Patent Application: US-82563204-A

Abstract:
the present invention provides nucleic acids that encodes a human dipeptidyl peptidase , which shares structural and functional characteristics with dipeptidyl peptidase iv and fibroblast activation protein . vectors and cells comprising the nucleic acids are also disclosed .

Description:
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 ™ 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 ). 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 μg . ml − 1 phytohaemagglutinin ( wellcome ) or 100 ng . ml − 1 okt3 ( orthoclone , fla ., usa ) for 72 h . the human cell lines jurkat , ccrf - cem , raji , daudi and hepg2 were grown to confluence in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( trace biosciences , nsw , australia ) supplemented with 10 % fetal bovine serum and 2 mm l - glutamine . liver and placental rna were prepared from snap - frozen human tissue as described previously [ 37 ]. however , rna was prepared from pbmcs and cell lines using an rna easy kit ( qiagen , germany ). blast programs [ 38 ] and all multiple sequence alignments were performed through the australian national genomic information service ( angis , sydney , nsw , australia ). pileup ( gcg version 8 , genetics computer group , madison , wis ., 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 ™ 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 ). estaa417787 was used to design forward ( caaatagaaattgacgatcag gtg ; seq id no : 24 ) and reverse ( tcttgaaggtagtgcaaaagatgc ; seq id no : 25 ) dpp8 primers for polymerase chain reaction ( pcr ) from estaa417787 . the pcr conditions were as follows : 94 ° c . for 5 min , followed by 35 cycles of 94 ° c . for 1 minute , 55 ° c . for 30 sec and 70 ° c . for 1 min . this 484 bp pcr product was gel purified , 32 p - α labelled using megaprime labeling kit ( amersham pharmacia biotec , uk ) and hybridized to a master rna blot ( clontech , palo alto , calif ., usa ) that contained poly a 30 from 50 adult and fetal tissues immobilized in dots as per manufacturers &# 39 ; 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 λ stretch plus library ( clontech , palo alto , calif ., usa ) for the presence of dpp8 cdna in the 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 λtripleex 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 . 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 gsp1 ( tccttccttcagcatcaatc ; seq id no : 26 ) and gsp2 ( cttaaaagtgactttaggatttgctgtacc ; seq id no : 27 ). 5 ′ race pcr products were cloned into pgem - t easy ® vector ( promega co ., madison , wis ., usa ) and sequenced by primer walking . reverse transcriptase pcr was carried out on atc rna using dpp8 - pr23 ( ggaagaagatgccagatcagctgg ; seq id no : 28 ) and dpp8 - pr19r ( tccgtgtatcctgta tcatagaag ; seq id no : 29 ) to span across the junction between the race product and the est and library clones . two gel purified products atcd3 - 2 - 1 ( 1603 bp ) and atc3 - 3 - 10 ( 1077 bp ) were cloned into pgem - t easy ® ( promega co ., madison , wis ., usa ) and sequenced . the atc race product , the atcd3 - 2 - 1 ( 1603 bp ) 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 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 . 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 ° 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 ° 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 ° c . a northern blot containing 10 ug of total liver rna per lane was made using standard methods [ 31 ]. 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 ° c . a 2 . 4 kb human dpp8 cdna ( pcr product ) was radiolabeled using the 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 with the blot overnight at 60 ° c . in express hybridization solution . washes were performed at low stringency ( 2 × ssc / 0 . 05 % sds for 1 h at 37 ° c . followed by 0 . 1 × ssc / 0 . 1 % sds for 30 min at 40 ° c .) and blots exposed to biomax ms film for three days with a biomax ms screen at − 70 ° c . mouse liver rna was reverse transcribed using the superscript ii enzyme kit ( gibco brl , gaithersburg , md .) as described previously [ 42 ]. the cdna was diluted 1 in 4 and stored in aliquots at − 70 ° c . pcr using mousedpp8 - pr1f ( atg att acc acc cag gaa gcg ) as the forward primer and mousedpp8 - pr2r ( atc tcc gac atc 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 × advantage 2 polymerase mix ( clontech ), 1 × advantage 2 pcr buffer ( clontech ) and 100 ng of each primer . the pcr involved an initial step of 95 ° c . for 1 min to inactivate the taqstart antibody . this was followed by 35 cycles ; denaturation at 95 ° c . for 30 sec , 68 ° c . for 1 min , followed by a final step of 68 ° c . for 1 min . the amplified products were analysed by electrophoresis of 10 μl of pcr reaction on a 3 : 1 nusieve gel ( fmc bioproducts , rockville , md .) plus 0 . 5 μg / ml ethidium bromide in tae buffer ( 0 . 04m tris acetate , 0 . 001m edta , ph 8 . 0 ). the gel was then southern blotted using standard techniques [ 31 ]. the southern blot was hybridized at 60 ° c . for 2 hr with the 2 . 4 kb human dpp8 cdna probe prepared as described above . washes were performed at low stringency ( 2 × ssc / 0 . 05 % sds for 1 h at 37 ° c . followed by 0 . 1 × ssc / 0 . 1 % sds for 40 min at 50 ° c .). the blot was exposed to xar5 kodak film for 30 min at rt . reverse transcriptase pcr was performed on human atc rna , human placental rna and human liver rna using ted primers dpp8 / pr3 ( gcactaccttcaagaaaaccttgg ; seq id no : 30 ) and dpp8 / pr20r ( tatggtattgctgggtctctcagg ; seq id no : 31 ) to give a 293 bp product . monkey kidney fibroblast ( cos - 7 ) cells ( american type culture collection , crl - 1651 ) were grown and transfected as described previously [ 39 ]. 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 [ 40 , 9 ]. the presence of dpp8 fused with the v5 epitope was detected using an anti - v5 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 [ 35 ], then double - stained 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 , respectively ( molecular probes , eugene , oreg ., usa ). flow cytometry and confocal scanning microscopy using a leica tcs - nt confocal microscope have been described previously [ 39 , 9 ]. cells ( 1 × 10 7 ) expressing each protein were sonicated in native buffer ( 50 mm sodium phosphate , 300 mm nacl ), 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 &# 39 ; s instructions for a batch / gravity flow procedure . the resin was washed with 50 mm sodium phosphate , containing 300 mm nacl 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 were performed as described previously [ 1 ]. either clarified cell extract from 1 × 10 4 sonicated cos - 7 cells or purified protein derived from 1 × 10 5 cells was incubated with substrate in 70 μl phosphate buffer , ph 7 . 4 , for 30 min at 37 ° c ., except where otherwise indicated . the specific dppiv substrates , gly - pro - toluenesulfonate , h - gly - pro - p - nitroanilide ( na )/ hcl ( sigma , st louis , mo ., usa ) and gly - pro - 7 - amino - 4 - trifluromethylcoumarin ( calbiochem , san diego , calif ., usa ) were tested . other substrates tested were h - ala - pro - pna / hcl , h - arg - pro - pna acetate salt , h - lys - ala - pna . 2hcl , h - asp - pro - pna , h - ala - ala - pna / hcl , h - ala - ala - pro - pna / hcl , h - ala - ala - phe - pna , succinyl - ala - pro - pna , h - ala - phe - pro - pna and z - ala - pro - p - na from bachem ( switzerland ). h - ala - pro - 4 - methoxyβna / hcl , z - lys - pro - 4 - methoxyβnaformate salt , h - lys - pro - 4 - methoxyβna / hcl , z - ala - pro - 4 - methoxyβna , h - gly - pro - βna and h - his - ser - 4 - methoxyβnaacetate 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 1 mm 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 ° 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 t8 clone . the probes were nick - translated with biotin - c 14 - datp and hybridized in situ at a final concentration of 10 ng / 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 . rna ( 1 μg ) was reverse - transcribed using the superscript ii enzyme kit ( gibco - brl ) as described previously [ 42 ]. pcr using dpp8 - pr18 ( ctgtgacgccactaattatctatg ; seq id no : 18 ) as the forward primer and dpp8 - pr26r ( cctagagaggctagggtattcaag ; seq id no : 19 ) as the reverse primer was used to detect full - length dpp8 mrna . the glyceraldehyde - 3 - phosphate dehydrogenase ( g3pdh ) control primer set was g3pdh for ( accacagtccatgccatcac ; seq id no : 20 ) and g3pdhrev ( tccaccaccctgttgctgta ; seq id no : 21 ) to give a 470 - bp product . cdna ( diluted 1 : 4 ; 1 μg ) was amplified in a 25 - μl pcr mixture which contained : 0 . 2 mm dntps , 0 . 125 unit amplitaq gold enzyme ( perkin - elmer ), 1 × buffer ii ( perkin - elmer ), 1 . 5 mm mgcl 2 and 100 ng ml − 1 each primer . the 35 - cycle pcr was performed as follows : denaturation at 94 ° c . for 1 min , primer annealing at 55 ° c . for 30 s , and an extension step at 72 ° c . for 1 min . the amplified products were analyzed by electrophoresis of 15 μl pcr mixture on a 3 : 1 nusieve gel ( fmc bioproducts , rockville , md ., usa ) plus 0 . 5 μg ml − 1 ethidium bromide in tris / acetate / edta buffer ( 0 . 04 m tris / acetate , 0 . 001 m edta , ph 8 . 0 ). methods followed are described in current protocols in immunology [ 35 ]. 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 . peptide name : tedda - n sequence : ctgyterymghpdqneqg - ( seq id no : 22 ) nh2 . this is amino acids 773 to 789 , plus a cys at the n - terminus . peptide name : teddr - c sequence : gkpydlqiypqerhsc - nh2 . ( seq id no : 23 ) this is amino acids 836 to 850 , plus a cys at the c - terminus . these sequences were taken from the c - terminal portion of dpp8 . standard methods were used for antibody production [ 35 ]. mice were immunized with 2 × 10 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 . 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 ( fig1 ). 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 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 ( fig1 ). 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 ( fig1 ). the first methionine within a kozak sequence was found 214 bp from the 5 ′ end of the activated t cell race product . this 5 ′ 211 bp region was 70 . 5 % gc rich and contained a number of potential promoter and enhancer elements ( sp1 , ap1 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 ( fig1 ). compared to t8 and t21 , both clones had an additional insert region of 144 bp ( 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 atcd3 - 3 - 10 was also found to represent another splice variant of dpp8 as it contained a 516 bp deletion at the 5 ′ 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 n - linked 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 ( fig2 ). 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 ( fig3 ). 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 different human tissues ( fig4 ). 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 β - actin probe and a common 2 . 0 kb transcript was seen in all tissues ( fig4 ). 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 ( fig9 a ). 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 537 bp pcr product . a southern blot of this product confirmed that the murine dpp8 cross - hybridizes with human dpp8 ( fig9 b ). 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 , pcdna3 . 1 - dpp8 / v5 / his expressed a protein with the v5 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 dpp8 / v5 / his expressing cells ( fig6 ). dpp8 / v5 / his protein was detected in the cytoplasmic compartment but not on the surface of ethanol fixed stable dpp8 / v5 / his expressing cos cells , using the anti - v5 mab . sequence homology between dppiv and dpp8 suggested functional similarities , so cell lysates of dpp8 - transfected 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 ( fig5 a ), 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 - 4mβna / hcl 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 possibly causes increased dipeptidase , tripeptidase and endopeptidase activities , similar to an effect of dppiv transfection of melanoma cells [ 18 ]. 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 [ 46 ]. the peptides ala - pro - gly and lys - pro mimic dpp8 substrates and probably competitively inhibited 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 ( fig5 ). 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 15 metaphases from the second normal male ( data not shown ). 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 ( fig1 ). at 35 cycles , amplification product of the expected size ( 783 bp ) was readily observed in okt3 - stimulated pbmcs ( six of six subjects ; fig8 ) but not in unstimulated pbmcs from most subjects ( four of five , fig8 a ), 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 ( fig8 b ). the sera of two rabbits were tested by elisa in peptide - coated 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 100 kda band , which is the size of dpp8 . ( data not shown ). table 2 inhibition of the peptidase activity of dpp8 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 . aebsf , 4 -( 2 - aminoethyl ) benzenesulfonylfluoride . residual activity (% of control ) type of inhibitor concentration dpp8 dppiv none 100 100 serine proteinase aebsf 4 mm 40 52 aprotinin 4 μg ml − 1 47 81 benzamidine / hcl 10 mm 82 89 peptides gly - gly - gly 10 mm 99 106 ala - pro - gly 10 mm 51 67 h - lys - pro - oh hcl salt 4 mm 63 45 zinc sulphate 2 mm 25 0 metalloproteinase edta 2 mm 115 99 aspartate ( acidic ) proteinase pepstatin 2 μg ml − 1 107 110 leupeptin 0 . 1 mm 93 104 cysteine ( thiol ) proteinase iodoacetamide 2 mm 100 115 dithiothreitol 2 mm 108 109 we describe the cloning , recombinant expression , biochemistry and tissue expression of a novel human dppiv - related 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 ( fig2 ). 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 ]. dppiv - specific 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 activity in lymphocyte activation [ 54 ]. in vivo , inhibitors of dppiv suppress arthritis [ 20 ] and prolong cardiac allograft survival in animal models [ 55 ]. 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 [ 9 , 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 α - amine lies n - terminal to the proline ( e . g . 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 β - propeller domain and an α / β - hydrolase domain [ 3 , 39 , 1 ]. 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 ]. 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 [ 65 ]. 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 [ 3 ]. cd26 - negative cell lines have residual dppiv enzyme activity and pbmc have non - dppiv derived activity against ala - pro substrates [ 69 ], indicating the existence of other peptidase ( s ) with dppiv - like activity . dppiv - β 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 ( fig1 ). 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 northern blots that are much larger than the cdna size [ 60 , 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 5 ′ 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 dpp8 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 . 1 . abbott c a , g w mccaughan & amp ; m d gorrell 1999 two highly conserved glutamic acid residues in the predicted beta propeller domain of dipeptidyl peptidase iv are required for its enzyme activity febs letters 458 : 278 – 84 . 2 . abbott c a , d m t yu , g w mccaughan & amp ; m d gorrell 2000 post proline peptidases having dp iv like enzyme activity advances in experimental medicine and biology 477 : 103 – 9 . 3 . mccaughan g w , m d gorrell , g a bishop , c a abbott , n a shackel , p h mcguinness , m t levy , a f sharland , d g bowen , d yu , l slaitini , w b church & amp ; j napoli 2000 molecular pathogenesis of liver disease : an approach to hepatic inflammation , cirrhosis and liver transplant tolerance immunological reviews 174 : 172 – 91 . 4 . scanlan m j , b k raj , b calvo , p garin - 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