043 proteins have roles in many disease states. Antagonists of the INTP041, INTP042 and INTP043 are of particular interest as they provide a way of modulating these disease states.

In a fourteenth aspect, the invention provides transgenic or knockout non-human animals that have been transformed to express higher, lower or absent levels of a polypeptide of the first aspect of the invention. Such transgenic animals are very useful models for the study of disease and may also be used in screening regimes for the identification of compounds that are effective in the treatment or diagnosis of such a disease.

As used herein, "functional equivalent" refers to a protein or nucleic acid molecule that possesses functional or structural characteristics that are substantially similar to a polypeptide or nucleic acid molecule of the present invention. A functional equivalent of a protein may contain modifications depending on the necessity of such modifications for the performance of a specific function. The term "functional equivalent" is intended to include the fragments, mutants, hybrids, variants, analogs, or chemical derivatives of a molecule.

Preferably, the "functional equivalent" may be a protein or nucleic acid molecule that exhibits any one or more of the functional activities of the polypeptides of the present invention.

Preferably, the "functional equivalent" may be a protein or nucleic acid molecule that displays substantially similar activity compared with INTP041, INTP042, INTP043, or fragments thereof in a suitable assay for the measurement of biological activity or function. Preferably, the "functional equivalent" may be a protein or nucleic acid molecule that displays identical or higher activity compared with INTP041, INTP042, INTP043, or fragments thereof in a suitable assay for the measurement of biological activity or function. Preferably, the "functional equivalent" may be a protein or nucleic acid molecule that displays 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100% or more activity compared with INTP041, INTP042, INTP043, or fragments thereof in a suitable assay for the measurement of biological activity or function.

Preferably, the "functional equivalent" may be a protein or polypeptide capable of exhibiting a substantially similar in vivo or in vitro activity as the polypeptides of the invention. Preferably, the "functional equivalent" may be a protein or polypeptide capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the corresponding portion of the polypeptides of the invention would. For example, a "functional equivalent" would be able, in an immunoassay, to diminish the binding of an antibody to the corresponding peptide {i.e., the peptide the amino acid sequence of which was modified to achieve the "functional equivalent") of the polypeptide of the invention, or to the polypeptide of the invention itself, where the antibody was raised against the corresponding peptide of the polypeptide of the invention. An equimolar concentration of the functional equivalent will diminish the aforesaid binding of the corresponding peptide by at least about 5%, preferably between about 5% and 10%, more preferably between about 10% and 25%, even more preferably between about 25% and 50%, and most preferably between about 40% and 50%.

For example, functional equivalents can be fully functional or can lack function in one or more activities. Thus, in the present invention, variations can affect the function, for example, of the activities of the polypeptide that reflect its possession of a glycoside hydrolase family 31 domain.

A summary of standard techniques and procedures which may be employed in order to utilise the invention is given below. It will be understood that this invention is not limited to the particular methodology, protocols, cell lines, vectors and reagents described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and it is not intended that this terminology should limit the scope of the present invention. The extent of the invention is limited only by the terms of the appended claims.

Standard abbreviations for nucleotides and amino acids are used in this specification.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology, which are within the skill of those working in the art. Such techniques are explained folly in the literature. Examples of particularly suitable texts for consultation include the following: Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and II (D.N Glover ed. 1985); Oligonucleotide Synthesis (MJ. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & SJ. Higgins eds. 1984); Transcription and Translation (B.D. Hames & SJ. Higgins eds. 1984); Animal Cell Culture (R.I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J.H. Miller and M.P. Calos eds. 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds. 1987, Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer Verlag, N.Y.); and Handbook of Experimental Immunology, Volumes I-IV (D.M. Weir and C. C. Blackwell eds. 1986).

As used herein, the term "polypeptide" includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. This term refers both to short chains (peptides and oligopeptides) and to longer chains (proteins).

The polypeptide of the present invention may be in the form of a mature protein or may be a pre-, pro- or prepro- protein that can be activated by cleavage of the pre-, pro- or prepro- portion to produce an active mature polypeptide. In such polypeptides, the pre-, pro- or prepro- sequence may be a leader or secretory sequence or may be a sequence that is employed for purification of the mature polypeptide sequence.

The polypeptide of the first aspect of the invention may form part of a fusion protein. For example, it is often advantageous to include one or more additional amino acid sequences which may contain secretory or leader sequences, pro-sequences, sequences which aid in purification, or sequences that confer higher protein stability, for example during recombinant production. Alternatively or additionally, the mature polypeptide may be fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol).

In a further preferred embodiment, a polypeptide of the invention, that may comprise a sequence having at least 85% of homology with INTP041, is a fusion protein.

These fusion proteins can be obtained by cloning a polynucleotide encoding a polypeptide comprising a sequence having at least 85% of homology with INTP041 in frame to the coding sequences for a heterologous protein sequence.

The term "heterologous", when used herein, is intended to designate any polypeptide other than a human INTP041 polypeptide.

Example of heterologous sequences, that can be comprised in the soluble fusion proteins either at N- or at C-terminus, are the following: extracellular domains of membrane-bound protein, immunoglobulin constant regions (Fc region), multimerization domains, domains of extracellular proteins, signal sequences, export sequences, or sequences allowing purification by affinity chromatography.

Many of these heterologous sequences are commercially available in expression plasmids since these sequences are commonly included in the fusion proteins in order to provide additional properties without significantly impairing the specific biological activity of the protein fused to them (Terpe K, Appl Microbiol Biotechnol, 60: 523-33, 2003). Examples of such additional properties are a longer lasting half-life in body fluids, the extracellular localization, or an easier purification procedure as allowed by the a stretch of Histidines forming the so-called "histidine tag"(Gentz et al., Proc Natl Acad Sci USA, 86: 821-4, 1989) or by the "HA"tag, an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell, 37: 767-78, 1994). If needed, the heterologous sequence can be eliinated by a proteolytic cleavage, for example by inserting a proteolytic cleavage site between the protein and the heterologous sequence, and exposing the purified fusion protein to the appropriate protease. These features are of particular importance for the fusion proteins since they facilitate their production and use in the preparation of pharmaceutical compositions. For example, the protein used in the examples (INTP041) can be purified by means of a hexa-histidine peptide fused at the C-terminus of INTP041. When the fusion protein comprises an immunoglobulin region, the fusion may be direct, or via a short linker peptide which can be as short as 1 to 3 amino acid residues in length or longer, for example, 13 amino acid residues in length. Said linker may be a tripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or a 13-amino acid linker sequence comprising Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met (SEQ ID NO: 199) introduced between the sequence of the substances of the invention and the immunoglobulin sequence. The resulting fusion protein has improved properties, such as an extended residence time in body fluids (half-life), increased specific activity, increased expression level, or the purification of the fusion protein is facilitated. In a preferred embodiment, the protein is fused to the constant region of an Ig molecule. Preferably, it is fused to heavy chain regions, like the CH2 and CH3 domains of human IgGl, for example. Other iso forms of Ig molecules are also suitable for the generation of fusion proteins according to the present invention, such as isoforms IgG₂ or IgG₄, or other Ig classes, like IgM or IgA, for example. Fusion proteins may be monomelic or multimeric, hetero- or homomultimeric.

In a further preferred embodiment, the functional derivative comprises at least one moiety attached to one or more functional groups, which occur as one or more side chains on the amino acid residues. Preferably, the moiety is a polyethylene (PEG) moiety. PEGylation may be carried out by known methods, such as the ones described in WO99/55377, for example.

Polypeptides may contain amino acids other than the 20 gene-encoded amino acids, modified either by natural processes, such as by post-translational processing or by chemical modification techniques which are well known in the art. Among the known modifications which may commonly be present in polypeptides of the present invention are glycosylation, lipid attachment, sulphation, gamma-carboxylation, for instance of glutamic acid residues, hydroxylation and ADP-ribosylation. Other potential modifications include acetylation, acylation, amidation, covalent attachment of flavin, covalent attachment of a haeme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulphide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, GPI anchor formation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. In fact, blockage of the amino or carboxyl terminus in a polypeptide, or both, by a covalent modification is common in naturally-occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention.

The modifications that occur in a polypeptide often will be a function of how the polypeptide is made. For polypeptides that are made recombinantly, the nature and extent of the modifications in large part will be determined by the post-translational modification capacity of the particular host cell and the modification signals that are present in the amino acid sequence of the polypeptide in question. For instance, glycosylation patterns vary between different types of host cell.

The polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally-occurring polypeptides (for example purified from cell culture), recombinantly-produced polypeptides (including fusion proteins), synthetically-produced polypeptides or polypeptides that are produced by a combination of these methods.

The functionally-equivalent polypeptides of the first aspect of the invention may be polypeptides that are homologous to the INTP041, INTP042 and INTP043 polypeptides. Two polypeptides are said to be "homologous", as the term is used herein, if the sequence of one of the polypeptides has a high enough degree of identity or similarity to the sequence of the other polypeptide. "Identity" indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. "Similarity" indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. Degrees of identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).

Homologous polypeptides therefore include natural biological variants (for example, allelic variants or geographical variations within the species from which the polypeptides are derived) and mutants (such as mutants containing amino acid substitutions, insertions or deletions) of the INTP041, INTP042 and INTP043 polypeptides. Such mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code. Typical such substitutions are among Ala, VaI, Leu and He; among Ser and Thr; among the acidic residues Asp and GIu; among Asn and GIn; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr. Particularly preferred are variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or added in any combination. Especially preferred are silent substitutions, additions and deletions, which do not alter the properties and activities of the protein. Also especially preferred in this regard are conservative substitutions. Such mutants also include polypeptides in which one or more of the amino acid residues includes a substituent group.

In accordance with the present invention, any substitution should be preferably a "conservative" or "safe" substitution, which is commonly defined a substitution introducing an amino acids having sufficiently similar chemical properties (e.g. a basic, positively charged amino acid should be replaced by another basic, positively charged amino acid), in order to preserve the structure and the biological function of the molecule.

The literature provide many models on which the selection of conservative amino acids substitutions can be performed on the basis of statistical and physico-chemical studies on the sequence and/or the structure of proteins (Rogov SI and Nekrasov AN, 2001). Protein design experiments have shown that the use of specific subsets of amino acids can produce foldable and active proteins, helping in the classification of amino acid "synonymous" substitutions which can be more easily accommodated in protein structure, and which can be used to detect functional and structural homologs and paralogs (Murphy LR et al., 2000). The groups of synonymous amino acids and the groups of more preferred synonymous amino acids are shown in Table 1.

Specific, non-conservative mutations can be also introduced in the polypeptides of the invention with different purposes. Mutations reducing the affinity of the glycoside hydrolase family 31 -like protein may increase its ability to be reused and recycled, potentially increasing its therapeutic potency (Robinson CR, 2002). Immunogenic epitopes eventually present in the polypeptides of the invention can be exploited for developing vaccines (Stevanovic S, 2002), or eliminated by modifying their sequence following known methods for selecting mutations for increasing protein stability, and correcting them (van den Burg B and Eijsink V, 2002; WO 02/05146, WO 00/34317, WO 98/52976).

Preferred alternative, synonymous groups for amino acids derivatives included in peptide mimetics are those defined in Table 2. A non-exhaustive list of amino acid derivatives also include aminoisobutyric acid (Aib), hydro xyproline (Hyp), 1,2,3,4-tetrahydro-isoquinoline- 3 -COOH, indoline-2carboxylic acid, 4-difluoro-proline, L- thiazolidine-4-carboxylic acid, L-homoproline, 3,4-dehydro-proline, 3,4-dihydroxy-phenylalanine, cyclohexyl-glycine, and phenylglycine.

By "amino acid derivative" is intended an amino acid or amino acid-like chemical entity other than one of the 20 genetically encoded naturally occurring amino acids. In particular, the amino acid derivative may contain substituted or non-substituted, linear, branched, or cyclic alkyl moieties, and may include one or more heteroatoms. The amino acid derivatives can be made de novo or obtained from commercial sources (Calbiochem- Novabiochem AG, Switzerland; Bachem, USA).

Various methodologies for incorporating unnatural amino acids derivatives into proteins, using both in vitro and in vivo translation systems, to probe and/or improve protein structure and function are disclosed in the literature (Dougherty DA, 2000). Techniques for the synthesis and the development of peptide mimetics, as well as non-peptide mimetics, are also well known in the art (Golebiowski A et al, 2001; Hruby VJ and Balse PM, 2000; Sawyer TK, in "Structure Based Drug Design", edited by Veerapandian P, Marcel Dekker Inc., pg. 557-663, 1997).

Typically, greater than 30% identity between two polypeptides is considered to be an indication of functional equivalence. Preferably, functionally equivalent polypeptides of the first aspect of the invention have a degree of sequence identity with the INTP041, INTP042 or INTP043 polypeptides, or with active fragments thereof, of greater than 80%. More preferred polypeptides have degrees of identity of greater than 85%, 90%, 95%, 98% or 99%, respectively.

The functionally-equivalent polypeptides of the first aspect of the invention may also be polypeptides which have been identified using one or more techniques of structural alignment. For example, the Inpharmatica Genome Threader technology that forms one aspect of the search tools used to generate the Biopendium™ search database may be used (see PCT application WO 01/69507) to identify polypeptides of presently-unknown function which, while having low sequence identity as compared to the INTP041, INTP042 and INTP043 polypeptides, are predicted to be members of the glycoside hydrolase family 31 family of proteins, by virtue of sharing significant structural homology with the INTP041, INTP042 and INTP043 polypeptide sequences. By "significant structural homology" is meant that the Inpharmatica Genome Threader predicts two proteins to share structural homology with a certainty of 10% and above. The polypeptides of the first aspect of the invention also include fragments of the INTP 041, INTP042 and INTP043 polypeptides and fragments of the functional equivalents of the INTP041, INTP042 and INTP043 polypeptides, provided that those fragments are members of the glycoside hydrolase family 31 family of proteins or have an antigenic determinant in common with the INTP041, INTP042 and INTP043 polypeptides.

As used herein, the term "fragment" refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of the amino acid sequence of the INTP041, INTP042 and INTP043 polypeptides or one of their functional equivalents. The fragments should comprise at least n consecutive amino acids from the sequence and, depending on the particular sequence, n preferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more). Small fragments may form an antigenic determinant.

Nucleic acid fragments according to the invention are preferably 10-5400 nucleotides in length, preferably 100-5000 nucleotides, preferably 500-4000 nucleotides, preferably 1000- 3000 nucleotides, preferably 1500-2000 nucleotides in length. Polypeptide fragments according to the invention are preferably 5-1800 amino acids in length, preferably 10-1500, preferably 50-1000, preferably 100-500 amino acids in length.

Fragments of the full length INTP041, INTP042 and INTP043 polypeptides may consist of combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46 of neighbouring exon sequences in the INTP041, INTP042 or INTP043 polypeptide sequences, respectively. Fragments may also consist of combinations of different domains of the INTP041, ΓNTP042 and/or INTP043 proteins, for instance a fragment may comprise or consist of the glycosyl transferase 31 domains, the INTP041-D1 and/or the INTP041-D2 domains (see example 4).

Such fragments may be "free-standing", i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region. When comprised within a larger polypeptide, the fragment of the invention most preferably forms a single continuous region. For instance, certain preferred embodiments relate to a fragment having a pre- and/or pro- polypeptide region fused to the amino terminus of the fragment and/or an additional region fused to the carboxyl terminus of the fragment. However, several fragments may be comprised within a single larger polypeptide. The polypeptides of the present invention or their immunogenic fragments (comprising at least one antigenic determinant) can be used to generate ligands, such as polyclonal or monoclonal antibodies, that are immunospecific for the polypeptides. Such antibodies may be employed to isolate or to identify clones expressing the polypeptides of the invention or to purify the polypeptides by affinity chromatography. The antibodies may also be employed as diagnostic or therapeutic aids, amongst other applications, as will be apparent to the skilled reader.

The term "immunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art. As used herein, the term "antibody" refers to intact molecules as well as to fragments thereof, such as Fab, F(ab')2 and Fv, which are capable of binding to the antigenic determinant in question. Such antibodies thus bind to the polypeptides of the first aspect of the invention.

By "substantially greater affinity" we mean that there is a measurable increase in the affinity for a polypeptide of the invention as compared with the affinity for known secreted proteins.

Preferably, the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 10³-fold, 10⁴- fold, 10⁵-fold or 10⁶-fold greater for a polypeptide of the invention than for known members of the glycoside hydrolase family 31 family of proteins. Preferably, there is a measurable increase in the affinity of a polypeptide of the invention as compared with known members of the glycoside hydrolase family 31 family of proteins. Preferably, there is a measurable increase in the affinity of a polypeptide of the invention as compared with natural members of the glycoside hydrolase family 31 family of proteins.

If polyclonal antibodies are desired, a selected mammal, such as a mouse, rabbit, goat or horse, may be immunised with a polypeptide of the first aspect of the invention. The polypeptide used to immunise the animal can be derived by recombinant DNA technology or can be synthesized chemically. If desired, the polypeptide can be conjugated to a carrier protein. Commonly used carriers to which the polypeptides may be chemically coupled include bovine serum albumin, thyroglobulin and keyhole limpet haemocyanin. The coupled polypeptide is then used to immunise the animal. Serum from the immunised animal is collected and treated according to known procedures, for example by immunoaffinity chromatography. Monoclonal antibodies to the polypeptides of the first aspect of the invention can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies using hybridoma technology is well known (see, for example, Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985).

Panels of monoclonal antibodies produced against the polypeptides of the first aspect of the invention can be screened for various properties, i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies are particularly useful in purification of the individual polypeptides against which they are directed. Alternatively, genes encoding the monoclonal antibodies of interest may be isolated from hybridomas, for instance by PCR techniques known in the art, and cloned and expressed in appropriate vectors.

Chimeric antibodies, in which non-human variable regions are joined or fused to human constant regions (see, for example, Liu et al., Proc. Natl. Acad. Sci. USA, 84, 3439 (1987)), may also be of use.

The antibody may be modified to make it less immunogenic in an individual, for example by humanisation (see Jones et al, Nature, 321, 522 (1986); Verhoeyen et al, Science, 239, 1534 (1988); Kabat et al, J. Immunol., 147, 1709 (1991); Queen et al, Proc. Natl Acad. Sci. USA, 86, 10029 (1989); Gorman et al, Proc. Natl Acad. Sci. USA, 88, 34181 (1991); and Hodgson et al, Bio/Technology, 9, 421 (1991)). The term "humanised antibody", as used herein, refers to antibody molecules in which the CDR amino acids and selected other amino acids in the variable domains of the heavy and/or light chains of a non-human donor antibody have been substituted in place of the equivalent amino acids in a human antibody. The humanised antibody thus closely resembles a human antibody but has the binding ability of the donor antibody. hi a further alternative, the antibody may be a "bispecific" antibody, that is an antibody having two different antigen binding domains, each domain being directed against a different epitope.

Phage display technology may be utilised to select genes which encode antibodies with binding activities towards the polypeptides of the invention either from repertoires of PCR amplified V-genes of lymphocytes from humans screened for possessing the relevant antibodies, or from naive libraries (McCafferty, J. et al, (1990), Nature 348, 552-554; Marks, J. et al, (1992) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al, (1991) Nature 352, 624-628).

Antibodies generated by the above techniques, whether polyclonal or monoclonal, have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). In these applications, the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme.

Preferred nucleic acid molecules of the second and third aspects of the invention are those which encode a polypeptide sequence as recited SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO.70, SEQ ID NO.72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:178, SEQ ID NO: 180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO: 190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO: 196, SEQ ID NO: 198 and functionally equivalent polypeptides. These nucleic acid molecules may be used in the methods and applications described herein. The nucleic acid molecules of the invention preferably comprise at least n consecutive nucleotides from the sequences disclosed herein where, depending on the particular sequence, n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).

The nucleic acid molecules of the invention also include sequences that are complementary to nucleic acid molecules described above (for example, for antisense or probing purposes).

Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance cDNA, synthetic DNA or genomic DNA. Such nucleic acid molecules may be obtained by cloning, by chemical synthetic techniques or by a combination thereof. The nucleic acid molecules can be prepared, for example, by chemical synthesis using techniques such as solid phase phosphoramidite chemical synthesis, from genomic or cDNA libraries or by separation from an organism. RNA molecules may generally be generated by the in vitro or in vivo transcription of DNA sequences.

The nucleic acid molecules may be double-stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the anti-sense strand.

The term "nucleic acid molecule" also includes analogues of DNA and RNA, such as those containing modified backbones, and peptide nucleic acids (PNA). The term "PNA", as used herein, refers to an antisense molecule or an anti-gene agent which comprises an oligonucleotide of at least five nucleotides in length linked to a peptide backbone of amino acid residues, which preferably ends in lysine. The terminal lysine confers solubility to the composition. PNAs may be pegylated to extend their lifespan in a cell, where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P. E. et al. (1993) Anticancer Drug Des. 8:53-63).

A nucleic acid molecule which encodes a polypeptide of this invention may be identical to the coding sequence of one or more of the nucleic acid molecules disclosed herein.

These molecules also may have a different sequence which, as a result of the degeneracy of the genetic code, encodes a polypeptide SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO.46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO.76, SEQ ID NO.78, SEQ ID NO.80, SEQ ID NO.82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO: 110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116. SEQ E) NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ E) NO: 132, SEQ ID NO: 134, SEQ ID NO:136, SEQ E) NO:138, SEQ E⁾ NO:140, SEQ E) NO:142, SEQ ID NO:144, SEQ ID NO: 146, SEQ E⁾ NO: 148, SEQ E) NO: 150, SEQ E) NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ TD NO: 158, SEQ E) NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ E) NO:166, SEQ ID NO:168, SEQ E) NO:170, SEQ ID NO:172 SEQ ID NO: 174, SEQ ID NO.-176, SEQ E) NO:178, SEQ ID NO: 180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ E) NO: 192, SEQ ID NO: 194, SEQ ID NO:186, SEQ E) NO:198. Such nucleic acid molecules may include, but are not limited to, the coding sequence for the mature polypeptide by itself; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pro-, pre- or prepro- polypeptide sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with further additional, non-coding sequences, including non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription (including termination signals), ribosome binding and mRNA stability. The nucleic acid molecules may also include additional sequences which encode additional amino acids, such as those which provide additional functionalities.

The nucleic acid molecules of the second and third aspects of the invention may also encode the fragments or the functional equivalents of the polypeptides and fragments of the first aspect of the invention. Such a nucleic acid molecule may be a naturally-occurring variant such as a naturally-occurring allelic variant, or the molecule may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the nucleic acid molecule may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.

Among variants in this regard are variants that differ from the aforementioned nucleic acid molecules by nucleotide substitutions, deletions or insertions. The substitutions, deletions or insertions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or insertions.

The nucleic acid molecules of the invention can also be engineered, using methods generally known in the art, for a variety of reasons, including modifying the cloning, processing, and/or expression of the gene product (the polypeptide). DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides are included as techniques which may be used to engineer the nucleotide sequences. Site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations and so forth.

Nucleic acid molecules which encode a polypeptide of the first aspect of the invention may be ligated to a heterologous sequence so that the combined nucleic acid molecule encodes a fusion protein. Such combined nucleic acid molecules are included within the second or third aspects of the invention. For example, to screen peptide libraries for inhibitors of the activity of the polypeptide, it may be useful to express, using such a combined nucleic acid molecule, a fusion protein that can be recognised by a commercially-available antibody. A fusion protein may also be engineered to contain a cleavage site located between the sequence of the polypeptide of the invention and the sequence of a heterologous protein so that the polypeptide may be cleaved and purified away from the heterologous protein.

The nucleic acid molecules of the invention also include antisense molecules that are partially complementary to nucleic acid molecules encoding polypeptides of the present invention and that therefore hybridize to the encoding nucleic acid molecules (hybridization). Such antisense molecules, such as oligonucleotides, can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide of the invention, as will be known by those of ordinary skill in the art (see, for example, Cohen, J.S., Trends in Pharm. ScL, 10, 435 (1989), Okano, J. Neurochem. 56, 560 (1991); O'Connor, J. Neurochem 56, 560 (1991); Lee et al, Nucleic Acids Res 6, 3073 (1979); Cooney et al, Science 241, 456 (1988); Dervan et al, Science 251, 1360 (1991).

The term "hybridization" as used here refers to the association of two nucleic acid molecules with one another by hydrogen bonding. Typically, one molecule will be fixed to a solid support and the other will be free in solution. Then, the two molecules may be placed in contact with one another under conditions that favour hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase molecule to the solid support (Denhardt's reagent or BLOTTO); the concentration of the molecules; use of compounds to increase the rate of association of molecules (dextran sulphate or polyethylene glycol); and the stringency of the washing conditions following hybridization (see Sambrook et al. [supra]).

The inhibition of hybridization of a completely complementary molecule to a target molecule may be examined using a hybridization assay, as known in the art (see, for example, Sambrook et al. [supra]). A substantially homologous molecule will then compete for and inhibit the binding of a completely homologous molecule to the target molecule under various conditions of stringency, as taught in Wahl, G.M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A.R. (1987; Methods Enzymol. 152:507-511).

"Stringency" refers to conditions in a hybridization reaction that favour the association of very similar molecules over association of molecules that differ. High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (15OmM NaCl, 15mM trisodium citrate), 5OmM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C. Low stringency conditions involve the hybridisation reaction being carried out at 35°C (see Sambrook et al. [supra]). Preferably, the conditions used for hybridization are those of high stringency.

Preferred embodiments of this aspect of the invention are nucleic acid molecules that are at least 70% identical over their entire length to a nucleic acid molecule encoding the INTP041, INTP042 and INTP043 polypeptides and nucleic acid molecules that are substantially complementary to such nucleic acid molecules. Preferably, a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to such coding sequences, or is a nucleic acid molecule that is complementary thereto. In this regard, nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98%, 99% or more identical over their entire length to the same are particularly preferred. Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the INTP041, INTP042 and INTP043 polypeptides.

The invention also provides a process for detecting a nucleic acid molecule of the invention, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting any such duplexes that are formed.

As discussed additionally below in connection with assays that may be utilised according to the invention, a nucleic acid molecule as described above may be used as a hybridization probe for RNA, cDNA or genomic DNA, in order to isolate full-length cDNAs and genomic clones encoding the INTP041, INTP042 and INTP043 polypeptides and to isolate cDNA and genomic clones of homologous or orthologous genes that have a high sequence similarity to the gene encoding this polypeptide.

In this regard, the following techniques, among others known in the art, may be utilised and are discussed below for purposes of illustration. Methods for DNA sequencing and analysis are well known and are generally available in the art and may, indeed, be used to practice many of the embodiments of the invention discussed herein. Such methods may employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase (US Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, IL), or combinations of polymerases and proof-reading exonucl eases such as those found in the ELONGASE Amplification System marketed by Gibco/BRL (Gaithersburg, MD). Preferably, the sequencing process may be automated using machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).

One method for isolating a nucleic acid molecule encoding a polypeptide with an equivalent function to that of the INTP041, INTP042 and INTP043 polypeptides is to probe a genomic or cDNA library with a natural or artificially-designed probe using standard procedures that are recognised in the art (see, for example, "Current Protocols in Molecular Biology", Ausubel et al. (eds). Greene Publishing Association and John Wiley Interscience, New York, 1989,1992). Probes comprising at least 15, preferably at least 30, and more preferably at least 50, contiguous bases that correspond to, or are complementary to, nucleic acid sequences from the appropriate encoding gene (SEQ ID NO:1, SEQ JJD NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ DD NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO:173, NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO: 195, SEQ ID NO: 197), are particularly useful probes. Such probes may be labelled with an analytically-detectable reagent to facilitate their identification. Useful reagents include, but are not limited to, radioisotopes, fluorescent dyes and enzymes that are capable of catalysing the formation of a detectable product. Using these probes, the ordinarily skilled artisan will be capable of isolating complementary copies of genomic DNA, cDNA or RNA polynucleotides encoding proteins of interest from human, mammalian or other animal sources and screening such sources for related sequences, for example, for additional members of the family, type and/or subtype.

In many cases, isolated cDNA sequences will be incomplete, in that the region encoding the polypeptide will be cut short, normally at the 5' end. Several methods are available to obtain full length cDNAs, or to extend short cDNAs. Such sequences may be extended utilising a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed is based on the method of Rapid Amplification of cDNA Ends (RACE; see, for example, Frohman et al, PNAS USA 85, 8998-9002, 1988). Recent modifications of this technique, exemplified by the Marathon™ technology (Clontech Laboratories Inc.), for example, have significantly simplified the search for longer cDNAs. A slightly different technique, termed "restriction-site" PCR, uses universal primers to retrieve unknown nucleic acid sequence adjacent a known locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). Inverse PCR may also be used to amplify or to extend sequences using divergent primers based on a known region (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic, 1, 111-119). Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991); Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PromoterFinder™ libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use libraries that have been size- selected to include larger cDNAs. Also, random-primed libraries are preferable, in that they will contain more sequences that contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.

In one embodiment of the invention, the nucleic acid molecules of the present invention may be used for chromosome localisation. In this technique, a nucleic acid molecule is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present invention is an important step in the confirmatory correlation of those sequences with the gene-associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library). The relationships between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localised by genetic linkage to a particular genomic region, any sequences mapping to that area may represent associated or regulatory genes for further investigation. The nucleic acid molecule may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.

The nucleic acid molecules of the present invention are also valuable for tissue localisation. Such techniques allow the determination of expression patterns of the polypeptide in tissues by detection of the mRNAs that encode them. These techniques include in situ hybridization techniques and nucleotide amplification techniques, such as PCR. Results from these studies provide an indication of the normal functions of the polypeptide in the organism. In addition, comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by a mutant gene provide valuable insights into the role of mutant polypeptides in disease. Such inappropriate expression may be of a temporal, spatial or quantitative nature.

Gene silencing approaches may also be undertaken to down-regulate endogenous expression of a gene encoding a polypeptide of the invention. RNA interference (RNAi) (Elbashir, SM et al, Nature 2001, 411, 494-498) is one method of sequence specific post- transcriptional gene silencing that may be employed. Short dsRNA oligonucleotides are synthesised in vitro and introduced into a cell. The sequence specific binding of these dsRNA oligonucleotides triggers the degradation of target mRNA, reducing or ablating target protein expression.

Efficacy of the gene silencing approaches assessed above may be assessed through the measurement of polypeptide expression (for example, by Western blotting), and at the RNA level using TaqMan-based methodologies.

The vectors of the present invention comprise nucleic acid molecules of the invention and may be cloning or expression vectors. The host cells of the invention, which may be transformed, transfected or transduced with the vectors of the invention may be prokaryotic or eukaryotic.

The polypeptides of the invention may be prepared in recombinant form by expression of their encoding nucleic acid molecules in vectors contained within a host cell. Such expression methods are well known to those of skill in the art and many are described in detail by Sambrook et al. (supra) and Fernandez & Hoeffler (1998, eds. "Gene expression systems. Using nature for the art of expression". Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto).

Generally, any system or vector that is suitable to maintain, propagate or express nucleic acid molecules to produce a polypeptide in the required host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well- known and routine techniques, such as, for example, those described in Sambrook et al, {supra). Generally, the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the transformed host cell.

Examples of suitable expression systems include, for example, chromosomal, episomal and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids. Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid. The vectors ρCR4-TOPO-INTP041-CP3/CP4 (Figure 11), pENTR_INTP041-Dl-6HIS (Figure 16), pEAK12d_ INTP041-D1-6HIS (Figure 17), pDEST12.2_INTP041-Dl-6HIS (Figure 18), ρENTR__INTP041-D2-6HIS (Figure 19), pEAK12dJNTP041-D2-6HIS (Figure 20), pDEST12.2_INTP041-D2-6HIS (Figure 21) are preferred examples of suitable vectors for use in accordance with the aspects of this invention relating to INTP041.

Particularly suitable expression systems include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (for example, baculovirus); plant cell systems transformed with virus expression vectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (for example, Ti or pBR322 plasmids); or animal cell systems. Cell-free translation systems can also be employed to produce the polypeptides of the invention.

Introduction of nucleic acid molecules encoding a polypeptide of the present invention into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al., (supra). Particularly suitable methods include calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection (see Sambrook et al, 1989 [supra]; Ausubel et al, 1991 [supra]; Spector, Goldman & Leinwald, 1998). In eukaryotic cells, expression systems may either be transient (for example, episomal) or permanent (chromosomal integration) according to the needs of the system.

The encoding nucleic acid molecule may or may not include a sequence encoding a control sequence, such as a signal peptide or leader sequence, as desired, for example, for secretion of the translated polypeptide into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals. Leader sequences can be removed by the bacterial host in post-translational processing.

In addition to control sequences, it may be desirable to add regulatory sequences that allow for regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those which cause the expression of a gene to be increased or decreased in response to a chemical or physical stimulus, including the presence of a regulatory compound or to various temperature or metabolic conditions. Regulatory sequences are those non-translated regions of the vector, such as enhancers, promoters and 5' and 3' untranslated regions. These interact with host cellular proteins to carry out transcription and translation. Such regulatory sequences may vary in their strength and specificity. Depending on the vector system and host utilised, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluescript phagemid (Stratagene, LaJoIIa, CA) or pSportl™ plasmid (Gibco BRL) and the like may be used. The baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (for example, heat shock, RUBISCO and storage protein genes) or from plant viruses (for example, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on SV40 or EBV may be used with an appropriate selectable marker.

An expression vector is constructed so that the particular nucleic acid coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the regulatory sequences being such that the coding sequence is transcribed under the "control" of the regulatory sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence. In some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.

The control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.

For long-term, high-yield production of a recombinant polypeptide, stable expression is preferred. For example, cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.

In the baculovirus system, the materials for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac" kit). These techniques are generally known to those skilled in the art and are described fully in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Particularly suitable host cells for use in this system include insect cells such as Drosophila S2 and Spodoptera Sf9 cells.

There are many plant cell culture and whole plant genetic expression systems known in the art. Examples of suitable plant cellular genetic expression systems include those described in US 5,693,506; US 5,659,122; and US 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30, 3861-3863 (1991).

In particular, all plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be utilised, so that whole plants are recovered which contain the transferred gene. Practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugar cane, sugar beet, cotton, fruit and other trees, legumes and vegetables.

Examples of particularly preferred bacterial host cells include streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells.

Examples of particularly suitable host cells for fungal expression include yeast cells (for example, S. cerevisiae) and Aspergillus cells.

Any number of selection systems are known in the art that may be used to recover transformed cell lines. Examples include the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11 :223-32) and adenine phosphoribosyltransferase (Lowy, I. et al (1980) Cell 22:817-23) genes that can be employed in tk^" or aprt* cells, respectively.

Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dihydrofolate reductase (DHFR) that confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al. (1981) J. MoI. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes have been described, examples of which will be clear to those of skill in the art.

Although the presence or absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the relevant sequence is inserted within a marker gene sequence, transformed cells containing the appropriate sequences can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a polypeptide of the invention under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

Alternatively, host cells that contain a nucleic acid sequence encoding a polypeptide of the invention and which express said polypeptide may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA- DNA or DNA-RNA hybridizations and protein bioassays, for example, fluorescence activated cell sorting (FACS) or immunoassay techniques (such as the enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]), that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein (see Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, MN) and Maddox, D.E. et al. (1983) J. Exp. Med, 158, 1211-1216).

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labelled hybridization or PCR probes for detecting sequences related to nucleic acid molecules encoding polypeptides of the present invention include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled polynucleotide. Alternatively, the sequences encoding the polypeptide of the invention may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesise RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labelled nucleotides. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, MI); Promega (Madison WI); and U.S. Biochemical Corp., Cleveland, OH)).

Suitable reporter molecules or labels, which may be used for ease of detection, include radionuclides, enzymes and fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Nucleic acid molecules according to the present invention may also be used to create transgenic animals, particularly rodent animals. Such transgenic animals form a further aspect of the present invention. This may be done locally by modification of somatic cells, or by genu line therapy to incorporate heritable modifications. Such transgenic animals may be particularly useful in the generation of animal models for drug molecules effective as modulators of the polypeptides of the present invention.

The polypeptide can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography is particularly useful for purification. Well known techniques for refolding proteins may be employed to regenerate an active conformation when the polypeptide is denatured during isolation and or purification.

Specialised vector constructions may also be used to facilitate purification of proteins, as desired, by joining sequences encoding the polypeptides of the invention to a nucleotide sequence encoding a polypeptide domain that will facilitate purification of soluble proteins. Examples of such purification-facilitating domains include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilised metals, protein A domains that allow purification on immobilised immunoglobulin, and the domain utilised in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the polypeptide of the invention may be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing the polypeptide of the invention fused to several histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilised metal ion affinity chromatography as described in Porath, J. et al. (1992), Prot. Exp. Purif. 3: 263-281) while the thioredoxin or enterokinase cleavage site provides a means for purifying the polypeptide from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll, DJ. et al. (1993; DNA Cell Biol. 12:441-453).

If the polypeptide is to be expressed for use in screening assays, generally it is preferred that it be produced at the surface of the host cell in which it is expressed. In this event, the host cells may be harvested prior to use in the screening assay, for example using techniques such as fluorescence activated cell sorting (FACS) or immunoaffinity techniques. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the expressed polypeptide. If polypeptide is produced intracellularly, the cells must first be lysed before the polypeptide is recovered.

As indicated above, the present invention also provides novel targets and methods for the screening of drug candidates or leads. These screening methods include binding assays and/or functional assays, and may be performed in vitro, in cell systems or in animals. In this regard, a particular object of this invention resides in the use of an INTP041, INTP042 or INTP043 polypeptide as a target for screening candidate drugs for treating or preventing glycoside hydrolase family 31 related disorders.

Another object of this invention resides in methods of selecting biologically active compounds, said methods comprising contacting a candidate compound with a INTP041 , INTP042 or INTP043 gene or polypeptide, and selecting compounds that bind said gene or polypeptide.

A further other object of this invention resides in methods of selecting biologically active compounds, said method comprising contacting a candidate compound with recombinant host cell expressing a INTP041, INTP042 or INTP043 polypeptide with a candidate compound, and selecting compounds that bind said INTP041, INTP042 or INTP043 polypeptide at the surface of said cells and/or that modulate the activity of the INTP041, INTP042 or INTP043 polypeptide.

A "biologically active" compound denotes any compound having biological activity in a subject, preferably therapeutic activity, more preferably a compound having glycoside hydrolase family 31 activity, and further preferably a compound that can be used for treating INTP041, INTP042 or INTP043 related disorders, or as a lead to develop drugs for treating glycoside hydrolase family 31 related disorder. A "biologically active" compound preferably is a compound that modulates the activity of INTP041, INTP042 or INTP043.

The above methods may be conducted in vitro, using various devices and conditions, including with immobilized reagents, and may further comprise an additional step of assaying the activity of the selected compounds in a model of glycoside hydrolase family 31 related disorder, such as an animal model.

Preferred selected compounds are agonists of INTP041, INTP042 or INTP043, i.e., compounds that can bind to INTP041, INTP042 or INTP043 and mimic the activity of an endogenous ligand thereof.

A further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a INTP041, INTP042 or INTP043 polypeptide according to the present invention and determining the ability of said test compound to modulate the activity of said INTP041, INTP042 or INTP043 polypeptide. A further object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a INTP041, INTP 042 or ESfTP 043 gene according to the present invention and determining the ability of said test compound to modulate the expression of said INTP 041, INTP042 or INTP043 gene, preferably to stimulate expression thereof.

In another embodiment, this invention relates to a method of screening, selecting or identifying active compounds, particularly compounds active on multiple sclerosis or related disorders, the method comprising contacting a test compound with a recombinant host cell comprising a reporter construct, said reporter construct comprising a reporter gene under the control of a INTP041, INTP042 or INTP043 gene promoter, and selecting the test compounds that modulate (e.g. stimulate or reduce, preferably stimulate) expression of the reporter gene.

The polypeptide of the invention can be used to screen libraries of compounds in any of a variety of drug screening techniques. Such compounds may activate (agonise) or inhibit (antagonise) the level of expression of the gene or the activity of the polypeptide of the invention and form a further aspect of the present invention. Preferred compounds are effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.

Agonist or antagonist compounds may be isolated from, for example, cells, cell-free preparations, chemical libraries or natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, enzymes, receptors or structural or functional mimetics. For a suitable review of such screening techniques, see Coligan et ah, Current Protocols in Immunology l(2):Chapter 5 (1991).

Binding to a target gene or polypeptide provides an indication as to the ability of the compound to modulate the activity of said target, and thus to affect a pathway leading to glycoside hydrolase family 31 related disorder in a subject. The determination of binding may be performed by various techniques, such as by labelling of the candidate compound, by competition with a labelled reference ligand, etc. For in vitro binding assays, the polypeptides may be used in essentially pure form, in suspension, immobilized on a support, or expressed in a membrane (intact cell, membrane preparation, liposome, etc.). Modulation of activity includes, without limitation, stimulation of the surface expression of the INTP(Ml. INTP042 or INTP043 receptor, modulation of multimerization of said receptor (e.g., the formation of multimeric complexes with other sub-units), etc. The cells used in the assays may be any recombinant cell (i.e., any cell comprising a recombinant nucleic acid encoding a INTP041, INTP042 or INTP043 polypeptide) or any cell that expresses an endogenous INTP041 , INTP042 or INTP043 polypeptide. Examples of such cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli, Pichia pastoήs, Hansenula polymorphs, Schizosaccharomyces pombe, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc).

Compounds that are most likely to be good antagonists are molecules that bind to the polypeptide of the invention without inducing the biological effects of the polypeptide upon binding to it. Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby inhibit or extinguish its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be inhibited, such that the normal biological activity of the polypeptide is prevented.

The polypeptide of the invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly. In general, such screening procedures may involve using appropriate cells or cell membranes that express the polypeptide that are contacted with a test compound to observe binding, or stimulation or inhibition of a functional response. The functional response of the cells contacted with the test compound is then compared with control cells that were not contacted with the test compound. Such an assay may assess whether the test compound results in a signal generated by activation of the polypeptide, using an appropriate detection system. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the test compound is observed.

A preferred method for identifying an agonist or antagonist compound of a polypeptide of the present invention comprises: (a) contacting a cell expressing (optionally on the surface thereof) the polypeptide according to the first aspect of the invention, the polypeptide being associated with a second component capable of providing a detectable signal in response to the binding of a compound to the polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and

(b) determining whether the compound binds to and activates or inhibits the polypeptide by measuring the level of a signal generated from the interaction of the compound with the polypeptide.

Methods for generating detectable signals in the types of assays described herein will be known to those of skill in the art. A particular example is cotransfecting a construct expressing a polypeptide according to the invention, or a fragment such as the LBD, in fusion with the GAL4 DNA binding domain, into a cell together with a reporter plasmid, an example of which is pFR-Luc (Stratagene Europe, Amsterdam, The Netherlands). This particular plasmid contains a synthetic promoter with five tandem repeats of GAL4 binding sites that control the expression of the luciferase gene. When a potential ligand is added to the cells, it will bind the GAL4-polypeptide fusion and induce transcription of the luciferase gene. The level of the luciferase expression can be monitored by its activity using a luminescence reader (see, for example, Lehman et al. JBC 270, 12953, 1995; Pawar et al. JBC, 277, 39243, 2002).

A further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises:

(a) contacting a labelled or unlabeled compound with the polypeptide immobilized on any solid support (for example beads, plates, matrix support, chip) and detection of the compound by measuring the label or the presence of the compound itself; or

(b) contacting a cell expressing on the surface thereof the polypeptide, by means of artificially anchoring it to the cell membrane, or by constructing a chimeric receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to the polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and

(c) determining whether the compound binds to and activates or inhibits the polypeptide by comparing the level of a signal generated from the interaction of the compound with the polypeptide with the level of a signal in the absence of the compound. For example, a method such as FRET detection of ligand bound to the polypeptide in the presence of peptide co-activators (Norris et al, Science 285, 744, 1999) might be used. A further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises:

(a) contacting a cell expressing (optionally on the surface thereof) the polypeptide, the polypeptide being associated with a second component capable of providing a detectable signal in response to the binding of a compound to the polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and

(b) determining whether the compound binds to and activates or inhibits the polypeptide by comparing the level of a signal generated from the interaction of the compound with the polypeptide with the level of a signal in the absence of the compound.

In further preferred embodiments, the general methods that are described above may further comprise conducting the identification of agonist or antagonist in the presence of labelled or unlabelled ligand for the polypeptide.

In another embodiment of the method for identifying agonist or antagonist of a polypeptide of the present invention comprises: determining the inhibition of binding of a ligand to cells which express a polypeptide of the invention (and which optionally have a polypeptide of the invention on the surface thereof), or to cell membranes containing such a polypeptide, in the presence of a candidate compound under conditions to permit binding to the polypeptide, and determining the amount of ligand bound to the polypeptide. A compound capable of causing reduction of binding of a ligand is considered to be an agonist or antagonist. Preferably the ligand is labelled.

More particularly, a method of screening for a polypeptide antagonist or agonist compound comprises the steps of:

(a) incubating a labelled ligand with a whole cell expressing a polypeptide according to the invention, optionally on the cell surface, or a cell membrane containing a polypeptide of the invention,

(b) measuring the amount of labelled ligand bound to the whole cell or the cell membrane;

(c) adding a candidate compound to a mixture of labelled ligand and the whole cell or the cell membrane of step (a) and allowing the mixture to attain equilibrium;

(d) measuring the amount of labelled ligand bound to the whole cell or the cell membrane after step (c); and (e) comparing the difference in the labelled ligand bound in step (b) and (d), such that the compound which causes the reduction in binding in step (d) is considered to be an agonist or antagonist.

Similarly, there is provided a method of screening for a polypeptide antagonist or agonist compound which comprises the steps of:

(a) incubating a labelled ligand with a polypeptide according to the invention on any solid support or the cell surface, or a cell membrane containing a polypeptide of the invention.

(b) measuring the amount of labelled ligand bound to the polypeptide on the solid support, whole cell or the cell membrane;

(c) adding a candidate compound to a mixture of labelled ligand and immobilized polypeptide on the solid support, the whole cell or the cell membrane of step (a) and allowing the mixture to attain equilibrium;

(d) measuring the amount of labelled ligand bound to the immobilized polypeptide or the whole cell or the cell membrane after step (c); and

(e) comparing the difference in the labelled ligand bound in step (b) and (d), such that the compound which causes the reduction in binding in step (d) is considered to be an agonist or antagonist.

The INTP041, INTP042 and INTP043 polypeptides of the present invention may modulate cellular growth and differentiation. Thus, the biological activity of the INTP041, INTP042 and INTP043 polypeptides can be examined in systems that allow the study of cellular growth and differentiation such as organ culture assays or in colony assay systems in agarose culture. Stimulation or inhibition of cellular proliferation may be measured by a variety of assays.

For example, for observing cell growth inhibition, one can use a solid or liquid medium. In a solid medium, cells undergoing growth inhibition can easily be selected from the subject cell group by comparing the sizes of colonies formed. In a liquid medium, growth inhibition can be screened by measuring culture medium turbity or incorporation of labelled thymidine in DNA. Typically, the incorporation of a nucleoside analog into newly synthesised DNA may be employed to measure proliferation {i.e., active cell growth) in a population of cells. For example, bromodeoxyuridine (BrdU) can be employed as a DNA labelling reagent and anti-BrdU mouse monoclonal antibodies can be employed as a detection reagent. This antibody binds only to cells containing DNA which has incorporated bromodeoxyuridine. A number of detection methods may be used in conjunction with this assay including immunofluorescence, immunohistochemical, ELISA, and colorimetric methods. Kits that include bromodeoxyuridine (BrdU) and anti-BrdU mouse monoclonal antibody are commercially available from Boehringer Mannheim (Indianapolis, IN).

The effect of the INTP041, INTP042 and INTP043 polypeptides upon cellular differentiation can be measured by contacting stem cells or embryonic cells with various amounts of the INTP041, INTP042 and INTP043 polypeptides and observing the effect upon differentiation of the stem cells or embryonic cells. Tissue-specific antibodies and microscopy may be used to identify the resulting cells.

Thus, the "functional equivalents" of the INTP041, INTP042 and INTP043 polypeptides include polypeptides that exhibit any of the same growth and differentiation regulating activities in the above-described assays in a dose-dependent manner. Although the degree of dose-dependent activity need not be identical to that of the INTP041, INTP042 and INTP043 polypeptides, preferably the "functional equivalents" will exhibit substantially similar dose-dependence in a given activity assay compared to the INTP041, INTP042 and INTP043 polypeptides.

In certain of the embodiments described above, simple binding assays may be used, in which the adherence of a test compound to a surface bearing the polypeptide is detected by means of a label directly or indirectly associated with the test compound or in an assay involving competition with a labelled competitor. In another embodiment, competitive drug screening assays may be used, in which neutralising antibodies that are capable of binding the polypeptide specifically compete with a test compound for binding. In this manner, the antibodies can be used to detect the presence of any test compound that possesses specific binding affinity for the polypeptide.

Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding the polypeptide in cells. For example, an ELISA may be constructed that measures secreted or cell-associated levels of polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. The formation of binding complexes between the polypeptide and the compound being tested may then be measured.

Assay methods that are also included within the terms of the present invention are those that involve the use of the genes and polypeptides of the invention in overexpression or ablation assays. Such assays involve the manipulation of levels of these genes/polypeptides in cells and assessment of the impact of this manipulation event on the physiology of the manipulated cells. For example, such experiments reveal details of signaling and metabolic pathways in which the particular genes/polypeptides are implicated, generate information regarding the identities of polypeptides with which the studied polypeptides interact and provide clues as to methods by which related genes and proteins are regulated.

Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564). In this method, large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with the polypeptide of the invention and washed. One way of immobilising the polypeptide is to use non-neutralising antibodies. Bound polypeptide may then be detected using methods that are well known in the art. Purified polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques.

The polypeptide of the invention may be used to identify membrane-bound or soluble receptors, through standard receptor binding techniques that are known in the art, such as ligand binding and crosslinking assays in which the polypeptide is labelled with a radioactive isotope, is chemically modified, or is fused to a peptide sequence that facilitates its detection or purification, and incubated with a source of the putative receptor (for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids). The efficacy of binding may be measured using biophysical techniques such as surface plasmon resonance and spectroscopy. Binding assays may be used for the purification and cloning of the receptor, but may also identify agonists and antagonists of the polypeptide, that compete with the binding of the polypeptide to its receptor. Standard methods for conducting screening assays are well understood in the art.

In another embodiment, this invention relates to the use of a INTP041, INTP042 or INTP043 polypeptide or fragment thereof, whereby the fragment is preferably a INTP041, INTP042 or INTP043 gene-specific fragment, for isolating or generating an agonist or stimulator of the INTP041, INTP042 or INTP043 polypeptide for the treatment of an immune related disorder, wherein said agonist or stimulator is selected from the group consisting of:

1. a specific antibody or fragment thereof including: a) a chimeric, b) a humanized or c) a fully human antibody, as well as;

2. a bispecific or multispecific antibody,

3. a single chain (e.g. scFv) or

4. single domain antibody, or

5. a peptide- or non-peptide mimetic derived from said antibodies or

6. an antibody-mimetic such as a) an anticalin or b) a fibronectin-based binding molecule (e.g. trinectin or adnectin).

The generation of peptide- or non-peptide mimetics from antibodies is known in the art (Saragovi et al, 1991 and Saragovi et al, 1992).

Anticalins are also known in the art (Vogt et al, 2004). Fibronectin-based binding molecules are described in US6818418 and WO2004029224.

Furthermore, the test compound may be of various origin, nature and composition, such as any small molecule, nucleic acid, lipid, peptide, polypeptide including an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin), etc., in isolated form or in mixture or combinations.

The invention also includes a screening kit useful in the methods for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, that are described above.

The invention includes the agonists, antagonists, ligands, receptors, substrates and enzymes, and other compounds which modulate the activity or antigenicity of the polypeptide of the invention discovered by the methods that are described above.

As mentioned above, it is envisaged that the various moieties of the invention (i.e. the polypeptides of the first aspect of the invention, a nucleic acid molecule of the second or third aspect of the invention, a vector of the fourth aspect of the invention, a host cell of the fifth aspect of the invention, a ligand of the sixth aspect of the invention, a compound of the seventh aspect of the invention) may be useful in the therapy or diagnosis of diseases. To assess the utility of the moieties of the invention for treating or diagnosing a disease one or more of the following assays may be carried out. Note that although some of the following assays refer to the test compound as being a protein/polypeptide, a person skilled in the art will readily be able to adapt the following assays so that the other moieties of the invention may also be used as the "test compound".

The invention also provides pharmaceutical compositions comprising a polypeptide, nucleic acid, ligand or compound of the invention in combination with a suitable pharmaceutical carrier. These compositions may be suitable as therapeutic or diagnostic reagents, as vaccines, or as other immunogenic compositions, as outlined in detail below. According to the terminology used herein, a composition containing a polypeptide, nucleic acid, ligand or compound [X] is "substantially free of impurities [herein, Y] when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95%, 98% or even 99% by weight.

The pharmaceutical compositions should preferably comprise a therapeutically effective amount of the polypeptide, nucleic acid molecule, ligand, or compound of the invention. The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

The precise effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drags or hormones.

A pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent. Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.

The pharmaceutical compositions utilised in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means. Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention. Typically, the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.

Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.

If the activity of the polypeptide of the invention is in excess in a particular disease state, several approaches are available. One approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition. Preferably, such antagonists are antibodies. Most preferably, such antibodies are chimeric and/or humanised to minimise their immunogenicity, as described previously.

In another approach, soluble forms of the polypeptide that retain binding affinity for the ligand, substrate, enzyme, receptor, in question, may be administered. Typically, the polypeptide may be administered in the form of fragments that retain the relevant portions.

In an alternative approach, expression of the gene encoding the polypeptide can be inhibited using expression blocking techniques, such as the use of antisense nucleic acid molecules (as described above), either internally generated or separately administered. Modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions (signal sequence, promoters, enhancers and introns) of the gene encoding the polypeptide. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al. (1994) In: Huber, B. E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY). The complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Such oligonucleotides may be administered or may be generated in situ from expression in vivo.

In addition, expression of the polypeptide of the invention may be prevented by using ribozymes specific to its encoding mRNA sequence. Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al, Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribozymes may be synthesised with non-natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases.

RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, queosine and butosine, as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine and uridine which are not as easily recognised by endogenous endonucleases.

For treating abnormal conditions related to an under-expression of the polypeptide of the invention and its activity, several approaches are also available. One approach comprises administering to a subject a therapeutically effective amount of a compound that activates the polypeptide, i.e., an agonist as described above, to alleviate the abnormal condition. Alternatively, a therapeutic amount of the polypeptide in combination with a suitable pharmaceutical earner may be administered to restore the relevant physiological balance of polypeptide.

Gene therapy may be employed to effect the endogenous production of the polypeptide by the relevant cells in the subject. Gene therapy is used to treat permanently the inappropriate production of the polypeptide by replacing a defective gene with a corrected therapeutic gene.

Gene therapy of the present invention can occur in vivo or ex vivo. Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene and introduction of the genetically altered cells back into the patient. In contrast, in vivo gene therapy does not require isolation and purification of a patient's cells.

The therapeutic gene is typically "packaged" for administration to a patient. Gene delivery vehicles may be non-viral, such as liposomes, or replication-deficient viruses, such as adenovirus as described by Berkner, K.L., in Curr. Top. Microbiol. Immunol., 158, 39-66 (1992) or adeno-associated virus (AAV) vectors as described by Muzyczka, N., in Curr. Top. Microbiol. Immunol., 158, 97-129 (1992) and U.S. Patent No. 5,252,479. For example, a nucleic acid molecule encoding a polypeptide of the invention may be engineered for expression in a replication-defective retroviral vector. This expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding the polypeptide, such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo (see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics (1996), T Strachan and A P Read, BIOS Scientific Publishers Ltd).

Another approach is the administration of "naked DNA" in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.

In situations in which the polypeptides or nucleic acid molecules of the invention are disease-causing agents, the invention provides that they can be used in vaccines to raise antibodies against the disease causing agent.

Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat disease after infection). Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with pharmaceutically-acceptable carriers as described above, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Additionally, these carriers may function as immunostimulating agents ("adjuvants"). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and other pathogens.

Since polypeptides may be broken down in the stomach, vaccines comprising polypeptides are preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.

The vaccine formulations of the invention may be presented in unit-dose or multi-dose containers. For example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

Genetic delivery of antibodies that bind to polypeptides according to the invention may also be effected, for example, as described in International patent application WO98/55607.

The technology referred to as jet injection (see, for example, www.powderject.com) may also be useful in the formulation of vaccine compositions.

A number of suitable methods for vaccination and vaccine delivery systems are described in International patent application WO00/29428.

This invention also relates to the use of nucleic acid molecules according to the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the nucleic acid molecules of the invention which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques.

Nucleic acid molecules for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), or other amplification techniques (see Saiki et al, Nature, 324, 163-166 (1986); Bej, et al, Crit. Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer et al, J. Virol. Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology, 8, 291-294 (1990)) prior to analysis.

In one embodiment, this aspect of the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to the invention and comparing said level of expression to a control level, wherein a level that is different to said control level is indicative of disease. The method may comprise the steps of: a)contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the invention and the probe; b)contacting a control sample with said probe under the same conditions used in step a); c)and detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease.

A further aspect of the invention comprises a diagnostic method comprising the steps of: a)obtaining a tissue sample from a patient being tested for disease; b)isolating a nucleic acid molecule according to the invention from said tissue sample; and c)diagnosing the patient for disease by detecting the presence of a mutation in the nucleic acid molecule which is associated with disease.

To aid the detection of nucleic acid molecules in the above-described methods, an amplification step, for example using PCR, may be included.

Deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labelled RNA of the invention or alternatively, labelled antisense DNA sequences of the invention. Perfectly-matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperatures. The presence or absence of the mutation in the patient may be detected by contacting DNA with a nucleic acid probe that hybridises to the DNA under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation in the corresponding portion of the DNA strand.

Such diagnostics are particularly useful for prenatal and even neonatal testing.

Point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by other well-known techniques, such as direct DNA sequencing or single-strand conformational polymorphism, (see Orita et al., Genomics, 5, 874-879 (1989)). For example, a sequencing primer may be used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotides or by automatic sequencing procedures with fluorescent-tags. Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. Further, point mutations and other sequence variations, such as polymorphisms, can be detected as described above, for example, through the use of allele-specific oligonucleotides for PCR amplification of sequences that differ by single nucleotides.

DNA sequence differences may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et al, Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and Sl protection or the chemical cleavage method (see Cotton et al., Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401).

In addition to conventional gel electrophoresis and DNA sequencing, mutations such as microdeletions, aneuploidies, translocations, inversions, can also be detected by in situ analysis (see, for example, Keller et al, DNA Probes, 2nd Ed., Stockton Press, New York, N.Y., USA (1993)), that is, DNA or RNA sequences in cells can be analysed for mutations without need for their isolation and/or immobilisation onto a membrane. Fluorescence in situ hybridization (FISH) is presently the most commonly applied method and numerous reviews of FISH have appeared (see, for example, Trachuck et al, Science, 250, 559-562 (1990), and Trask et al, Trends, Genet., 7, 149-154 (1991)).

In another embodiment of the invention, an array of oligonucleotide probes comprising a nucleic acid molecule according to the invention can be constructed to conduct efficient screening of genetic variants, mutations and polymorphisms. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al, Science (1996), VoI 274, pp 610-613).

In one embodiment, the array is prepared and used according to the methods described in PCT application WO95/11995 (Chee et al); Lockhart, D. J. et al. (1996) Nat. Biotech. 14: 1675-1680); and Schena, M. et al (1996) Proc. Natl. Acad. Sci. 93: 10614-10619). Oligonucleotide pairs may range from two to over one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support. In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/25116 (Baldeschweiler et aϊ). In another aspect, a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number between two and over one million which lends itself to the efficient use of commercially-available instrumentation.

In addition to the methods discussed above, diseases may be diagnosed by methods comprising determining, from a sample derived from a subject, an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.

Assay techniques that can be used to determine levels of a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and are discussed in some detail above (including radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays). This aspect of the invention provides a diagnostic method which comprises the steps of: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand- polypeptide complex; and (b) detecting said complex.

Protocols such as ELISA, RIA, and FACS for measuring polypeptide levels may additionally provide a basis for diagnosing altered or abnormal levels of polypeptide expression. Normal or standard values for polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably humans, with antibody to the polypeptide under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, such as by photometric means.

Antibodies which specifically bind to a polypeptide of the invention may be used for the diagnosis of conditions or diseases characterised by expression of the polypeptide, or in assays to monitor patients being treated with the polypeptides, nucleic acid molecules, ligands and other compounds of the invention. Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for the polypeptide include methods that utilise the antibody and a label to detect the polypeptide in human body fluids or extracts of cells or tissues. The antibodies may be used with or without modification, and may be labelled by joining them, either covalently or non-covalently, with a reporter molecule. A wide variety of reporter molecules known in the art may be used, several of which are described above.

Quantities of polypeptide expressed in subject, control and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease. Diagnostic assays may be used to distinguish between absence, presence, and excess expression of polypeptide and to monitor regulation of polypeptide levels during therapeutic intervention. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.

A diagnostic kit of the present invention may comprise:

(a) a nucleic acid molecule of the present invention;

(b) a polypeptide of the present invention; or

(c) a ligand of the present invention.

In one aspect of the invention, a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease. The kit may further comprise a third container holding an agent for digesting unhybridised RNA.

In an alternative aspect of the invention, a diagnostic kit may comprise an array of nucleic acid molecules, at least one of which may be a nucleic acid molecule according to the invention.

To detect polypeptide according to the invention, a diagnostic kit may comprise one or more antibodies that bind to a polypeptide according to the invention; and a reagent useful for the detection of a binding reaction between the antibody and the polypeptide.

Such kits will be of use in diagnosing a disease or disorder or susceptibility to disease or disorder in members of the glycoside hydrolase family 31 family of proteins are implicated. Such diseases and disorders may include reproductive disorders, cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis¹ sarcoma; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, multiple sclerosis, peripheral nervous system disease and pain; developmental disorders; metabolic disorders including diabetes mellitus, hypoglycemia, osteoporosis, and obesity, AIDS and renal disease; infections including viral infection, bacterial infection, fungal infection and parasitic infection and other pathological conditions. Preferably, the diseases are those in which glycoside hydrolase family 31 proteins are implicated, particularly diabetes and sugar metabolism related disorders.

Thus, embodiments of the invention provide for the use of the polypeptides in kits for diagnosing the existence of or susceptibility to sugar metabolism related disorders (in particular diabetes) or cancer.

In one embodiment of the invention, the cancer is selected from carcinoma, including adenocarcinoma, lymphoma, blastema, melanoma, sarcoma or leukemia. In one embodiment of the invention, the cancer is selected from lung, colorectal, breast, pancreas and head and neck carcinomas.

Various aspects and embodiments of the present invention will now be described in more detail by way of example, with particular reference to the INTP041, INTP042 and INTP043 polypeptides.

It will be appreciated that modification of detail may be made without departing from the scope of the invention.

Brief description of the Figures

Figure 1: ClustalX alignment of INTP041 and INTP042 nucleotide sequences. Figure 2: ClustalX alignment of INTP041 and INTP042 protein sequences.

Figure 3: Top ten results from BLAST against NCBI non-redundant database using SEQ ID NO:96 (INTP041 full-length polypeptide sequence).

Figure 4: Alignment generated by BLAST between SEQ ID NO:96 (INTP041 full-length polypeptide sequence) and the top hit, Maltase-glucoamylase, Homo sapiens.

Figure 5: Top ten results from BLAST against NCBI non-redundant database using SEQ ID NO:100 (INTP042 polypeptide sequence).

Figure 6: Alignment generated by BLAST between SEQ ID NO: 100 (INTP042 polypeptide sequence) and the top hit, Maltase-glucoamylase, Homo sapiens.

Figure 7: Top ten results from BLAST against NCBI non-redundant database using SEQ ID NO: 156 (INTP043 partial polypeptide sequence).

Figure 8: Alignment generated by BLAST between SEQ ID NO: 156 (INTP043 partial polypeptide sequence) and the top hit, Maltase-glucoamylase, Homo sapiens.

Figure 9: INTP041 predicted sequence with translation of the coding sequence (up to the last confidently predicted splice site) showing the positions of the PCR primers

Figure 10: Nucleotide sequence with translation of INTP041 PCR product cloned using primers INTP041-CP3 and INTP041-CP4

Figure 11: Map of pCR4-TOPO-INTP041-CP3/CP4

Figure 12: INTP041 3'-RACE products showing the alternative potential 3' ends, with the 5 '-most stop codon in each sequence highlighted and potential polyadenylation signal sequence underlined . Figure 13: Map of pDONR 221

Figure 14: Map of Expression vector pEAK12d Figure 15: Map of Expression vector pDEST12.2 Figure 16: Map of pENTR_INTP041-Dl-6HIS Figure 17: Map of pEAK12d_INTP041-Dl-6HIS Figure 18: Map of pDEST12.2_INTP041-Dl-6HIS Figure 19: Map of pENTR_INTP041-D2-6HIS Figure 20: Map of ρEAK12d_ INTP041-D2-6HIS Figure 21: Map of pDEST12.2_ INTP041-D2-6HIS

TABLE 1

TABLE 2

Examples

The INTP041 polypeptide sequence (SEQ ID NO: 96) and the INTP042 polypeptide sequence (SEQ ID NO: 100) were aligned (Figure 2). From this alignment, the similarities and differences in the sequences can be clearly seen. It is clear to see that INTP042 is a splice variant of INTP041 in that it splices in an additional exon 2 in the INTP042 polypeptide.

Example 1: INTP041 Protein BLAST Results

The INTP041 polypeptide sequence, shown in SEQ ID NO:96, was used as a BLAST query against the NCBI non-redundant sequence database. As can be seen in Figure 3, the top three hits are for Maltase-glucoamylases from Homo sapiens, and these three hits have an expectation value of zero which is therefore very significant. It can therefore be inferred that INTP041 is a member of the glycoside hydrolase family 31 family of proteins.

Example 2: INTP042 Protein BLAST Results

The INTP042 polypeptide sequence, shown in SEQ ID NO: 100, was used as a BLAST query against the NCBI non-redundant sequence database. As can be seen in Figure 5, the top three hits are for Maltase-glucoamylases from Homo sapiens, and these three hits have an expectation value of zero which is therefore very significant. It can therefore be inferred that INTP042 is a member of the glycoside hydrolase family 31 family of proteins.

Example 3: INTP043 Protein BLAST Results

The INTP043 partial polypeptide sequence, shown in SEQ ID NO: 156, was used as a BLAST query against the NCBI non-redundant sequence database. As can be seen in Figure 7, the top two hits are for Maltase-glucoamylases from Homo sapiens, and these three hits have very low and therefore significant expectation values (e^~155 and e^"154). It can therefore be inferred that INTP043 is a member of the glycoside hydrolase family 31 family of proteins.

Example 4: Cloning of INTP041

4.1 Preparation of human cDNA templates

First strand cDNA was prepared from Human Universal Reference Total RNA (a mixture of RNA from 10 cancer cell lines) (Stratagene) using Superscript II RNase H^" Reverse Transcriptase (Invitrogen) according to the manufacturer's protocol. Oligo (dT)i₅ primer (lμl at 500 μg/ml) (Promega). 2 μg human total RNA, 1 μl 10 niM dNTP mix (10 mM each of dATP, dGTP, dCTP and dTTP at neutral pH) and sterile distilled water to a final volume of 12 μl were combined in a 1.5 ml Eppendorf tube, heated to 65 ⁰C for 5 min and then chilled on ice. The contents were collected by brief centrifugation and 4 μl of 5X First-Strand Buffer, 2 μl 0.1 M DTT, and 1 μl RnaseOUT Recombinant Ribonuclease Inhibitor (40 units/μl, Invitrogen) were added. The contents of the tube were mixed gently and incubated at 42 ⁰C for 2 min; then 1 μl (200 units) of Superscript II enzyme was added and mixed gently by pipetting. The mixture was incubated at 42 ⁰C for 50 min and then inactivated by heating at 70 ⁰C for 15 min. To remove RNA complementary to the cDNA, lμl (2 units) of E. coli RNase H (Invitrogen) was added and the reaction mixture incubated at 37 ⁰C for 20 min. The final 21 μl reaction mix was diluted by adding 179 μl sterile water to give a total volume of 200 μl.

4.2 Gene specific cloning primers for PCR

Three pair of PCR primers having a length of between 18 and 25 bases were designed for amplifying the complete coding sequence of the virtual cDNA using Primer Designer Software (Scientific & Educational Software, PO Box 72045, Durham, NC 27722-2045, USA). PCR primers were optimized to have a Tm close to 55 + 10 ⁰C and a GC content of 40-60%. Primers were selected which had high selectivity for the target sequence (INTP041) with little or no none specific priming.

4.3 PCR amplification ofINTP041-CPl/INTP-CP2 products from a Stratagem Human Universal Reference cDNA template

Gene-specific cloning primers (INTP041-CP1 and INTP041-CP2, Figure 9 and Table 3) were designed to amplify a cDNA fragment of 835 bp from exons 1-3 of the INTP041 coding sequence prediction. Interrogation of the GeneSeq database of patented nucleotide sequences with the INTP041 sequence identified a number of short sequences which corresponded to various regions of the INTP041 prediction. These sequences were mostly derived from tumour cDNA samples, and so the INTP041-CP1/INTP041-CP2 primer pair was tested on the Stratagene Human Universal Reference cDNA sample. The PCR reactions were performed in a final volume of 50 μl containing IX Platinum^® Taq High Fidelity PCR buffer, 2 rtiM MgSO₄, 200 μM dNTPs, 0.2 μM of each cloning primer, 1 unit of Platinum^® Taq DNA High Fidelity (Invitrogen) DNA polymerase and 100 ng of human cDNA template using an MJ Research DNA Engine, programmed as follows: 94 ⁰C, 2 min; 39 cycles of 94 ⁰C, 30 sec, 55 ⁰C, 30 sec, 68 ⁰C, 30 sec, followed by 1 cycle at 68 ⁰C for 7 min and a holding cycle at 4 ⁰C. The amplification product was visualized on a 0.8 % agarose gel in 1 X TAE buffer (Invitrogen) and a single PCR product was seen migrating at approximately the predicted molecular mass. This product was purified using the Wizard PCR Preps DNA Purification system (Promega). The PCR product was eluted in 50 μl of water and subcloned directly.

4.4 Subcloning of PCR Products

The PCR product was subcloned into the topoisomerase I modified cloning vector (pCR4- TOPO) using the TA cloning kit purchased from the Invitrogen Corporation using the conditions specified by the manufacturer. Briefly, 4 μl of gel purified PCR product was incubated for 15 min at room temperature with 1 μl of TOPO vector and 1 μl salt solution. The reaction mixture was then transformed into E. coli strain TOPlO (Invitrogen) as follows: a 50 μl aliquot of One Shot TOPlO cells was thawed on ice and 2 μl of TOPO reaction was added. The mixture was incubated for 15 min on ice and then heat shocked by incubation at 42 ⁰C for exactly 30 s. Samples were returned to ice and 250 μl of warm (room temperature) SOC media was added. Samples were incubated with shaking (220 rpm) for 1 h at 37 ⁰C. The transformation mixture was then plated on L-broth (LB) plates containing ampicillin (100 μg/ml) and incubated overnight at 37 C.

4.5 Colony PCR

Colonies were inoculated into 50 μl sterile water using a sterile toothpick. A 10 μl aliquot of the inoculum was then subjected to PCR in a total reaction volume of 20 μl containing IX AmpliTaq™ buffer, 200 μM dNTPs, 20 pmoles T7 primer, 20 pmoles of T3 primer, 1 unit of AmpliTaq™ (Perkin Elmer) using an MJ Research DNA Engine. The cycling conditions were as follows: 94 ⁰C, 2 min; 30 cycles of 94 ⁰C, 30 sec, 48 ⁰C, 30 sec and 72 ⁰C for 1 min. Samples were maintained at 4 ⁰C (holding cycle) before further analysis.

PCR reaction products were analyzed on 1 % agarose gels in 1 X TAE buffer. Colonies which gave the expected PCR product size (835 bp cDNA + 105 bp due to the multiple cloning site or MCS) were grown up overnight at 37 ⁰C in 5 ml L-Broth (LB) containing ampicillin (100 μg /ml), with shaking at 220 rpm.

4.6 Plasmid DNA preparation and sequencing

Miniprep plasmid DNA was prepared from the 5 ml culture using a Qiaprep Turbo 9600 robotic system (Qiagen) or Wizard Plus SV Minipreps kit (Promega cat. no. 1460) according to the manufacturer's instructions. Plasmid DNA was eluted in 100 μl of sterile water. The DNA concentration was measured using an Eppendorf BO photometer or a Spectramax 190 photometer (Molecular Devices). Plasmid DNA (200-500 ng) was subjected to DNA sequencing with the T7 primer and T3 primer using the BigDyeTerminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. The primer sequences are shown in Table 3. Sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.

Sequence analysis identified clones which matched to the predicted INTP041 sequence. This identified cDNA from the Stratagene Human Universal Reference sample as a suitable template for further INTP 041 amplifications.

4.7 PCR amplification of INTP041-CP3/INTP-CP4 and INTP041-CP5/INTP041-CP6 products from a Stratagene Human Universal Reference cDNA template Gene-specific cloning primers (INTP041-CP3 and INTP041-CP4, Figure 9, Figure 10 and Table 3) were designed to amplify a cDNA fragment of 5316 bp from exons 1-46 of the INTP041 coding sequence prediction. Gene-specific cloning primers (INTP041-CP5 and INTP041-CP6, Figure 9 and Table 3) were designed as a inner nested pair to be used on the INTP041-CP3/INTP041-CP4 PCR product to amplify a cDNA fragment of 5134 bp from exons 1-46 of the INTP041 coding sequence. The INTP041-CP3/INTP041-CP4 primer pair were tested on the Stratagene Human Universal Reference cDNA sample. The PCR reactions were performed in a final volume of 50 μl containing IX Platinum^® Taq High Fidelity PCR buffer, 2 niM MgSO₄, 200 μM dNTPs, 0.2 μM of each cloning primer, 2.5 unit of Platinum^® Taq DNA High Fidelity (Invitrogen) DNA polymerase, 100 ng of human cDNA template, and either OX, 0.5X, IX or 2X final concentration of PCRx Enhancer solution (Invitrogen), using an MJ Research DNA Engine, programmed as follows: 94 ⁰C, 2 min; 40 cycles of 94 ⁰C, 30 sec, 55 ⁰C, 30 sec, 68 ⁰C₃ 6 min, followed by 1 cycle at 68 ⁰C for 10 min and a holding cycle at 4 C.

The INTP041-CP5/INTP041-CP6 primer pair was tested on the products from the INTP041-CP3/INTP041-CP4 amplification, using the same concentration of PCRx Enhancer solution in PCR2 as had been used in PCRl. The PCR reactions were performed in a final volume of 50 μl containing IX Platinum^® Taq High Fidelity PCR buffer, 2 mM MgSO₄, 200 μM dNTPs, 0.2 μM of each cloning primer, 2.5 unit of Platinum^® Taq DNA High Fidelity (Invitrogen) DNA polymerase, lμl PCRl product, and either OX, 0.5X, IX or 2X final concentration of PCRx Enhancer solution (Invitrogen), using an MJ Research DNA Engine, programmed as follows: 94 ⁰C, 2 min; 40 cycles of 94 ⁰C, 30 sec, 61 ⁰C, 30 sec, 68 ⁰C, 6 min, followed by 1 cycle at 68 ⁰C for 10 min and a holding cycle at 4 ⁰C.

The amplification products were visualized on a 0.8 % agarose gel in 1 X TAE buffer (Invitrogen) and PCR products migrating at approximately the predicted molecular mass were purified from the reactions containing 0.5X PCRx Enhancer solution for both the INTP041-CP3/INTP041-CP4 and INTP041-CP5/INTP041-CP6 amplifications. The products were purified using the Qiaquick Gel Extraction Kit (Qiagen). The PCR products were eluted in 30 μl of water and subcloned directly.

4.8 Subcloning of PCR Products

PCR products were subcloned into the topoisomerase I modified cloning vector for long PCR products (pCR-XL-TOPO) using the TA cloning kit purchased from the Invitrogen Corporation using the conditions specified by the manufacturer. Briefly, 4 μl of purified PCR product was incubated for 5 min at room temperature with 1 μl of pCR-XL-TOPO vector. 1 μl of 6X TOPO Cloning Stop Solution was then added and the reagents mixed, centrifuged briefly and then placed on ice. The reaction mixture was then transformed into E. coli strain TOPlO (Invitrogen) as follows: a 50 μl aliquot of One Shot TOPlO electrocompetent cells was thawed on ice and 2 μl of TOPO reaction was added. The mixture was transferred to a chilled 0.1 cm electroporation cuvette and the cells electroporated using a BioRad Gene-Pulser™ according to the manufacturer's recommended protocol. 450 μl of warm SOC media (room temperature) was added immediately after electroporation and the solution transferred to a 15 ml snap-cap tube and incubated with shaking (220 rpm) for 1 h at 37 ⁰C. 20 μl, 50 μl and 200 μl of the transformation mixture was then plated on L-broth (LB) plates containing kanamycin (40 μg/ml) and incubated overnight at 37 ⁰C.

4.9 Plasmid DNA preparation and sequencing

Eight colonies from the INTP041-CP3/INTP041-CP4 product transformation and eight from the INTP041-CP5/INTP041-CP6 product transformation were grown up overnight at 37 ⁰C in 5 ml L-Broth (LB) containing kanamycin (40 μg /ml), with shaking at 220 rpm. Miniprep plasmid DNA was prepared from the 5 ml culture using a Qiaprep Turbo 9600 robotic system (Qiagen) or Wizard Plus SV Minipreps kit (Promega cat. no. 1460) according to the manufacturer's instructions. Plasmid DNA was eluted in 100 μl of sterile water. The DNA concentration was measured using an Eppendorf BO photometer or a Spectramax 190 photometer (Molecular Devices). Plasmid DNA (100-200 ng) was subjected to DNA sequencing with the T7 primer and M13R primer using the BigDyeTerminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. The primer sequences are shown in Table 3. Sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer. Three clones containing the INTP041-CP3/INTP041-CP4 product were further sequenced with 9 internal sequencing primers (INTP041-SP1 - INTP041-SP9, Table 3) within the INTP041 predicted sequence. One clone was identified which contained the INTP041 predicted sequence. This clone contained amino acid substitutions H678R, V741G and M742V compared with the original INTP041 prediction, but these 'substitutions' were also found in the published Celera genomic sequence and so were considered likely to reflect polymorphisms rather than PCR-induced errors. The sequence of the cloned cDNA fragment is shown in Figure 2. The plasmid map of the cloned PCR product (pCR-XL- TOPO-INTP041-CP3/CP4) (plasmid ID.15453) is shown in Figure 11.

4.10 3 '-RA CE (Rapid Amplification ofcDNA Ends

The INTP041 prediction did not include a predicted stop codon. A nested pair of 3'-RACE amplification primers, INTP041-GR1-3' and INTP041-GRlnest-3' were designed within the rNTP041-CP3/INTP041-CP4 product to attempt to identify the 3' end of the INTP041 coding sequence. These amplification primers were tested using a cDNA template derived from the Stratagene Universal Reference RNA sample.

4.11 3 ' RA CE amplification reactions

The 3' RACE was earned out using the GeneRacer™ system (Invitrogen) in accordance with the manufacturer's instructions. All reactions components, except the RNA templates, were supplied with the system. Human Universal Reference RNA (Stratagene) was converted to 3 ' RACE-ready first strand cDNA using the supplied GeneRacer™ Oligo dT primer (Table 3) and the Superscript II RNase H^" Reverse Transcriptase (Invitrogen) according to the manufacturer's protocol. Briefly, 1 μl GeneRacer™ Oligo dT primer (50 μM), 1 μl dNTP mix (1OmM), 5 μg total RNA sample and DEPC-treated sterile water were mixed in a final volume of 12 μl in a 1.5 ml Eppendorf tube, heated at 65 ⁰C for 5 min and then chilled on ice for 2 min. The contents were collected by brief centrifugation and 4 μl of 5X First-Strand Buffer, 2 μl 0.1 M DTT, 1 μl RnaseOUT Recombinant Ribonuclease Inhibitor (40 units/μl) and 1 μl Superscript II RT (200 U/μl) were added. The contents were mixed gently and collected by brief centrifugation, incubated at 42 ⁰C for 50 min, then inactivated by heating at 70 ⁰C for 15 min. The mixture was then chilled on ice for 2 min, the contents collected again by brief centrifugation, and lμl (2 units) of E. coli RNase H added to remove RNA complementary to the cDNA. The mixture was incubated at 37 ⁰C for 20 min, then chilled on ice. The first strand cDNA was stored at -20 ⁰C before being used in RACE reactions.

A pair of gene specific nested 3' RACE primers (INSTP041-GR1-3' and INTP041- GRlnest-3', Table 3) were designed within exon 44 and exon 45/46, respectively, of the INTP 041 sequence. These primers were used in consecutive RACE PCRs in conjunction with the GeneRacer™ 3' primer and the GeneRacer™ 3' Nested Primer, respectively. For the first amplification reaction, IX Platinum^® Taq High Fidelity PCR buffer, 2 mM MgSO₄, 200 μM dNTPs, 0.2 μM of INTP041-GR1-3' primer, 0.6 μM of GeneRacer™ 3' Primer, 2.5 units of Platinum^® Taq High Fidelity DNA polymerase (Invitrogen), 2 μl of 3' GeneRacer™-ready first strand cDNA template were combined in a final volume of 50 μl. Thermal cycling was carried out using an MJ Research DNA Engine programmed as follows: 94 ⁰C, 2 min; 5 cycles of 94 ⁰C, 30 sec, 72 ⁰C, 3 min; 5 cycles of 94 ⁰C, 30 sec, 70 ⁰C, 3 min; 25 cycles of 94 ⁰C, 30 sec, 61 ⁰C, 30 sec, 68 ⁰C, 3 min; followed by 1 cycle at 68 ⁰C for 8 min and a holding cycle at 4 ⁰C.

For the secondary PCR, 1 μl of PCRl product was combined with IX Platinum^® Taq High Fidelity PCR buffer, 2 mM MgSO₄, 200 μM dNTPs, 0.2 μM of INTP041-GRlnest-3' primer, 0.2 μM of GeneRacer™ Nested 3' Primer, and 2.5 units of Platinum^® Taq High Fidelity DNA polymerase (Invitrogen) in a final volume of 50 μl. Thermal cycling was carried out using an MJ Research DNA Engine programmed as follows: 94 ⁰C, 2 min; 25 cycles of 94 ⁰C, 30 sec, 60 ⁰C, 30 sec, 68 ⁰C, 3 min; followed by 1 cycle at 68 ⁰C for 8 min and a holding cycle at 4 ⁰C.

All 50 μl of each amplification product was visualized on a 0.8 % agarose gel in 1 X TAE buffer (Invitrogen). Several bands were observed on the gel in the products of PCRl. Three major bands were excised from the PCRl product lane, and one band from the PCR2 product lane. These PCR products were purified using the Qiagen MinElute DNA Purification System (Qiagen). Each product was eluted in 10 μl of EB buffer (1OmM Tris.Cl, pH 8.5) and subcloned directly. 4.12 Subcloning of 3 ' RA CE PCR Products

Each RACE PCR product was subcloned into the topoisomerase I modified cloning vector (pCR4-TOPO) using the TA cloning kit purchased from the Invitrogen Corporation using the conditions specified by the manufacturer. Briefly, 4 μl of gel purified PCR product was incubated for 15 min at room temperature with 1 μl of TOPO vector and 1 μl salt solution. The reaction mixture was then transformed into E. coli strain TOPlO (Invitrogen) as follows: a 50 μl aliquot of One Shot TOPlO cells was thawed on ice and 2 μl of TOPO reaction was added. The mixture was incubated for 15 min on ice and then heat shocked by incubation at 42 ⁰C for exactly 30 s. Samples were returned to ice and 250 μl of warm (room temperature) SOC media was added. Samples were incubated with shaking (220 rpm) for 1 h at 37 ⁰C. The transformation mixture was then plated on L-broth (LB) plates containing ampicillin (100 μg/ml) and incubated overnight at 37 ⁰C.

4.13 Colony PCR of subcloned 3 ' RACE Products

Colonies were inoculated into 50 μl sterile water using a sterile toothpick. A 10 μl aliquot of the inoculum was then subjected to PCR in a total reaction volume of 20 μl containing IX AmpliTaq™ buffer, 200 μM dNTPs, 20 pmoles T7 primer, 20 pmoles of T3 primer, 1 unit of AmpliTaq™ (Perkin Elmer) using an MJ Research DNA Engine. The cycling conditions were as follows: 94 ⁰C, 2 min; 30 cycles of 94 ⁰C, 30 sec, 48 ⁰C, 30 sec and 72 ⁰C for 2 min. Samples were maintained at 4 ⁰C (holding cycle) before further analysis.

PCR reaction products were analyzed on 1 % agarose gels in 1 X TAE buffer. Colonies which appeared to contain an insert, i.e. gave a PCR product size greater than the 105 bp due to the multiple cloning site, were grown up overnight at 37 ⁰C in 5 ml L-Broth (LB) containing ampicillin (100 μg /ml), with shaking at 220 rpm.

4.14 Plasmid DNA preparation and sequencing

Miniprep plasmid DNA was prepared from the 5 ml culture using a Qiaprep Turbo 9600 robotic system (Qiagen) or Wizard Plus SV Minipreps kit (Promega cat. no. 1460) according to the manufacturer's instructions. Plasmid DNA was eluted in 100 μl of sterile water. The DNA concentration was measured using an Eppendorf BO photometer or Spectramax 190 photometer (Molecular Devices). Plasmid DNA (100-200 ng) was subjected to DNA sequencing with the T7 primer and T3 primer using the BigDye Terminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. The primer sequences are shown in Table 3. Sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.

Only the RACE product purified from the PCR2 reaction produced clones which corresponded to the INTP041 sequence after the initial sequencing. PCR2 corresponded to the inner nested RACE product and so should have been more specific than the PCRl product. Further colonies were picked from the plate containing the cloned PCR2 product band, grown up overnight in 5 ml L-Broth + Ampicillin, the plasmid DNA prepared and sequenced, as above.

Sequence analysis identified three clones which contained the 3' end of the predicted INTP041 coding sequence and then continued into downstream sequence. These three clones represented different potential 3' ends of the INTP041 coding sequence and were called INTP041-V1, INTP041-V2 and INTP041-V3. The foil insert sequence in the INTP041-V3 clone had not been sequenced using the T7 and T3 vector primers, so it was sequenced with two further primers, INTP041-SP12 and INTP041-SP13, designed within the sequence from the vector primers (Table 3). The sequences of the 3 different versions of potential INTP041 coding sequence 3' ends are shown in Figure 12. Only INTP041-V1 contains a potential polyadenylation signal sequence downstream of the 5 '-most stop codon and upstream of the polyA tail (Figure 12). In INTP041-V2 and INTP041-V3, the 5 '-most stop codon is immediately upstream of the polyA tail, suggesting these sequences may be RACE artifacts rather than true alternative 3' ends.

PCR primers were designed to amplify the foil length versions of INTP041 from the predicted start codon to the 3 possible stop codons identified by 3'-RACE. These primers were tested on the Stratagene Human Universal Reference cDNA sample but no products of the expected sizes were produced after analysis on agarose gel.

4.15 Identification ofINTP041 predicted functional domains

The three potential 3' ends of the INTP041 coding sequence identified by RACE were analysed and found to contain only low complexity regions. The interesting functional domains of the prediction were considered to be the trefoil domain (amino acids 42-87 of the INTP041 sequence), the first glycosyl transferase family 31 domain (amino acids 156- 865 of the INTP041 sequence) and the second glycosyl transferase family 31 domain (amino acids 1023-1767 of the INTP041 sequence). These regions had already been cloned in plasmid ID 15453, and so amplification of the full length versions of the coding sequence as determined by RACE was not pursued.

Expression constructs containing either the trefoil domain and the first glycosyl transferase family 31 domain (INTP041-D1), or the trefoil domain and the first and second glycosyl transferase family 31 domains (INTP041-D2) were produced.

Example 5 Construction of mammalian cell expression vectors for INTP041-D1 and INTP041-D2

Plasmid 15453 was used as a PCR templates to generate pEAK12d (Figure 17, 20) and ρDEST12.2 (Figure 18, 21) expression clones containing the INTP041-D1, INTP041-D2 ORF sequences respectively with a 3 ' sequence encoding a 6HIS tag using the Gateway™ cloning methodology (Invitrogen).

5.1 Generation of Gateway compatible INTP041-D1 and INTP041-D2 ORF fused to an in frame 6HIS tag sequence.

The first stage of the Gateway cloning process involves a two step PCR reaction which generates the ORFs of INTP041-D1 and INTP041-D2 flanked at the 5" end by an attBl recombination site and Kozak sequence, and flanked at the 3 ' end by a sequence encoding an in frame 6 histidine (6HIS) tag, a stop codon and the attB2 recombination site (Gateway compatible cDNA). The first PCR reaction (in a final volume of 50 μl) contains: 1 μl (40 ng) of plasmid 15453, 1.5 μl dNTPs (10 mM), 10 μl of 1OX Pfx polymerase buffer, 1 μl MgSO4 (50 mM), 0.5 μl each of gene specific primer (100 μM) (INTP041-D1-EX1 and INTP041-D1-EX2 for INTP041-D1, INTP041-D2-EX1 and INTP041-D2-EX2 for INTP041-D2), and 0.5 μl Platinum Pfx DNA polymerase (Invitrogen). The PCR reaction was performed using an initial denaturing step of 95 C for 2 min, followed by 12 cycles of 94 ⁰C for 15 s; 55 ⁰C for 30 s and 68 ⁰C for 5.5 min; and a holding cycle of 4 ⁰C. The amplification products were visualized on 0.8 % agarose gel in 1 X TAE buffer (Invitrogen) and the products migrating at the predicted molecular mass (2683 bp for INTP041-D1-6HIS and 5389 bp for INTP041-D2-6HIS) were purified from the gel using the Wizard PCR Preps DNA Purification System (Promega) and recovered in 50 μl sterile water according to the manufacturer's instructions.

The second PCR reaction (in a final volume of 50 μl) contained 10 μl purified PCR 1 product, 1.5 μl dNTPs (10 mM), 5 μl of 1OX Pfx polymerase buffer, 1 μl MgSO4 (50 mM), 0.5 μl of each Gateway conversion primer (100 μM) (GCP forward and GCP reverse) and 0.5 μl of Platinum Pfx DNA polymerase. The conditions for the 2nd PCR reaction were: 95 ⁰C for 1 min; 4 cycles of 94 ⁰C, 15 sec; 50 ⁰C, 30 sec and 68 ⁰C for 6 min; 25 cycles of 94 ⁰C, 15 sec; 55 ⁰C , 30 sec and 68 ⁰C, 5.5 min; followed by a holding cycle of 4 ⁰C. PCR products were gel purified using the Wizard PCR prep DNA purification system (Promega) according to the manufacturer's instructions.

5.2 Subcloning of Gateway compatible INTP041-D1 and INTP041-D2 ORFs into Gateway entry vector pDONR221 and expression vectors pEAK12d andpDEST12.2

The second stage of the Gateway cloning process involves subcloning of the Gateway modified PCR product into the Gateway entry vector pDONR221 (Invitrogen, Figure 13) as follows: 5 μl of purified product from PCR2 were incubated with 1.5 μl pDONR221 vector (0.1 μg/μl), 2 μl BP buffer and 1.5 μl of BP clonase enzyme mix (Invitrogen) in a final volume of 10 μl at RT for 3 h. The reaction was stopped by addition of proteinase K 1 μl (2 μg/μl) and incubated at 37 ⁰C for a further 10 min. An aliquot of this reaction (1 μl) was used to transform E. coli DHlOB cells by electroporation as follows: a 25 μl aliquot of DHlOB electrocompetent cells (Invitrogen) was thawed on ice and 1 μl of the BP reaction mix was added. The mixture was transferred to a chilled 0.1 cm electroporation cuvette and the cells electroporated using a BioRad Gene-Pulser™ according to the manufacturer's recommended protocol. SOC media (0.5 ml) which had been pre-warmed to room temperature was added immediately after electroporation. The mixture was transferred to a 15 ml snap-cap tube and incubated, with shaking (220 rpm) for 1 h at 37 ⁰C. Aliquots of the transformation mixture (10 μl and 50 μl) were then plated on L-broth (LB) plates containing kanamycin (40 μg/ml) and incubated overnight at 37 ⁰C.

Plasmid mini-prep DNA was prepared from 5 ml cultures from 6 of the resultant colonies using a Qiaprep Turbo 9600 robotic system (Qiagen). Plasmid DNA (150-200 ng) was subjected to DNA sequencing with 21M13, M13Rev, and INTP041-SP1 to INTP041-SP11 primers using the BigDyeTerminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. The primer sequences are shown in Table 3. Sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer. Plasmid eluate (2 μl or approx. 150 ng) from one of the clones which contained the correct sequence (pENTR_INTP041-Dl-6HIS, plasmid ID 15972, Figure 16 and pENTR_INTP041-D2-6HIS, plasmid ID 15973, Figure 19 respectively) were then used in recombination reactions containing 1.5 μl of either pEAK12d vector or pDEST12.2 vector (Figures 14 & 15) (0.1 μg / μl), 2 μl LR buffer and 1.5 μl of LR clonase (Invitrogen) in a final volume of 10 μl. The mixture was incubated at RT for 3 h, stopped by addition of proteinase K (2 μg) and incubated at 37 ⁰C for a further 10 min. An aliquot of this reaction (1 ul) was used to transform E. coli DHlOB cells by electroporation as follows: a 25 μl aliquot of DHlOB electrocompetent cells (Invitrogen) was thawed on ice and 1 μl of the LR reaction mix was added. The mixture was transferred to a chilled 0.1 cm electroporation cuvette and the cells electroporated using a BioRad Gene-Pulser™ according to the manufacturer's recommended protocol. SOC media (0.5 ml) which had been pre-warmed to room temperature was added immediately after electroporation. The mixture was transferred to a 15 ml snap-cap tube and incubated, with shaking (220 rpm) for 1 h at 37 ⁰C. Aliquots of the transformation mixture (10 μl and 50 μl) were then plated on L-broth (LB) plates containing ampicillin (100 μg/ml) and incubated overnight at 37 ⁰C.

Plasmid mini-prep DNA was prepared from 5 ml cultures from 6 of the resultant colonies subcloned in each vector using a Qiaprep Turbo 9600 robotic system (Qiagen). Plasmid DNA (200-500 ng) in the pEAK12d vector was subjected to DNA sequencing with pEAK12F, pEAK12R and INTP041-SP1 to INTP041-SP11 primers as described above. Plasmid DNA (200-500 ng) in the ρDEST12.2 vector was subjected to DNA sequencing with 21M13, M13Rev and INTP041-SP1 to INTP041-SP11 primers as described above. Primer sequences are shown in Table 3.

CsCl gradient purified maxi-prep DNA was prepared from a 500 ml culture of one of each of the sequence verified clones (pEAK12d_INTP041-Dl-6HIS and ρEAK12d_ INTP041- D2-6HIS plasmid ID numbers 15978 and 15979 and Figures 17 and 20 respectively and pDEST12.2_INTP041-Dl-6HIS, pDEST12.2_INTP041-D2-6HIS, plasmid IDs 15982 and 15983 and Figures 18 and 21 respectively using the method described by Sambrook J. et al., 1989 (in Molecular Cloning, a Laboratory Manual, 2^nd edition, Cold Spring Harbor Laboratory Press). Plasmid DNA was resuspended at a concentration of 1 μg/μl in sterile water (or 10 mM Tris-HCl pH 8.5) and stored at -20 ⁰C. Endotoxin-free maxi-prep DNA was prepared from a 500 ml culture of one of each of the sequence verified clones (pDEST12.2_INTP041-Dl-6HIS and pDEST12.2_INTP041-D2- 6HIS, plasmid IDs 15982 and 15983 and Figures 18 and 21 respectively) using the EndoFree Plasmid Mega kit (Qiagen).

Full details of the plasmids produced are provided in Table 4.

Table 3: Primers for INTP041 cloning, sequencing and 3'-RACE

Primer Sequence (5 '-3 )

INTP041-CP1 TCC TCT TGC TGC TTC TTG TG (SEQ ID NO: 200)

INTP041-CP2 GTC AAG AAT GCC TCC AAT CG (SEQ ID NO: 201)

INTP041-CP3 CGA GGA AGC TCA GTG TAT TGG (SEQ ID NO : 202)

INTP041-CP4 ATT GCC GGT TGT CAT AGG TG (SEQ ID NO: 203)

INTP041-CP5 CTT GCT GCT TCT TGT GTT GGA G (SEQ ID NO: 204)

INTP041-CP6 GGT CTG GAT TTG CAG GAT GTT C (SEQ ID NO: 205)

INTP041-GR1-3' CCA GGT GTT CTG GGA TGA TGG ACA AAG (SEQ ID NO: 206)

INTP041-GRlnest- GCA GCT CAG AAC ATC CTG CAA ATC C (SEC ID NO: 207) 3'

INTP041-D1-EX1 GCA GGC TTC GCC ACC ATG GCG AGG AAG CTC AGT GTA TTG

(SEQ ID NO: 208)

INTP041-D1-EX2 GTG ATG GTG ATG GTG GAC GAT AAA ATT AGC TGG CT (SEQ ID NO: 209)

INTP041-D2-EX1 GCA GGC TTC GCC ACC ATG GCG AGG AAG CTC AGT GTA TTG

(SEQ ID NO: 210)

INTP041-D2-EX2 GTG ATG GTG ATG GTG GAA ACT GAC TTG TGT CAC AT (SEQ ID NO: 211)

GCP Forward G GGG ACA AGT TTG TAC AAA AAA GCA GGC TTC GCC i ^QLCC (SEQ

ID NO: 212)

GCP Reverse GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TCA ATG GTG ATG GTG ATG GTG (SEC ID NO: 213)

INTP041-SP1 GGT TGA TGG GAT TGC TGA TG (SEQ ID NO: 214)

INTP041-SP2 AGC TTG TTG GAC GGC CAT TC (SEQ ID NO: 215)

INTP041-SP3 CTC TGC ATG GAC ACG GAG TT (SEQ ID NO: 216)

INTP041-SP4 ATC TGA ACA TCC GCT ACA CC (SEQ ID NO: 217)

INTP041-SP5 GGA CAC TGA CAA CCT CAT GT (SEQ ID NO: 218)

INTP041-SP6 CAG CAA CCA TGC TGC AGG TC (SEQ ID NO: 219)

INTP041-SP7 GCT CTG ACA TAC CGC ACC AC (SEQ ID NO: 220)

INTP041-SP8 CAC AGC TGC GTG GTG GAA GA (SEQ ID NO: 221)

INTP041-SP9 TGG CAT GAT GGA GTT CAG TC (SEQ ID NO: 222)

INTP041-SP10 aka GTC AAG AAT GCC TCC AAT CG (SEQ ID NO: 223) INTP041-CP2

INTP041-SP11 aka GGT CTG GAT TTG CAG GAT GTT C (SEQ ID NO: 224) INTP041-CP6

INTP041-SP12 CAC TGG CAC TAC TGA TAC TG (SEQ ID NO: 225)

INTP041-SP13 TGC TAC TGT TCC CGA TAC (SEQ ID NO: 226) pEAK12F GCC AGC TTG GCA CTT GAT GT (SEQ ID SIO: 227) pEAK12R GAT GGA GGT GGA CGT GTC AG (SEQ ID MO: 228)

21M13 TGT AAA ACG ACG GCC AGT (SEQ ID NO: 229)

Ml3REV CAG GAA ACA GCT ATG ACC (SEQ ID NO: 230)

T7 primer TAA TAC GAC TCA CTA TAG GG (SEQ ID 1 MO: 231)

T3 primer CTC CCT TTA GTG AGG GTA ATT (SEQ ID NO: 232)

Underlined sequence = Kozak sequence Bold = Stop codon

Italic sequence = His tag

Table 4: Plasmid details

Example 6: Expression and purification of INTP041

Further experiments may now be performed to determine the tissue distribution and expression levels of the INTP041 polypeptides in vivo, on the basis of the nucleotide and amino acid sequence disclosed herein. The presence of the transcripts for INTP041 may be investigated by PCR of cDNA from different human tissues. The INTP041 transcripts may be present at very low levels in the samples tested. Therefore, extreme care is needed in the design of experiments to establish the presence of a transcript in various human tissues as a small amount of genomic contamination in the RNA preparation will provide a false positive result. Thus, all RNA should be treated with DNAse prior to use for reverse transcription. In addition, for each tissue a control reaction may be set up in which reverse transcription was not undertaken (a -ve RT control).

For example, lμg of total RNA from each tissue may be used to generate cDNA using Multiscript reverse transcriptase (ABI) and random hexamer primers. For each tissue, a control reaction is set up in which all the constituents are added except the reverse transcriptase (-ve RT control). PCR reactions are set up for each tissue on the reverse transcribed RNA samples and the minus RT controls. INTP041 -specific primers may readily be designed on the basis of the sequence information provided herein. The presence of a product of the correct molecular weight in the reverse transcribed sample together with the absence of a product in the minus RT control may be taken as evidence for the presence of a transcript in that tissue. Any suitable cDNA libraries may be used to screen for the INTP041 transcripts, not only those generated as described above.

The tissue distribution pattern of the INTP041 polypeptides will provide further useful information in relation to the function of those polypeptides. In addition, further experiments may now be performed using the pEAK12d_INTP041-Dl- 6HIS, pEAK12d_INTP041-D2-6HIS, pDEST12.2_INTP041-Dl-6HIS and pDEST12.2_INTP041-D2-6HIS expression vectors. Transfection of mammalian cell lines with these vectors may enable the high level expression of the INTP041 proteins and thus enable the continued investigation of the functional characteristics of the INTP041 polypeptides. The following material and methods are an example of those suitable in such experiments: Cell Culture

Human Embryonic Kidney 293 cells expressing the Epstein-Barr virus Nuclear Antigen (HEK293-EBNA, Invitrogen) are maintained in suspension in Ex-cell VPRO serum-free medium (seed stock, maintenance medium, JRH). Sixteen to 20 hours prior to transfection (Day-1), cells are seeded in 2x T225 flasks (50ml per flask in DMEM / F12 (1 :1) containing 2% FBS seeding medium (JRH) at a density of 2x10⁵ cells/ml). The next day (transfection day 0) transfection takes place using the JetPEITM reagent (2μl/μg of plasmid DNA, PolyPlus-transfection). For each flask, plasmid DNA is co-transfected with GFP (fluorescent reporter gene) DNA. The transfection mix is then added to the 2xT225 flasks and incubated at 37°C (5%CO₂) for 6 days. Confirmation of positive transfection may be carried out by qualitative fluorescence examination at day 1 and day 6 (Axiovert 10 Zeiss).

On day 6 (harvest day), supernatants from the two flasks are pooled and centrifuged (e.g. 4°C, 40Og) and placed into a pot bearing a unique identifier. One aliquot (500μl) is kept for QC of the 6His-tagged protein (internal bioprocessing QC). Scale-up batches may be produced by following the protocol called "PEI transfection of suspension cells", referenced BP/PEI/HH/02/04, with PolyEthylenelmine from Polysciences as transfection agent.

Purification process

The culture medium sample containing the recombinant protein with a C-terminal 6His tag is diluted with cold buffer A (5OmM NaH₂PO₄; 60OmM NaCl; 8.7 % (w/v) glycerol, pH 7.5). The sample is filtered then through a sterile filter (Millipore) and kept at 4°C in a sterile square media bottle (Nalgene).

The purification is performed at 4⁰C on the VISION workstation (Applied Biosystems) connected to an automatic sample loader (Labomatic). The purification procedure is composed of two sequential steps, metal affinity chromatography on a Poros 20 MC (Applied Biosystems) column charged with Ni ions (4.6 x 50 mm, 0.83ml), followed by gel filtration on a Sephadex G-25 medium (Amersham Pharmacia) column (1,0 x 10cm).

For the first chromatography step the metal affinity column is regenerated with 30 column volumes of EDTA solution (10OmM EDTA; IM NaCl; pH 8.0), recharged with Ni ions through washing with 15 column volumes of a 10OmM NiSO₄ solution, washed with 10 column volumes of buffer A, followed by 7 column volumes of buffer B (50 mM NaH₂PO₄; 60OmM NaCl; 8.7 % (w/v) glycerol, 40OmM; imidazole, pH 7.5), and finally equilibrated with 15 column volumes of buffer A containing 15mM imidazole. The sample is transferred, by the Labomatic sample loader, into a 200ml sample loop and subsequently charged onto the Ni metal affinity column at a flow rate of 10ml/min. The column is washed with 12 column volumes of buffer A, followed by 28 column volumes of buffer A containing 2OmM imidazole. During the 2OmM imidazole wash loosely attached contaminating proteins are eluted from the column. The recombinant His-tagged protein is finally eluted with 10 column volumes of buffer B at a flow rate of 2ml/min, and the eluted protein is collected. For the second chromatography step, the Sephadex G-25 gel-filtration column is regenerated with 2ml of buffer D (1.137M NaCl; 2.7mM KCl; 1.5mM KH₂PO₄; 8mM Na₂HPO₄; pH 7.2), and subsequently equilibrated with 4 column volumes of buffer C (137mM NaCl; 2.7mM KCl; 1.5mM KH₂PO₄; 8mM Na₂HPO₄; 20% (w/v) glycerol; pH 7.4). The peak fraction eluted from the Ni-column is automatically loaded onto the Sephadex G-25 column through the integrated sample loader on the VISION and the protein is eluted with buffer C at a flow rate of 2 ml/min. The fraction was filtered through a sterile centrifugation filter (Millipore), frozen and stored at — 80°C. An aliquot of the sample is analyzed on SDS-PAGE (4-12% NuPAGE gel; Novex) Western blot with anti- His antibodies. The NuPAGE gel may be stained in a 0.1 % Coomassie blue R250 staining solution (30% methanol, 10% acetic acid) at room temperature for Ih and subsequently destained in 20% methanol, 7.5% acetic acid until the background is clear and the protein bands clearly visible.

Following the electrophoresis the proteins are electrotransferred from the gel to a nitrocellulose membrane. The membrane is blocked with 5% milk powder in buffer E (137mM NaCl; 2.7mM KCl; 1.5mM KH₂PO₄; 8mM Na₂HPO₄; 0.1 % Tween 20, pH 7.4) for Ih at room temperature, and subsequently incubated with a mixture of 2 rabbit polyclonal anti-His antibodies (G-18 and H-15, 0.2μg/ml each; Santa Cruz) in 2.5% milk powder in buffer E overnight at 4⁰C. After a further 1 hour incubation at room temperature, the membrane is washed with buffer E (3 x 1 Omin), and then incubated with a secondary HRP-conjugated anti-rabbit antibody (DAKO₅ HRP 0399) diluted 1/3000 in buffer E containing 2.5% milk powder for 2 hours at room temperature. After washing with buffer E (3 x 10 minutes), the membrane is developed with the ECL kit (Amersham Pharmacia) for 1 min. The membrane is subsequently exposed to a Hyperfilm (Amersham Pharmacia), the film developed and the western blot image visually analysed.

For samples that showed detectable protein bands by Coomassie staining, the protein concentration may be determined using the BCA protein assay kit (Pierce) with bovine serum albumin as standard.

Furthermore, overexpression or knock-down of the expression of the polypeptides in cell lines may be used to determine the effect on transcriptional activation of the host cell genome. Dimerisation partners, co-activators and co-repressors of the INTP041 polypeptide may be identified by immunoprecipitation combined with Western blotting and immunoprecipitation combined with mass spectroscopy.

Example 7: Microarray studies

Custom microarrays have been manufactured using Agilent Technologies' (Agilent Technologies Inc, Palo Alto, CA) non-contact in situ synthesis process of printing 60-mer length oligonucleotide probes, base-by-base, from digital sequence files. This is achieved with an inkjet process which delivers extremely small, accurate volumes (picoliters) of the chemicals to be spotted. Standard phosphoramidite chemistry used in the reactions allows for very high coupling efficiencies to be maintained at each step in the synthesis of the full- length oligonucleotide. Precise quantities are reproducibly deposited "on the fly." This engineering feat is achieved without stopping to make contact with the slide surface and without introducing surface-contact feature anomalies, resulting in consistent spot uniformity and traceability. (Hughes et al. (2001) Nat. Biotech. Apr; 19(4): 342-7. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer). Probe Synthesis

Methodologies are carried out according to Agilent instructions. Essentially, cDNA synthesis and subsequent T7 polymerase amplification of Cyanine 3(5)-CTP labeled cRNA probe was carried out using Agilent's low RNA input fluorescent linear amplification kit from a template of 5μg of total RNA according to the kit protocol (version 2 August 2003, Agilent, Palo Alto, CA). cRNA is then fragmented using Agilent's In Situ hybridization kit-plus and hybridized both according to Agilent's protocol (Agilent 60-mer oligo microarray processing protocol version 4.1 April 2004, Agilent, Palo Alto, CA). Microarray Chip Design

• 10,536 probes are on the array

• 5557 of the probes designed specifically to detect secreted sequences of primary interest » 1000 probes designed as negative controls

• 500 probes designed as positive controls

• Remainder of the probes were designed to public domain sequences which are known to be either secreted soluble extracellular proteins or membrane bound proteins with an extracellular domain in contact with the extracellular milieu.

Example 8: Hydrolytic activity of the polypeptides of the present invention

The biological activity of the polypeptides of the present invention can be evaluated by measuring the hydrolytic activity using various p-nitrophenyl (NP) glycosides as substrate. The reaction mixture contains, in a total volume of 40μl, 5OmM sodium acetate buffer (pH 5.0), 0.25mM substrate, and the polypeptide. After incubation for an appropriate time at 37' C, the reaction is stopped by adding 60μl of IM sodium carbonate, and the released p-nitrophenol (p NP) is measured by the the absorbence at 400nm. One unit of enzyme activity is defined as the amount of enzyme releasing lμmol of p NP per minute (Fujita et al. (2005) J.Biol.Chem, 280:37415-22).

Claims

1. A polypeptide, which polypeptide:

(i) comprises the amino acid sequence as recited in SEQ ID NO:96, SEQ ID NO: 158, SEQ ID NO:162, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180 or SEQ ID NO: 182;

(ii) is a fragment thereof which is a member of the glycoside hydrolase family 31 family of proteins, or having an antigenic determinant in common with the polypeptide of (i); or (iii)is a functional equivalent of (i) or (ii).

2. A polypeptide according to claim 1 which:

(i) consists of the amino acid sequence as recited in SEQ ID NO:96, SEQ ID NO: 158,

SEQ ID NO: 162, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID

NO: 174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180 or SEQ ID NO:182;

3. A polypeptide, which polypeptide:

4. A polypeptide according to claim 3 which:

(i) consists of the amino acid sequence as recited in SEQ ID NO: 100 or SEQ ID

NO: 160;

(ii) is a fragment thereof which is a member of the glycoside hydrolase family 31 family of proteins, or having an antigenic determinant in common with the polypeptide of (i); or

(iii)is a functional equivalent of (i) or (ii).

5. A polypeptide, which polypeptide:

(i) comprises the amino acid sequence as recited in SEQ ID NO: 156; (ii) is a fragment thereof which is a member of the glycoside hydrolase family 31 family of proteins, or having an antigenic determinant in common with the polypeptide of (i); or (iii)is a functional equivalent of (i) or (ii).

6. A polypeptide according to claim 5 which:

(i) consists of the amino acid sequence as recited in SEQ ID NO: 156;

7. A polypeptide which is a functional equivalent according to part (iii) of any of the above claims, characterised in that it is homologous to the amino acid sequence as recited in SEQ ID NO:96, SEQ ID NO: 100, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182 and is a member of the glycoside hydrolase family 31 family of proteins.

8. A polypeptide which is a fragment or a functional equivalent as recited in any one of claims 1 to 7, which has greater than 80% sequence identity with the amino acid sequence recited in SEQ ID NO:96, SEQ ID NO: 100, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172,

SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182 or with an active fragment thereof, preferably greater than 85%, 90%, 95%, 98% or 99% sequence identity.

9. A polypeptide which is a functional equivalent as recited in any one of claims 1 to 8, which exhibits significant structural homology with a polypeptide having the amino acid sequence recited in SEQ ID NO:96, SEQ ID NO: 100, SEQ ID NO: 156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182.

10. A polypeptide which is a fragment as recited in claims 1-6 and claim 8 having an antigenic determinant in common with the polypeptide of part (i) of any one of claim 1 to claim 6 which consists of 7 or more amino acid residues from the amino acid sequence recited in SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO.180, SEQ ID NO:182.

11. A fusion protein comprising a polypeptide according to any previous claim.

12. A purified nucleic acid molecule which encodes a polypeptide according to any one of the preceding claims.

13. A purified nucleic acid molecule according to claim 12, which comprises the nucleic acid sequence as recited in SEQ ID NO:95, SEQ ID NO: 99, SEQ ID NO: 155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179 or SEQ ID NO: 181 , or is a redundant equivalent or fragment thereof.

14. A purified nucleic acid molecule according to claim 13 which consists of the nucleic acid sequence as recited in SEQ ID NO:95, SEQ ID NO: 99, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179 or SEQ ID NO: 181 , or is a redundant equivalent or fragment thereof.

15. A purified nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule according to any one of claims 12 to 14.

16. A vector comprising a nucleic acid molecule as recited in any one of claims 13 to 15.

17. A host cell transformed with a vector according to claim 16.

18. A ligand which binds specifically to the glycoside hydrolase family 31 family polypeptide according to any one of claims 1 to 11.

19. A ligand according to claim 18, which is an antibody.

20. A compound that either increases or decreases the level of expression or activity of a polypeptide according to any one of claims 1 to 11.

21. A compound according to claim 20 that binds to a polypeptide according to any one of claims 1 to 11 without inducing any of the biological effects of the polypeptide.

22. A compound according to claim 21, which is a natural or modified substrate, ligand, enzyme, receptor or structural or functional mimetic.

23. A polypeptide according to any one of claims 1 to 11, a nucleic acid molecule according to any one of claims 12 to 15, a vector according to claim 16, a host cell according to claim 17, a ligand according to claim 18 or claim 19, or a compound according to any one of claims 20 to 22, for use in therapy or diagnosis of disease.

24. A method of diagnosing a disease or disorder in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to any one of claims 1 to 11 , or assessing the activity of a polypeptide according to any one of claims 1 to 11, in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease.

25. A method according to claim 24 that is carried out in vitro.

26. A method according to claim 24 or claim 25, which comprises the steps of: a) contacting a ligand according to claim 18 or claim 19 with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and b) detecting said complex.

27. A method according to claim 24 or claim 25, comprising the steps of: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule according to any one of claims 12 to 15 and the probe; b) contacting a control sample with said probe under the same conditions used in step a); and c) detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease.

28. A method according to claim 24 or claim 25, comprising: a) contacting a sample of nucleic acid from tissue of the patient with a nucleic acid primer under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule according to any one of claims 12 to 15 and the primer; b) contacting a control sample with said primer under the same conditions used in step a); and c) amplifying the sampled nucleic acid; and d) detecting the level of amplified nucleic acid from both patient and control samples; wherein detection of levels of the amplified nucleic acid in the patient sample that differ significantly from levels of the amplified nucleic acid in the control sample is indicative of disease.

29. A method according to claim 24 or claim 25 comprising: a) obtaining a tissue sample from a patient being tested for disease; b) isolating a nucleic acid molecule according to any one of claims 12 to 15 from said tissue sample; and c) diagnosing the patient for disease by detecting the presence of a mutation which is associated with disease in the nucleic acid molecule as an indication of the disease.

30. The method of claim 29, further comprising amplifying the nucleic acid molecule to form an amplified product and detecting the presence or absence of a mutation in the amplified product.

31. The method of claim 29 or claim 30, wherein the presence or absence of the mutation in the patient is detected by contacting said nucleic acid molecule with a nucleic acid probe that hybridises to said nucleic acid molecule under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation.

32. A method according to any one of claims 24 to 31, wherein said disease or disorder includes, but is not limited to, reproductive disorders, cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis' sarcoma; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, multiple sclerosis, peripheral nervous system disease and pain; developmental disorders; metabolic disorders including diabetes mellitus, hypoglycemia, osteoporosis, and obesity, AIDS and renal disease; infections including viral infection, bacterial infection, fungal infection, parasitic infection and sugar metabolism related disorders.

33. A method according to any one of claims 24 to 31, wherein said disease is a disease in which the glycoside hydrolase family 31 family of proteins are implicated.

34. Use of a polypeptide according to any one of claims 1 to 11 as a glycoside hydrolase family 31 protein.

35. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 11, a nucleic acid molecule according to any one of claims 12 to 15, a vector according to claim 16, a host cell according to claim 17, a ligand according to claim 18 or claim 19, or a compound according to any one of claims 20 to 22.

36. A vaccine composition comprising a polypeptide according to any one of claims 1 to 11 or a nucleic acid molecule according to any one of claims 12 to 15.

37. A polypeptide according to any one of claims 1 to 11, a nucleic acid molecule according to any one of claims 12 to 15, a vector according to claim 16, a host cell according to claim 17, a ligand according to claim 18 or claim 19, or a compound according to any one of claims 20 to 22, or a pharmaceutical composition according to claim 35, for use in the manufacture of a medicament for the treatment of reproductive disorders, cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis' sarcoma; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, multiple sclerosis, peripheral nervous system disease and pain; developmental disorders; metabolic disorders including diabetes mellitus, hypoglycemia, osteoporosis, and obesity, AIDS and renal disease; infections including viral infection, bacterial infection, fungal infection, parasitic infection, sugar metabolism related disorders and other pathological conditions.

38. A polypeptide according to any one of claims 1 to 11, a nucleic acid molecule according to any one of claims 12 to 15, a vector according to claim 16, a host cell according to claim 17, a ligand according to claim 18 or claim 19, or a compound according to any one of claims 20 to 22, or a pharmaceutical composition according to claim 35, for use in the manufacture of a medicament for the treatment of a disease in which the glycoside hydrolase family 31 family of proteins is implicated.

39. A method of treating a disease in a patient, comprising administering to the patient a polypeptide according to any one of claims 1 to 11, a nucleic acid molecule according to any one of claims 12 to 15, a vector according to claim 16, a host cell according to claim 17, a ligand according to claim 18 or claim 19, or a compound according to any one of claims 20 to 22, or a pharmaceutical composition according to claim 35.

40. A method according to claim 39, wherein, for diseases in which the expression of the natural gene or the activity of the polypeptide is lower in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, vector, ligand, compound or composition administered to the patient is an agonist.

41. A method according to claim 39, wherein, for diseases in which the expression of the natural gene or activity of the polypeptide is higher in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, vector, ligand, compound or composition administered to the patient is an antagonist.

42. A method of monitoring the therapeutic treatment of disease in a patient, comprising monitoring over a period of time the level of expression or activity of a polypeptide according to any one of claims 1 to 11 , or the level of expression of a nucleic acid molecule according to any one of claims 12 to 15 in tissue from said patient, wherein altering said level of expression or activity over the period of time towards a control level is indicative of regression of said disease.

43. A method for the identification of a compound that is effective in the treatment and/or diagnosis of disease, comprising contacting a polypeptide according to any one of claims 1 to 11, or a nucleic acid molecule according to any one of claims 12 to 15 with one or more compounds suspected of possessing binding affinity for said polypeptide or nucleic acid molecule, and selecting a compound that binds specifically to said nucleic acid molecule or polypeptide.

44. A kit useful for diagnosing disease comprising a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to any one of claims 12 to 15; a second container containing primers useful for amplifying said nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease.

45. The kit of claim 44, further comprising a third container holding an agent for digesting unhybridised RNA.

46. A kit comprising an array of nucleic acid molecules, at least one of which is a nucleic acid molecule according to any one of claims 12 to 15.

47. A kit comprising one or more antibodies that bind to a polypeptide as recited in any one of claims 1 to 11; and a reagent useful for the detection of a binding reaction between said antibody and said polypeptide.

48. A transgenic or knockout non-human animal that has been transformed to express higher, lower or absent levels of a polypeptide according to any one of claims 1 to 11.

49. A method for screening for a compound effective to treat disease, by contacting a non- human transgenic animal according to claim 48 with a candidate compound and determining the effect of the compound on the disease of the animal.

50. The use of an INTP041 polypeptide, INTP042 polypeptide or INTP043 polypeptide as a target for screening cadidadte drugs for treating or preventing a glycoside hydrolase 31 family related disorder.

51. Method of selecting biologically active compounds comprising:

(i) contacting a candidate compound with recombinant host cells expressing an INTP041 , INTP042 or INTP043 polypeptide;

(ii) selecting compounds that bind said INTP041, INTP042 or INTP043 polypeptide, respectively, at the surface of said cells and/or that modulate the activity of the INTP041, INTP042 or INTP043 polypeptide, respectively.