Patent Application: US-21455599-A

Abstract:
the present invention relates to the cloning of human pro - protein converting enzyme 5 cdna isolated from human adrenal gland messenger rna . additionally , this invention relates to a method for reducing restenosis occurring at an injured vascular site comprising delivering to the injured site an antisense nucleic acid to suppress the expression of human pc5 .

Description:
cdna library construction and screening : a cdna library derived from total human adrenal rna was constructed by stratagene ( la jolla , calif .) in the phage vector uni - zap xr . six hundred thousand phage plaques were screened initially using radioactive probes and standard methodologies . 14 the initial hybridization probe was a 320 base pair dna fragment derived from reverse - transcriptase pcr of human brain rna using information derived from an unidentified human cdna sequence tag in genbank ( accession # m85522 ) with a high degree of similarity to the previously cloned mouse pcs . 15 fragment labeling was carried out using 32 p dctp and a random primer labeling kit ( boehringer - mannheim canada , laval , quebec , canada ) according to manufacturer &# 39 ; s instructions . one positive hybridizing phage ( hpc5a ) was identified . its insert was sequenced in its entirety using the dideoxy - chain termination method and found to code for an 1150 base pair cdna with a high degree of sequence similarity to mouse pcs ( data not shown ). a 1070 base pair fragment ( excluding the poly a tail ) was excised from hpc5a , labeled and used to re - screen an additional 600 , 000 phage from the cdna library . a second phage clone ( hpc5b ) was isolated and found to contain an 1807 base pair cdna insert overlapping hpc5a and extending toward the 5 ′ end of the cdna ( fig1 ). reverse - transcriptase pcr : one microgram of poly a + rna from total human adrenal ( clontech laboratories , palo alto , calif .) was subjected to reverse transcriptase polymerase chain reaction ( rt - pcr ) using a published procedure 16 and the following oligonucleotides : forward oligonucleotide ; derived from a region corresponding to the signal peptide of mouse pc5 . 15 an artificial hindiii restriction enzyme cleavage site added to the 5 ′- end of the amplified fragment for the purpose of cloning is underlined : reverse oligonucleotide ; derived from the 5 ′- end of the phage hpc5b . an internal bgiii restriction enzyme site is underlined : the entire rt - pcr reaction was repeated 4 times and 4 independently derived clones of the amplified fragment were sequenced and the sequences were compared . the sequence submitted to genbank ( accession # u49114 ) represents the consensus sequence , defined as any nucleotide appearing in ¾ clones . northern blot analysis : tissue distribution of pc5 mrna was determined by hybridizing commercially purchased nitrocellulose filters containing aliquots ( 2 μg ) of poly - a rna from various human tissues ( clontech laboratories , palo alto , calif .). the probe used was a complementary rna derived from the full length hpc5 cdna . probe labeling and hybridization were carried out as previously described . 17 expression vector construction : a cdna fragment from the kpni site ( fig1 ) to just past the stop codon was excised from the phage hpc5b and combined with a kpni to hindiii ( see above ) fragment derived from portions of two independent rt - pcr clones ( so as to eliminate errors arising from the taq polymerase ). a region corresponding to the first 16 amino acids of the signal peptide derived from mpc5 was attached to the 5 ′- end by overlap - extension pcr . 18 thus , the entire cdna , encoding amino acids 1 - 16 derived from the mpc5 signal peptide and the remainder from hpc5 , was subcloned into the expression vector rsv - globin 19 which places the cdna under the control of the rsv promoter and provides a 3 ′ intron and polyadenylation signal from the rabbit beta globin gene . the entire subcloned fragment was subsequently verified by dna sequencing . cell culture and transfection : gh 4 c1 cells were plated in 6 - well culture dishes at a density of 5 × 10 5 cells per well . twenty four hours later , medium was changed and the cells were transfected by the deae - dextran method using a commercial kit ( cellphect transfection kit , pharmacia biotech , baie d &# 39 ; urfe , quebec , canada ) according to manufacturer &# 39 ; s instructions . each well received 0 . 18 μg of either the hpc5 expression vector or a neutral plasmid vector ( puc18 ) and 0 . 18 μg of an expression vector for human prorenin ( prhr1100 ) or its equivalents in which amino acids 42 or 43 of the prorenin prosegment were mutated to alanine ( k / a - 2 and r / a - 1 , respectively 20 ). supernatants were collected 30 hrs . after transfection and assayed for prorenin and renin content as previously described . 20 to verify that conversion of the prorenin occurred in the secretory granules , gh 4 c1 transfected with the human prorenin and hpc5 expression vectors were stimulated to release secretory granules by depolarization using a previously published technique . 21 forty hrs . after co - transfection , the culture medium in parallel wells of transfected cells was replaced with pre - warmed medium supplemented to a final concentration of 50 mmol / l with either nacl ( control ) or kcl ( secretagogue ). the media were collected after 20 min . and assayed for renin / prorenin . a potassium - dependent increase in the percent active renin contained in cell supernatants was taken as an indication of active renin release from the secretory granules of the transfected cells . results shown in fig5 represent the mean of three independent transfection experiments . immunolocalization of hpc5 in human tissues : human tissue was obtained post - mortem ( kidney and adrenal gland ) or post - partum ( placental cotyledon ), fixed in bouin &# 39 ; s solution and embedded in paraffin . for immunolocalization , 5 μm sections were mounted on gelatin - coated slides , deparaffinized and incubated with a 1 : 50 dilution of a polyclonal rabbit antiserum raised against a peptide corresponding to the n - terminal 16 amino acids of rat pc5 ( pc5 . map antibody ) or a 1 : 200 dilution of a polyclonal rabbit antiserum against recombinant human prorenin . for kidney and placental specimens , immune complexes were revealed by incubation with protein a - colloidal gold ( 15 nm particles ) synthesized from tetra - chloroauric acid ( bdh ) according to the method of ghitescu and bendayan . 22 gold particles were enhanced for viewing in the light microscope by incubation with silver ( intense ™ m silver enhancement kit , amersham life science , oakville , ontario , canada ) and sections were counter - stained with hematoxylin and methyl green . immune complexes on human adrenal sections were detected with a 1 : 200 dilution of biotin - labeled donkey anti - rabbit igg and a 1 : 300 dilution of streptavidin - horseradish peroxidase complex ( amersham life science , oakville , ontario , canada ) and were incubated with diaminobenzidine and hydrogen peroxide ( sigma chemicals , st . louis , mo .) as chromogen . all positive staining patterns were subsequently verified for specificity by omission of the first antibody . the primary sequence of human pc5 is shown in fig2 . we were unable to clone the extreme 5 ′- end of the cdna either by the race protocol 16 or by using oligonucleotides based on the published sequence of mouse pc5 15 , 23 , possibly due to a high g / c content of the cdna in this region . however , based on the published cdna sequences for rat and mouse pc5 15 , we are confident that we have isolated all but the 5 ′- most portion of the cdna corresponding to the first 12 amino acids of the signal peptide . by comparison with the published sequence of mouse pc5 , we predict that the cdna isolated would code for a prepropc5 of 915 amino acids , including a signal peptide and a prosegment of 32 and 84 amino acids , respectively . the deduced sequence of hpc5 is 88 % identical to the previously published mouse pc5 cdna and 96 % identical to the mouse pc5 protein . northern analysis of poly a rna from a variety of human tissues reveals a major band of approximately 6 . 6 kb and a minor band at approximately 3 . 8 kb ( fig3 ). pc5 rna is detected in the brain , heart , placenta , lung , thyroid gland and testes and at lower levels in the skeletal muscle , kidney and pancreas , small intestine and stomach . in the adrenal gland , pc5 is particularly enriched in the cortex ( fig3 ). because pc5 rna appears to be expressed in a number of tissues previously reported to contain active renin , we have tested the ability of hpc5 to cleave human prorenin in a cell co - transfection assay ( fig4 a ). as has been previously reported 10 , when cultured rat sommatotrophic gh 4 c1 cells are co - transfected with an expression vector encoding human prorenin and a neutral plasmid vector , only unprocessed prorenin is secreted into the culture supernatant . in contrast , if the human prorenin expression vector is co - transfected with an expression vector encoding human pc5 , a portion of the expressed prorenin is secreted as active renin . co - expression of human pc5 with prorenin mutated at either of the basic residues forming the native cleavage site ( lysine 42 or arginine 43 ) prevents activation . these results suggest that human pc5 activates human prorenin by proteolytic cleavage at the site previously reported for activation of renin in humans . 5 while human pc5 cleaves human prorenin in gh 4 c1 cells , there is no apparent increase in active renin secretion when co - transfections are carried out in chinese hamster ovary ( cho ) cells ( fig4 b ). one obvious difference in the cho cell line as compared to gh 4 c1 cells is their lack of secretory granules , suggesting that either human pc5 or human prorenin or both require the secretory granule environment for this proteolytic step . this conclusion is supported by the acute increase in active renin detected in the supernatants of co - transfected gh 4 c1 cells treated for 20 min . with potassium chloride ( fig5 ), a de - polarizing agent which causes the release of secretory granules . 21 using a polyclonal antibody raised against a peptide derived from mouse pc5 , we have studied the distribution of human pc5 in several human tissues ( fig6 ). to date , we have been unable to detect staining for pc5 in the human kidney , although our sections stain positively for renin . in the placental cotyledon , pc5 is located in the syncitiotrophoblast layer of the chorionic villi while anti - renin antibody stains primarily the chorionic mesoderm . in the adrenal gland , the antibodies against both renin and pc5 show a preferential staining of zona glomerulosa cells in the adrenal cortex ( g ) with very little staining of the capsule ( c ) and zona fasciculata ( f ). no staining was evident with omission of the first antibody ( data not shown ). thus , our immunohistochemical studies would suggest that , of the three tissues studied , it is likely that prorenin and pc5 are only clearly co - localized in the zona glomerulosa of the human adrenal cortex . in the present study , we describe the cloning and expression of the human prohormone convertase pc5 and its activity as a human ppe . co - transfection assays in cultured cells demonstrates that hpc5 activates human prorenin with the expected site - specificity and that this cleavage most likely takes place in dense core secretory granules . in addition , immunohistochemistry of human tissues shows co - localization of hpc5 with renin in the zona glomerulosa of the adrenal cortex . several lines of evidence suggest that the human adrenal gland contains a physiologically important local ras : first , rna encoding angiotensinogen and renin have been detected in preparations from the human adrenal zona glomerulosa , fasciculata and medulla 24 , 25 , confirming that both renin and its substrate are synthesized within the human adrenal gland . second , ace inhibition or blockade of angiotensin receptors inhibits aldosterone release from human adrenal tissue explants 26 , suggesting that the local ras plays an active role in the regulation of aldosterone secretion from the adrenal gland . third , tissue explants of human adrenal cortex and aldosterone - secreting adenomas secrete small quantities of active renin 24 , 26 , 27 , suggesting that the adrenal cortex expresses a ppe capable of activating human prorenin . our current results suggest that pc5 could be the ppe responsible for activation of renin in the human adrenal cortex as both renin and hpc5 are immuno - detectable in the zona glomerulosa . additional circumstantial evidence supports this conclusion : first , centrifugal fractionation of adrenal cortical cells reveals that renin is contained in the “ granular ” fraction , of intermediate density between vesicles and lysosomes . 28 as our current study suggests that pc5 only cleaves human prorenin in cells containing secretory granules , renin would be in the appropriate intracellular compartment to be activated by pc5 in the adrenal cortex . second , rats transgenic for mouse ren - 2 renin [ tgr ( mren - 2 ) 27 ] display fulminant hypertension 29 which correlates best with the expression of the mouse prorenin in the adrenal gland . 30 - 32 as previous studies have demonstrated that pc5 is capable of activating mouse ren - 2 prorenin , but not rat prorenin ( 23 and data not shown ) it is possible that the tgr ( mren - 2 ) 27 transgenic rat is a model for activation of a tissue ras by the fortuitous juxtaposition of prorenin with an appropriate ppe in the adrenal cortex . these results also raise the possibility that the tissue - distribution of ppes and their apparent selectivity in activating prorenin from different species could lead to differing functions of the tissue ras between rodents and humans . the principal source of circulating active renin in humans is the jg cells of the kidney . although low levels of hpc5 rna can be detected by northern blot analysis in a sample of total kidney poly - a rna ( fig3 ), we were unable to localize pc5 immunostaining in kidney sections ( fig6 ) raising the possibility that pc5 is expressed at low levels in diffuse cell types in the kidney . thus , while these results do not formally rule out pc5 as a ppe in the kidney , our inability to detect it in jg cells makes it unlikely that it plays a major role in the production of renal renin . in contrast , relatively abundant amounts of pc5 mrna and protein were detected in the placenta although evidence suggests that placental cells in culture 33 and in vivo 34 only secrete prorenin . however , immunostaining revealed that the cells producing pc5 and prorenin in the human placenta are distinct . it is also unlikely that pc5 would activate prorenin once the two proteins are secreted due to the apparent requirement of a granular environment for the cleavage of prorenin by hpc5 in transfected cells . thus , in contrast to the case in the adrenal gland , it is unlikely that pc5 expressed in the human placenta would activate placental prorenin . in the mouse , two forms of hpc5 have been predicted based on cloned cdnas ; the first would be analogous to the hpc5 cdna described in this study and to that cloned from rat tissues 15 , 23 while the second , called pc6b , is extended at its 3 ′- end due to a differential rna splicing event . 35 although the hpc5 cdna we have cloned is only roughly 3 kb in length , the major rna band seen in human tissues is of approximately 6 . 6 kb . the identity of the longer band hybridizing to the hpc5 probe is currently unknown . it should be noted that neither of the cdna clones isolated from a screening of 1 . 2 million phage from the adrenal library was extended at its 3 ′- end ( fig1 ), although the probes used in their isolation cover the region of homology with the mouse pc6b variant . 35 in mouse tissues , expression of the pc6b variant is restricted to few tissues 35 while the abundance of the 6 . 6 kb detected with the hpc5 probe is directly proportional to the abundance of the 3 . 8 kb band . hybridization of rna blots from rodent tissues using a pc5 probe also reveals rna bands of 3 . 8 , 6 . 5 and 7 . 5 kb 15 , 35 and use of a pc5 - specific probe reveals a band at 6 . 5 kb . thus , it is possible that additional pc5 rna species exist in mammals that are extended at their 5 ′- ends . alternatively , human tissues may be particularly enriched in a homologue to pc6b which was not picked up in our screenings . recent data suggest that the alternate c - terminal tail present on pc6b may serve to retain the enzyme in the golgi network , while the “ short ” form of mouse pc5 is targeted to dense core secretory granules ( n . g . seidah , unpublished ). these data and the results of our co - transfection assays ( fig4 ) would suggest that the “ short ” form of hpc5 described here is the form which would be active in renin processing in secretory granules . the pc5 enzymes isolated from humans and mice show a remarkably high degree of conservation at the nucleotide and protein sequence levels . this degree of similarity is higher than that seen for the other mammalian pc enzymes which seem to diverge in the c - terminal half of the enzyme . 36 , 37 this high degree of sequence conservation may reflect an essential function of pc5 ( and the c - terminus of pc5 ) in mammals . to investigate which pc could be a potential target of smooth muscle cell proliferation , we tested if any of the pcs were affected in the process of restenosis , wherein such proliferation is observed . changes in pc levels in the process of restenosis is a distinct possibility since in previous studies using animal models or cell lines , we have shown that pc levels can be regulated or even be induced . we thus obtained human restenosed coronary tissues from patients . these tissues were screened for each of the pc mrnas using in vivo hybridization histochemistry in order to obtain information within an anatomical context . coronaries with partial or total occlusions demonstrated dramatically increased pc5 mrna levels within smooth muscle tissues , whereas coronary tissue without occlusions were pc5 negative . these results indicate that pc5 is either strongly up - regulated or induced in the human coronary arteries during the active process of stenosis ( fig7 ). to our knowledge this is the first indication that a specific pc is directly linked to smooth muscle proliferation . these results suggested that if pc5 enzymatic activity could somehow be inhibited or the upregulation of pc5 mrna could be prevented , this may attenuate or stop the process of restenosis . this could occur through the inhibition of the processing function of this enzyme on the numerous growth factors that are involved in the formation of the coronary lesion . if these growth factors are not processed they will remain biologically inactive . our approach was to test the effectiveness of pc5 antisense inhibition on smooth muscle proliferation in vitro . a specific antisense oligonucleotide ( odn ) was shown to drastically inhibit smooth muscle proliferation using an in vitro model of rabbit smooth muscle in culture . incubating rabbit smooth muscle cells with a pc5 antisense 17 - mer oligonucleotide shown in table 1 caused a dose - dependent inhibition smooth muscle proliferation with a maximal inhibitory effect of 81 . 6 %+ 1 . 6 % at 10 mm ( mean of three experiments done in quadruplicates ). this inhibitory effect is highly significant ( p = 0 . 0001 ) as compared to controls which included either a sense or a mismatched oligonucleotide used at the same concentration ( see table 1 ). in addition we found that the expression of pc5 is decreased in the affected cells ( fig8 ). when compared to other targets , such as c - myc , this approach was much more effective in inhibiting smooth muscle proliferation , as the best effects of antisense c - myc resulted in 71 . 7 + 3 . 5 % ( means of three experiment done in quadruplicate ) inhibition ( mean of three experiment done in quadruplicates ). these results are indicative of an in vivo effect since silencing pc5 would impede muscle cell proliferation and restenosis . phosphorothioate antisense odns were synthesized on a dna / rna synthesizer following standard procedure ( applied biosystems ). conjugation of oligomers with cholesterol was achieved with 3 ′- cholesterol - vn cpg ( clontech ), a virtual nucleotide ( vn ) glass reagent that introduces a cholesterol label to the 3 ′ terminus of an oligonucleotide via solid - phase synthesis . when odn synthesis . when odn synthesis was completed , oligomers were removed from the column with 30 % nh 4 oh ( 1 hour at room temperature ), and then deprotected for 8 hours at 60 ° c . oligos were purified and detritylated with oligonucleotide purification cartridges ( applied biosystems ), and then lyophilized with a centrifugal evaporator ( savant speedvac ). new zealand rabbits male or female ( 2 kg ) were intramuscularly sedated with xylazine ( 2 mg / kg ) and anesthetized with ketamine ( 100 mg / kg ) prior to surgical exposure of left carotid artery . segments of 10 mm of carotids were transiently isolated by temporary ligatures and rinsed with 0 . 9 % sodium chloride via a cannula until there was no more visible evidence of blood components . carotid arteries were transfected with 80 μmol / l of antisense odns in a 1 cm portion either alone or conjugated to cholesterol for a period of 30 minutes . the volume infused was 100 μl , and no visible loss of volume was noted throughout the incubation period . following transfection , the treated segments were rinsed with 0 . 9 % sodium chloride ( 3 × 100 μl ) and upon cannula removal , the arteriotomy site was repaired with microsutures , restoring normal blood flow and the neck wound closed . all experimental protocols in this project were approved by the institutional committee for animal protection of the louis - charles simard research center . a total of 36 new zealand white rabbit carotid arteries were injured with a 2 . 5 mm balloon catheter serially inflated for 1 minute to 4 , 6 , 8 and 10 atm with gentle traction , allowing 45 seconds between inflations . two weeks later , a second injury was imposed at the same arterial site which was then transfected in a 1 cm portion with 80 μmol / l ( 100 μl of volume injected ) of therapeutic molecules or with 100 μl of nacl 0 . 9 % as control . intimal / medial areas were evaluated by computer analysis on histological sections derived from transfected arteries two weeks following the second injury and transfection procedure . the addition of a pc5 antisense 17 - mer odn shown in table 1 at the time of a second carotid injury with a balloon catheter decreased carotid stenosis , measured as area ratio intima / media , by 40 % ( area ratio intima / media sense odn minus area ratio antisense odn divided by area ratio sense odn ; see fig9 ). this inhibitory effect is highly significant ( p 0 . 0118 and p = 0 . 0078 ) as compared to the sense and random controls , respectively . these results are the basis of a method of preventing stenosis comprising administering an effective stenosis inhibitory dose of a pc5 antisense to a subject in need for such a treatment . other antisense oligonucleotides may be added to optimize this method of preventing restenosis , such as those silencing the expression of other convertases , namely pc2 , which are also observed in atherectomy specimens ( see fig1 ). the development of drugs based on the inhibition or the inactivation of the convertases is of great interest because the drugs can easily be delivered directly at the affected site during the intervention by the cardiologist . in addition we claim that this therapeutic approach , based on the inhibition of cell growth by antisense against one of the convertases will be applicable to all proliferative diseases involving maturation of a given proteic precursor into an active protein : as mentioned above , pc5 , pc7 and furin are known to be present in cd4 + t lymphocytes . all three enzymes cleave hiv gp160 to gp120 and gp41 as well as a synthetic peptide covering the junction wherein cleavage occurs in gp160 . since the 17 - mer antisense odn defined in seq id . no . 3 successfully silenced the expression of pc5 and prevented restenosis , the same oligonucleotide as well as any other oligonucleotide or construct having an equivalent silencing function will find use in inhibiting the action of pc5 on hiv gp160 in cd4 + t lymphocytes . to optimize the inhibition of conversion of gp160 into its fusiogenic form , a cocktail comprising antisense molecules to pc5 , pc7 and furin is contemplated as part of the present invention . appropriate vehicles such as liposomes may be used to deliver these antisense molecules to the target tissues . this invention has been described hereinbelow . it may be apparent to the skilled reader that modifications can be made thereto without departing from the above teachings . these modifications are considered as part of the scope of the present invention 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j clin invest . 1993 ; 91 : 2058 - 2064 26 . fallo f , pistorello m , pedini f , d &# 39 ; agostino d , mantero f , boscaro m . in vitro evidence for local generation of renin and angiotensin ii / iii immunoreactivity by the human adrenal gland . acta endocrinologica . 1991 ; 125 : 319 - 330 27 . sarzani r , fallo f , dessi - fulgheri p , pistorello m , lanari a , paci vm , mantero f , rappelli a . local renin - angiotensin system in human adrenals and aldosteronomas . hypertension . 1992 ; 19 : 702 - 707 28 . mizuno k , hoffman l h , mckenzie j c , inagami t . presence of renin secretory granules in rat adrenal gland and stimulation of renin secretion by angiotensin ii but not by adrenocorticotropin . j clin invest . 1988 ; 82 : 1007 - 1016 29 . mullins j j , peters j , ganten d . fulminant hypertension in transgenic rats harbouring the mouse ren - 2 gene . nature . 1990 ; 344 : 541 - 544 30 . yamaguchi t , tokita y , franco - saenz r , mulrow p j , peters j , ganten d . zonal distribution and regulation of adrenal renin in a transgenic model of hypertension in the rat . endocrinology . 1992 ; 131 : 1955 - 1962 31 . rocco s , rebuffat p , cimolato m , opocher g , peters j , mazzocchi g , ganten d , mantero f , nussdorfer g g . zona glomerulosa of the adrenal gland in a transgenic strain of rat : a morphologic and functional study . cell & amp ; tiss res . 1994 ; 278 : 21 - 28 32 . tokita y , franco - saenz r , mulrow p j , ganten d . effects of nephrectomy and adrenalectomy on the renin - angiotensin system of transgenic rats tgr ( mren2 ) 27 . endocrinology . 1994 ; 134 : 253 - 257 33 . duncan k g , haidar m a , baxter j d , reudelhuber t l . regulation of human renin expression in chorion cell primary cultures . proc natl acad sci usa . 1990 ; 87 : 7588 - 7592 34 . lenz t , james g d , laragh j h , sealey j e . prorenin secretion from human placenta perfused in vitro . am j physiol . 1991 ; 260 : e876 - e882 35 . nakagawa t , murakami k , nakayama k . identification of an isoform with an extremely large cys - rich region of pc6 , a kex2 - like processing endoprotease . febs lett . 1993 ; 327 : 165 - 171 36 . seidah n g , hamelin j , gaspar a m , day r , chretien m . the cdna sequence of the human pro - hormone and pro - protein convertase pc1 . dna & amp ; cell biology . 1992 ; 11 : 283 - 289 37 . seidah n g , day r , marcinkiewicz m , chretien m . mammalian paired basic amino acid convertases of prohormones and proproteins . ann ny acad sci . 1993 ; 680 : 135 - 146 38 . miranda l , wolf j , pichuantes s , duke r , franzusoff a . isolation of the human pc6 gene encoding the putative host proteas for hiv - 1 gp160 processing in cd4 + t lymphocytes . proc . natl . acad . sci . usa 1996 ; 93 : 7695 - 7700 . atg gac tgg gac tgg ggg aac cgc tgc agc cgc ccg gga cgg cgg gat 48 ctg ctg tgc gtg ctg gcg ctg ctc ggg ggc tgc ctg ctc ccc gtg tgt 96 cgg acg cgc gtc tac acc aac cac tgg gca gtc aaa atc gcc ggg ggc 144 arg thr arg val tyr thr asn his trp ala val lys ile ala gly gly ttc ccg gag gcc aac cgt atc gcc agc aag tac gga ttc atc aac ata 192 phe pro glu ala asn arg ile ala ser lys tyr gly phe ile asn ile gga cag ata ggg gcc ctg aag gac tac tac cac ttc tac cat agc agg 240 gly gln ile gly ala leu lys asp tyr tyr his phe tyr his ser arg acg att aaa agg tca gtt atc tcg agc aga ggg acc cac agt ttc att 288 tca atg gaa cca aag gtg gaa tgg atc caa cag caa gtg gta aaa aag 336 cgg aca aag agg gat tat gac ttc agt cgt gcc cag tct acc tat ttc 384 aat gat ccc aag tgg ccc agc atg tgg tat atg cac tgc agt gac aat 432 aca cat ccc tgc cag tct gac atg aat atc gaa gga gcc tgg aag aga 480 thr his pro cys gln ser asp met asn ile glu gly ala trp lys arg ggc tac acg gga aag aac att gtg gtc act atc ctg gat gac gga att 528 gag aga acc cat cca gat ctg atg caa aac tac gat gct ctg gca agt 576 glu arg thr his pro asp leu met gln asn tyr asp ala leu ala ser tgc gac gtg aat ggg aat gac ttg gac cca atg cct cgt tat gat gca 624 cys asp val asn gly asn asp leu asp pro met pro arg tyr asp ala agc aac gag aac aag cat ggg act cgc tgt gct gga gaa gtg gca gcc 672 ser asn glu asn lys his gly thr arg cys ala gly glu val ala ala gct gca aac aat tcg cac tgc aca gtc gga att gct ttc aac gcc aag 720 ala ala asn asn ser his cys thr val gly ile ala phe asn ala lys atc gga gga gtg cga atg ctg gac gga gat gtc acg gac atg gtt gaa 768 gca aaa tca gtt agc ttc aac ccc cag cac gtg cac att tac agc gcc 816 ala lys ser val ser phe asn pro gln his val his ile tyr ser ala agc tgg ggc ccg gat gat gat ggc aag act gtg gac gga cca gcc ccc 864 ctc acc cgg caa gcc ttt gaa aac ggc gtt aga atg ggg cgg aga ggc 912 leu thr arg gln ala phe glu asn gly val arg met gly arg arg gly ctc ggc tct gtg ttt gtt tgg gca tct gga aat ggt gga agg agc aaa 960 gac cac tgc tcc tgt gat ggc tac acc aac agc atc tac acc atc tcc 1008 atc agc agc act gca gaa agc gga aag aaa cct tgg tac ctg gaa gag 1056 ile ser ser thr ala glu ser gly lys lys pro trp tyr leu glu glu tgt tca tcc acg ctg gcc aca acc tac agc agc ggg gag tcc tac gat 1104 aag aaa atc atc act aca gat ctg agg cag cgt tgc acg gac aac cac 1152 act ggg acg tca gcc tca gcc ccc atg gct gca ggc atc att gcg ctg 1200 gcc ctg gaa gcc aat ccg ttt ctg acc tgg aga gac gta cag cat gtt 1248 ala leu glu ala asn pro phe leu thr trp arg asp val gln his val att gtc agg act tcc cgt gcg gga cat ttg aac gct aat gac tgg aaa 1296 ile val arg thr ser arg ala gly his leu asn ala asn asp trp lys acc aat gct gct ggt ttt aag gtg agc cat ctt tat gga ttt gga ctg 1344 thr asn ala ala gly phe lys val ser his leu tyr gly phe gly leu atg gac gca gaa gcc atg gtg atg gag gca gag aag tgg acc acc gtt 1392 ccc cgg cag cac gtg tgt gtg gag agc aca gac cga caa atc aag aca 1440 pro arg gln his val cys val glu ser thr asp arg gln ile lys thr atc cgc cct aac agt gca gtg cgc tcc atc tac aaa gct tca ggc tgc 1488 ile arg pro asn ser ala val arg ser ile tyr lys ala ser gly cys tcg gat aac ccc aac cgc cat gtc aac tac ctg gag cac gtc gtt gtg 1536 cgc atc acc atc acc cac ccc agg aga gga gac ctg gcc atc tac ctg 1584 acc tcg ccc tct gga act agg tct cag ctt ttg gcc aac agg cta ttt 1632 gat cac tcc atg gaa gga ttc aaa aac tgg gag ttc atg acc att cat 1680 asp his ser met glu gly phe lys asn trp glu phe met thr ile his tgc tgg gga gaa aga gct gct ggt gac tgg gtc ctt gaa gtt tat gat 1728 act ccc tct cag cta agg aac ttt aag act cca ggt aaa ttg aaa gaa 1776 thr pro ser gln leu arg asn phe lys thr pro gly lys leu lys glu tgg tct ttg gtc ctc tac ggc acc tcc gtg cag cca tat tca cca acc 1824 aat gaa ttt ccg aaa gtg gaa cgg ttc cgc tat agc cga gtt gaa gac 1872 ccc aca gac gac tat ggc aca gag gat tat gca ggt ccc tgc gac cct 1920 gag tgc agt gag gtt ggc tgt gac ggg cca gga cca gac cac tgc aat 1968 gac tgt ttg cac tac tac tac aag ctg aaa aac aat acc agg atc tgt 2016 gtc tcc agc tgc ccc cct ggc cac tac cac gcc gac aag aag cgc tgc 2064 val ser ser cys pro pro gly his tyr his ala asp lys lys arg cys agg aag tgt gcc ccc aac tgt gag tcc tgc ttt ggg agc cat ggt gac 2112 arg lys cys ala pro asn cys glu ser cys phe gly ser his gly asp caa tgc atg tcc tgc aaa tat gga tac ttt ctg aat gaa gaa acc aac 2160 gln cys met ser cys lys tyr gly tyr phe leu asn glu glu thr asn agc tgt gtt act cac tgc cct gat ggg tca tat cag gat acc aag aaa 2208 ser cys val thr his cys pro asp gly ser tyr gln asp thr lys lys aat ctt tgc cgg aaa tgc agt gaa aac tgc aag aca tgt act gaa ttc 2256 cat aac tgt aca gaa tgt agg gat ggg tta agc ctg cag gga tcc cgg 2304 his asn cys thr glu cys arg asp gly leu ser leu gln gly ser arg tgc tct gtc tcc tgt gaa gat gga cgg tat ttc aac ggc cag gac tgc 2352 cys ser val ser cys glu asp gly arg tyr phe asn gly gln asp cys cag ccc tgc cac cgc ttc tgc gcc act tgt gct ggg gca gga gct gat 2400 ggg tgc att aac tgc aca gag ggc tac ttc atg gag gat ggg aga tgc 2448 gly cys ile asn cys thr glu gly tyr phe met glu asp gly arg cys gtg cag agc tgt agt atc agc tat tac ttt gac cac tct tca gag aat 2496 val gln ser cys ser ile ser tyr tyr phe asp his ser ser glu asn gga tac aaa tcc tgc aaa aaa tgt gat atc agt tgt ttg acg tgc aat 2544 ggc cca gga ttc aag aac tgt aca agc tgc cct agt ggg tat ctc tta 2592 gac tta gga atg tgt caa atg gga gcc att tgc aag gat gca acg gaa 2640 asp leu gly met cys gln met gly ala ile cys lys asp ala thr glu gag tcc tgg gcg gaa gga ggc ttc tgt atg ctt gtg aaa aag aac aat 2688 glu ser trp ala glu gly gly phe cys met leu val lys lys asn asn ctg tgc caa cgg aag gtt ctt caa caa ctt tgc tgc aaa aca tgt aca 2736 arg thr arg val tyr thr asn his trp ala val lys ile ala gly gly phe pro glu ala asn arg ile ala ser lys tyr gly phe ile asn ile gly gln ile gly ala leu lys asp tyr tyr his phe tyr his ser arg thr his pro cys gln ser asp met asn ile glu gly ala trp lys arg glu arg thr his pro asp leu met gln asn tyr asp ala leu ala ser cys asp val asn gly asn asp leu asp pro met pro arg tyr asp ala ser asn glu asn lys his gly thr arg cys ala gly glu val ala ala ala ala asn asn ser his cys thr val gly ile ala phe asn ala lys ala lys ser val ser phe asn pro gln his val his ile tyr ser ala leu thr arg gln ala phe glu asn gly val arg met gly arg arg gly ile ser ser thr ala glu ser gly lys lys pro trp tyr leu glu glu ala leu glu ala asn pro phe leu thr trp arg asp val gln his val ile val arg thr ser arg ala gly his leu asn ala asn asp trp lys thr asn ala ala gly phe lys val ser his leu tyr gly phe gly leu pro arg gln his val cys val glu ser thr asp arg gln ile lys thr ile arg pro asn ser ala val arg ser ile tyr lys ala ser gly cys asp his ser met glu gly phe lys asn trp glu phe met thr ile his thr pro ser gln leu arg asn phe lys thr pro gly lys leu lys glu val ser ser cys pro pro gly his tyr his ala asp lys lys arg cys arg lys cys ala pro asn cys glu ser cys phe gly ser his gly asp gln cys met ser cys lys tyr gly tyr phe leu asn glu glu thr asn ser cys val thr his cys pro asp gly ser tyr gln asp thr lys lys his asn cys thr glu cys arg asp gly leu ser leu gln gly ser arg cys ser val ser cys glu asp gly arg tyr phe asn gly gln asp cys gly cys ile asn cys thr glu gly tyr phe met glu asp gly arg cys val gln ser cys ser ile ser tyr tyr phe asp his ser ser glu asn asp leu gly met cys gln met gly ala ile cys lys asp ala thr glu glu ser trp ala glu gly gly phe cys met leu val lys lys asn asn agcgtcggga cc atg gat tgg gat tgg ggg aac cgc tgc agc cgc ccg gga 51 cgg cgg gac ctg ctg tgc gtg ctg gca ctg ctc gcc ggc tgt ctg ctc 99 ccg gta tgc cgg acg cgc gtc tac acc aac cac tgg gca gtg aag atc 147 pro val cys arg thr arg val tyr thr asn his trp ala val lys ile gcc ggc ggc ttc gcg gag gca gat cgc ata gcc agc aag tac gga ttc 195 atc aac gta gga cag atc ggt gca ctg aag gac tac tat cac ttc tac 243 ile asn val gly gln ile gly ala leu lys asp tyr tyr his phe tyr cat agt agg acc att aaa agg tct gtt ctc tcg agc aga gga acc cac 291 agt ttc att tca atg gaa cca aag gtg gag tgg atc caa cag caa gtg 339 gtg aaa aaa aga acc aag agg gat tat gac ctc agc cat gcc cag tca 387 val lys lys arg thr lys arg asp tyr asp leu ser his ala gln ser acc tac ttc aat gat ccc aag tgg cca agt atg tgg tac atg cac tgt 435 thr tyr phe asn asp pro lys trp pro ser met trp tyr met his cys agc gac aat aca cat ccc tgc cag tct gac atg aat atc gaa gga gcc 483 ser asp asn thr his pro cys gln ser asp met asn ile glu gly ala tgg aag aga ggc tac acg gga aag aac att gtg gtc act atc ctg gat 531 trp lys arg gly tyr thr gly lys asn ile val val thr ile leu asp gac gga att gag aga acc cat cca gat ctg atg caa aac tac gat gct 579 asp gly ile glu arg thr his pro asp leu met gln asn tyr asp ala ctg gca agt tgc gac gtg aat ggg aat gac ttg gac cca atg cct cgt 627 leu ala ser cys asp val asn gly asn asp leu asp pro met pro arg tat gat gca agc aac gag aac aag cat ggg act cgc tgt gct gga gaa 675 tyr asp ala ser asn glu asn lys his gly thr arg cys ala gly glu gtg gca gcc gct gca aac aat tcg cac tgc aca gtc gga att gct ttc 723 aac gcc aag atc gga gga gtg cga atg ctg gac gga gat gtc acg gac 771 asn ala lys ile gly gly val arg met leu asp gly asp val thr asp atg gtt gaa gca aaa tca gtt agc ttc aac ccc cag cac gtg cac att 819 met val glu ala lys ser val ser phe asn pro gln his val his ile tac agc gcc agc tgg ggc ccg gat gat gat ggc aag act gtg gac gga 867 cca gcc ccc ctc acc cgg caa gcc ttt gaa aac ggc gtt aga atg ggg 915 pro ala pro leu thr arg gln ala phe glu asn gly val arg met gly cgg aga ggc ctc ggc tct gtg ttt gtt tgg gca tct gga aat ggt gga 963 agg agc aaa gac cac tgc tcc tgt gat ggc tac acc aac agc atc tac 1011 arg ser lys asp his cys ser cys asp gly tyr thr asn ser ile tyr acc atc tcc atc agc agc act gca gaa agc gga aag aaa cct tgg tac 1059 ctg gaa gag tgt tca tcc acg ctg gcc aca acc tac agc agc ggg gag 1107 tcc tac gat aag aaa atc atc act aca gat ctg agg cag cgt tgc acg 1155 gac aac cac act ggg acg tca gcc tca gcc ccc atg gct gca ggc atc 1203 att gcg ctg gcc ctg gaa gcc aat ccg ttt ctg acc tgg aga gac gta 1251 ile ala leu ala leu glu ala asn pro phe leu thr trp arg asp val cag cat gtt att gtc agg act tcc cgt gcg gga cat ttg aac gct aat 1299 gln his val ile val arg thr ser arg ala gly his leu asn ala asn gac tgg aaa acc aat gct gct ggt ttt aag gtg agc cat ctt tat gga 1347 asp trp lys thr asn ala ala gly phe lys val ser his leu tyr gly ttt gga ctg atg gac gca gaa gcc atg gtg atg gag gca gag aag tgg 1395 acc acc gtt ccc cgg cag cac gtg tgt gtg gag agc aca gac cga caa 1443 atc aag aca atc cgc cct aac agt gca gtg cgc tcc atc tac aaa gct 1491 tca ggc tgc tcg gat aac ccc aac cgc cat gtc aac tac ctg gag cac 1539 ser gly cys ser asp asn pro asn arg his val asn tyr leu glu his gtc gtt gtg cgc atc acc atc acc cac ccc agg aga gga gac ctg gcc 1587 atc tac ctg acc tcg ccc tct gga act agg tct cag ctt ttg gcc aac 1635 ile tyr leu thr ser pro ser gly thr arg ser gln leu leu ala asn agg cta ttt gat cac tcc atg gaa gga ttc aaa aac tgg gag ttc atg 1683 arg leu phe asp his ser met glu gly phe lys asn trp glu phe met acc att cat tgc tgg gga gaa aga gct gct ggt gac tgg gtc ctt gaa 1731 thr ile his cys trp gly glu arg ala ala gly asp trp val leu glu gtt tat gat act ccc tct cag cta agg aac ttt aag act cca ggt aaa 1779 val tyr asp thr pro ser gln leu arg asn phe lys thr pro gly lys ttg aaa gaa tgg tct ttg gtc ctc tac ggc acc tcc gtg cgg cca tat 1827 leu lys glu trp ser leu val leu tyr gly thr ser val arg pro tyr tca cca acc aat gaa ttt ccg aaa gtg gaa cgg ttc cgc tat agc cga 1875 gtt gaa gac ccc aca gac gac tat ggc aca gag gat tat gca ggt ccc 1923 tgc gac cct gag tgc agt gag gtt ggc tgt gac ggg cca gga cca gac 1971 cac tgc aat gac tgt ttg cac tac tac tac aag ctg aaa aac aat acc 2019 agg atc tgt gtc tcc agc tgc ccc cct ggc cac tac cac gcc gac aag 2067 arg ile cys val ser ser cys pro pro gly his tyr his ala asp lys aag cgc tgc agg aag tgt gcc ccc aac tgt gag tcc tgc ttt ggg agc 2115 cat ggt gac caa tgc atg tcc tgc aaa tat gga tac ttt ctg aat gaa 2163 his gly asp gln cys met ser cys lys tyr gly tyr phe leu asn glu gaa acc aac agc tgt gtt act cac tgc cct gat ggg tca tat cag gat 2211 glu thr asn ser cys val thr his cys pro asp gly ser tyr gln asp acc aag aaa aat ctt tgc cgg aaa tgc agt gaa aac tgc aag aca tgt 2259 act gaa ttc cat aac tgt aca gaa tgt agg gat ggg tta agc ctg cag 2307 thr glu phe his asn cys thr glu cys arg asp gly leu ser leu gln gga tcc cgg tgc tct gtc tcc tgt gaa gat gga cgg tat ttc aac ggc 2355 cag gac tgc cag ccc tgc cac cgc ttc tgc gcc act tgt gct ggg gca 2403 gga gct gat ggg tgc att aac tgc aca gag ggc tac ttc atg gag gat 2451 gly ala asp gly cys ile asn cys thr glu gly tyr phe met glu asp ggg aga tgc gtg cag agc tgt agt atc agc tat tac ttt gac cac tct 2499 gly arg cys val gln ser cys ser ile ser tyr tyr phe asp his ser tca gag aat gga tac aaa tcc tgc aaa aaa tgt gat atc agt tgt ttg 2547 acg tgc aat ggc cca gga ttc aag aac tgt aca agc tgc cct agt ggg 2595 tat ctc tta gac tta gga atg tgt caa atg gga gcc att tgc aag gat 2643 gca acg gaa gag tcc tgg gcg gaa gga ggc ttc tgt atg ctt gtg aaa 2691 ala thr glu glu ser trp ala glu gly gly phe cys met leu val lys aag aac aat ctg tgc caa cgg aag gtt ctt caa caa ctt tgc tgc aaa 2739 arg thr arg val tyr thr asn his trp ala val lys ile ala gly gly phe ala glu ala asp arg ile ala ser lys tyr gly phe ile asn val gly gln ile gly ala leu lys asp tyr tyr his phe tyr his ser arg arg thr lys arg asp tyr asp leu ser his ala gln ser thr tyr phe thr his pro cys gln ser asp met asn ile glu gly ala trp lys arg glu arg thr his pro asp leu met gln asn tyr asp ala leu ala ser cys asp val asn gly asn asp leu asp pro met pro arg tyr asp ala ser asn glu asn lys his gly thr arg cys ala gly glu val ala ala ala ala asn asn ser his cys thr val gly ile ala phe asn ala lys ala lys ser val ser phe asn pro gln his val his ile tyr ser ala leu thr arg gln ala phe glu asn gly val arg met gly arg arg gly ile ser ser thr ala glu ser gly lys lys pro trp tyr leu glu glu ala leu glu ala asn pro phe leu thr trp arg asp val gln his val ile val arg thr ser arg ala gly his leu asn ala asn asp trp lys thr asn ala ala gly phe lys val ser his leu tyr gly phe gly leu pro arg gln his val cys val glu ser thr asp arg gln ile lys thr ile arg pro asn ser ala val arg ser ile tyr lys ala ser gly cys asp his ser met glu gly phe lys asn trp glu phe met thr ile his thr pro ser gln leu arg asn phe lys thr pro gly lys leu lys glu val ser ser cys pro pro gly his tyr his ala asp lys lys arg cys arg lys cys ala pro asn cys glu ser cys phe gly ser his gly asp gln cys met ser cys lys tyr gly tyr phe leu asn glu glu thr asn ser cys val thr his cys pro asp gly ser tyr gln asp thr lys lys his asn cys thr glu cys arg asp gly leu ser leu gln gly ser arg cys ser val ser cys glu asp gly arg tyr phe asn gly gln asp cys gly cys ile asn cys thr glu gly tyr phe met glu asp gly arg cys val gln ser cys ser ile ser tyr tyr phe asp his ser ser glu asn asp leu gly met cys gln met gly ala ile cys lys asp ala thr glu glu ser trp ala glu gly gly phe cys met leu val lys lys asn asn