Patent Application: US-90416807-A

Abstract:
peptides including hisglytrpsertyrglyglypheleu ; 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 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 .

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 . primers gdd pr1f 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 jm 109 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 . an analysis of the nucleotide sequence of fragments dpp4 - like 2a , 2b and 2c with the sequencher ™ version 3 . 0 computer program , and the 5 ′ fragment isolated by primers dpp9 - 22f and gdd3 ′ end , revealed the nucleotide sequence shown in fig3 . the predicted amino acid sequence shown in fig3 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 fig3 to the nucleotide sequence of the predicted open reading frame of gdd ( fig4 ). 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 fig3 . however , as noted in fig4 , there are regions of amino acid sequence encoded by the sequence of fig3 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 fig3 are different nucleotide sequences . as described further herein , the predicted amino acid sequence encoded by the cdna sequence shown in fig3 is homologous to the amino acid sequence of dpp8 . accordingly , and as a cdna consisting of the nucleotide sequence shown in fig3 was not known , the sequence shown in fig3 was named cdna dpp9 . the predicted amino acid sequence encoded by cdna dpp9 ( called dpp9 ) is 969 amino acids and is shown in fig3 . the alignment of dpp9 and dpp8 amino acid sequences suggests that the nucleotide sequence shown in fig3 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 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 glu 259 lys 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 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 hybridized 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 ( ggctgagaggatggccaccaccggg ), ( seq id no : 37 ), as the forward primer and gdd3 ′ end ( gggcgggacaaagtgcctccactgg ), ( seq id no : 35 ), as the reverse primer . the hybridization was carried out according to the manufacturers &# 39 ; instructions at 60 ° c . to detect cross - species hybridization . after overnight hybridization the blot was washed at room temperature ( 2 × ssc , 0 . 1 % sds ) then at 40 ° c . ( 0 . 1 . times . 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 fig6 . 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 . b57b16 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 . mu . g of liver rna was reverse - transcribed using superscript ii rnase h - reverse transcriptase ( gibco brl ). 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 72 ° 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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