Patent Application: US-41512203-A

Abstract:
peptides which comprise sequences as shown in seq id no : 2 or hisglytrpsertypglyglypheleu ; leuaspgluasnvalhisphephe ; gluarghisserilearg 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 , h - gly - pro - pna ans h - arg - pro - pna , peptides having sequence id no : 7 are also claimed . nucleic acids , vectors , antibodies and hybridoma cells are also claimed with reference to the above sequences and there abilities .

Description:
restriction enzymes and other enzymes used in cloning were obtained from boehringer mannheim roche . standard molecular biology techniques were used unless indicated otherwise . the nucleotide sequence of dpp8 shown in fig1 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 gdd 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 pr 15f and gdd pr 7r ( table 1 ) were then used to isolate a cdna clone encoding a dpp4 - like 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 ′). primer 22f was used in conjunction with primer gdd3 ′ end ( 5 ′ gggcgggacaaagtgc ctcactgg3 ′) on cdna made from the human cem cell line to produce a 3000 bp product as expected fig1 . an analysis of the nucleotide sequence of fragments dpp4 - like 2a , 2b and 2c with the sequencher ™ version 3 . 0 computer program ( fig3 ), and the 5 ′ fragment isolated by primers dpp9 - 22f and gdd3 ′ end , revealed the nucleotide sequence shown in fig4 . the predicted amino acid sequence shown in fig4 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 fig4 to the nucleotide sequence of the predicted open reading frame of gdd ( fig5 ). 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 fig4 . however , as noted in fig5 there are regions of amino acid sequence encoded by the sequence of fig4 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 fig4 are different nucleotide sequences . as described further herein , the predicted amino acid sequence encoded by the cdna sequence shown in fig4 is homologous to the amino acid sequence of dpp8 ( fig6 ). accordingly , and as a cdna consisting of the nucleotide sequence shown in fig4 was not known , the sequence shown in fig4 was named cdna dpp9 . the predicted amino acid sequence encoded by cdna dpp9 ( called dpp9 ) is 969 amino acids and is shown in fig4 . the alignment of dpp9 and dpp8 amino acid sequences suggests that the nucleotide sequence shown in fig4 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 , 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 e 259 , e 260 . these are equivalent to the residues glu 205 and glu 206 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 : glu 354 , glu 355 , ser 136 , asp 914 and his 946 . 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 . dpp4 - like - 2a was used to probe a human master rna blot ™ ( 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 blots ™ ( 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 fig8 . 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 mrna 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 . 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 [ 32 p ] dctp ( amersham international plc , amersham , uk ). the dpp9 pcr product used to make the probe was generated using met3f ( ggctgagag gat ggccaccac cggg ) as the forward primer and gdd 3 ′ end ( gggcgggacaaagtgc ctcactgg ) as the reverse primer . the hybridisation was carried out according to the manufacturers &# 39 ; 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 . 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 sequencher ™ version 3 . 0 computer program revealed the nucleotide sequence shown in fig9 . 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 - prlf ( 5 ′ acctgggaggaagcaccccactgtg3 ′) and mgdd - pr4r ( 5 ′ ttccacctggtcctcaatctcc3 ′) were designed from this sequence and used to amplify a 452 bp product as expected from liver mouse cdna , as described below . 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 . 2 μg of liver rna was reverse - transcribed using superscript ii rnase h - reverse transcriptase ( gibco brl ). pcr using mdpp9 - 1f ( acctgggaggaagcaccccactgtg ) as the forward primer and mdpp9 - 2r ( ctctccacatgcagggctacagac ) as the reverse primer was used to synthesise a 550 base 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 . 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 [ 32 p ] 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 ). 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pyrrolidides and thiazolidides : new inhibitors of proline specific peptidases biochimica et biophysica acta — protein structure & amp ; molecular enzymology 1479 : 15 - 31 . 34 . schön , i born , h u demuth , j faust , k neubert , t steinmetzer , a barth & amp ; s ansorge 1991 dipeptidyl peptidase iv in the immune system . effects of specific enzyme inhibitors on activity of dipeptidyl peptidase iv and proliferation of human lymphocytes biological chemistry hoppe seyler 372 : 305 - 11 . 35 . coligan j e , a m kruisbeek , d h margulies , e m shevach & amp ; w strober , eds . current protocols in immunology . 1998 , john wiley & amp ; sons : usa . 36 . fibroblast activation protein . rettig w j . 1998 , in handbook of proteolytic enzymes , a j barrett , n d rawlings & amp ; j f woessner , editor . academic press : san diego . p . 387 - 9 . ser val ser his ala cys ser trp asn gly gly ser leu asp pro leu val lys lys leu arg leu asp lys glu asn thr gly ser trp arg ser arg phe gln val gln lys his ser trp asp gly leu arg ser ile ile his gly ser arg lys tyr ser gly leu ile val asn lys ala pro his asp phe gln phe val gln lys thr asp glu ser gly pro his ser his leu ser trp lys gln met leu asp his phe gln ala thr pro his his gly val phe gly ile thr ser tyr asp phe his ser glu ser gly leu phe leu phe gln ala ser asn ser leu phe his cys arg asp gly gly lys asn gly phe met val ser pro met lys pro leu glu ile lys thr gln cys ser gly pro arg met asp pro lys ile cys pro ala asp pro asn val leu asp asp pro lys ser ala gly val ala thr phe val ile gln glu glu phe asp arg phe thr gly tyr trp trp cys pro thr ala ser trp glu gly ser gln gly leu lys thr leu arg ile leu tyr glu lys asn pro lys ile ala leu lys leu ala glu phe gln thr asp ser gln gly lys ile val ser thr gln glu lys glu leu val gln pro phe ser ser leu phe pro lys val glu tyr ile ala arg ala gly trp thr arg asp gly lys tyr ala trp ala met phe leu asp arg pro gln gln val his asp ile phe tyr pro phe pro gln ser glu gly glu asp glu leu cys phe leu arg ala asn glu cys lys thr gly phe cys his leu tyr lys val thr ala val leu lys ser gln gly tyr asp trp ser glu ile ala leu thr ser gly glu trp glu val leu ala arg his gly ser lys ile trp val asn glu glu thr lys leu val tyr phe gln gly thr lys asp thr pro leu glu his his leu tyr val val ser tyr glu ala ala gly glu ile val arg leu thr thr pro gly phe ser his ser cys ser thr pro pro cys val his val tyr lys leu ser gly pro asp asp asp pro leu his lys gln pro arg phe trp ala ser met met glu ala ala ser cys pro pro asp tyr val pro pro glu ile phe his phe his thr arg ser asp val arg leu tyr gly met ile tyr lys pro his ala pro gln val gln leu val asn asn ser phe lys gly ile lys tyr leu arg leu asn thr leu ala ser leu gly tyr ala val val val ile asp phe val ala glu lys tyr gly phe ile asp leu ser arg val ala ile trp met ala tyr asp thr gly tyr thr glu arg tyr met asp val pro leu ile arg ala gly lys pro tyr gln leu gln ile tyr pro asn glu arg his ser ile arg cys pro glu ser gly glu his tyr glu val thr lys ser ser gly leu ile val ser lys ala pro his asp phe gln phe val gln lys pro asp glu ser gly pro his ser his arg leu tyr tyr gln met leu asp his phe gln ala thr pro his his gly val tyr ser ile thr ser tyr asp phe his ser glu ser gly leu phe leu phe gln ala ser asn ser leu phe his cys arg asp gly gly lys asn gly phe met val ser pro met lys pro leu glu ile lys thr gln cys ser gly phe ile asn asn ser asp leu trp val ala asn ile glu thr gly glu glu arg arg leu thr phe cys his gln gly ser ala gly val leu asp asn pro lys ser ala gly val ala thr phe val ile gln glu glu phe asp arg phe thr gly cys trp trp cys pro thr ala ser trp glu gly ser glu gly leu lys thr leu arg ile leu tyr glu glu val asp glu pro lys val glu tyr ile ala arg ala gly trp thr arg asp gly lys arg ala asn glu cys lys thr gly phe cys his leu tyr arg val thr thr glu gly glu phe lys cys pro ile lys glu glu val ala leu thr asn glu gln thr lys leu val tyr phe gln gly thr lys asp thr pro val arg leu thr thr leu gly phe ser his ser cys ser met ser gln lys gln pro arg phe trp ala ser met met glu ala ala asn cys pro pro asp tyr val pro pro glu ile phe his phe his thr arg ala asp val gln leu tyr gly met ile tyr lys pro his thr leu gln pro gly arg lys his pro thr val leu phe val tyr gly gly pro gln val gln leu val asn asn ser phe lys gly ile lys tyr leu arg leu asn thr cys gln arg gly leu his phe glu gly ala leu lys asn gln met gly lys tyr gly phe ile asp leu ser arg val ala ile his gly trp ser tyr gly gly phe leu ser leu met gly leu ile his lys pro gln val phe lys val ala ile ala gly ala pro val thr val trp met ala tyr asp thr gly tyr thr glu arg tyr met asp val pro glu asn asn gln gln gly tyr glu ala gly ser val ala leu his val glu lys leu pro val his phe phe his thr asn phe leu val ser gln leu ile arg ala gly lys pro tyr gln leu gln ile tyr pro asn glu arg his ser ile arg cys arg glu ser gly glu his tyr glu val thr leu leu his phe thr ala asp cys glu glu asn ile glu ser gln asp arg pro lys leu glu pro phe tyr val glu arg tyr ser trp ser gln leu lys lys leu leu ala asp thr arg lys tyr his gly tyr met met ala lys ala pro his asp phe met phe val lys arg asn asp pro asp gly pro his ser asp arg ile tyr tyr leu ala met ser gly glu asn arg glu asn thr leu phe tyr ser glu ile pro lys thr ile asn arg ala ala val leu met leu ser trp lys pro leu leu asp leu phe gln ala thr leu asp thr phe leu phe gln ala gly ser gly ile tyr his val lys asp gly thr ser cys pro asn ile arg met asp pro lys leu cys pro ala asp ala asn met glu glu asp ala arg ser ala gly val ala thr phe val leu gln glu glu phe asp arg tyr ser gly tyr trp trp cys pro lys ala glu thr thr pro ser gly gly lys ile leu arg ile leu tyr glu leu glu thr arg arg ala asp ser phe arg tyr pro lys thr gly thr ala asn pro lys val thr phe lys met ser glu ile met ile asp ala glu gly arg ile ile asp val ile asp lys glu leu ile gln pro phe glu ile leu phe glu gly val glu tyr ile ala arg ala gly trp thr pro glu gly lys tyr ala trp ser ile leu leu asp arg ser gln thr glu phe ile phe ala ser glu cys lys thr gly phe arg his leu tyr gly leu pro ala pro ser asp phe lys cys pro ile lys glu glu ile ala ile thr ser gly glu trp glu val leu gly arg his gly ser asn ile gln val asp glu val arg arg leu val tyr phe glu gly thr lys gly glu val thr arg leu thr asp arg gly tyr ser his ser cys cys asn pro his cys val ser leu tyr lys leu ser ser pro glu asp asp pro thr cys lys thr lys glu phe trp ala thr ile leu asp ser ala arg gly ser cys his arg gly leu lys phe glu gly ala phe lys tyr leu ala ser arg tyr asp phe ile asp leu asp arg val gly ile his ser asp ile phe arg val ala ile ala gly ala pro val thr leu trp ile phe tyr asp thr gly tyr thr glu arg tyr met gly his pro asp asp glu asn val his phe ala his thr ser ile leu leu ser phe leu val arg ala gly lys pro tyr asp leu gln ile tyr pro gln glu arg leu his tyr leu gln glu asn leu gly ser arg ile ala ala leu lys asn lys ala pro his asp phe gln phe val gln lys thr asp glu ser glu ala leu leu leu leu ser trp lys gln met leu asp his phe gln ala thr pro his his gly val tyr ser arg glu glu glu leu leu arg glu arg lys arg leu gly val phe gly ile thr ser tyr asp phe his cys arg asp gly gly lys asn gly phe met val ser pro met lys pro leu glu ile lys thr gln cys ser gly pro arg met asp pro lys ile trp val ala asn ile glu thr gly glu glu arg arg leu thr phe cys his gln gly leu ser asn val leu asp asp pro lys ser ala gly val ala thr phe val ile gln glu glu phe asp arg phe thr gly tyr trp trp cys pro thr ala ser trp glu gly ser gln gly leu lys thr leu val pro ser pro ala leu glu glu arg lys thr asp ser tyr arg tyr pro arg thr gly ser lys asn pro lys ile ala leu lys leu ala glu leu val gln pro phe ser ser leu phe pro lys val glu tyr ile ala arg ala gly trp thr arg asp gly lys tyr ala trp ala met phe leu phe ile pro ser thr glu asn glu glu gln arg leu ala ser ala arg asn val trp ile asn val his asp ile phe tyr pro phe pro gln ser glu gly glu asp glu leu cys phe leu arg ala 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