AU767005B2 - Dipeptidyl peptidases - Google Patents

Description

WO 01/19866 PCT/AU00/01085 1

TITLE

DIPEPTIDYL PEPTIDASES FIELD OF INVENTION The invention relates to a dipeptidyl peptidase, to a nucleic acid molecule which encodes it, and to uses of the peptidase.

BACKGROUND OF THE INVENTION The dipeptidyl peptidase (DPP) IV-like gene family is a family of molecules which have related protein structure and function The gene family includes the following molecules: DPPIV (CD26), dipeptidyl aminopeptidase-like protein (DPP6) and fibroblast activation protein (FAP) Another possible member is DPPIV-p[6].

The molecules of the DPPIV-like gene family are serine proteases, they are members of the peptidase family S9b, and together with prolyl endopeptidase (S9a) and acylaminoacyl peptidase (S9c), they are comprised in the prolyl oligopeptidase family[5,7].

DPPIV and FAP both have similar postproline dipeptidyl amino peptidase activity, however, unlike DPPIV, FAP also has gelatinase activity[8,9].

DPPIV substrates include chemokines such as RANTES, eotaxin, macrophage-derived chemokine and stromal-cellderived factor 1; growth factors such as glucagon and glucagon-like peptides 1 and 2; neuropeptides including neuropeptide Y and substance P; and vasoactive peptides[10-12].

DPPIV and FAP also have non-catalytic activity; DPPIV binds adenosine deaminase, and FAP binds to c3pi and asP 1 integrin[13-14].

WO 01/19866 PCT/AU00/01085 2 In view of the above activities, the DPPIV-like family members are likely to have roles in intestinal and renal handling of proline containing peptides, cell adhesion, peptide metabolism, including metabolism of cytokines, neuropeptides, growth factors and chemokines, and immunological processes, specifically T cell stimulation[3,11,12].

Consequently, the DPPIV-like family members are likely to be involved in the pathology of disease, including for example, tumour growth and biology, type II diabetes, cirrhosis, autoimmunity, graft rejection and HIV infection[3,15-18].

Inhibitors of DPPIV have been shown to suppress arthritis, and to prolong cardiac allograft survival in animal models in vivo[19,20]. Some DPPIV inhibitors are reported to inhibit HIV infection[21]. It is anticipated that DPPIV inhibitors will be useful in other therapeutic applications including treating diarrhoea, growth hormone deficiency, lowering glucose levels in non insulin dependent diabetes mellitus and other disorders involving glucose intolerance, enhancing mucosal regeneration and as immunosuppressants[3,21-24].

There is a need to identify members of the DPPIV-like gene family as this will allow the identification of inhibitor(s) with specificity for particular family member(s), which can then be administered for the purpose of treatment of disease. Alternatively, the identified member may of itself be useful for the treatment of disease.

WO 01/19866 PCT/AU00/01085 3 SUMMARY OF THE INVENTION The present invention seeks to address the above identified need and in a first aspect provides a peptide which comprises the amino acid sequence shown in SEQ ID NO:1.

This peptide has substrate specificity for the following compounds: H-Ala-Pro-pNA, H-Gly-Pro-pNA and H-Arg-Pro-pNA.

Therefore, it is a prolyl oligopeptidase and a dipeptidyl peptidase, because it is capable of hydrolysing the peptide bond C-terminal to proline in each of these compounds.

The peptide is homologous with human DPPIV, and importantly, identity between the sequences of DPPIV and SEQ ID NO: 1 is observed at the region of DPPIV containing the catalytic triad residues and the two glutamate residues of the P-propeller domain essential for DPPIV enzyme activity. The observation of amino acid sequence homology means that the peptide which has the amino acid sequence shown in SEQ ID NO:1 is a member of the DPPIVlike gene family. Accordingly the peptide was provisionally named DPPIVL1, and is now named and described herein as DPP8.

The following sequences of the human DPPIV amino acid sequence are important for the catalytic activity of DPPIV: Tyr 62 GlyTrpSerTyrGlyGlyTyrVal; (ii) Ala 7 0 7 AspAspAsnValHisPhe; (iii) Glu 738 AspHisGlyIleAlaGln; and (iv) Tyr 20 ValTyrGluGluGluVal [25-28]. As described herein, the alignment of the following sequences of DPP8: His 736 GlyTrpSerTyrGlyGlyTyrLeu; LeuB16AspGluAsnValHisPheAla; Glu 847 ArgHisSerIleArg and Phe 2 SSValLeuGlnGluGluPhe with sequences to (iv) above, respectively, suggests that these sequences of DPP8 are likely to confer the catalytic activity of DPP8. Thus, in a second aspect, the invention provides a peptide comprising the following amino acid WO 01/19866 PCT/AUOO/01085 4 sequences: His7 36 GlyTrpSerTyrGlyGlyTyrLeu Leu 8 6 AspGluAsnValHisPheAlaHis; Glu 847 ArgHisSerIleArg and Phe 2 SSValLeuGlnGluGluPhe; which has the substrate specificity of the sequence shown in SEQ ID NO:1.

Also described herein, using multiple sequence alignment, it is observed that DPP8 has 55% amino acid similarity and 32% amino acid identity with a C. elegans protein.

Further, as shown herein, a nucleic acid molecule which encodes DPP8, is capable of hybridising specifically with DPP8 sequences derived from non-human species. Together these data suggest that DPP8 is expressed in non-human species. Thus in a third aspect, the invention provides a peptide which has at least 60% amino acid identity with the amino acid sequence shown in SEQ ID NO:1, and which has the substrate specificity of the sequence shown in SEQ ID NO:1. Preferably, the amino acid identity is More preferably, the amino acid identity is 95%. Amino acid identity is calculated using GAP software [GCG Version 8, Genetics Computer Group, Madison, WI, USA] as described further herein. Typically, the non-human DPP8 comprises the following sequences: His36GlyTrpSerTyrGlyGlyTyrLeu; Leu 1 6AspGluAsnValHisPheAlaHis; Glu 847 ArgHisSerIleArg and Phe 255 ValLeuGlnGluGluPhe.

In view of the homology between DPPIV and DPP8 amino acid sequences, it is expected that these sequences will have similar tertiary structure. This means that the tertiary structure of DPP8 is likely to include the seven-blade Ppropeller domain and the cd/ hydrolase domain of DPPIV.

These structures in DPP8 are likely to be conferred by the regions comprising p-propeller, Gly 180 to Asp' 06

C/P

hydrolase, Ser 607 to Ile 882 and about 70 to 100 residues in the region Arg 39 to Gln 179 As it is known that the Ppropeller domain regulates proteolysis mediated by the catalytic triad in the a/P hydrolase domain of prolyl WO 01/19866 PCT/AU00/01085 5 oligopeptidase, [29] it is expected that truncated forms of DPP8 can be produced, which have the substrate specificity of the sequence shown in SEQ ID NO:1, comprising the regions referred to above (His 736 GlyTrpSerTyrGlyGlyTyrLeu; Leu 816 AspGluAsnValHisPheAlaHis; Glu 84 ArgHisSerIleArg and Phe 255 ValLeuGlnGluGluPhe) which confer the catalytic specificity of DPP8. Examples of truncated forms of DPP8 which might be prepared are those in which the region conferring the P-propeller domain and the o/P hydrolase domain are spliced together. Other examples of truncated forms include those which are encoded by splice variants of DPP8 mRNA. Thus although, as described herein, the biochemical characterisation of DPP8 shows that DPP8 consists of 882 amino acids and has a molecular weight of about 100kDa, it is recognised that truncated forms of DPP8 which have the substrate specificity of the sequence shown in SEQ ID NO:1, may be prepared using standard techniques [30,31]. Thus in a fourth aspect, the invention provides a fragment of the sequence shown in SEQ ID NO: 1, which has the substrate specificity of the sequence shown in SEQ ID NO:1. Preferably, the fragment has an amino acid sequence shown in SEQ ID NO: 3, 5 or 7.

As described herein, the sequence shown in SEQ ID NO:1 does not contain a consensus sequence for N-linked glycosylation. Therefore it is unlikely that DPP8 is associated with N-linked glycosylation. In this regard, DPP8 is distinguished from other DPPIV-like gene family members, which contain between 6 and 9 consensus sequences for N-linked glycosylation. Thus in one embodiment, an asparagine residue in the peptide of the first aspect of the invention is not linked to a carbohydrate molecule.

The analysis of DPPB expression described herein shows that it is likely that DPPB is expressed as a cytoplasmic protein. The expression of DPP8 is therefore WO 01/19866 PCT/AU00/01085 6 distinguished from other DPPIV-like gene family members, which are expressed on the cytoplasmic membrane, or in other words, the cell surface membrane. Thus in another embodiment, the peptide of the first aspect of the invention is not expressed on a cell surface membrane of a cell.

It is recognised that DPP8 may be fused, or in other words, linked to a further amino acid sequence, to form a fusion protein which has the substrate specificity of the sequence shown in SEQ ID NO:1. An example of a fusion protein is described herein which comprises the sequence shown in SEQ ID NO:1 which is linked to a further amino acid sequence: a "tag" sequence which consists of an amino acid sequence encoding the V5 epitope and a His tag. An example of another further amino acid sequence which may be linked with DPP8 is a glutathione S transferase (GST) domain Another example of a further amino acid sequence is a portion of CD8x Thus in one aspect, the invention provides a fusion protein comprising the amino acid sequence shown in SEQ ID NO:1 linked with a further amino acid sequence, the fusion protein having the substrate specificity of the sequence shown in SEQ ID NO:1.

It is also recognised that the peptide of the first aspect of the invention may be comprised in a polypeptide, so that the polypeptide has the substrate specificity of DPP8. The polypeptide may be useful, for example, for altering the protease susceptibility of DPP8, when used in in vivo applications. An example of a polypeptide which may be useful in this regard, is albumin. Thus in another embodiment, the peptide of the first aspect is comprised in a polypeptide which has the substrate specificity of DPP8.

WO 01/19866 PCT/AU00/01085 7 As described above, the isolation and characterisation of DPP8 is necessary for identifying inhibitors of DPP8 catalytic activity, which may be useful for the treatment of disease. A method for identifying inhibitors of DPP8 catalytic activity, described herein, has identified that various inhibitors of DPPIV and serine proteases, zinc and mimetic peptides, Ala-Pro-Gly and Lys-Pro, but not inhibitors of metalloproteinases, aspartyl proteinases or cysteinyl proteinases, inhibit DPP8 catalytic activity.

Accordingly, in a fifth aspect, the invention provides a method of identifying a molecule capable of inhibiting cleavage of a substrate by DPP8, the method comprising the following steps: contacting DPP8 with the molecule; contacting DPP8 of step with a substrate capable of being cleaved by DPP8, in conditions sufficient for cleavage of the substrate by DPP8; and detecting substrate not cleaved by DPP8, to identify that the molecule is capable of inhibiting cleavage of the substrate by DPP8.

It is recognised that although inhibitors of DPP8 may also inhibit DPPIV and other serine proteases, as described herein, the alignment of the DPP8 amino acid sequence with most closely related molecules,(i.e. DPPIV), reveals that the DPP8 amino acid is distinctive, particularly at the regions controlling substrate specificity. Accordingly, it is expected that it will be possible to identify inhibitors which inhibit DPP8 catalytic activity specifically, which do not inhibit catalytic activity of DPPIV-like gene family members, or other serine proteases.

Thus, in a sixth aspect, the invention provides a method of identifying a molecule capable of inhibiting specifically, the cleavage of a substrate by DPP8, the method comprising the following steps: WO 01/19866 PCT/AUOO/01085 -8 contacting DPPB and a further protease with the molecule; contacting DPP8 and the further protease of step with a substrate capable of being cleaved by DPP8 and the further protease, in conditions sufficient for cleavage of the substrate by DPP8 and the further protease; and detecting substrate not cleaved by DPP8, but cleaved by the further protease, to identify that the molecule is capable of inhibiting specifically, the cleavage of the substrate by DPP8.

In a seventh aspect, the invention provides a method of reducing or inhibiting the catalytic activity of DPP8, the method comprising the step of contacting DPP8 with an inhibitor of DPP8 catalytic activity. As various inhibitors of DPPIV catalytic activity are shown herein to inhibit DPP8 catalytic activity, it is recognised that other inhibitors of DPPIV may be useful for inhibiting DPP8 catalytic activity. Examples of inhibitors suitable for use in the seventh aspect are described in [21,32,33].

Other inhibitors useful for inhibiting DPP8 catalytic activity can be identified by the methods of the fifth or sixth aspects of the invention, which methods are exemplified herein.

In one embodiment, the catalytic activity of DPP8 is reduced or inhibited in a mammal by administering the inhibitor of DPP8 catalytic activity to the mammal. It is recognised that these inhibitors have been used to reduce or inhibit DPPIV catalytic activity in vivo, and therefore, may also be used for inhibiting DPP8 catalytic activity in vivo. Examples of inhibitors useful for this purpose are disclosed in the following [21,32-34].

Preferably, the catalytic activity of DPP8 in a mammal is reduced or inhibited in the mammal, for the purpose of WO 01/19866 PCT/AU00/01085 9 treating a disease in the mammal. Diseases which are likely to be treated by an inhibitor of DPP8 catalytic activity are those in which DPPIV-like gene family members are associated [3,10,11,17,21,36], including for example, neoplasia, type II diabetes, cirrhosis, autoimmunity, graft rejection and HIV infection.

Preferably, the inhibitor for use in the seventh aspect of the invention is one which inhibits the cleavage of a peptide bond C-terminal adjacent to proline. As described herein, examples of these inhibitors are 4-(2aminoethyl)benzenesulfonylfluoride, aprotinin, benzamidine/HCl, Ala-Pro-Gly, H-Lys-Pro-OH HC1 salt and zinc ions, for example, zinc sulfate or zinc chloride.

More preferably, the inhibitor is one which specifically inhibits DPP8 catalytic activity, and which does not inhibit the catalytic activity of other serine proteases, including, for example DPPIV or FAP.

In an eighth aspect, the invention provides a method of cleaving a substrate which comprises contacting the substrate with DPP8 in conditions sufficient for cleavage of the substrate by DPP8, to cleave the substrate.

Examples of molecules which can be cleaved by the method are H-Ala-Pro-pNA, H-Gly-Pro-pNA and H-Arg-Pro-pNA. The conditions sufficient for cleaving the substrate are described herein. Molecules which are cleaved by DPPIV including RANTES, eotaxin, macrophage-derived chemokine, stromal-cell-derived factor 1, glucagon and glucagon-like peptides 1 and 2, neuropeptide Y, substance P and vasoactive peptide are also likely to be cleaved by DPP8 [11,12]. In one embodiment, the substrate is cleaved by cleaving a peptide bond C-terminal adjacent to proline in the substrate. The molecules cleaved by DPP8 may have Ala, or Trp, Ser, Gly, Val or Leu in the P1 position, in place of Pro [11,12].

WO 01/19866 PCT/AUOO/01085 10 As described herein, DPP8 gene expression is upregulated in stimulated lymphocyte and lymphocytic cell lines which suggests that DPP8 may have a functional role in T cell costimulation and proliferation. It is recognised therefore that measuring DPP8 gene expression is useful for detecting T cell activation. Thus in a ninth aspect, the invention provides a method of detecting an activated T cell, the method comprising the step of detecting the level of DPP8 gene expression in a T cell. In one embodiment, the level of DPP8 gene expression is detected by measuring the amount of DPP8 mRNA in the cell, as described herein.

The inventors have characterised the sequence of a nucleic acid molecule which encodes the amino acid sequence shown in SEQ ID NO:1. Thus in a tenth aspect, the invention provides a nucleic acid molecule which encodes the amino acid sequence shown in SEQ ID NO:1.

In an eleventh aspect, the invention provides a nucleic acid molecule which consists of the sequence shown in SEQ ID NO:2.

As described herein, at least three splice variants of DPP8 RNA which have an open reading frame from 2.6 to 3.1 kb in length are observed. As a frame shift mutation or termination signal was not observed in the sequence of these splice variants, and as the coding sequence of two of the splice variants includes a sequence which encodes the amino acid sequence associated with catalytic activity, it is recognised that some of the peptides encoded by the splice variants are likely to have the substrate specificity of DPP8. Thus in an embodiment, the nucleic acid molecule is a fragment of the sequence shown in SEQ ID NO: 1 which is about 2.6 to 3.1 kb in length and which encodes a peptide which has the substrate WO 01/19866 PCT/AU00/01085 11 specificity of the sequence shown in SEQ ID NO:1.

Preferably, the nucleic acid molecule has a sequence shown in any one of SEQ ID NO.s: 4, 6 and 8.

In a twelfth aspect, the invention provides a nucleic acid molecule which is capable of hybridising to a nucleic acid molecule consisting of the sequence shown in SEQ ID NO:2 in stringent conditions, and which encodes a peptide which has the substrate specificity of the sequence shown in SEQ ID NO:1. As shown in the Northern blot analysis described herein, DPP8 mRNA hybridises specifically to the sequence shown in SEQ ID NO:2, after washing in 2XSSC/ 1.0%SDS at 37 0 C, or after washing in 0.1XSSC/0.1% SDS at 50 0

C.

"Stringent conditions" are conditions in which the nucleic acid molecule is exposed to 2XSSC/ 1.0% SDS. Preferably, the nucleic acid molecule is capable of hybridising to a molecule consisting of the sequence shown in SEQ ID NO:2 in high stringent conditions. "High stringent conditions" are conditions in which the nucleic acid molecule is exposed to 0.1XSSC/ 0.1%SDS at 50 0

C.

As described herein, the inventors believe that the gene which encodes DPP8 is located at band q22 on human chromosome 15. The location of the DPPB gene is distinguished from genes encoding other prolyl oligopeptidases, which are located on chromosome 2, at bands 2q24.3 and 2q23, or chromosome 7. Thus in an embodiment, the nucleic acid molecule is one capable of hybridising to a gene which is located at band q22 on human chromosome It is recognised that a nucleic acid molecule which encodes the amino acid sequence shown in SEQ ID NO:1, or which comprises has the sequence shown in SEQ ID NO:2, could be made by producing the fragment of the sequence which is translated, using standard techniques [30,31].

WO 01/19866 PCT/AUO0/01085 12 Thus in an embodiment, the nucleic acid molecule does not contain 5' or 3' untranslated sequences.

In a thirteenth aspect, the invention provides a vector which comprises a nucleic acid molecule of the tenth aspect of the invention. In one embodiment, the vector is capable of replication in a COS-7 cell, CHO cell or 293T cell, or E.coli. In another embodiment, the vector is selected from the group consisting of XTripleEx, pTripleEx, pGEM-T Easy Vector, pSecTag2Hygro, petl5b, pEE14.HCMV.gs and pCDNA3.1/V5/His.

In a fourteenth aspect, the invention provides a cell which comprises a vector of the thirteenth aspect of the invention. In one embodiment, the cell is an E.coli cell.

Preferably, the E. coli is MC1061, DH5a, JM109, BL21DE3, pLysS. In another embodiment, the cell is a COS-7, COS-1, 293T or CHO cell.

In a fifteenth aspect, the invention provides a method for making a peptide of the first aspect of the invention comprising, maintaining a cell according to the fourteenth aspect of the invention in conditions sufficient for expression of the peptide by the cell. The conditions sufficient for expression are described herein. In one embodiment, the method comprises the further step of isolating the peptide.

In a sixteenth aspect, the invention provides a peptide when produced by the method of the fifteenth aspect.

In a seventeenth aspect, the invention provides a composition comprising a peptide of the first aspect and a pharmaceutically acceptable carrier.

WO 01/19866 PCT/AU00/01085 13 In an eighteenth aspect, the invention provides an antibody which is capable of binding a peptide according to the first aspect of the invention. The antibody can be prepared by immunising a subject with purified DPP8 or a fragment thereof according to standard techniques As described herein, an antibody was prepared by immunising with transiently transfected DPP8 cells. It is recognised that the antibody is useful for inhibiting activity of DPP8, or for detecting increased gene expression of DPP8, for the purpose of identifying an activated T cell. In one embodiment, the antibody of the eighth aspect of the invention is produced by a hybridoma cell.

In a nineteenth aspect, the invention provides a hybridoma cell which secretes an antibody of the nineteenth aspect.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Cloning strategy for isolating full-length DPP8 cDNA and the alternative splicing variants of DPP8 observed. Representation of three splice variants is shown including loss of serine recognition site by one splice variant (T8).

Figure 2. Nucleotide sequence and amino acid sequence of human DPP8. The nucleotide and predicted one letter code amino acid sequence are shown. This sequence shows no putative membrane spanning domain (deduced from hydrophobicity plots) or potential N-linked glycosylation sites. The putative serine recognition site and aspartic acid and histidine which form the Ser-Asp-His catalytic triad are marked. Base pairs are numbered in the right margin.

WO 01/19866 PCT/AU00/01085 14 Figure 3. Alignment of the deduced amino acid residue sequence of DPP8 with the C. elegans homolog of DPP8 and human DPPIV. Amino-acid residues are numbered in the right margin. Amino-acid residues identical in all three proteins are boxed. Asterisks mark the putative catalytic triad residues and two glutamates of the P-propeller domain essential for DPPIV enzyme activity. The grey shading denotes the al/phydrolase domain of these proteins. Filled triangles joined by lines indicate starts and ends of alternatively spliced transcripts, stPBMCdy3-3-10 (solid lines), T8(dashed lines) and T21 (solid lines). The alignment was constructed using the PILEUP program in GCG.

Figure 4. Northern Blot analysis of DPP8 expression. Human multiple tissue Northern blots (CLONTECH) containing 2 pg per lane of poly A* RNA were hybridized with a 32 P labeled DPP8 probe at 68 0 C and washed at high stringency. The autoradiograph was exposed for 1 day at -70 0 C with a BIOMAX MS screen. Molecular mass markers are indicated in base pairs on the left side of each autoradiogram. Figure 4a.

Master RNA (CLONTECH) blot of poly A RNA was hybridized with a 32 P labelled DPP8 probe at 65 0 C and washed at high stringency. The autoradiograph was exposed for 3 days at 0 C with BIOMAX MS screen. DPP8 mRNA was detected in all tissues examined.

Figure 5. Chromosomal localization of human DPP8.

Metaphase showing FISH with the biotinylated DPP8 cDNA probe. Normal male chromosomes stained with DAPI.

Hybridization sites on chromosome 15 are indicated by an arrow.

Figure 6. Western blot analysis of transfected cell lines.

Analysis of lysates of stable cell lines. DPP8 protein was WO 01/19866 PCT/AU00/01085 15 seen in DPP8 /V5/His stable cell lines but not in DPP4 or vector-only stable cell lines. The electrophoretic mobility of the protein was not altered when samples were boiled. The band of greater mobility was probably a breakdown product of intact DPP8.

Figure 7. DPP8 enzyme activity. pH-dependence of DPP8 enzyme activity. DPP8 and DPPIV enzyme kinetics.

Means SD of absorbance change per minute, multiplied by 1000 are shown. Curve fitting assumed Michaelis-Menten kinetics.

Figure 8. RT-PCR analysis of DPP8 expression. PCR amplifications with primers specific for either a portion of human DPP8 that contained no alternate splicing, Va1416 to Gly 679 (top of each gel) or glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (bottom of each gel. Top gel, lanes 1-5 contain PCR products from unstimulated PBMC cDNA from five subjects. Bottom gel, lanes 6 to 11 contain PCR products from OKT3-stimulated PBMC cDNA from six subjects.

PCR products are from cDNA from lymphocytic cell lines, liver or placenta as indicated. Negative control amplifications contained reaction mix, enzyme and no cDNA template. Each PCR was performed for 35 cycles. The PCR products were electrophoresed on agarose gels and stained with ethidium bromide. The left lane of each gel contains PUC19 digested with HaeIII as size markers.

Figure 9. Northern blot analysis of murine DPP8 expression. A murine Northern blot containing 10 jg per lane of total RNA was hybridized with a 32 P-labeled human DPP8 probe at 60°C and washed at low stringency.

Autoradiographic exposure was for 3 days at -70 0 C with a BIOMAX MS screen.

WO 01/19866 PCT/AUOO/01085 16 DETAILED DESCRIPTION OF THE INVENTION

EXAMPLES

General Restriction enzymes and other enzymes used in cloning were obtained from Boehringer Mannheim Roche. Standard molecular biology techniques were used [31] unless indicated otherwise.

An EST clone (GENBANK TM accession number AA417787) was obtained from American Type Culture Collection. The DNA insert of this clone was sequenced on both strands using automated sequencing at SUPAMAC (Sydney, Australia).

Cell culture and RNA preparation Human peripheral blood monocytes (PBMCs) were isolated by Ficoll-Hypaque density-gradient centrifugation (Pharmacia, Uppsala, Sweden) of blood obtained from healthy donors.

The PBMCs were incubated in AIM-V medium (Life Technologies, Gaithersburg, MD, USA) supplemented with 2 mM L-glutamine and were stimulated with either 1 gg.mL 1 phytohaemagglutinin (Wellcome) or 100ng.mL 1 OKT3 (Orthoclone, FL, USA) for 72 h. The human cell lines Jurkat, CCRF-CEM, Raji, Daudi and HepG2 were grown to confluence in Dulbecco's modified Eagle's medium (Trace Biosciences, NSW, Australia) supplemented with 10% fetal bovine serum and 2mM L-glutamine.

Liver and placental RNA were prepared from snap-frozen human tissue as described previously However, RNA was prepared from PBMCs and cell lines using an RNAeasy kit (Qiagen, Germany).

Bioinformatics BLAST programs [38] and all multiple sequence alignments WO 01/19866 PCT/AU00/01085 17 were performed through the Australian National Genomic Information Service (ANGIS, Sydney, NSW, Australia).

PILEUP (GCG Version 8, Genetics Computer Group, Madison, WI, USA) was used for multiple sequence alignments of proteins.

A BLAST search was performed on the public expressed sequence tag (EST) database using the complete human DPPIV (GenBank T M accession number X60708) and FAP (accession number U09278) nucleotide sequences as query sequences.

An EST clone (accession number AA417787) was obtained from the American Type Culture Collection. The DNA insert of this clone was sequenced on both strands using automated sequencing at SUPAMAC (Sydney, NSW, Australia). Because of its homology with DPPIV, this new gene was named dipeptidyl peptidase 8 (DPP8).

DPP8 Cloning ESTAA417787 was used to design forward (caa ata gaa att gac gat cag gtg) and reverse (tct tga agg tag tgc aaa aga tgc) DPP8 primers for polymerase chain reaction (PCR) from ESTAA417787. The PCR conditions were as follows: 94°C for min, followed by 35 cycles of 94 0 C for 1 minute, 55 0 C for sec and 70 0 C for 1 min. This 484 bp PCR product was gel purified, 32 P-a labelled using Megaprime Labeling Kit (Amersham Pharmacia Biotec, UK) and hybridized to a Master RNA blot (CLONTECH, Palo Alto, CA, USA) that contained poly A' from 50 adult and fetal tissues immobilized in dots as per manufacturers' instructions. This Master RNA blot was also probed with DPP4 for comparison of mRNA tissue expression.

The forward and reverse DPP8 primers were used for PCR to screen a human placental X STRETCH PLUS library (CLONTECH, Palo Alto, CA, USA) for the presence of DPP8 cDNA in the WO 01/19866 PCT/AU00/01085 18 library. The library was then screened by standard molecular biology techniques [30,31]. After primary screening, 23 clones were selected for secondary screening, after which 22 remained positive. For the tertiary screen the clones contained in XTripleEx were converted into pTriplEx plasmids and transformed into BM25.8 E. coli recipient bacteria. The plated bacteria were screened and it was confirmed that all 22 clones were positive. Two of these clones, T8 and T21 were selected for further study.

(Rapid amplification of cDNA ends) A 5' RACE Version 2.0 kit (Gibco BRL, Life technologies) was applied on activated T cell (ATC) and placental RNA as prescribed in the kit instructions. The T8 DNA sequence was used to design GSP 1 (TCC TTC CTT CAG CAT CAA TC) and GSP2 (CTT AAA AGT GAC TTT AGG ATT TGC TGT ACC). 5' RACE PCR products were cloned into pGEM-T Easy®Vector (Promega Co., Madison, WI, USA) and sequenced by primer walking.

Confirmation of identity of RACE product Reverse transcriptase PCR was carried out on ATC RNA using DPP8-pr23 (GGA AGA AGA TGC CAG ATC AGC TGG) and DPP8-prl9r (TCC GTG TAT CCT GTA TCA TAG AAG) to span across the junction between the RACE product and the EST and library clones. Two gel purified products ATCd3-2-1 (1603bp) and ATC3-3-10 (1077bp) were cloned into pGEM-T Easy® (Promega Co., Madison, WI, USA) and sequenced.

Subcloning of DPP8 cDNA into a pcDNA3.1/V5/His Expression Vector The ATC RACE product, the ATCd3-2-1 (1603bp) junction fragment and the library clone T21 were joined together and cloned into the expression vector pcDNA3.1/V5/His A (Invitrogen, the Netherlands) to form a DPP8 cDNA of 3.1 WO 01/19866 PCT/AU00/01085 19 kb with an open reading frame of 882 aa. The first construct was made using three sequential cloning steps.

Firstly, a Eco RV/Xba I fragment of T21 (containing 3' DPP8, stop codon and 3' untranslated region on DPP8 cDNA) was ligated into the vector pcDNA3.1/V5/His A which had been digested with Eco RV/Xba I. An Eco RI/Eco RV fragment of ATCd3-2-1 was then added to this construct digested with Eco RI/Eco RV. Finally the RACE product was cut with Eco RI and cloned into the Eco RI site of the previous construct to form the complete 3.1 kb DPP8 cDNA. This construct pcDNA3.1-DPP8 expressed protein with no detectable tag. In addition the stop codon in the DPP8 expression construct in pcDNA3.1/V5/His V5 was genetically altered using PCR to create a C-terminal fusion with the V5 and His tag contained in the vector. This construct was named pcDNA3.1- DPP8/V5/His. All expression constructs subcloned into pcDNA3.1/V5/His were verified by full sequence analysis.

DPP8 gene expression by Northern Blot Human multiple tissue Northern blots (CLONTECH) containing 2 ug of poly A' RNA were prehybridized in Express Hybridization solution (CLONTECH) for 30 min at 68 0

C.

Both the DPP8 484 bp product and the 5' RACE ATC product were radiolabeled using a Megaprime Labeling kit (Amersham Pharmacia Biotech) and 32 P]dCTP (NEN Dupont).

Unincorporated label was removed using a NICK column (Amersham Pharmacia Biotech) and the denatured probe was incubated for 2 hrs at 68 0 C in Express Hybridization solution. Washes were performed at high stringency and blots exposed to BIOMAX MS film for overnight with a BIOMAX MS screen at -70 0

C.

DPP8 qene expression in mice by Northern Blot WO 01/19866 PCT/AU00/01085 20 A Northern blot containing 10 ug of total liver RNA per lane was made using standard methods The RNA was derived from male and female mice of two strains, C57B16 and Balb/c. The Northern blot was prehybridized in Express Hybridization solution (CLONTECH, Palo Alto, USA) for 1 hr at 60 0 C. A 2.4kb human DPP8 cDNA (PCR product) was radiolabeled using the Megaprime Labeling kit (Amersham Pharmacia Biotech) and 32 "PdCTP (NEN Dupont).

Unincorporated label was removed using a NICK column (Amersham Pharmacia Biotech) and the denatured probe was incubated with the blot overnight at 60 0 C in Express Hybridization solution. Washes were performed at low stringency (2 x SSC/0.05% SDS for 1 h at 37°C followed by 0.1x SSC/0.1% SDS for 30 min at 40 0 C) and blots exposed to BIOMAX MS film for three days with a BIOMAX MS screen at 0

C.

Expression of DPP8 in mouse liver using rtPCR Mouse liver RNA was reverse transcribed using the Superscript II enzyme kit (Gibco BRL, Gaithersburg, MD) as described previously The cDNA was diluted 1 in 4 and stored in aliquots at 70 0 C. PCR using mouseDPP8-prlF (atg att acc acc cag gaa gcg) as the forward primer and mouseDPP8-pr2R (atc tcc gac ate ttg aaa gtg acc) as the reverse primer was used to detect mouse DPP8 mRNA.

One ul of diluted cDNA was amplified in a 50 ul PCR reaction which contained: 0.2 mM dNTPs, 1 ul of 50 x Advantage 2 Polymerase Mix (Clontech), 1 X Advantage 2 PCR buffer (Clontech) and 100 ng of each primer. The PCR involved an initial step of 95 0 C for 1 min to inactivate the TaqStart Antibody. This was followed by 35 cycles; denaturation at 95°C for 30 sec, 68 0 C for 1 min, followed by a final step of 68 0 C for 1 min. The amplified products were analysed by electrophoresis of 10 p1 of PCR reaction on a 3:1 Nusieve gel (FMC Bioproducts, Rockville, MD) plus WO 01/19866 PCT/AU00/01085 21 g/ml ethidium bromide in TAE buffer (0.04M Tris acetate, 0.001 M EDTA, pH The gel was then Southern Blotted using standard techniques The Southern blot was hybridized at 60 0 C for 2hr with the 2.4 kb human DPP8 cDNA probe prepared as described above. Washes were performed at low stringency (2 x SSC/0.05% SDS for 1 h at 37 0 C followed by 0.1x SSC/0.1% SDS for 40 min at 50 0 The blot was exposed to XAR5 Kodak film for 30 min at RT.

DPP8 expression by RT-PCR Reverse transcriptase PCR was performed on human ATC RNA, human placental RNA and human liver RNA using TED primers DPP8/pr3 (GCA CTA CCT TCA AGA AAA CCT TGG) and DPP8/pr20R (TAT GGT ATT GCT GGG TCT CTC AGG) to give a 293 bp product.

Transfection, Western blot, immunocytochemistry, cytochemistry and flow cytometry Monkey kidney fibroblast (COS-7) cells (American Type Culture Collection, CRL-1651) were grown and transfected as described previously For making stable cell lines, Geneticin (G418; Gibco-BRL) was added to the medium, beginning 24 h after transfection.- COS cell extracts were prepared by sonication followed by differential centrifugation and neither boiled nor reduced before SDS/PAGE (10% gel) and transfer to nitrocellulose, as described previously The presence of DPP8 fused with the V5 epitope was detected using an mAb (Invitrogen). COS cell monolayers were fixed in cold ethanol before staining with anti-V5 mAb [39,41,9]. Some monolayers were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 then doublestained with wheat germ agglutinin to label Golgi apparatus and with goat anti-mouse IgG to label DPP8, conjugated to Alexa Fluor 488 and Alexa Fluor 594, WO 01/19866 PCT/AU00/01085 22 respectively (Molecular Probes, Eugene, OR, USA). Flow cytometry and confocal scanning microscopy using a Leica TCS-NT confocal microscope have been described previously [39,9].

Purification of recombinant DPP8/V5/His and Cells (1 x 107) expressing each protein were sonicated in native buffer (50m sodium phosphate, 300 mM NaC1), then treated with 700 U DNAse for 20 min at room temperature.

DPPIV is expressed at the cell surface, so 1% Triton X-100 was used to solubilize DPPIV/V5/His. Insoluble material was removed by centrifugation. The supernatant was incubated with 1 mL Talon® Metal Affinity Resin (Clontech) following the manufacturer's instructions for a batch/gravity flow procedure. The resin was washed with mM sodium phosphate, containing 300 mM NaC1 and 5 mM imidazole, and proteins were eluted using the same buffer containing 150 mM imidazole. Enzyme activity was used to monitor eluted fractions.

Enzyme assays Enzyme assays were performed as described previously Either clarified cell extract from 1 x 104 sonicated COS-7 cells or purified protein derived from 1 x 10 s cells was incubated with substrate in 70pL phosphate buffer, pH 7.4, for 30 min at 37 0 C, except where otherwise indicated. The specific DPPIV substrates, Gly-Pro-toluenesulfonate, H- Gly-Pro-p-nitroanilide (NA)/HC1 (Sigma, St Louis, MO, USA) and Gly-Pro-7-amino-4-trifluromethylcoumarin (Calbiochem, San Diego, CA, USA) were tested. Other substrates tested were H-Ala-Pro-pNA/HC1, H-Arg-Pro-pNA acetate salt, H-Lys- Ala-pNA.2HC1, H-Asp-Pro-pNA, H-Ala-Ala-pNA/HC1, H-Ala-Ala- Pro-pNA/HC1, H-Ala-Ala-Phe-pNA, succinyl-Ala-Pro-pNA, H- Ala-Phe-Pro-pNA and Z-Ala-Pro-p-NA from Bachem WO 01/19866 PCT/AU00/01085 23 (Switzerland). H-Ala-Pro-4-methoxypNA/HCl, Z-Lys-Pro-4methoxypNAformate salt, H-Lys-Pro-4-methoxypNA/HCl, Z-Ala- Pro-4-methoxypNA, H-Gly-Pro-PNA and H-His-Ser-4methoxypNAacetate salt (Bachem) were tested for their ability to stain unfixed transfected cells.

All inhibitors were (see Table 2) incubated with each purified enzyme in phosphate buffer, pH 7.4, for 15 min before the addition of substrate. After the addition of 1mM H-Ala-Pro-pNA substrate for purified DPP8 and 1 mM H- Gly-Pro-pNA substrate for purified DPPIV, samples were incubated for 60 min at 37 0 C. All enzyme assays were performed in triplicate.

Chromosomal localization of DPP8 by Fluorescence in situ Hybridization (FISH) analysis DPP8 was localized using two different probes, the DPP8 EST and the TB clone. The probes were nick-translated with biotin-C 14 -dATP and hybridized in situ at a final concentration of 10ng/ul to metaphases from two normal males. The FISH method was modified from that previously described [37] in that chromosomes were stained before analysis with both propidium iodide (as counterstain) and DAPI (for chromosomal identification). Images of metaphase preparations were captured by a cooled CCD camera using the Cyto Vision Ultra image collection and enhancement system (Applied Imaging International Ltd). FISH signals and the DAPI banding pattern were merged for figure preparation.

Expression of DPP8 in human lymphocytes and cell lines RNA (lpg) was reverse-transcribed using the Superscript II enzyme kit (Gibco-BRL) as described previously PCR using DPP8-prl8 (CTGTGACGCCACTAATTATCTATG) as the forward primer and DPP8-pr26R (CCTAGAGAGGCTAGGGTATTCAAG) as the WO 01/19866 PCT/AU00/01085 24 reverse primer was used to detect full-length DPP8 mRNA.

The glyceraldehyde-3-phosphate dehydrogenase (G3PDH) control primer set was G3PDH for (ACCACAGTCCATGCCATCAC) and G3PDHrev (TCCACCACCCTGTTGCTGTA) to give a 470-bp product.

cDNA (diluted 1 4; lg) was amplified in a 25-gL PCR mixture which contained: 0.2 mM dNTPs, 0.125 unit Amplitaq Gold enzyme (Perkin-Elmer), 1 x buffer II (Perkin-Elmer), 1.5 mM MgC12 and 100ng mL' 1 each primer. The 35-cycle PCR was performed as follows: denaturation at 94 0 C for 1 min, primer annealing at 55 0 C for 30 s, and an extension step at 72 0 C for 1 min. The amplified products were analyzed by electrophoresis of 15tL PCR mixture on a 3 1 Nusieve gel (FMC Bioproducts, Rockville, MD, USA) plus 0.5 ig mL ethidium bromide in Tris/acetate/EDTA buffer (0.04 M Tris/acetate, 0.001 M EDTA, pH Anti-peptide antibody Methods followed are described in Current Protocols in Immunology Two peptides were chosen using the software MacVector to predict antigenicity. The two peptides were custom synthesized (Auspep, Melbourne) and conjugated to diptheria toxin (Auspep, Melbourne).

Rabbits were immunized with both peptides and serum collected at time zero and after each injection (IMVS, Adelaide.

The two peptides used were: PEPTIDE Name: TEDDA-N SEQUENCE: CTGYTERYMGHPDQNEQG-NH2 This is amino acids 773 to 789, plus a Cys at the N- WO 01/19866 PCT/AU00/01085 25 terminus.

PEPTIDE Name: TEDDR-C SEQUENCE: GKPYDLQIYPQERHSC-NH2 This is amino acids 836 to 850, plus a Cys at the Cterminus.

These sequences were taken from the C-terminal portion of DPP8.

Monoclonal antibody to DPP8 Standard methods were used for antibody production Mice were immunized with 2 x10 7 live COS-7 (African Green Monkey Kidney) cells that had been transiently transfected with the DPP8 cDNA in the pcDNA3 vector. The final immunisation was with CHO (Chinese Hamster Ovary) cells stably transfected with DPP8 cDNA in the pEE14 vector.

Spleen cells were fused with a standard fusion partner, X63Ag8 myeloma cells. Hybridoma culture supernatants were tested by immunoperoxidase histochemistry on monolayers of the DPP8-transfected CHO cell line, using untransfected CHO cells as the negative control. Hybridomas that produced antibody activity were cloned.

RESULTS

Molecular cloning and sequence analysis of DPP8 The insert in ATCC EST AA417787 was 795 bp in length, containing 527 bp of coding sequence, a TAA stop codon and 258 bp of 3' noncoding sequence (Figure 1).

The hybridization of the Master RNA blot revealed that the gene comprising ESTAA417787 has ubiquitous tissue expression, with high levels of expression in testis and placenta. Based on this expression pattern, a placental WO 01/19866 PCT/AU00/01085 26 cDNA library was screened with a 484 bp PCR product produced by the forward and reverse DPP8 primers.

Sequence homology analysis revealed that only 2 of 23 clones contained 5' sequence additional to the sequence of ESTAA417787. These cDNA clones were designated T8 and T21, and were 1669 bp and 1197 bp respectively (Figure 1).

In addition, comparison of these sequences to ESTAA417787 revealed that T8 cDNA lacked a 153 bp (51aa) region that was present in T21 cDNA and ESTAA417787. This deletion would result in the loss of the catalytic serine (GWSYGG) in T8 cDNA. Many of the other clones characterized appeared to contain unrelated sequence which are probably intronic sequences as a result of incomplete splicing.

The 5' RACE technique was utilized on both ATC RNA and placental RNA to obtain the 5' end of the DPP8 gene. The RACE product obtained from activated T cell RNA was 0.2 kb larger than that from placental RNA but otherwise identical (Figure The first methionine within a Kozak sequence was found 214 bp from the 5' end of the activated T cell RACE product. This 5' 211bp region was 70.5 GC rich and contained a number of potential promoter and enhancer elements (Spl, Apl and ETF sites) and so was deduced to be the 5' flanking region of the DPP8 gene. In order to confirm the identity of the 5' RACE product as the 5' end of DPP8, RT-PCR was carried out to span across the junction between the RACE product and T8 cDNA library clone. The RT-PCR on ATC RNA produced two clones ATCd3-2- 1 and ATC3-3-10 (Figure Compared to T8 and T21, both clones had an additional insert region of 144bp (48 aa) immediately adjacent to the splice site of T8. Sequence homology analysis of this additional insert region found a homologous region in both the C. elegans homologue and DPP4. This clearly showed that T8 and T21 library clones represented splice variants of DPP8. The smaller clone WO 01/19866 PCT/AU00/01085 27 ATCd3-3-10 was also found to represent another splice variant of DPP8 as it contained a 516 bp deletion at the end which would result in a deletion of 175 aa.

A full-length DPP8 clone was created using the larger RACE product, ATC3-2-1 and the T21 library clone. This generated a putative DPP8 cDNA of 3.1 kb (including 5' and 3' untranslated regions) with an open reading frame of 882 aa for further sequence analysis and examining DPP8 function. This 882 putative DPP8 protein contained no Nlinked glycosylation sites and Kyte-Doolittle hydrophobicity analyses revealed it lacked a transmembrane domain, unlike DPP4, FAP and DPP6. Thus it is likely that DPP8 is a cytoplasmic protein (Figure The predicted DPP8 protein shared 51 amino acid similarity and 27 amino acid identity with human DPP4; the C termini of these proteins exhibited the most homology (Figure 3).

Tissue distribution of DPP8 as determined by Master RNA and Northern Blot A master RNA blot was probed with a 484 nt PCR product produced by the forward and reverse DDP8 primers as mentioned previously. The mRNA tissue expression of DPP8 was ubiquitous in all human adult and fetal tissues. A similar ubiquitous expression pattern was observed using DPP4 cDNA as a probe (data not shown). However, by visual assessment the greatest levels of expression using each gene specific probe were in different tissues. The most intense signals using the DPP8 probe were in testis followed by placenta whereas the most intense signals using the DPP4 probe were in salivary gland and prostate gland followed by placenta (data not shown). The probes did not bind any of the negative controls on the blot.

Northern blot analysis was performed on mRNA derived from WO 01/19866 PCT/AU00/01085 28 different human tissues (Figure Two DPP8 specific probes indicated the presence of transcripts in all tissues examined. A transcript approximately 3.0 kb in size consistent with the approximate expected size of DPP8 message was detected only in the testis. However, two transcripts of 8.0 and 5.0 kb respectively were present in testis, spleen, peripheral blood leukocytes and ovary at high levels; in prostrate, small intestine, and colonic mucosa at moderate levels; and in the thymus at lower levels. The Multiple tissue Northern blot was also probed with radiolabeled human P-actin probe and a common 2.0 kb transcript was seen in all tissues (Figure 4).

Expression of DPP8 in mice determined by Northern Blot and rtPCR.

The human DPP8 cDNA sequence cross-hybridized with murine derived liver RNA. The Northern blot containing total RNA from mouse liver hybridized to a human DPP8 probe, showing that DPP8 mRNA is expressed in mouse liver (Figure 9A).

Two mRNA transcripts of murine DPP8 were present. This is a similar pattern to that observed for human DPP8. These transcripts probably represent different length 5' and 3' untranslated regions of the murine DPP8 gene. The presence of DPP8 mRNA in the mouse liver was also demonstrated using rt-PCR. The primers tested generated a 537bp PCR product. A Southern blot of this product confirmed that the murine DPP8 cross-hybridizes with human DPP8 (Figure 9B).

Expression and functional activity of DPP8 To assess the function of DPP8 protein, the full length DPP8 cDNA of 3.1 kb was cloned into the Xba I site of pcDNA3.1A/V5/His expression vector to produce two constructs. The first construct, pcDNA3.1-DPP8, expressed DPP8 protein on its own whilst the second construct, WO 01/19866 PCT/AU00/01085 29 pcDNA3.1-DPP8/V5/His expressed a protein with the epitope and His tag fused to the C-terminus of DPP8 to facilitate analysis of protein expression. Mammalian expression constructs were stably transfected into COS-7 cells and cellular sonicates prepared. Consistent with the molecular weight predicted from the amino acid sequence a 100 kDa monomer was detected by Western blotting of stable expressing cells (Figure protein was detected in the cytoplasmic compartment but not on the surface of ethanol fixed stable expressing COS cells, using the anti-V5 mAb.

DPP8 is a dipeptidyl peptidase Sequence homology between DPPIV and DPP8 suggested functional similarities, so cell lysates of DPP8transfected cells were examined for proline-specific peptidase activity. DPPIV expressed in COS-7 cells with or without the V5/His tag were positive controls, and negative controls included vector-only transfected COS07 cells. Extracts of DPP8-transfected COS-7 cells hydrolyzed H-Ala-Pro-pNA and H-Arg-Pro-pNA but not H-Gly- Pro-pNA, H-Gly-Arg-pNA, H-Gly-Pro-toluenesulfonate or H- Gly-Pro-7-amino-4-trifluoromethylcoumarin above the levels exhibited by untransfected COS-7 cells (data not shown).

The pH optimum of DPP8 enzyme activity was 7.4 (Fig. similar to the pH 7.8 optimum DPPIV enzyme activity [43,44]. DPP8 exhibited little activity below pH 6.3, suggesting that it is not an enzyme of the lysosome/endosome compartment. Of all the substrates tested on cell monolayers, only Ala-Pro-4MONA/HC1 stained DPP8-transfected COS cells and CHO cells (data not shown).

Both purified recombinant DPP8/V5/His and purified recombinant DPPIV/V5/His hydrolyzed H-Ala-Pro-pNA, G-Gly- Pro-pNA and H-Arg-Pro-pNA. Transfection with DPP8 WO 01/19866 PCT/AUOO/01085 30 possibly causes increased dipeptidase, tripeptidase and endopeptidase activities, similar to an effect of DPPIV transfection of melanoma cells Indeed, our results showed that DPP8 transfected COS-7 cells, but not purified recombinant DPP8, exhibited tripeptidyl peptidase activity using the substrate H-Ala-Ala-Pro-pNA and endopeptidase activity using the substrate Z-Ala-Pro-pNA (data not shown). This was investigated further, and neither of the tripeptidyl peptidase substrates H-Ala-Ala-Phe-pNA or H- Ala-Phe-Pro-pNA [45] nor the prolyl endopeptidase substrates Z-Ala-Pro-pNA or succinyl-Ala-Pro-pNA were cleaved by purified DPP8. Our data clearly demonstrate that DPP8 is a dipeptidyl peptidase and lacks tripeptidyl peptidase or endopeptidase activities.

The nature of the catalytic mechanism of DPP8 was further investigated using various inhibitors. DPP8 enzyme activity was significantly inhibited by serine proteinase inhibitors and was insensitive to inhibitors of metalloproteinases, aspartyl proteinases and cysteine proteinases. DPP8 enzyme activity was significantly inhibited by zinc, which completely inhibits DPPIV enzyme activity The peptides Ala-Pro-Gly and Lys-Pro mimic DPP8 substrates and probably competitively inhibited DPP8.

Chromosomal localization of DPP8 Two probes were used for FISH analysis, ESTAA417787 and the T8 clone from the placental library. Seventeen metaphases from the first normal male were examined for fluorescent signal. All of these metaphases showed signal on one or both chromatids of 15 at band q22 (Figure There were a total of 2 non-specific background dots observed in these metaphases. A similar result was obtained from the hybridization of the probe to metaphases from the second normal male (data not shown).

WO 01/19866 PCT/AU00/01085 31 Analysis of DPP8 gene expression by RT-PCR DPPIV is expressed by most lymphocytes and lymphocytic cell lines but upregulated on activated lymphocytes [47, 41, 48, 49]. The various splice variants of DPP8 might not encode functional protein, so the PCR was designed to detect only mRNA that contained full-length sequence (Fig.

At 35 cycles, amplification product of the expected size (783 bp) was readily observed in OKT3-stimulated PBMCs (six of six subjects; Fig 8) but not in unstimulated PBMCs from most subjects (four of five, Fig. 8A), suggesting that more DPP8 mRNA is expressed in activated T cells than in unstimulated PBMCs. Similar RT-PCR data were obtained from PBMCs stimulated with phytohaemagglutinin (data not shown). In addition, DPP8 mRNA was expressed in all B and T cell lines examined and in both liver and placenta( Fig. 8B).

Anti-peptide antibody The sera of two rabbits were tested by ELISA in peptidecoated wells. Both sera bound both peptides whereas the pre-immunisation serum samples did not exhibit specific binding. Western blots on extracts of cell lines, cell lines transfected with DPP8 cDNA and activated human lymphocytes showed that a rabbit antiserum to the two DPP8 peptides binds a 100kDa band, which is the size of DPP8.

(Data not shown) Table 1. K. and Vx values for DPP8 and DPPIV Kn (m Vmax (AA min' x 1000) DPPIV DPP8 DPPIV DPP8 H-Ala-Pro-pNA 0.374 0.134 0.991 0.171 9.6 1.0 12.4 0.9 H-Gly-Pro-pNA 0.347 0.088 0.467 0.064 7.2 0.49 3.5 0.14 WO 01/19866 WO 0119866PCTAUOO/01 085 -32 Table 2. Inhibition of the peptidase activity of DPPB in comparison with DPPIV. Common proteinase inhibitors of various enzyme types were incubated with the purified peptidases before assay with the substrates H-Ala-Pro-pNA on DPP8 or H-Gly-Pro-pNA on DPPIV. AEBSE, 4-(2aminoethyl) benzenesulfonylfluoride.

Residual activity of control) DPPS DPPIV 100 100 Type of inhibitor None Serine proteinase

AEBSF

Aprotinin Benzamidine/H-C1 Peptides Gly-Gly-Gly Ala-Pro-Gly H-Lys--Pro-OH HC1 salt Zinc sulphate Metalloproteinase

EDTA

Aspartate (acidic) proteinase Pepstatin Leupept in Cysteine (thiol) proteinase Iodoacetamide Dithiothreitol Concentration mm J.Lg r-L 1 mM 106 67 4S 0 2 mM 2 ,tg rnL' 0. 1 M 2 M 2 mMv 115 109 WO 01/19866 PCT/AUOO/01085 33 Discussion We describe the cloning, recombinant expression, biochemistry and tissue expression of a novel human DPPIVrelated postproline peptidase that we have named DPP8.

DPP8 exhibited dipeptidyl aminopeptidase but not tripeptidyl peptidase or endopeptidase activity. Like DPPIV, DPP8 was found to exhibit significant mRNA expression in activated T cells. Clear indications that DPP8 is a monomeric, nonglycosylated, soluble, cytoplasmic protein, which are characteristics of PEP but not of DPPIV, FAP or DPP6, were provided by our sequence and localisation data. DPP8 enzyme activity had a neutral pH optimum, suggesting that it is not active in the acidic lysosome/endosome compartment.

By homology with DPPIV, DPP8 is a member of the DPPIV-like gene family, a member of the prolyl oligopeptidase family S9b, and a member of the enzyme clan SC. The residues in DPP8 that potentially form the charge-relay system are Ser739, Asp817 and His849 (Fig.2). The dipeptidyl peptidase activity of DPP8 and the absence of detectable tripeptidyl peptidase or endopeptidase activities by purified DPP8 further support its placement in the S9b family. Furthermore, the DPP8 substrate specificity was indistinguishable from that of the structurally related peptidases DPPIV and FAP.

The role of DPPIV in human lymphocytes has been studied in detail using enzyme inhibitors [49, 50-54]. DPPIVspecific inhibitors suppress both DNA synthesis and cytokine production in vitro [48, 49, 52]. In addition, DPPIV-specific inhibitors decrease phorbol myristate acetate-induced tyrosine phosphorylation in human lymphocytes, further suggesting a role for DPPIV enzyme WO 01/19866 PCT/AU00/01085 34 activity in lymphocyte activation In vivo, inhibitors of DPPIV suppress arthritis [20] and prolong cardiac allograft survival in animal models The ability of DPP8 to cleave DPPIV substrates indicates that DPPIV inhibitors may also inhibit DPP8 and that inhibitor studies may require further interpretation. Indeed, DPP8 may be responsible for some of the physiological functions that have been assigned to DPPIV.

FAP and DPPIV are integral membrane glycoproteins and require dimerization for catalytic activity 56, 57].

In contrast, DPP8 and PEP are non-glycosylated cytosolic proteins that are catalytically active as monomers [58] and cleave Pro-Xaa bonds [43,59]. However, the substrate specificity of PEP is distinct from DPP8. PEP is an endopeptidase that does not cleave if a free a-amine lies N-terminal to the proline it does not cleave H-Ala- Pro). Recently we have proposed that the tertiary structure of DPPIV is similar to that of PEP in having a seven-blade p-propeller domain and an a/P-hydrolase domain 39, The significant sequence identity between DPP8 and DPPIV indicates that the tertiary structures of DPP8 and DPPIV are similar. However, DPP8 contains 110 amino acids more than DPPIV, so it could have an additional element of tertiary structure such as an eighth propeller blade.

The ancestral relationships between DPP8, DPPIV and FAP are reflected in their chromosomal localization. While DPPIV and FAP have both been localized to the long arm of chromosome 2, 2q24.3 [60] and 2q23 [61] respectively, DPP8 was localized to 15q22. The related genes DPP6 and PEP have been localized to chromosome 7 [62] and 6q22 respectively [63].

WO 01/19866 PCT/AU00/01085 35 Two human disease loci have been mapped to 15q22. These loci are an autosomal recessive deafness locus [64] and a form of Bardet-Biedl syndrome, type 4 Two of the clinical manifestations of Bardet-Biedl syndrome are obesity and diabetes. Attractin [66] and DPPIV have roles in obesity [67] and diabetes [22, 68, 15] respectively and as their substrate specificities overlap with that of DPP8, it is possible that DPP8 may be involved in Bardet- Biedl syndrome.

DPPIV is expressed on the surface of T cells and is a costimulatory molecule called CD26 CD26-negative cell lines have residual DPPIV enzyme activity and PBMC have non-DPPIV derived activity against Ala-Pro substrates indicating the existence of other peptidase(s) with DPPIV-like activity. DPPIV-0 exhibits a peptidase activity similar to DPPIV but is a 70-80 kDa cell surface glycoprotein [70] and is therefore distinct from DPP8.

The biological significance of the three splice variants of DPP8 that we discovered is unknown. None of these splice variants result in a frame shift or premature protein termination (Fig. Two of the splice variants contain all the predicted catalytic triad residues and thus potentially produce proteins with peptidase activity.

Alternate splice forms of FAP mRNA have also been observed [71, 72]. It is possible that expression of splice variants may be used to regulate the levels of active protein. DPP8 Northern blots revealed a number of differently sized transcripts. The predicted sizes of splice variants of DPP8 ranged from 2.6 to 3.1 kb whereas the large transcripts seen in most tissues examined in the Northern blots were 8.5 kb and 5.0 kb respectively.

Similarly, two other members of the DPPIV-like gene family, DPPIV and DPP6, exhibit mRNA transcripts in 1 WO 01/19866 PCT/AU00/01085 36 Northern blots that are much larger than the cDNA size 61]. We propose that the major transcripts for DPP8 mRNA and its splice variants lie within the 5 kb band while the 8.5 kb transcript(s) may contain additional and 3' untranslated sequences. DPP8 appears to be like DPPIV in having a ubiquitous mRNA expression pattern by Northern analysis while being upregulated in activated T cells. The similarities between DPP8 and DPPIV suggest that DPP8 may, like DPPIV, play a role in T cell costimulation and proliferation. The development of DPP8 specific antibodies or inhibitors will facilitate work in this area.

In summary, we have identified and characterized a novel human dipeptidyl aminopeptidase DPPB with structural and functional similarities to DPPIV and FAP. With many diverse biological roles suggested for DPPIV, particularly in the immune system, and the roles of FAP in tumor growth and liver disease, it will be interesting to investigate the roles of this new member of the DPPIV-like gene family in these systems. Further work in understanding this novel protein and the elucidation of inhibitors and physiological substrates will help identify the specific functions of individual members of this gene family.

WO 01/19866 PCT/AU00/01085 37

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It is to be understood that a reference herein to a prior art document does not constitute an admission that the document forms part of the common general knowledge in the art in Australia or in any other country.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprising" and grammatical variations thereof, is used in the sense of "including" i.e. the features specified may be associated with further features in various embodiments of the invention.

EDITORIAL NOTE APPLICATION NUMBER 73946/00 The following Sequence Listing pages 1 to 15 are part of the description. The claims pages follow on pages 48 to 52.

WO 01/19866 WO 01/ 9866PCT/AUOO/01 085 1. ST25. txt SEQUENCE LISTING <110> THE UNIVERSITY OF SYDNEY <120> DIPEPTIDYL PEPTIDASE <130> P37354 <150> AU PQ2762 <151> 1999-09-10 <150> AU PQS709 <151> 2000-02-18 <160> 8 <170> Patentln version <210> 1 <211> 882 <213> H <400>1 Met Ala 1 Thr Ala Giu Pro Leu Ala His Asp Asp Arg Leu Phe Met Leu Tyr Gly 130 Ile Gly 145

RT

omo sapiens Ala Asp Phe Asp Phe Ile Tyr Ser 115 Met Thr Ala Cys Tyr Thr Met Tyr Ser 100 Trp Tyr Val1 Met 5 Glu Val1 Arg Phe Tyr Glu Lys Ser Gly Glu Thr Glu Gin Leu Gly Val Glu Ile Phe Glu Glu Giu Lys Val 70 Leu Ile Pro Arg Ile 150 Asn Arg Tyr 55 Lys Ala Pro Leu Giu 135 Ala Ile Giu Ser 25 Tyr Ser Trp, 40 His Gly Tyr Arg Asn Asp Met Ser Gly 90 Lys Thr Ile 105 Leu Asp Leu 120 Giu Giu Leu Ser Tyr Asp Gin Asp Ser Gin Met Met Pro Asp 75 Glu Asn Asn Arg Phe Gin Leu Arg 140 Arg Leu Ala Gly Arg Ala Ala 125 Glu Pro Lys Lys Pro Glu Ala 110 Thr Arg Lys Lys Ala His Asn Val Leu Lys Leu Leu Pro Ser Thr Leu Asp Arg Gly 160 Tyr 155 His Gin Gly Ser Page 1 WO 01119866 PCTIAUOO/0I 085 1.ST25.txt Thr Phe Leu Phe Gin Ala Gly Ser Gly Ile Tyr His Val 165 Gly Thr Pro 2 Ile 225 Ala Leu Ala Glu Leu 305 Ala Glu Glu Pro Arg 385 Asp Val Ile ?ro 3er %sp 210 Jal Asn ln Glu Asn 290 Glu Asn Gly Ile Glu 370 Leu Asp Thr His Gin C 1 Cys I 195 Trp I Thr I Met Glu Thr 275 Asp 4 Thr Pro Arg Leu 355 Gly Gin Val Pro Asp 435 ily .80 !ro Ele %rg 3lu lu 260 rhr Glu Arg Lys Ile 340 Phe Lys IlE Met Let 42( Il Phe 1 Asn Ala I Glu Glu 245 Phe Pro Ser Arg Val 325 Ile Glu Tyr Val Glu 405 ;i Ile E Phe hr le ?he :lu 230 Asp Asp Ser Glu Ala 310 Thr Asp Gly Ala Leu 390 Arg Ile His Gln Arg Ile 215 Arg Ala Arg Gly Val 295 Asp Phe Val Val Trr 375 ilE Gl Ty] Va Gin I Met 200 His Arg Arg Tyr Gly 280 Glu Ser Lys Ile Glu 360 Ser Ser i Arg Glu i Phe 440 ?ro 185 ksp Ser Leu Ser Ser 265 Lys Ile Phe Met Asp 345 Tyr Ile Prc Let Gli 42E Pr( 170 Leu 2 Pro I Asn Thr Ala 250 Gly Ile Ile Arg Ser 330 Lys Ile Leu Glu Ile 410 i Thr Gin krg -ys Asp Tyr 235 Gly Tyr Leu His Tyr 315 Glu Glu Ala Leu Leu 395 Glu Thy Se2 Pro 2 Leu Ile 220 Vai Val Trp Arg Va1 300 Pro Ile Leu Arg Asp 380 Phe Ser Asp His sn :ys 205 rrp His Ala Trp Ile 285 Thr Lys Met Ile Ala 365 Arc IlE Va Il Gli Lys Leu 190 Pro Ile Asn Thr Cys 270 Leu Ser Thr Ile Gin 350 Gly Ser Pro L Pro Trp 430 u Glu ksp 175 Val Ala Ser Glu Phe 255 Pro Tyr Pro Gly Asp 335 Prc Trp Glr Val AsI 41E IlE Gli Gly Glu Asp Asn Leu 240 Val Lys Glu Met Thr 320 Ala Phe Thr Thr Glu 400 Ser Asn Ile 445 Glu Phe Ile Phe Ala Ser Giu Cys Lys Thr Giy Phe Arg His Leu Tyr Page 2 WO 01/19866 PCT/AU00/01085 1.ST25.txt 450 455 460 Lys 465 Ile Thr Ser Gly Leu Pro Ala Ala Ile Asp Gly 545 Ile Asn Pro Gly Thr 625 Gin Gin Leu Arg Lys 705 Ile Gin Ser 530 Glu Ser Pro Thr Pro 610 Thr Pro Val Asn Gly 690 Met Thr Val 515 Pro Val Gin His Cys 595 Leu Gly Gly Gin Thr 675 Ser Gly Ser 500 Asp Leu Thr His Cys 580 Lys Pro Phe Lys Leu 660 Leu Cys Gin Arg Ile Leu Lys Glu Ser 470 Pro Ser Asp Phe Lys 485 Gly Glu Trp Glu Val 505 Glu Val Arg Arg Leu 520 Glu His His Leu Tyr 535 Arg Leu Thr Asp Arc 550 Cys Asp Phe Phe Ii 565 Val Ser Leu Tyr Lys 58 Thr Lys Glu Phe Tr 600 Asp Tyr Thr Pro Pr 615 Thr Leu Tyr Gly Me 630 Lys Tyr Pro Thr Va 645 Val Asn Asn Arg Ph 66 Ala Ser Leu Gly Ty 680 His Arg Gly Leu Ly 695 Ile Glu Ile Asp As 710 Tyr Asp Phe Ile As 1 e 5 r 5 r s p p Lys Tyr 475 Cys Pro 490 Leu Gly Val Tyr Val Val Gly Tyr 555 Ser Lys 570 Leu Ser Ala Thr Glu Ile Leu Tyr 635 Leu Phe 650 Lys Gly Val Val Phe Glu Gin Val 715 SLeu Asp 730 Lys Arg Ser Ser Gly 480 Ile Lys Glu Glu Ile 495 Arg His Gly Ser Asn 510 Phe Glu Gly Thr Lys 525 Ser Tyr Val Asn Pro 540 Ser His Ser Cys Cys 560 Tyr Ser Asn Gin Lys 575 Ser Pro Glu Asp Asp 590 Ile Leu Asp Ser Ala 605 Phe Ser Phe Glu Ser 620 Lys Pro His Asp Leu 640 Ile Tyr Gly Gly Pro 655 Val Lys Tyr Phe Arg 670 Val Val Ile Asp Asn 685 SGly Ala Phe Lys Tyr 700 Glu Gly Leu Gin Tyr 720 Arg Val Gly Ile His 735 Leu Ala Ser 725 Gly Trp Ser Tyr Gly 740 Gly Tyr Leu Ser Leu Met 745 Page 3 Ala Leu Met Gin Arg 750 WO 01/19866 WO 01/ 9866PCT/AUOO/01 085 Ser Asp Ile 755 Ile Phe Tyr 770 Gin Asn Giu 785 Lys Phe Pro Asp Giu Asn Vai Arg Aia 835 His Ser Ile 850 Leu His Tyr 865 Vai Ile <210> 2 <211> 3i20 <2i2> DNA <213> Homo <400> 2 aagtgc taaa cgttcgccgc 120 gagtggaggc 180 tccgggcggg 240 ctgggtgttg 300 cctaaattgg 360 gccgatacca 420 Phe Asp Gin Ser Val1 820 Gly Arg Leu Arg Thr Gly Giu 805 His Lys Val1 Gin Val Gly Tyr 790 Pro Phe Pro Pro Giu 870 Ala Tyr 775 Tyr Asn Ala Tyr Giu 855 Asn 1. ST25 .txt Ile Ala Gly 760 Thr Giu Arg Leu Gly Ser Arg Leu Leu 810 His Thr Ser 825 Asp Leu Gin 840 Ser Gly Glu Leu Gly Ser Ala Pro Tyr Met 780 Val Ala 795 Leu Leu Ile Leu Ile Tyr His Tyr 860 Arg Ile 875 Vai 765 Gly Met His Leu Pro 845 Giu Ala Thr His Gin Gly Ser 830 Gin Leu Ala Leu Pro Ala Phe 815 Phe Giu His Leu Trp Asp Giu 800 Leu Leu Arg Leu Lys 880 sapiens gcctccgagg ctgggttgtc ggcgcagcat gccgggggga agatatttga agcc tt t tta gaaaatatca ccaaggccgc accggcgccg gaagcggcgc aggaaaatgc aactgcggac tgttgagcgg tggc tacatg tgctactgcc ccgccgagga aggcccgctc aacatggcag tgtgaggaga tattcctgga atggc taagg gccgctgctt agccactgca catagcgcac cagcaatgga atattgaatc gtcagcttaa caccacatga cttagtgccg accaggaccg g tcgggacgg aacagaacag acaggatcgg aaagctgctt tttcatgttt Page 4 WO 01/19866 WO 01/ 9866PCT/AUOO/0 1085 gtgaagagga atgatccaga 480 ggtgagaaca gagaaaatac 540 gcagtcttaa tgctctcttg 600 ggaatgtatt ctcgagaaga 660 attgcttctt acgattatca 720 atttatcacg taaaagatgg tggacctcat tcagacagaa tctattacct tgccatgtct 780 c tagtggaaa 840 gactggattg 900 gaaaggagac 960 gctggagtcg 1020 tgtccaaaag 1080 aatgatgaat 1140 gcagattcat 1200 tcagaaataa 1260 caacc ttttg 1320 c tagt tgtc c cttttataca tcacttatgt c tacc t ttg t ctgaaacaac c tgaggtgga tccgttatcc tgattgatgc agattctatt actgttttat gaagcctctt agaactatta ccaaggaagt agggccacaa caacatacgg tagcaacgat gcacaatgag tc tccaagaa tcccagtggt aattattcat taaaacaggt tgaaggaagg tgaaggagtt catcctacta tatcccagta tctgaaattc ttggatcttt agagaaagaa ggaacatttc ggatt tacgc atggatccaa atttggatat c tagccaac a gaatttgata.

ggtaaaattc gttacatccc acagcaaatc atcatagatg gaatatattg gatcgctccc Lgaaqatgatc ccaaaactat ttcaggcaac aacgcattgg tgtttcaagc aacaaccttt aattatgccc c taacatcgt tggaagaaga gatattctgg ttagaattct ctatgttgga ctaaagtcac tcatagataa ccagagc tgg agactcgcct fttatggaaag atgaagaaac caatagagca actggactat aacagtcgga cggtagtgga aaggcccaat cgctgatcca aaccagagaa tgccagatca ctattggtgg atatgaagaa aacaaggagg ttttaagatg ggaactaatt atggactcct acagatagtg gcagagactc aacagacatc gagggaaaat atgcttggtc 1380 ttgatctcac ctgaattatt 1440 attgagtcag tgcctgattc 1500 tgtgacgcca ctaattatct tggataaata tccatgacat ctttcatgtt tttccccaaa Page gtcacgaaga ggaaattgag WO 01/19866 WO 0119866PCT/AUOO101085 1. ST25 .txt 1560 tttatttttg cctctgaatg caaaacaggt 1620 t taaaggaaa 1680 tgtcctatca 1740 ggatctaata 1800 tcccctttag 1860 ctgac tgacc 1920 agtaagtata 1980 gaagatgacc 2040 cctcttcctg 2100 ttgtatggga 2160 ctgttcatat 2220 tatttccgct 2280 ggatcc tgtc 2340 gaaattgacg 2400 ttagatcgtg 2460 atgcagaggt 2520 2580 tattacttac 2640 gcaaatataa acgatccagt aagaggagat tccaagttga agcatcacct gtggctactc gtaaccagaa caacttgcaa actatactcc tgctctacaa atggtggtcc tgaataccc t accgagggc t atcaggtgga tgggcatcca cagatatct~t Lcaggatacac rgatctgtggc agcaattacc tgaagtcaga gtacgtagtc acattcttgc gaatccacac aacaaaggaa tccagaaatt gcctcatgat tcaggtgcag agcctctcta taaatttgaa aggactccaa cggctggtcc cagggttgct ggaacgttat catgcaagcE ttccgtcatt ggtgggc tgc agtggtgaat aggctggtat agttacgtaa tgcatcagtc tgtgtgtcc ttttgggcca ttctcttttg ctacagcctg ttggtgaata ggttatgtgg *ggcgccttta *tatctagctt *tatggaggat -attgctgggg -atgggtcacc i gaaaagttcc ctgctccaag gggaagt tct attttgaagg atcctggaga agcac tgtga tttacaagct ccattttgga aaagtac tac gaaagaaata atcggtttaa ttgtagtgat aatataaaat c tcgatatga acctctccct ccccagtcac ctgaccagaa cctctgaacc tatacaaaat tacatctatt tgatttcaag tggccggcat caccaaagac gg tgacaagg cttctttata atcaagtcct ttcagcaggt tggatttaca tcctactgtg aggagtcaag agacaacagg gggtcaaata tttcattgac gatggcat ta tctgtggatc tgaacagggc aaatcgttta Page 6 WO 01/19866 WO 01/ 9866PCT/AUOO/01 085 1. ST2 ctgctcttac atggtttcct ggatgagaat gtccattttg 2700 agttttttag tgagggctgg aaagccatat gatttacaga 2760 agcataagag ttcctgaatc gggagaacat tatgaactgc 2820 gaaaaccttg gatcacgtat tgctgctcta aaagtgatat 2880 tctctggtat acactggcta tttaaccaaa tgaggaggtt 2940 attgatcatc acattttgat acctgccatg taacatctac 3000 ccatgcaggg gtctacggtt tgtggtagta atctaatacc 3060 tcaaatgata catattcctg agagacccag caataccata 3120 <210> 3 <211> 310 <212> PRT <213> Homo sapiens <400> 3 Phe Giu Gly Thr Lys Asp Ser Pro Leu Giu His 1 5 10 Ser Tyr Val Asn Pro Gly Glu Val Thr Arg Leu 25 Ser His Ser Cys Cys Ile Ser Gin His Cys Asp 40 Tyr Ser Asn Gin Lys Asn Pro His Cys Val Ser 55 Ser Pro Glu Asp Asp Pro Thr Cys Lys Thr Lys 70 75 Ile Leu Asp Ser Ala Gly Pro Leu Pro Asp Tyr 90 Phe Ser Phe Glu Ser Thr Thr Gly Phe Thr Leu 100 105 Lys Pro His Asp Leu Gin Pro Gly Lys Lys Tyr Page 7 cacataccag tctatcc tca atcttttgca aattttgacc taatcaacag tcc tgaaaat ttaaccccac agaattacta His Leu Ty Thr Asp Ar Phe Phe Il Leu Tyr Ly Glu Phe Tr *Thr Pro Pr Tyr Gly Me 11 *Pro Thr Va tatattactg ggagagac ac ctaccttcaa tgtgtagaac aaaacacaga aaatgtggtg atgc tcaaaa aaaaaaaaaa r Val Val g Giy Tyr Ser Lys s Leu Ser p Ala Thr o Glu Ile :t Leu Tyr 0 .I Leu Phe WO 01/19866 WO 0119866PCTAUOOIOI 085 1. ST25 .txt 115 120 Ile Tyr G 130 Gly Leu G 145 Val Gly I Leu Met G Val Thr L.

1 Gly His 1 210 Met Gin 7Z 225 His Gly Leu Ser Pro Gin Glu Leu 290 Ala Ala 305 <210> 4 ly Gly Pro in le in 5 eu .95 ~ro lia ?he ?he 31u 275 H*is Leu Tyr His Arg 180 Trp Asp Glu Leu Leu 260 Arg Leu Lys Leu Gly 165 Ser Ile Gin Lys Asp 245 Val His Leu Val Gin Gly 135 Ala Ser 150 Trp Ser Asp Ile Phe Tyr Asn Glu 215 Phe Pro 230 Giu Asn Arg Ala Ser Ile His Tyr 295 Ile 310 Arg Tyr Phe Asp 200 Gin Ser Val Gly Arg 280 Gln Ile Giu Tyr Gly Arg 185 Thr Gly Gia His Lys 265 Val Asp Gly 170 Val1 Gly Tyr Pro Phe 250 Pro Pro Ile Phe 155 Tyr Ala Tyr Tyr As n 235 Ala Tyr Gia Asp 140 Ile Lea Ile Thr Leu 220 Arg His Asp Ser Asp Asp Ser Al a Gla 205 Gly Lea Thr Lea Gly 285 Gin Leu Lea G ly 190 Arg Ser Leu Ser Gin 270 Gia Val Asp Met 175 Ala Tyr Val Leu Ile 255 Ile His Gia Arg 160 Ala Pro Met Ala Leu 240 Lea Tyr Tyr Lea Gin Glu Asn Leu Gly Ser Arg Ile 300 <211> 1197 <212> DNA <213> Homo sapiens <400> 4 attttgaagg caccaaagac tcccctttag agcatcacct. gtacgtagtc agttacgtaa atcctggaga ggtgacaagg ctgactgacc gtggctactc acattcttgc tgcatcagtc 120 agcactgtga cttctttata agtaagtata gtaaccagaa gaatccacac tgtgtgtCc 180 tttacaagct atcaagtcct gaagatgacc caacttgcaa aacaaaggaa ttttgggcca 240 Page 8 WO 01/19866 WO 0119866PCT/AUOO/01085 1. ST25 .txt ccattttgga 300 aaagtac tac 360 gaaagaaata 420 acgatcaggt 480 gtgtgggcat 540 ggtcagatat 600 atacaggata 660 taggatc tgt 720 tacatggttt 780 tagtgagggc 840 gagttcctga 900 ttggatcacg 960 tatacactgg 1020 atcacatttt 1080 ggggtc tacg 1140 atacatattc 1197 <210> <211> 465 <212> PRJ ttcagcaggt cctcttcctg actatactcc tccagaaatt ttctcttttg tggatttaca tcctactgtg ggaaggactc ccacggctgg cttcagggtt cacggaacgt ggccatgcaa cctggatgag tggaaagcca atcgggagaa tattgctgct ctatttaacc gatacctgcc gtttgtggta ttgtatggga ctgttcatat caatatctag tcctatggag gctattgctg tatatgggtc gcagaaaagt aatgtccatt tatgatttac cattatgaac c taaaagtga aaatgaggag atgtaacatc gtaatctaat tgc tc tacaa atggtggt~cc cttctcgata gatacctctc gggccccagt accctgacca tcccctctga ttgcacatac agatctatcc tgcatctttt tataattttg gtttaatcaa tactcctgaa accttaaccc gcctcatgat tcagggtcaa tgatttcatt cctgatggca cactctgtgg gaatgaacag accaaatcgt cagtatatta tcaggagaga gcactacctt acctgtgtag cagaaaacac aataaatgtg cacatgctca ctacagcctg atagaaattg gac ttagatc ttaatgcaga atcttctatg ggctattact ttactgctct ctgagttttt cacagcataa caagaaaacc aactctctgg agaattgatc gtgccatgca aaatcaaatg ctgagagacc cagcaatacc ataagaatta ctaaaaaaaa aaaaaaa <213> Homo sapiens Page 9 WO 01119866 PCT/AUOO/01085 1.ST25.txt <400> Thr C 1 Ile I Gin I Gly Ser Pro Pro Trp Glu His 145 Ser Glu Gly Gly Val 225 Ser Asn ly ~sp ?ro Prp 31n Val Asp Ile Glu 130 Leu Ser Glu Ser Thy 21C Asr Cys Gl Thr 2 Ala Phe Thr Thr Glu Ser Asn 115 Ile Tyr Gly Ile Asn 195 Lys Pro Cys I Lys kia 3lu lu Pro Arg Asp Val 100 Ile Glu Lys Gly Ala 180 Ile Asp Gi Ile Asr 26( Asn 5 Gly Ile Glu Leu Asp Thr His Phe Ile Leu 165 Ile Gin Ser Glu Ser 245 i Pro Pro Arg Leu Gly Gin 70 Val Pro Asp Ile Thr 150 Pro Thr Val Pro Val 230 Gin His Lys Ile Phe I Lys 55 Ile Met Leu Ile Phe 135 Ser Ala Ser Asp Leu 215 Thr His Cys ial Thr Phe Lys Met Ser Glu Ile Met lie lu 40 Tyr Val Glu Ile Phe 120 Ala Ile Pro Gly Glu 200 Glu Arg Cys Val Asp 25 Gly Ala Leu Arg Ile 105 His Ser Leu Ser Glu 185 Va1 His Leu Asr Sei 26E 10 Val Val Trp Ile Gin 90 Tyr Val Glu Lys Asp 170 Trp Arg His Thr Phe 250 Leu Ile flu Ser Ser 75 Arg Glu Phe Cys Glu 155 Phe Glu Arg Leu Asp 235 Phe Tyr Asp Tyr Ile Pro Leu Glu Pro Lys 140 Ser Lys Val Leu Tyr 220 Arg Ile Lys Lys Ile Leu Glu Ile Thr Gin 125 Thr Lys Cys Leu Val 205 Val Gly Ser Leu Glu Ala Leu Leu Glu Thr 110 Ser Gly Tyr Pro Gly 190 Tyr Val Tyr Lys Ser 270 Leu Arg Asp Phe Ser Asp His Phe Lys Ile 175 Arg Phe Ser Ser Tyr 255 Ser Ile Ala Arg Ile Val Ile Glu Arg Arg 160 Lys His Glu Tyr His 240 Ser Pro Glu Asp Asp Pro Thr Cys Lys Thr 275 280 Lys Giu Phe Trp Ala Thr Ile Leu 285 Page WO 01/19866 WO 0119866PCTAUOO/01 085 1. ST25 .txt Asp Ser Ala Gly Pro Leu Pro Asp Tyr Thr Pro 290 295 Phe 305 His Giy Phe Asn Phe 385 Giu Arg Ser Giu Asp Gly Tyr Giu 370 Pro Asn Ala Ile Ser Leu Pro Asp 355 Gln Ser Val Gly Arg 435 Thr Gin Gin 340 Thr Gly Giu His Lys 420 Val1 Thr Pro 325 Val Gly Tyr Pro Phe 405 Pro Pro Gly 310 Gly Ala Tyr Tyr As n 390 Ala Tyr Glu Phe Lys Ile Thr Leu 375 Arg His Asp Ser Thr Lys Ala Glu 360 Gly Leu Thr Leu Gly 440 Leu Tyr Gly 345 Arg Ser Leu Ser Gin 425 Glu Tyr Pro 330 Ala Tyr Val Leu Ile 410 Ile His Gly 315 Thr Pro Met Ala Leu 395 Leu Tyr Tyr Pro Giu Ile 300 Met Leu Tyr Val Leu Phe Vai Thr Leu 350 Giy His Pro 365 Met Gin Ala 380 His Gly Phe Leu Ser Phe Pro Gin Glu 430 Glu Leu His 445 Phe Ser Lys Pro 320 Ile Tyr 335 Trp Ile Asp Gin Giu Lys Leu Asp 400 Leu Val 415 Arg His Leu Leu His Tyr Leu Gin Giu Asn Leu Gly Ser Arg Ie Ala Ala Leu Lys Val 450 455 460 Ile 465 <210> 6 <211> 1669 <212> DNA <213> Homo sapiens <400> 6 aacaggtaca gcaaatccta aagtcacttt taagatgtca gaaataatga ttgatgctga aggaaggatc atagatgtca tagataagga, actaattcaa ccttttgaga ttctatttga 120 aggagttgaa tatattgcca gagctggatg gactcctgag ggaaaatatg cttggtccat 180 cctactagat cgctcccaga ctcgcctaca, gatagtgttg atctcacctg aattatttat 240 Page 11 WO 01/19866 WO 0119866PCT/AUOO/O 1085 1. ST25 .txt gatgatgtta tggaaaggca gagactcatt gagtcagtgc ctgattctgt cccagtagaa 300 gacgccac ta 360 tcatgttttt 420 aacaggtttc 480 atccagtggt 540 aattaccagt 600 agtcagaagg 660 cgtag tcag t 720 ttcttgctgc 780 tccacactgt 840 aaaggaattt 900 agaaattttc 960 tcatgatcta.

1020 ggt tgc tat t 1080 acgttatatg 1140 gcaagcagaa 1200 tgagaatgtc 1260 gccatatgat 1320 attatctatg aagaaacaac ccccaaagtc acgaagagga cgtcatttat gggctgcctg ggtgaatggg ctggtatatt tacgtaaatc atcagtcagc gtgtCccttt tgggccacca tcttttgaaa cagcctggaa.

gctggggcc ggtcaccctg aagttcccct cattttgcac ttacagatct acaaaattac ctccaagtga aagttcttgg4 ttgaaggcac ctggagaggt actgtgactt acaagc tatc ttt tggattc gtac tac tgg agaaatatcc cagtcac tc t accagaatga c tgaaccaaa.

ataccagtat atcctcagga agacatctgg ataaatatcc atgacatctt aattgagttt atttttgcct ctgaatgcaa atctatttta. aaggaaagca aatataaacg tttcaagtgt cctatcaaag aggagatagc ccggcatgga. tctaatatcc aagttgatga caaagactcc cctttagagc atcacctgta gacaaggctg actgaccgtg gctactcaca ctttataagt aagtatagta accagaagaa aagtcctgaa. gatgacccaa cttgcaaaac agcaggtcct cttcctgact atactcctcc atttacattg tatgggatgc tctacaagcc tactgtgctg ttcatatatg gtggtcctca gtggatcttc tatgatacag gatacacgga acagggctat tacttaggat ctgtggccat tcgtttactg ctcttacatg gtttcctgga attactgagt tttttagtga gggctggaaa gagacacagc ataagagttc ctgaatcggg agaacattat gaactgcatc ttttgcacta ccttcaagaa aaccttggat cacgtattgc Page 12 WO 01119866 WO 0119866PCT/AUOO/01085 1. ST25. txt 1380 tgctctaaaa gtgatataat tttgacctgt gtagaactct ctggtataca ctggctattt 1440 aaccaaatga ggaggtttaa tcaacagaaa acacagaatt gatcatcaca ttttgatacc 1500 tgccatgtaa catctactcc tgaaaataaa tgtggtgcca tgcaggggtc tacggtttgt 1560 ggtagtaatc taatacctta accccacatg ctcaaaatca aatgatacat attcctgaga 1620 gacccagcaa taccataaga attactaaaa aaaaaaaaaa aaaaaaaaa 1669 <210> 7 <211> 360 <212> PRT <213> Homo sapiens <400> 7 Glu Glu Asp Ala Arg 1 Glu Phe Asp Arg Tyr Thr Pro Ser Gly Gly Glu Ser Glu Val Glu Arg Arg Ala Asp Ser Lys Val Thr Phe Lys Ile Ile Val Asp Glu 100 Asp Ser Pro Leu Glu 115 Gly Glu Val Thr Arg 130 Ile Ser Gin His Cys 145 Ser Ala Gly Val Ala Thr Phe Val Leu Gin Glu Ser Lys Ile Phe 70 Met Val His Leu Asp 150 Gly Ile Ile 55 Arg Ser Arg His Thr 135 Phe Tyr Leu 40 His Tyr Glu Arg Leu 120 Asp Phe Trp 25 Arg Val Pro Ile Leu 105 Tyr Arg Ile Trp Cys Ile Leu Thr Ser Lys Thr 75 Met Ile 90 Val Tyr Val Val Gly Tyr Ser Lys 155 Pro Lys Tyr Glu Pro Met Gly Thr Asp Ala Phe Giu Ser Tyr 125 Ser His 140 Tyr Ser Ala Glu Leu Ala Giu Gly 110 Val Ser Asn Giu Asn Glu Asn Gly Thr Asn Cys Gin Thr

ASP

Thr Pro Arg Lys Pro Cys Lys 160 Page 13 WO 01/19866 WO 0119866PCT/AUOO/01085 1. ST2S .txt Asn Pro His Cys Val Ser Leu Tyr Lys Leu Ser Ser Pro Pro Gly Thr Gin 225 Gin Leu Arg Lys ILeu 305 Gly Ser Thr Pro Thr 210 Pro Val Asia Gly Met 290 Ala Trp Asp Cys Leu 195 Gly Gly Gin Thr Ser 275 Gly Ser Ser Ie L~ys 180 Pro Phe Lys Leu Leu 260 Cys Gin Arg Tyr Phe 340 165 Thr ,Asp Thr Lys Val 245 Ala His Ile Tyr Giy 325 Arg 170 Lys Tyr Leu Tyr 230 Asn Ser Arg Giu Asp 310 Gly Val Giu Thr Tyr 215 Pro Asn Leu Gly Ile 295 Phe Tyr Ala Phe Pro 200 Giy Thr Arg Gly Leu 280 Asp Ile Leu Trp Aia Thr Ile 185 Pro Met Val Phe Tyr 265 Lys Asp Asp Ser G iu Leu Leu Lys 250 Val Phe Gin Leu Leu 330 Ile Tyr Phe 235 Giy Val Giu Val1 Asp 315 Met Phe Lys 220 Ile Val Val1 Gly Glu 300 Arg Ala Leu Ser 205 Pro Tyr Lys Val Ala 285 Gly Val Leu Glu Asp 190 Phe His Gly Tyr Ile 270 Phe Leu Gly Met Asp 175 Ser Glu Asp Gly Phe 255 Asp Lays Gin Ile Gin 335 Asp Ala Ser Leu Pro 240 Arg Asn Tyr Tyr His 320 Arg Ile Ala Gly Ala 345 Pro Val Thr Leu Trp 350 Ile Phe Tyr Asp Thr Gly Tyr Thr 355 360 <210> 8 <211> 1083 <212> DNA <213> Homo sapiens <400> 8 ggaagaagat gccagatcag ctggagtcgc tacctttgtt ctccaagaag aatttgatag atattctggc tattggtggt gtccaaaagc tgaaacaact cccagtggtg gtaaaattct 120 tagaattcta tatgaagaaa atgatgaatc tgaggtggaa attattcatg ttacatcccc 180 tatgttggaa acaaggaggg cagattcatt ccgttatcct aaaacaggta cagcaaatcc Page 14 WO 01/19866 WO 0119866PCT/AUOO/01085 240 taaagtcact tttaagatgt cagaaataat gattgatgct gaaggaagga tcatagttga.

300 tgaagtcaga 360 gtacgtagtc 420 acattcttgc 480 gaatccacac 540 aacaaaggaa 600 tccagaaatt 660 gcc tcatgat 720 tcaggtgcag 780 agcctctcta 840 taaatttgaa 900 aggactccaa 960 cggc tggtcc 1020 cagggttgct 1080 gga 1083 aggctggtat agttacgtaa tgcatcagtc tgtgtgtccc ttttgggcca ttctcttttg ctacagcctg ttggtgaata ggttatgtgg ggcgccttta tatctagctt tatggaggat attgctgggg attttgaagg atcctggaga agoac tgtga tttacaagct ccattttgga aaagtactac gaaagaaata atcggtttaa ttgtagtgat aatataaaat ctcgatatga acctctccct ccccagtcac caccaaagac ggtgacaagg cttctttata atcaagtcc t ttcagcaggt tggat ttac a tcc tactgtg aggagtcaag agac aacagg gggtcaaata tttcattgac gatggcatta tctgtggatc tcccctttag agcatcacct c tgac tgacc agtaagtata gaagatgac cctcttcctg ttgtatggga ctgttcatat tatttccgct ggatcctgtc gaaattgacg ttagatcgtg a tgcagagg t ttctatgata gtggctaCtC gtaaccagaa caacttgcaa actatactcc tgctctacaa atggtggtcc tgaataccct accgagggct atcaggtgga tgggcatcca cagatatctt caggatacac Page

Claims (28)

1. A peptide which comprises: the sequence shown in SEQ ID NO:1; or the amino acid sequences: His736GlyTrpSerTyrGlyGlyTyrLeu; Leu 8 6AspGluAsnValHisPheAlaHis; Glu 847 ArgHisSerIleArg and Phe 2 SSValLeuGlnGluGluPhe, and which has the substrate specificity of the sequence shown in SEQ ID NO:1; or the sequence which has at least 60% identity with the sequence shown in SEQ ID NO:1, and which has the substrate specificity of the sequence shown in SEQ ID NO:1.

2. A peptide according to claim 1 wherein the amino acid identity is at least

3. A peptide according to claim 1 wherein the amino acid identity is at least

4. A fragment of the sequence shown in SEQ ID NO:1 which has the substrate specificity of the sequence shown in SEQ ID NO:1.

5. A fragment according to claim 4 which consists of the sequence shown in SEQ ID NO.s: 3, 5 or 7.

6. A peptide according to claim 1, wherein an asparagine residue in the peptide is not linked to a carbohydrate molecule.

7. A peptide according to claim 1, wherein the peptide is not expressed on the cell surface membrane of a cell. WO 01/19866 PCT/AU00/01085 49

8. A fusion protein comprising the amino acid sequence shown in SEQ ID NO:1 linked with a further amino acid sequence, the fusion protein having the substrate specificity of the sequence shown in SEQ ID NO:1.

9. A fusion protein according to claim 8 wherein the further amino acid sequence is selected from the group consisting of GST, V5 epitope and His tag.

10. A method of identifying a molecule capable of inhibiting cleavage of a substrate by DPP8 comprising the following steps: contacting DPP8 with the molecule; contacting DPP8 of step with a substrate capable of being cleaved by DPP8, in conditions sufficient for cleavage of the substrate by DPP8; and detecting substrate not cleaved by DPP8, to identify that the molecule is capable of inhibiting cleavage of the substrate by DPP8.

11. A method of identifying a molecule capable of inhibiting specifically, the cleavage of a substrate by DPP8, the method comprising the following -steps: contacting DPP8 and a further protease with the molecule; contacting DPP8 and the further protease of step with a substrate capable of being cleaved by DPP8 and the further protease, in conditions sufficient for cleavage of the substrate by DPP8 and the further protease; and detecting substrate not cleaved by DPP8, but cleaved by the further protease, to identify that the molecule is capable of inhibiting specifically, the cleavage of the substrate by DPP8. WO 01/19866 PCT/AU00/01085 50

12. A method of reducing or inhibiting the catalytic activity of DPP8, the method comprising the step of contacting DPP8 with an inhibitor of DPP8 catalytic activity.

13. A method of cleaving a substrate comprising the step of contacting the substrate with DPP8 in conditions sufficient for cleavage of the substrate by DPP8.

14. A method of detecting an activated T cell, the method comprising the step of measuring the level of DPP8 gene expression in a T cell. A method according to claim 14, wherein the level of DPP8 gene expression is detected by detecting the amount of DPP8 RNA in the cell.

16. A nucleic acid molecule which: encodes the sequence shown in SEQ ID NO:1; or consists of the sequence shown in SEQ ID NO:2; or is capable of hybridizing to a nucleic acid molecule consisting of the sequence shown in SEQ ID NO:2 in stringent conditions, and which encodes a peptide which has the substrate specificity of the sequence shown in SEQ ID NO:1.

17. A nucleic acid molecule according to claim 16 wherein the molecule is capable of hybridising in high stringent conditions.

18. A nucleic acid molecule according to claim 16 which is capable of hybridising to a gene which is located at band q 22 on human chromosome

19. A nucleic acid molecule according to claim 16 WO 01/19866 PCT/AU00/01085 51 which does not contain 5' or 3' untranslated regions. A fragment of a nucleic acid molecule consisting of the sequence shown in SEQ ID NO:2, which encodes a peptide which has the substrate specificity of the sequence shown in SEQ ID NO:1.

21. A fragment according to claim 20 which consists of the sequence shown in any one of SEQ ID NO.s: 4, 6 or 8.

22. according A vector comprising a nucleic acid molecule to claim 16.

23. A cell comprising a vector according to claim 22.

24. A composition comprising a peptide according to claim 1. An antibody which is capable of binding to a peptide according to claim 1.

26. An antibody according to claim 25 which is produced by a hybridoma cell.

27. A hybridoma cell capable of making an antibody according to claim 26. 004479235

28. A peptide which comprises: a. the sequence shown in SEQ ID NO: 1 b. a sequence which has at least 95% identity with the sequence shown in SEQ ID NO: 1, and which has the substrate speciality of the sequence shown in SEQ ID NO: 1.

29. A peptide according to claim 1, the peptide being substantially as described herein with reference to the Examples. A nucleic acid molecule encoding a peptide according to claim 29.

31. A cell or composition comprising a peptide according to claim 29 or a nucleic acid according to claim

32. An antibody for binding to a peptide according to claim 29. Dated this 2 8 th day of August 2003 The University of Sydney Sby its attorneys Freehills Carter Smith Beadle