# Patent

## Title
Dipeptidyl peptidases

## Abstract

Peptides including HisGlyTrpSerTyrGlyGlyPheLeu; LeuAspGluAsnValHisPhePhe; GluArgHisSerIle Arg and PheValIleGlnGluGluPhe which show peptidase ability and have substrate specificity for at least one of the compounds H-Ala-Pro-pNA, H-Gly-Pro-pNA and H-Arg-Pro-pNA are disclosed. Nucleic acids, vectors, antibodies and hybridoma cells are also claimed with reference to the above sequences and their abilities.

## Background
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 [1-3]. The gene family includes the following molecules: DPPIV (CD26), dipeptidyl amino-peptidase-like protein 6 (DPP6), dipeptidyl amino-peptidase-like protein 8 (DPP8) and fibroblast activation protein (FAP) [1,2,4,5]. Another possible member is DPPIV-β[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-cell-derived 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 α3β1, and α5β1 integrin[13-14].
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.
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:2.
As described herein, the inventors believe that the peptide is a prolyl oligopeptidase and a dipeptidyl peptidase, because it has substantial and significant homology with the amino acid sequences of DPPIV and DPP8. As homology is observed between DPP8, DPPIV and DPP9, it will be understood that DPP9 has a substrate specificity for at least one of the following compounds: H-Ala-Pro-pNA, H-Gly-Pro-pNA and H-Arg-Pro-pNA.
The peptide is homologous with human DPPIV and DPP8, and importantly, identity between the sequences of DPPIV and DPP8 and SEQ ID NO: 2 is observed at the regions of DPPIV and DPP8 containing the catalytic triad residues and the two glutamate residues of the β-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:2 is a member of the DPPIV-like gene family. Accordingly the peptide is now named and described herein as DPP9.
The following sequences of the human DPPIV amino acid sequence are important for the catalytic activity of DPPIV: (i) Trp617GlyTrpSerTyrGlyGlyTyrVal, (SEQ ID NO:42); (ii) Ala707AspAspAsnValHisPhe, (SEQ ID NO:43); (iii) Glu738AspHisGlyIleAlaSer, (SEQ ID NO:44); (iv) Trp201ValTyrGluGluGluVal, (SEQ ID NO:45) [25-28]. As described herein, the alignment of the following sequences of DPP9: His833GlyTrpSerTyrGlyGlyPheLeu, (SEQ ID NO:46); Leu913AspGluAsnValHisPhePhe, (SEQ ID NO:47); Glu944ArgHisSerIleArg (SEQ ID NO:48) and Phe350ValIleGlnGluGluPhe (SEQ ID NO:49) with sequences (i) to (iv) above, respectively, suggests that these sequences of DPP9 are likely to confer the catalytic activity of DPP9. This is also supported by the alignment of DPP9 and DPP8 amino acid sequences. More specifically, DPP8 has substrate specificity for H-Ala-Pro-pNA, H-Gly-Pro-pNA and H-Arg-Pro-pNA, and shares near identity, with only one position of amino acid difference, in each of the above described sequences of DPP9.
Thus, in a second aspect, the invention provides a peptide comprising the following amino acid sequences: HisGlyTrpSerTyrGlyGlyPheLeu, (SEQ ID NO:50); LeuAspGluAsnValHisPhePhe, (SEQ ID NO:51); GluArgHisSerIleArg, (SEQ ID NO:52) and PheCalIleGlnGluGluPhe, (SEQ ID NO:53); which has the substrate specificity of the sequence shown in SEQ ID NO:2.
Also described herein, using the GAP sequence alignment algorithm, it is observed that DPP9 has 53% amino acid similarity and 29% amino acid identity with a C. elegans protein. Further, as shown herein, a nucleic acid molecule which encodes DPP9, is capable of hybridising specifically with DPP9 sequences derived from non-human species, including rat and mouse. Further, the inventors have isolated and characterised a mouse homologue of human DPP9. Together these data demonstrate that DPP9 is expressed in non-human species. Thus in a third aspect, the invention provides a peptide which has at least 91% amino acid identity with the amino acid sequence shown in SEQ ID NO:2, and which has the substrate specificity of the sequence shown in SEQ ID NO:2. Typically the peptide has the sequence shown in SEQ ID NO:4. Preferably, the amino acid identity is 75%. More preferably, the amino acid identity is 95%. Amino acid identity is calculated using GAP software [GCG Version 8, Genetics Computer Group, Madison, Wis., USA] as described further herein. Typically, the peptide comprises the following sequences: HisGlyTrpSerTyrGlyGlyPheLeu, (SEQ ID NO:54); LeuAspGluAsnValHisPhePhe, (SEQ ID NO:55); GluArgHisSerIleArg, (SEQ ID NO:56); PheValIleGinGluGLuPhe, (SEQ ID NO:57).
In view of the homology between DPPIV, DPP8 and DPP9 amino acid sequences, it is expected that these sequences will have similar tertiary structure. This means that the tertiary structure of DPP9 is likely to include the seven-blade β-propeller domain and the α/β hydrolase domain of DPPIV. These structures in DPP9 are likely to be conferred by the regions comprising β-propeller, Val226 to Ala705, α/β hydrolase, Ser706 to Leu969 and about 70 to 90 residues in the region Ser136 to Gly225.
As it is known that the β-propeller domain regulates proteolysis mediated by the catalytic triad in the α/β hydrolase domain of prolyl oligopeptidase, [29] it is expected that truncated forms of DPP9 can be produced, which have the substrate specificity of the sequence shown in SEQ ID NO:2, comprising the regions referred to above (His833GlyTrpSerTyrGlyGlyPheLeu, (SEQ ID NO:46); Leu913spGluAsnValHisPhePhe, (SEQ ID NO:47) Glu944ArgHisSerIleArg (SEQ ID NO:48) and Phe350ValIleGlnGluGluPhe (SEQ ID NO:49), which confer the catalytic specificity of DPP9. Examples of truncated forms of DPP9 which might be prepared are those in which the region conferring the β-propeller domain and the α/β hydrolase domain are spliced together. Other examples of truncated forms include those that are encoded by splice variants of DPP9 mRNA. Thus although, as described herein, the biochemical characterisation of DPP9 shows that DPP9 consists of 969 amino acids and has a molecular weight of about 110 kDa, it is recognised that truncated forms of DPP9 which have the substrate specificity of the sequence shown in SEQ ID NO:2, 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: 2, which has the substrate specificity of the sequence shown in SEQ ID NO:2. The inventors believe that a fragment from Ser136 to Leu969 (numbered according to SEQ ID NO:2) would have enzyme activity.
It is recognised that DPP9 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:2. An example of a fusion protein is one which comprises the sequence shown in SEQ ID NO:2 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 DPP9 is a glutathione S transferase (GST) domain [30]. Another example of a further amino acid sequence is a portion of CD8α [8]. Thus in one aspect, the invention provides a fusion protein comprising the amino acid sequence shown in SEQ ID NO:2 linked with a further amino acid sequence, the fusion protein having the substrate specificity of the sequence shown in SEQ ID NO:2.
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 DPP9. The polypeptide may be useful, for example, for altering the protease susceptibility of DPP9, 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 DPP9.
In one aspect, the invention provides a peptide which includes the amino acid sequence shown in SEQ ID NO:7. In one embodiment the peptide consists of the amino acid sequence shown in SEQ ID NO:7.
As described further herein, the amino acid sequence shown in SEQ ID NO:7, and the amino acid sequences of DPPIV, DPP8 and FAP are homologous. DPPIV, DPP8 and FAP have dipeptidyl peptidase enzymatic activity and have substrate specificity for peptides which contain the di-peptide sequence, Ala-Pro. The inventors note that the amino acid sequence shown in SEQ ID NO:7 contains the catalytic triad, Ser-Asp-His. Accordingly, it is anticipated that the amino acid sequence shown in SEQ ID NO:7 has enzymatic activity in being capable of cleaving a peptide which contains Ala-Pro by hydrolysis of a peptide bond located C-terminal adjacent to proline in the di-peptide sequence.
In one embodiment, the peptide comprises an amino acid sequence shown in SEQ ID NO:7 which is capable of cleaving a peptide bond which is C-terminal adjacent to proline in the sequence Ala-Pro. The capacity of a dipeptidyl peptidase to cleave a peptide bond which is C-terminal adjacent to proline in the di-peptide sequence Ala-Pro can be determined by standard techniques, for example, by observing hydrolysis of a peptide bond which is C-terminal adjacent to proline in the molecule Ala-Pro-p-nitroanilide.
The inventors recognise that by using standard techniques it is possible to generate a peptide which is a truncated form of the sequence shown in SEQ ID NO:7, which retains the proposed enzymatic activity described above. An example of a truncated form of the amino acid sequence shown in SEQ ID NO:7 which retains the proposed enzymatic activity is a form which includes the catalytic triad, Ser-Asp-His. Thus a truncated form may consist of less than the 831 amino acids shown in SEQ ID NO:7. Accordingly, in a further embodiment, the peptide is a truncated form of the peptide shown in SEQ ID NO:7, which is capable of cleaving a peptide bond which is C-terminal adjacent to proline in the sequence Ala-Pro.
It will be understood that the amino acid sequence shown in SEQ ID NO:7 may be altered by one or more amino acid deletions, substitutions or insertions of that amino acid sequence and yet retain the proposed enzymatic activity described above. It is expected that a peptide which is at least 47% similar to the amino acid sequence of SEQ ID NO:7, or which is at least 27% identical to the amino acid sequence of SEQ ID NO:7, will retain the proposed enzymatic activity described above. The % similarity can be determined by use of the program/algorithm “GAP” which is available from Genetics Computer Group (GCG), Wisconsin. Thus in another embodiment of the first aspect, the peptide has an amino acid sequence which is at least 47% similar to the amino acid sequence shown in SEQ ID NO:7, and is capable of cleaving a peptide bond which is C-terminal adjacent to proline in the sequence Ala-Pro.
As described above, the isolation and characterisation of DPP9 is necessary for identifying inhibitors of DPP9 catalytic activity, which may be useful for the treatment of disease. Accordingly, in a fifth aspect, the invention provides a method of identifying a molecule capable of inhibiting cleavage of a substrate by DPP9, the method comprising the following steps:

    
    
        (a) contacting DPP9 with the molecule;
        (b) contacting DPP9 of step (a) with a substrate capable of being cleaved by DPP9, in conditions sufficient for cleavage of the substrate by DPP9; and
        (c) detecting substrate not cleaved by DPP9, to identify that the molecule is capable of inhibiting cleavage of the substrate by DPP9.
    
    


It is recognised that although inhibitors of DPP9 may also inhibit DPPIV and other serine proteases, as described herein, the alignment of the DPP9 amino acid sequence with most closely related molecules, (i.e. DPPIV), reveals that the DPP9 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 DPP9 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 DPP9, the method comprising the following steps:

    
    
        (a) contacting DPP9 and a further protease with the molecule;
        (b) contacting DPP9 and the further protease of step (a) with a substrate capable of being cleaved by DPP9 and the further protease, in conditions sufficient for cleavage of the substrate by DPP9 and the further protease; and
        (c) detecting substrate not cleaved by DPP9, but cleaved by the further protease, to identify that the molecule is capable of inhibiting specifically, the cleavage of the substrate by DPP9.
    
    


In a seventh aspect, the invention provides a method of reducing or inhibiting the catalytic activity of DPP9, the method comprising the step of contacting DPP9 with an inhibitor of DPP9 catalytic activity. In view of the homology between DPP9 and DPP8 amino acid sequences, it will be understood that inhibitors of DPPB activity may be useful for inhibiting DPP9 catalytic activity. Examples of inhibitors suitable for use in the seventh aspect are described in [21,32,33]. Other inhibitors useful for inhibiting DPP9 catalytic activity can be identified by the methods of the fifth or sixth aspects of the invention.
In one embodiment, the catalytic activity of DPP9 is reduced or inhibited in a mammal by administering the inhibitor of DPP9 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 DPP9 catalytic activity in vivo. Examples of inhibitors useful for this purpose are disclosed in the following [21,32-34].
Preferably, the catalytic activity of DPP9 in a mammal is reduced or inhibited in the mammal, for the purpose of treating a disease in the mammal. Diseases which are likely to be treated by an inhibitor of DPP9 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-(2-aminoethyl)benzenesulfonylfluoride, aprotinin, benzamidine/HCl, Ala-Pro-Gly, H-Lys-Pro-OH HCl salt and zinc ions, for example, zinc sulfate or zinc chloride. More preferably, the inhibitor is one which specifically inhibits DPP9 catalytic activity, and which does not inhibit the catalytic activity of other serine proteases, including, for example DPPIV, DPP8 or FAP.
In an eighth aspect, the invention provides a method of cleaving a substrate which comprises contacting the substrate with DPP9 in conditions sufficient for cleavage of the substrate by DPP9, 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. 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 DPP9 [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 DPP9 may have Ala, or Trp, Ser, Gly, Val or Leu in the P1 position, in place of Pro [11,12].
The inventors have characterised the sequence of a nucleic acid molecule which encodes the amino acid sequence shown in SEQ ID NO:2. Thus in a tenth aspect, the invention provides a nucleic acid molecule which encodes the amino acid sequence shown in SEQ ID NO:2.
In an eleventh aspect, the invention provides a nucleic acid molecule which consists of the sequence shown in SEQ ID NO:1.
In another aspect, the invention provides a nucleic acid molecule which encodes a peptide comprising the amino acid sequence shown in SEQ ID NO:7.
The inventors have characterised the nucleotide sequence of the nucleic acid molecule encoding SEQ ID NO:7. The nucleotide sequence of the nucleic acid molecule encoding DPP4-like-2 is shown in SEQ ID NO:8. Thus, in one embodiment, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:8. In another embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:8.
The inventors recognise that a nucleic acid molecule which has the nucleotide sequence shown in SEQ ID NO:8 could be made by producing only the fragment of the nucleotide sequence which is translated. Thus in an embodiment, the nucleic acid molecule does not contain 5′ or 3′ untranslated nucleotide sequences.
As described herein, the inventors observed RNA of 4.4 kb and aminor band of 4.8 kb in length which hybridised to a nucleic acid molecule comprising sequence shown in SEQ ID NO:8. It is possible that these mRNA species are splice variants. Thus in another embodiment, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:8 and which is approximately 4.4 kb or 4.8 kb in length.
In another embodiment, the nucleic acid molecule is selected from the group of nucleic acid molecules consisting of DPP4-like-2a, DPP4-like-2b and DPP4-like-2c, as shown in FIG. 2.
In another aspect, the invention provides a nucleic acid molecule having a sequence shown in SEQ ID NO: 3.
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:1 in stringent conditions, and which encodes a peptide which has the substrate specificity of the sequence shown in SEQ ID NO:2. As shown in the Northern blot analysis described herein, DPP9 mRNA hybridises specifically to the sequence shown in SEQ ID NO:1, after washing in 2×SSC/1.0% SDS at 37° C., or after washing in 0.1×SSC/0.1% SDS at 50° C. “Stringent conditions” are conditions in which the nucleic acid molecule is exposed to 2×SSC/1.0% SDS. Preferably, the nucleic acid molecule is capable of hybridising to a molecule consisting of the sequence shown in SEQ ID NO:1 in high stringent conditions. “High stringent conditions” are conditions in which the nucleic acid molecule is exposed to 0.1×SSC/0.1% SDS at 50° C.
As described herein, the inventors believe that the gene which encodes DPP9 is located at band p13.3 on human chromosome 19. The location of the DPP9 gene is distinguished from genes encoding other prolyl oligopeptidases, which are located on chromosome 2, at bands 2q24.3 and 2q23, chromosome 7 or chromosome 15q22. Thus in an embodiment, the nucleic acid molecule is one capable of hybridising to a gene which is located at band p13.3 on human chromosome 19.
It is recognised that a nucleic acid molecule which encodes the amino acid sequence shown in SEQ ID NO:2, or which comprises the sequence shown in SEQ ID NO:1, could be made by producing the fragment of the sequence which is translated, using standard techniques [30,31]. 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 % TripleEx, λTripleEx, pGEM-T Easy Vector, pSecTag2Hygro, pet15b, pEE14.HCMV.gs and pcDNA3.1/VS/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, DH5α, 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.
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 DPP9 or a fragment thereof according to standard techniques [35]. An antibody may be prepared by immunising with transiently transfected DPP9+ cells. It is recognised that the antibody is useful for inhibiting activity of DPP9. In one embodiment, the antibody of the eighteenth 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
FIG. 1. Nucleotide sequence of DPP8 (SEQ ID NO:5).
FIG. 2. Schematic representation of the cloning of human CDNA DPP9.
FIG. 3. Schematic representation of the assembly of nucleotide sequences of human cDNA DPP9.
FIG. 4. Nucleotide sequence of human cDNA DPP9 (SEQ ID NO:1) and amino acid sequence of human DPP9 (SEQ ID NO:2).
FIG. 5. Alignment of human DPP9 amino acid sequence beginning with residue 140 of SEQ ID NO:2 (lower sequence) with the amino acid sequence encoded by a predicted open reading frame of GDD (SEQ ID NO:9, upper sequence).
FIG. 6. Alignment of human DPP8 (SEQ ID NO:6), DPP9 (SEQ ID NO:2), DPP4 (SEQ ID NO:10), and FAP (SEQ ID NO:11) amino acid sequences.
FIG. 7. Northern blot analysis of human DPP9 RNA.
FIG. 8. Alignment of a human (upper) DPP9 amino acid sequence (SEQ ID NO:2) with a predicted amino acid sequence of murine (lower) DPP9 (SEQ ID NO:4) from expressed sequence tags.
FIG. 9. Alignment of murine (lower; SEQ ID NO:3) and human (upper) DPP9 cDNA (SEQ ID NO:1) nucleotide sequences.
FIG. 10. Northern blot analysis of rat DPP9 RNA.
FIG. 11. Detection of DPP9 cDNA in CEM cells.
FIG. 12. Detection of murine DPP9 nucleotide sequence in two panels, where FIG. 12A is a gel showing DPP9 liver cDNA and FIG. 12B is a Southern blot of the gel of FIG. 12A.



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 unless indicated otherwise.
DPP9 Cloning
The nucleotide sequence of DPP8 shown in FIG. 1 was used to search the GenBank database for homologous nucleotide sequences. Nucleotide sequences referenced by GenBank accession numbers AC005594 and AC005783 were detected and named GDD. The GDD nucleotide sequence is 39.5 kb and has 19 predicted exons. The analysis of the predicted exon-intron boundaries in GDD suggests that the predicted open reading frame of GDD is 3.6 kb in length.
In view of the homology of DPP8 and the GDD nucleotide sequences, we hypothesised the existence of DPPIV-like molecules other than DPP8. We used oligonucleotide primers derived from the nucleotide sequence of GDD and reverse transcription PCR (RT-PCR) to isolate a cDNA encoding DPPIV-like molecules.
RT-PCR amplification of human liver RNA derived from a pool of 4 patients with autoimmune hepatitis using the primers ODD pr 1f and GDD pr 1r (Table 1) produced a 500 base pair product. This suggested that DPPIV-like molecules are likely to be expressed in liver cells derived from individuals with autoimmune hepatitis and that RNA derived from these cells is likely to be a suitable source for isolating cDNA clones encoding DPPIV-like molecules.
Primers GDD pr 3f and GDD pr 1r (Table 1) were then used to isolate a cDNA clone encoding a DPP4-like molecule. A 1.6 kb fragment was observed named DPP4-like-2a. Primers GDD pr1f and GDD pr 7r (Table 1) were then used to isolate a cDNA clone encoding a DPP4like molecule. A 1.9 kb product was observed and named DPP4-like-2b. As described further herein, the sequence of DPP4-like-2b overlaps with the sequence of DPP4-like-2a.
The DPP4-like-2a and 2b fragments were gel purified using WIZARD® PCR preps kit and cloned into the pGEM®-T-easy plasmid vector using the EcoRI restriction sites. The ligation reaction was used to transform JM109 competent cells. The plasmid DNA was prepared by miniprep. The inserts were released by EcoRI restriction digestion. The DNA was sequenced in both directions using the M13Forward and M13Reverse sequencing primers. The complete sequence of DPP4-like-2a and 2b fragments was derived by primer walking.
The nucleotide sequence 5′ adjacent to DPP4-like-2b was obtained by 5′RACE using dC tailing and the gene specific primers GDD GSP1.1 and 2.1 (Table 1). A fragment of 500 base pairs (DPP4-like-2c) was observed. The fragment was gel purified using WIZARD® PCR preps kit and cloned into the pGEM®-T-easy plasmid vector using the EcoRI restriction sites. The ligation reaction was used to transform JM109 competent cells. The plasmid DNA was prepared by miniprep. The inserts were released by EcoRI restriction digestion. The DNA was sequenced in both directions using the M13Forward and M13Reverse sequencing primers.
We identified further sequences, BE727051 and BE244612, with identity to the 5′ end of DPP9. These were discovered while performing BLASTn with the 5′ end of the DPP9 nucleotide sequence. BE727051 contained further 5′ sequence for DPP9, which was also present in the genomic sequence for DPP9 on chromosome 19p13.3. This was used to design primer DPP9-22F (5′GCCGGCGGGTCCCCTGTGTCCG3′), (SEQ ID NO: 34). Primer 22F was used in conjunction with primer GDD3′end (5′GGGCGGGACAAAGTGCCTCACTGG3′), (SEQ ID NO:35). on cDNA made from the human CEM cell line to produce a 3000 bp product as expected FIG. 11.
Nucleotide Sequence Analysis of DPP4-like-2a, 2b, and 2c Fragments.
An analysis of the nucleotide sequence of fragments DPP4-like 2a, 2b and 2c with the SequencherTM version 3.0 computer program (FIG. 3), and the 5′ fragment isolated by primers DPP9-22F and GDD3′end, revealed the nucleotide sequence shown in FIG. 4.
The predicted amino acid sequence shown in FIG. 4 was compared to a predicted amino acid sequence encoded by a predicted open reading frame of GDD (predicted from the nucleotide sequence referenced by GenBank Accession Nos. AC005594 and AC005783), to determine the relatedness of the nucleotide sequence of FIG. 4 to the nucleotide sequence of the predicted open reading frame of GDD (FIG. 5). Regions of amino acid identity were observed suggesting that there may be regions of nucleotide sequence identity of the predicted open reading frame of GDD and the sequence of FIG. 4. However, as noted in FIG. 5, there are regions of amino acid sequence encoded by the sequence of FIG. 4 and the amino acid sequence encoded by the predicted open reading frame of GDD which are not identical, demonstrating that the nucleotide sequences encoding the predicted open reading frame of GDD and the sequence shown in FIG. 4 are different nucleotide sequences.
As described further herein, the predicted amino acid sequence encoded by the cDNA sequence shown in FIG. 4 is homologous to the amino acid sequence of DPP8 (FIG. 6). Accordingly, and as a cDNA consisting of the nucleotide sequence shown in FIG. 4 was not known, the sequence shown in FIG. 4 was named cDNA DPP9.
The predicted amino acid sequence encoded by cDNA DPP9 (called DPP9) is 969 amino acids and is shown in FIG. 4. The alignment of DPP9 and DPP8 amino acid sequences suggests that the nucleotide sequence shown in FIG. 4 may be a partial length clone. Notwithstanding this point, as discussed below, the inventors have found that the alignment of DPP9 amino acid sequence with the amino acid sequences of DPP8, DPP4 and FAP shows that DPP9 comprises sequence necessary for providing enzymolysis and utility. In view of the similarity between DPP9 and DPP8, a full length clone may be of the order of 882 amino acids. A full length clone could be obtained by standard techniques, including for example, the RACE technique using an oligonucleotide primer derived from the 5′ end of cDNA DPP9.
In view of the homology between the DPP8 and DPP9 amino acid sequences, it is likely that cDNA DPP9 encodes an amino acid sequence which has dipeptidyl peptidase enzymatic activity. Specifically, it is noted that the DPP9 amino acid sequence contains the catalytic triad Ser-Asp-His in the order of a non-classical serine protease as required for the charge relay system. The serine recognition site characteristic of DPP4 and DPP4-like family members, GYSWGG, (SEQ ID NO:36), surrounds the serine residue also suggesting that DPP9 cDNA will encode a DPP4-like enzyme activity.
Further, DPP9 amino acid sequence also contains the two glutamic acid residues located at positions 205 and 206 in DPPIV. These are believed to be essential for the dipeptidyl peptidase enzymatic activity. By sequence alignment with DPPIV, the residues in DPP8 predicted to play a pivotal role in the pore opening mechanism in Blade 2 of the propeller are E259, E260. These are equivalent to the residues Glu205 and Glu206 in DPPIV which previously have been shown to be essential for DPPIV enzyme activity. A point mutation Glu259Lys was made in DPP8 cDNA using the Quick Change Site directed Mutagenesis Kit (Stratagene, La Jolla). COS-7 cells transfected with wildtype DPP8 cDNA stained positive for H-Ala-Pro4 MbNA enzyme activity while the mutant cDNA gave no staining. Expression of DPP8 protein was demonstrated in COS cells transfected with wildtype and mutant cDNAs by immunostaining with anti-VS mAB. This mAB detects the V5 epitope that has been tagged to the C-terminus of DPP8 protein. Point mutations were made to each of the catalytic residues of DPP8, Ser739A, Asp817Ala and His849Ala, and each of these residues were also determined to be essential for DPP8 enzyme activity. In summary, the residues that have been shown experimentally to be required for enzyme activity in DPPIV and DPP8 are present in the DPP9 amino acid sequence: Glu354, Glu355, Ser136, Asp914 and His946.
The DPP9 amino acid sequence shows the closest relatedness to DPP8, having 77% amino acid similarity and 60% amino acid identity. The relatedness to DPPIV is 25% amino acid identity and 47% amino acid similarity. The % similarity was determined by use of the program/algorithm “GAP” which is available from Genetics Computer Group (GCG), Wisconsin.
DPP9 mRNA Expression Studies
DPP4-like-2a was used to probe a Human Master RNA BlotTM (CLONTECH Laboratories Inc., USA) to study DPP9 tissue expression and the relative levels of DPP9 mRNA expression.
The DPP4-like-2a fragment hybridised to all tissue mRNA samples on the blot. The hybridisation also indicated high levels of DPP9 expression in most of the tissues samples on the blot (data not shown).
The DPP4-like-2a fragment was then used to probe two Multiple Tissue Northern BlotsTM (CLONTECH Laboratories Inc., USA) to examine the mRNA expression and to determine the size of DPP9 mRNA transcript.
The autoradiographs of the DPP9 Multiple Tissue Northern blot are shown in FIG. 7. The DPP9 transcript was seen in all tissues examined confirming the results obtained from the Master RNA blot. A single major transcript 4.4 kb in size was seen in all tissues represented on two Blots after 16 hours of exposure. Weak bands could also be seen in some tissues after 6 hours of exposure. The DPP9 transcript was smaller than the 5.1 kb mPA transcript of DPP8. A minor, very weak transcript 4.8 kb in size was also seen in the spleen, pancreas, peripheral blood leukocytes and heart. The highest mRNA expression was observed in the spleen and heart. Of all tissues examined the thymus had the least DPP9 mRNA expression. The Multiple Tissue Northern Blots were also probed with a β-actin positive control. A 2.0 kb band was seen in all tissues. In addition as expected a 1.8 kb β-actin band was seen in heart and skeletal muscle.
Rat DPP9 Expression
A Rat Multiple Tissue Northern Blot (CLONTECH Laboratories, Inc., USA;catalogue #: 7764-1) was hybridised with a human DPP9 radioactively labeled probe, made using Megaprime DNA Labeling kit and [32P] dCTP (Amersham International plc, Amersham, UK). The DPP9 PCR product used to make the probe was generated using Met3F (GGCTGAGAGGATGGCCACCACCGGG), (SEQ ID NO:37), as the forward primer and GDD3′ end (GGGCGGGACAAAGTGCCTCCACTGG), (SEQ ID NO:35), as the reverse primer. The hybridisation was carried out according to the manufacturers' instructions at 60° C. to detect cross-species hybridisation. After overnight hybridization the blot was washed at room temperature (2×SSC, 0.1% SDS) then at 40° C.(0.1×SSC, 0.1% SDS).
The human cDNA probe identified two bands in all tissues examined except in testes. A major transcript of 4 kb in size was seen in all tissues except testes. This 4 kb transcript was strongly expressed in the liver, heart and brain. A second weaker transcript 5.5 kb in size was present in all tissues except skeletal muscle and testes. However in the brain the 5.5 kb transcript was expressed at a higher level than the 4.4 kb transcript. In the testes only one transcript approximately 3.5 kb in size was detected. Thus, rat DPP9 mRNA hybridised with a human DPP9 probe indicating significant homology between DPP9 of the two species. The larger 5.5 kb transcript observed may be due to crosshybridisation to rat DPP8.
Mouse DPP9 Expression
A Unigene cluster for Mouse DPP9 was identified (UniGene Cluster Mm.33185) by homology to human DPP9. An analysis of expressed sequence tags contained in this cluster and mouse genomic sequence (AC026385) for Chromosome 17 with the SequencherTM version 3.0 computer program revealed the nucleotide sequence shown in FIG. 9. This 3517 bp cDNA encodes a 869 aa mouse DPP9 protein (missing N-terminus) with 91% amino acid identity and 94% amino acid similarity to human DPP9. The mouse DPP9 amino acid sequence also has the residues required for enzyme activity, Ser, Asp and His and the two Glu residues.
The primers mgdd-pr1F (5′ACCTGGGAGGAAGCACCCCACTGTG3′), (SEQ ID NO:38), and mgdd-pr4R (5′TTCCACCTGGTCCTCAATCTCC3′), SEQ ID NO:39), were designed from this sequence and used to amplify a 452 bp product as expected from liver mouse cDNA, as described below.
RNA Preparation
B57Bl6 mice underwent carbon tetrachloride treatment to induce liver fibrosis. Liver RNA were prepared from snap-frozen tissues using the TRIzol® Reagent and other standard methods.
cDNA Synthesis
2 μg of liver RNA was reverse-transcribed using SuperScript II RNase H-Reverse Transcriptase (Gibco BRL).
PCR
PCR using mDPP9-1F (ACCTGGGAGGAAGCACCCCACTGTG), (SEQ ID NO:40), as the forward primer and mDPP9-2R (CTCTCCACATGCAGGGCTACAGAC), (SEQ ID NO:41), as the reverse primer was used to synthesize a 550 bas pair mouse DPP9 fragment. The PCR products were generated using AmpliTaq Gold® DNA Polymerase. The PCR was performed as follows: denaturation at 95° C. for 10 min, followed by 35 cycles of denaturation at 95° C. for 30 seconds, primer annealing at 60° C. for 30 seconds, and an extension 720 C for 1 min.
Southern Blot
DPP9 PCR products from six mice as well as the largest human DPP9 PCR product were run on a 1% agarose gel. The DNA on the gel was then denatured using 0.4 M NaOH and transferred onto a Hybond-N+ membrane (Amersham International plc, Amersham, UK). The largest human DPP9 PCR product was radiolabeled using the Megaprime DNA Labeling kit and [32P] dCTP (Amersham International plc, Amersham, UK). Unincorporated label was removed using a NAP column (Pharmacia Biotech, Sweden) and the denatured probe was incubated with the membrane for 2 hours at 60° C. in Express Hybridisation solution (CLONTECH Laboratories, Inc., USA). (FIG. 12). Thus, DPP9 mRNA of appropriate size was detected in fibrotic mouse liver using rt-PCR. Furthermore, the single band of mouse DPP9 cDNA hybridised with a human DPP9 probe indicating significant homology between DPP9 of the two species.
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Primer length
Primer sequence (5′-3′)
















FORWARD Primer name









GDD pr 1f
24mer
GTG GAG ATC GAG GAC CAG GTG GAG


 


GDD pr 2f
24mer
CAA AGT GAG GAA AAA TGC ACT CCG


 


GDD pr 2a
24mer
TGA GGA AAA ATG CAC TCC GAG CAG


 


GDD pr 3f
24mer
AAA CTG GCT GAG TTC CAG ACT GAC


 


GDD pr 5f
24mer
CGG GGA AGG TGA GCA GAG CCT GAC


 


GDD pr 6f
24mer
AGA AGC ACC CCA CCG TCC TCT TTG


 


GDD pr 11f
24mer
GAG AAG GAG CTG GTG CAG CCC TTC


 


GDD pr 12f
24mer
TCA GAG GGA GAG GAC GAG CTC TGC


 


GDD pr 14f
24mer
CCG CTT CCA GGT GCA GAA GCA CTC


 


GDD pr 15f
24mer
CTA CGA CTT CCA CAG CGA GAG TGG


 


GDD pr 16f
25mer
GAT GAG TCC GAG GTG GAG GTC ATT C


 







REVERSE Primer name









GDD pr 1r
24mer
GCT CAG AGG TAT TCC TGT AGA AAG


 


GDD pr 4r
24mer
CCC ATG TTG GCC AGG CTG GTC TTG


 


GDD pr 7r
24mer
AGG ACC AGC CAT GGA TGG CAA CTC


 


GDD pr 8r
24mer
CCG CTC AGC TTG TAG ACG TGC ACG


 


GDD pr 9r
24mer
TCA TTC TCT GTG CTC GGG ATG AAC


 


GDD pr 13r
24mer
GCA CAT CCG AGC GCG TGT GGA AAT


 


GDD pr 17r
24mer
TGG GAG AAG CCG GGC GTG GTG AGG


 


GDD pr 18r
25mer
GCG GTC GAA CTC TTC CTG TAT GAC G


 







5′RACE Primer name









GDD GSP 1.1
18mer
TGA AGG AGA AGA AGG CAG


 


GDD GSP 2.1
24mer
CCT GAG CAC TGG GTC TTG ATT TCC


 


5′ RACE Abridged Anchor Primer (AAP)
36mer
GGC CAC GCG TCG ATC ATG ACG GGI IGG GII GGG IIG

## Claims

1. An isolated fragment of the sequence shown in SEQ ID NO:2, wherein the fragment has prolyl dipeptidyl peptidase activity and has the substrate specificity of the sequence shown in SEQ ID NO:2.

2. A fusion protein comprising the amino acid sequence shown in SEQ ID NO:2 linked with a further amino acid sequence, the fusion protein having prolyl dipeptidyl peptidase activity and having the substrate specificity of the sequence shown in SEQ ID NO:2.

3. An isolated protein comprising the polypeptide having the sequence of SEQ ID NO: 2, wherein the polypeptide has prolyl dipeptidyl peptidase activity.

4. An isolated protein comprising a polypeptide having at least 95% homology to SEQ ID NO: 4, wherein the polypeptide has prolyl dipeptidyl peptidase activity.

5. The isolated protein of claim 4, wherein the polypeptide has the sequence set forth by SEQ ID NO: 4.

6. An isolated prolyl dipeptidyl peptidase polypeptide consisting of the sequence set forth by SEQ ID NO: 2 or SEQ ID NO: 4.

7. An isolated fragment of the polypeptide set forth by SEQ ID NO: 2 or SEQ ID NO: 4, wherein the fragment has prolyl dipeptidyl peptidase activity.

8. A fusion protein comprising the prolyl dipeptidyl peptidase polypeptide set forth by SEQ ID NO: 2 or SEQ ID NO: 4 linked to a tag.

9. A composition comprising any one of the protein of claim 3, 4, or 5, the prolyl dipeptidyl peptidase polypeptide of claim 6, the fragment of claim 7, or the fusion protein of claim 8.