Patent Application: US-54024500-A

Abstract:
disclosed are methods and compositions for identifying agents which modulate the interaction of robo and a robo ligand and for modulating the interaction of robo and a robo ligand . the methods for identifying robo : ligand modulators find particular application in commercial drug screens . these methods generally comprise combining a robo polypeptide , a slit polypeptide and a candidate agent under conditions whereby , but for the presence of the agent , the robo and slit polypeptides engage in a first interaction , and determining a second interaction of the robo and slit polypeptides in the presence of the agent , wherein a difference between the first and second interactions indicates that the agent modulates the interaction of the robo and slit polypeptides . the subject methods of modulating the interaction of robo and a robo ligand involve combining a robo polypeptide , a slit polypeptide and a modulator under conditions whereby , but for the presence of the modulator , the robo and slit polypeptides engage in a first interaction , whereby the robo and slit polypeptides engage in a second interaction different from the first interaction . in a particular embodiment , the modulator is dominant negative form of the robo or slit polypeptide .

Description:
the subject methods include screens for agents which modulate robo : ligand interactions and methods for modulating robo : ligand interactions . robo activation is found to regulate a wide variety of cell functions , including cell - cell interactions , cell mobility , morphology , etc . slit polypeptides are disclosed as specific activators and inactivators of robo polypeptides . accordingly , the invention provides methods for modulating targeted cell function comprising the step of modulating robo activation by contacting the cell with a modulator of a robo : slit interaction .. the targeted robo polypeptide is generally naturally expressed on the targeted cells . the nucleotide sequences of exemplary natural cdnas encoding drosophila 1 , drosophila 2 , c . elegans , human 1 , human 2 and mouse 1 robo polypeptides and their translates are described in kidd et al . ( 1998 ) cell 92 , 205 - 215 and u . s . ser . no . 08 / 971 , 172 . the targeted robo polypeptides comprise at least a finctional robo domain , which domain has robo - specific amino acid sequence and binding specificity or function . preferred robo domains comprise at least 8 , preferably at least 16 , more preferably at least 32 , most preferably at least 64 consecutive residues of a natural full length robo . in a particular embodiment , the domains comprise one or more structural / functional robo immunoglobulin , fibronectin or cytoplasmic motif domains described herein . the subject domains provide robo - specific antigens and / or immunogens , especially when coupled to carrier proteins . for example , peptides corresponding to robo - and human robo - specific domains are covalently coupled to keyhole limpet antigen ( klh ) and the conjugate is emulsified in freunds complete adjuvant . laboratory rabbits are immunized according to conventional protocol and bled . the presence of robo - specific antibodies is assayed by solid phase immunosorbant assays using immobilized robo polypeptides . generic robo - specific peptides are readily apparent as conserved regions in aligned robo polypeptide sequences . in addition , species - specific antigenic and / or immunogenic peptides are readily apparent as diverged extracellular or cytosolic regions in alignments human robo - specific antibodies are characterized as uncross - reactive with non - human robo polypeptides . the subject domains provide robo domain specific activity or function , such as robo - specific cell , especially neuron modulating or modulating inhibitory activity , robo - ligand - binding or binding inhibitory activity . robo - specific activity or function may be determined by convenient in vitro , cell - based , or in vivo assays : e . g . in vitro binding assays , cell culture assays , in animals ( e . g . gene therapy , transgenics , etc . ), etc . the binding target may be a natural intracellular binding target , a robo regulating protein or other regulator that directly modulates robo activity or its localization ; or non - natural binding target such as a specific immune protein such as an antibody , or a robo specific agent such as those identified in screening assays such as described below . robo - binding specificity may be assayed by binding equilibrium constants ( usually at least about 10 7 m − 1 , preferably at least about 10 8 m − 1 , more preferably at least about 10 9 m − 1 ), by the ability of the subject polypeptide to function as negative mutants in robo - expressing cells , to elicit robo specific antibody in a heterologous host ( e . g a rodent or rabbit ), etc . similarly , the slit polypeptide is conveniently selected from slit polypeptides which specifically activate or inhibit the activation of the robo polypeptide . exemplary suitable slit polypeptides ( a ) comprises a vertebrate slit sequence disclosed herein , especially human slit - 1 ( seq id no : 02 ), or a deletion mutant thereof which specifically modulates robo expression or a sequence about 60 - 70 %, preferably about 70 - 80 %, more preferably about 80 - 90 %, more preferably about 90 - 95 %, most preferably about 95 - 99 % similar to a vertebrate slit sequence disclosed herein as determined by best fit analysis using default settings and is other than a natural drosophila slit sequence , preferably other than a natural invertebrate slit sequence , and / or ( b ) is encoded by a nucleic acid comprising a natural slit encoding sequence ( such as a natural human slit - 1 encoding sequence , seq id no : 01 ) or a fragment thereof at least 36 , preferably at least 72 , more preferably at least 144 , most preferably at least 288 nucleotides in length which specifically hybridizes thereto . suitable deletion mutants are readily screened in robo binding or activation assays as described herein . preferred slit domains / deletion mutants / fragments comprise at least 8 , preferably at least 16 , more preferably at least 32 , most preferably at least 64 consecutive residues of a disclosed vertebrate slit sequences and provide a slit specific activity , such as slit - specific antigenicity and / or immunogenicity , especially when coupled to carrier proteins as described above for robo above . suitable natural slit encoding sequence fragements are of length sufficient to encode such slit domains . in a particular embodiment , the slit fragments comprise species specific fragments ; such fragments are readily discerned from alignments of the disclosed sequences , see , e . g . shown as unboxed sequences in tables 1 and 2 . exemplary such human slit - 1 immunogenic and / or antigenic peptides are shown in table 3 . the subject domains provide slit domain specific activity or function , such as slit - specific cell , especially neuron modulating or modulating inhibitory activity , slit - ligand - binding or binding inhibitory activity . slit - specific activity or function may be determined by convenient in vitro , cell - based , or in vivo assays : e . g . in vitro binding assays , cell culture assays , in animals ( e . g . gene therapy , transgenics , etc . ), etc . the binding target may be a natural intracellular binding target , a slit regulating protein or other regulator that directly modulates slit activity or its localization ; or non - natural binding target such as a specific immune protein such as an antibody , or a slit specific agent such as those identified in screening assays such as described below . slit - binding specificity may be assayed by binding equilibrium constants ( usually at least about 10 7 m − 1 , preferably at least about 10 8 m − 1 , more preferably at least about 10 9 m − 1 ), by the ability of the subject polypeptide to function as negative mutants in slit - expressing cells , to elicit slit specific antibody in a heterologous host ( e . g a rodent or rabbit ), etc . in one embodiment , the slit polypeptides are encoded by a nucleic acid comprising seq id no : 01 or a fragment thereof which hybridizes with a full - length strand thereof , preferably under stringent conditions . such nucleic acids comprise at least 36 , preferably at least 72 , more preferably at least 144 and most preferably at least 288 nucleotides of seq id no : 01 . demonstrating specific hybridization generally requires stringent conditions , for example , hybridizing in a buffer comprising 30 % formamide in 5 × sspe ( 0 . 18 m nacl , 0 . 01 m napo 4 , ph7 . 7 , 0 . 001 m edta ) buffer at a temperature of 42 ° c . and remaining bound when subject to washing at 42 ° c . with 0 . 2 × sspe ( conditions i ); preferably hybridizing in a buffer comprising 50 % formamide in 5 × sspe buffer at a temperature of 42 ° c . and remaining bound when subject to washing at 42 ° c . with 0 . 2 × sspe buffer at 42 ° c . ( conditions ii ). exemplary nucleic acids which hybridize with a strand of seq id no : 01 are shown in table 4 . a wide variety of cell types express robo polypeptides subject to regulation by the disclosed methods , including many neuronal cells , transformed cells , infected ( e . g . virus ) cells , etc . ascertaining robo binding or activation is readily effected by binding assays or cells function assays as disclosed herein or in the cited copending applications . accordingly , indications for the subject methods encompass a wide variety of cell types and function , including axon outgrowth , tumor cell invasion or migration , etc . the target cell may reside in culture or in situ , i . e . within the natural host . for in situ applications , the compositions are added to a retained physiological fluid such as blood or synovial fluid . for cns administration , a variety of techniques are available for promoting transfer of the therapeutic across the blood brain barrier including disruption by surgery or injection , drugs which transiently open adhesion contact between cns vasculature endothelial cells , and compounds which facilitate translocation through such cells . slit polypeptides may also be amenable to direct injection or infusion , topical , intratracheal / nasal administration e . g . through aerosol , intraocularly , or within / on implants e . g . fibers e . g . collagen , osmotic pumps , grafts comprising appropriately transformed cells , etc . a particular method of administration involves coating , embedding or derivatizing fibers , such as collagen fibers , protein polymers , etc . with therapeutic polypeptides . other useful approaches are described in otto et al . ( 1989 ) j neuroscience research 22 , 83 - 91 and otto and unsicker ( 1990 ) j neuroscience 10 , 1912 - 1921 . generally , the amount administered will be empirically determined , typically in the range of about 10 to 1000 μg / kg of the recipient and the concentration will generally be in the range of about 50 to 500 μg / ml in the dose administered . other additives may be included , such as stabilizers , bactericides , etc . will be present in conventional amounts . in one embodiment , the invention provides administering the subject slit polypeptides in combination with a pharmaceutically acceptable excipient such as sterile saline or other medium , gelatin , an oil , etc . to form pharmaceutically acceptable compositions . the compositions and / or compounds may be administered alone or in combination with any convenient carrier , diluent , etc . and such administration may be provided in single or multiple dosages . useful carriers include solid , semi - solid or liquid media including water and non - toxic organic solvents . in another embodiment , the invention provides the subject compounds in the form of a pro - drug , which can be metabolically converted to the subject compound by the recipient host . a wide variety of prodrug formulations for polypeptide - based therapeutics are known in the art . the compositions may be provided in any convenient form including tablets , capsules , troches , powders , sprays , creams , etc . as such the compositions , in pharmaceutically acceptable dosage units or in bulk , may be iincorporated into a wide variety of containers . for example , dosage units may be included in a variety of containers including capsules , pills , etc . the compositions may be advantageously combined and / or used in combination with other therapeutic or prophylactic agents , different from the subject compounds . in many instances , administration in conjunction with the subject compositions enhances the efficacy of such agents , see e . g . goodman & amp ; gilman &# 39 ; s the pharmacological basis of therapeutics , 9 th ed ., 1996 , mcgraw - hill . in another aspect , the invention provides methods of screening for agents which modulate robo - slit interactions . these methods generally involve forming a mixture of a robo - expressing cell , a slit polypeptide and a candidate agent , and determining the effect of the agent on the amount of robo expressed by the cell . the methods are amenable to automated , cost - effective high throughput screening of chemical libraries for lead compounds . identified reagents find use in the pharmaceutical industries for animal and human trials ; for example , the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development . cell and animal based neural guidance / repulsion assays are described in detail in the experimental section below . the amino acid sequences of the disclosed vertebrate slit polypeptides are used to back - translate slit polypeptide - encoding nucleic acids optimized for selected expression systems ( holler et al . ( 1993 ) gene 136 , 323 - 328 ; martin et al . ( 1995 ) gene 154 , 150 - 166 ) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural slit - encoding nucleic acid sequences (“ gcg ” software , genetics computer group , inc , madison wis .). slit - encoding nucleic acids used in slit - expression vectors and incorporated into recombinant host cells , e . g . for expression and screening , transgenic animals , e . g . for finctional studies such as the efficacy of candidate drugs for disease associated with slit - modulated cell function , etc . the invention also provides nucleic acid hybridization probes and replication / amplification primers having a vertebrate slit cdna specific sequence comprising a fragment of a disclosed vertebrate cdna sequence , and sufficient to effect specific hybridization thereto . such primers or probes are at least 12 , preferably at least 24 , more preferably at least 36 and most preferably at least 96 nucleotides in length . demonstrating specific hybridization generally requires stringent conditions , for example , hybridizing in a buffer comprising 30 % formamide in5 × sspe ( 0 . 18 m nacl , 0 . 01 m napo 4 , ph7 . 7 , 0 . 001 m edta ) buffer at a temperature of 42 ° c . and remaining bound when subject to washing at 42 ° c . with 0 . 2 × sspe ; preferably hybridizing in a buffer comprising 50 % formamide in 5 × sspe buffer at a temperature of 42 ° c . and remaining bound when subject to washing at 42 ° c . with 0 . 2 × sspe buffer at 42 ° c . slit nucleic acids can also be distinguished using alignment algorithms , such as blasix ( altschul et al . ( 1990 ) basic local alignment search tool , j mol biol 215 , 403 - 410 ). in addition , the invention provides nucleic acids having a sequence about 60 - 70 %, preferably about 70 - 80 %, more preferably about 80 - 90 %, more preferably about 90 - 95 %, most preferably about 95 - 99 % similar to a vertebrate slit sequence disclosed herein as determined by best fit analysis using default settings and is other than a natural drosophila slit sequence , preferably other than a natural invertebrate slit sequence . in a particular embodiment , the slit polynucleotide fragments comprise species specific fragments ; such fragments are readily discerned from alignments of the disclosed sequences . the subject nucleic acids are of synthetic / non - natural sequences and / or are recombinant , meaning they comprise a non - natural sequence or a natural sequence joined to nucleotide ( s ) other than that which it is joined to on a natural chromosome . the subject recombinant nucleic acids comprising the nucleotide sequence of disclosed vertebrate slit nucleic acids , or fragments thereof , contain such sequence or fragment at a terminus , immediately flanked by ( i . e . contiguous with ) a sequence other than that which it is joined to on a natural chromosome , or flanked by a native flanking region fewer than 10 kb , preferably fewer than 2 kb , more preferably fewer than 500 bp , which is at a terminus or is immediately flanked by a sequence other than that which it is joined to on a natural chromosome . while the nucleic acids are usually rna or dna , it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability , etc . the subject nucleic acids find a wide variety of applications including use as translatable transcripts , hybridization probes , pcr primers , diagnostic nucleic acids , etc . ; use in detecting the presence of slit genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional slit homologs and structural analogs . in diagnosis , slit hybridization probes find use in identifying wild - type and mutant slit alleles in clinical and laboratory samples . mutant alleles are used to generate allele - specific oligonucleotide ( aso ) probes for high - throughput clinical diagnoses . in therapy , therapeutic slit nucleic acids are used to modulate cellular expression or intracellular concentration or availability of active slit . exemplary humain slit - 1 probes and primers are shown in table 5 and table 6 . leucine rich repeats ( lrrs ) are predicted by comparison with known proteins and by the presence of a leucine rich core sequence . in slit proteins , the lrrs are flanked by conserved sequences referred to as the amino - and carboxy - flanking regions . these flanking regions are found in other known proteins , but only in a few instances are both the amino - and carboxy - flank regions present in a single protein . the so called “ 99aa spacer .” is actually ˜ 200 amino acids in the drosophila protein and 174 amino acids in human slit - 1 . this region shows homology to the g - loops of laminin a chains . cysteine knots are dimerisation domains defined by the presence of six cysteine residues between which disulphide bridges form . the only absolutely conserved residues are the six cysteines , and spacing between them is highly variable , apart from between cysteines 2 and 3 , and 5 and 6 . the glycine between cysteines 2 and 3 is only present in a subset of cysteine knots . drosophila slit and human slit - 1 both have an extra cysteine after cysteines 5 and 6 : this may serve as an intermolecular bond . human slit - 1 gene displays the overall structure of the drosophila gene , and amino acid conservation is found along the entire length of the protein ( 48 % homology at the amino acid sequence excluding the signal sequence ; see below ). the human gene has an extra lrr between lrr2 and lrr3 of the first set of lrrs ; in the third set , the human gene has an extra lrr between lrr3 and lrr4 . the human gene has two extra egf repeats , on either side of the seventh egf repeat in drosophila slit . searching of the est database revealed an est , ab16g10 . rl1 , with homology to the 99aa spacer region of drosophila slit . this est was used to probe a human fetal brain library ( stratagene ), and clones for human slit - 1 were isolated . the following examplary assay is offered by way of illustration and not by way of limitation : expression construct : cdnas encoding targeted slit polypeptides are tagged with the fc portion of human igg and subcloned into a 293 expression vector ( pcep4 : in vitrogen ). transfection : 293 ebna cells are transfected ( capo 4 method ) with the slit expression constructs . after 24 h recovery , transfected cells are selected with g418 ( geneticin , 250 ug / ml , gibco ) and hygromycin ( 200 ug / ml ). once the selection process is complete , cells are maintained in dulbecco &# 39 ; s modified eagles medium ( dme )/ 10 % fcs under selection . preparation of conditioned medium : serum - containing media is replaced with optimem with glutamax - 1 ( gibco ) and 300 ng / ml heparin ( sigma ), and the cells are conditioned for 3 days . the media is collected and spun at 3 , 000xg for 10 minutes . the supernatant is filtered ( 0 . 45 um ) and stored with 0 . 1 % azide at 4 ° c . for no more than 2 weeks . expression construct : cdna encoding a corresponding robo c - terminal deletion mutant comprising the extracellular domain ( truncated immediately n - terminal to the transmembrane region ) is subdloned into a 293 expression vector ( pcep4 : in vitrogen ). transfection : 293 ebna cells are transfected ( capo 4 method ) with the receptor mutant expression construct . after 24 h recovery , transfected cells are selected with g418 ( geneticin , 250 ug / ml , gibco ) and hygromycin ( 200 ug / ml ). once the selection process is complete , cells are maintained in dulbecco &# 39 ; s modified eagles medium ( dme )/ 10 % fcs under selection . preparation of conditioned medium : serumn - containing media is replaced with optimem with glutamax - 1 ( gibco ) and 300 ng / ml heparin ( sigma ), and the cells are conditioned for 3 days . the media is collected and spun at 3 , 000xg for 10 minutes . the supernatant is filtered ( 0 . 45 um ) and stored with 0 . 1 % azide at 4 ° c . for no more than 2 weeks . seed cos cells ( 250 , 000 ) on 35 mm dishes in 2 ml dme / 10 % fcs . 18 - 24 h later , dilute 1 ug of robo - encoding dna ( cdna cloned into pmt21 expression vector ) into 200 ul serum - free media and add 6 ul of lipofectamine ( gibco ). incubate this solution at room temperature for 15 - 45 min . wash the cells 2x with pbs . add 800 ul serum - free media to the tube containing the lipid - dna complexes . overlay this solution onto the washed cells . incubate for 6 h . stop the reaction by adding 1 ml dma / 20 % fcs . refeed cells . assay cells 12 hr later . wash plates of transfected cos cells 1x with cold pbs ( plus ca / mg )/ 1 % goat serum . add 1 ml conditioned media neat and incubate 90 min at room temp . wash plates 3x with pbs ( plus ca / mg ). on the 4th wash , add 1 ml 50 % methanol to 1 ml pbs . then add 1 ml methanol . evacuate and add 1 ml methanol . add secondary antibody ( 1 - to - 2 , 000 anti - human fc conjugated to alkaline phosphatase ( jackson lab )) in pbs / 1 % goat serum . incubate 30 - 40 min room temp . wash 3x with pbs . wash 1x alkaline phosphatase buffer ( 100 mm tris - cl , ph 9 . 5 , 100 mm nacl , 5 mm mgcl 2 ). prepare alkaline phosphatase reagents : 4 . 5 ul / ml nbt and 3 . 5 ul / ml bcip ( gibco ) in alkaline phosphatase buffer . incubate 10 - 30 min , quench with 20 mm edta in pbs . cells that have bound slit polypeptides are visible by the presence of a dark purple reaction product . in parallel incubations , positive controls are provided by titrating slit binding with serial dilutions of the mutant receptor conditioned medium . cell expressing mammalian slit polypeptides were shown to bind robo . no reactivity was observed with control cos cells or with receptor - expressing cos cells in the presence of the secondary antibody but in the absence of the slit - fc fusion . binding was observed to receptor - expression cells using a construct in which a slit polypeptide is fused directly to alkaline phosphatase , for which a secondary antibody is not required . receptor deletion imutants titrate the slit - robo binding , serving as a positive control for inhibition assays . blocking buffer : 5 % bsa , 0 . 5 % tween 20 in pbs ; 1 hour at room temperature . assay buffer : 100 mm kcl , 20 mm hepes ph 7 . 6 , 1 mm mgcl 2 , 1 % glycerol , 0 . 5 % np - 40 , 50 mm β - mercaptoethanol , 1 mg / ml bsa , cocktail of protease inhibitors . 33 p robo polypeptide 10x stock : 10 − 8 - 10 − 6 m “ cold ” robo polypeptide specific robo domain supplemented with 200 , 000 - 250 , 000 cpm of labeled robo ( beckman counter ). place in the 4 ° c . microfridge during screening . protease inhibitor cocktail ( 1000x ): 10 mg trypsin inhibitor ( bmb # 109894 ), 10 mg aprotinin ( bmb # 236624 ), 25 mg benzamidine ( sigma # b - 6506 ), 25 mg leupeptin ( bmb # 1017128 ), 10 mg apmsf ( bmb # 917575 ), and 2mm navo 3 ( sigma # s - 6508 ) in 10 ml of pbs . slit : 10 − 7 - 10 − 5 m biotinylated slit in pbs . coat with 120 μl of stock n - avidin per well overnight at 4 ° c . add 10 μl 33 p - robo ( 20 - 25 , 000 cpm / 0 . 1 - 10 pmoles / well = 10 − 9 - 10 − 7 m final conc ). add 40 μm biotinylated slit ( 0 . 1 - 10 pmoles / 40 ul in assay buffer ) stop the reaction by washing 4 times with 200 μm pbs . all publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference . although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding , it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims . atg cgc ggc gtt ggc tgg cag atg ctg tcc ctg tcg ctg ggg tta gtg 48 ctg gcg atc ctg aac aag gtg gca ccg cag gcg tgc ccg gcg cag tgc 96 tct tgc tcg ggc agc aca gtg gac tgt cac ggg ctg gcg ctg cgc agc 144 gtg ccc agg aat atc ccc cgc aac acc gag aga ctg gat tta aat gga 192 aat aac atc aca aga att acg aag aca gat ttt gct ggt ctt aga cat 240 asn asn ile thr arg ile thr lys thr asp phe ala gly leu arg his cta aga gtt ctt cag ctt atg gag aat aag att agc acc att gaa aga 288 leu arg val leu gln leu met glu asn lys ile ser thr ile glu arg gga gca ttc cag gat ctt aaa gaa cta gag aga ctg cgt tta aac aga 336 aat cac ctt cag ctg ttt cct gag ttg ctg ttt ctt ggg act gcg aag 384 asn his leu gln leu phe pro glu leu leu phe leu gly thr ala lys cta tac agg ctt gat ctc agt gaa aac caa att cag gca atc cca agg 432 leu tyr arg leu asp leu ser glu asn gln ile gln ala ile pro arg aaa gct ttc cgt ggg gca gtt gac ata aaa aat ttg caa ctg gat tac 480 lys ala phe arg gly ala val asp ile lys asn leu gln leu asp tyr aac cag atc agc tgt att gaa gat ggg gca ttc agg gct ctc cgg gac 528 asn gln ile ser cys ile glu asp gly ala phe arg ala leu arg asp ctg gaa gtg ctc act ctc aac aat aac aac att act aga ctt tct gtg 576 gca agt ttc aac cat atg cct aaa ctt agg act ttt cga ctg cat tca 624 ala ser phe asn his met pro lys leu arg thr phe arg leu his ser aac aac ctg tat tgt gac tgc cac ctg gcc tgg ctc tcc gac tgg ctt 672 cgc aaa agg cct cgg gtt ggt ctg tac act cag tgt atg ggc ccc tcc 720 arg lys arg pro arg val gly leu tyr thr gln cys met gly pro ser cac ctg aga ggc cat aat gta gcc gag gtt caa aaa cga gaa ttt gtc 768 his leu arg gly his asn val ala glu val gln lys arg glu phe val tgc agt gat gag gaa gaa ggt cac cag tca ttt atg gct cct tct tgt 816 cys ser asp glu glu glu gly his gln ser phe met ala pro ser cys agt gtt ttg cac tgc cct gcc gcc tgt acc tgt agc aac aat atc gta 864 gac tgt cgt ggg aaa ggt ctc act gag atc ccc aca aat ctt cca gag 912 asp cys arg gly lys gly leu thr glu ile pro thr asn leu pro glu acc atc aca gaa ata cgt ttg gaa cag aac aca atc aaa gtc atc cct 960 cct gga gct ttc tca cca tat aaa aag ctt aga cga att gac ctg agc 1008 pro gly ala phe ser pro tyr lys lys leu arg arg ile asp leu ser aat aat cag atc tct gaa ctt gca cca gat gct ttc caa gga cta cgc 1056 asn asn gln ile ser glu leu ala pro asp ala phe gln gly leu arg tct ctg aat tca ctt gtc ctc tat gga aat aaa atc aca gaa ctc ccc 1104 ser leu asn ser leu val leu tyr gly asn lys ile thr glu leu pro aaa agt tta ttt gaa gga ctg ttt tcc tta cag ctc cta tta ttg aat 1152 gcc aac aag ata aac tgc ctt cgg gta gat gct ttt cag gat ctc cac 1200 ala asn lys ile asn cys leu arg val asp ala phe gln asp leu his aac ttg aac ctt ctc tcc cta tat gac aac aag ctt cag acc atc gcc 1248 aag ggg acc ttt tca cct ctt cgg gcc att caa act atg cat ttg gcc 1296 lys gly thr phe ser pro leu arg ala ile gln thr met his leu ala cag aac ccc ttt att tgt gac tgc cat ctc aag tgg cta gcg gat tat 1344 gln asn pro phe ile cys asp cys his leu lys trp leu ala asp tyr ctc cat acc aac ccg att gag acc agt ggt gcc cgt tgc acc agc ccc 1392 leu his thr asn pro ile glu thr ser gly ala arg cys thr ser pro cgc cgc ctg gca aac aaa aga att gga cag atc aaa agc aag aaa ttc 1440 cgt tgt tca ggt aca gaa gat tat cga tca aaa tta agt gga gac tgc 1488 ttt gcg gat ctg gct tgc cct gaa aag tgt cgc tgt gaa gga acc aca 1536 phe ala asp leu ala cys pro glu lys cys arg cys glu gly thr thr gta gat tgc tct aat caa aag ctc aac aaa atc ccg gag cac att ccc 1584 val asp cys ser asn gln lys leu asn lys ile pro glu his ile pro cag tac act gca gag ttg cgt ctc aat aat aat gaa ttt acc gtg ttg 1632 gaa gcc aca gga atc ttt aag aaa ctt cct caa tta cgt aaa ata aac 1680 glu ala thr gly ile phe lys lys leu pro gln leu arg lys ile asn ttt agc aac aat aag atc aca gat att gag gag gga gca ttt gaa gga 1728 gca tct ggt gta aat gaa ata ctt ctt acg agt aat cgt ttg gaa aat 1776 gtg cag cat aag atg ttc aag gga ttg gaa agc ctc aaa act ttg atg 1824 val gln his lys met phe lys gly leu glu ser leu lys thr leu met ttg aga agc aat cga ata acc tgt gtg ggg aat gac agt ttc ata gga 1872 leu arg ser asn arg ile thr cys val gly asn asp ser phe ile gly ctc agt tct gtg cgt ttg ctt tct ttg tat gat aat caa att act aca 1920 gtt gca cca ggg gca ttt gat act ctc cat tct tta tct act cta aac 1968 val ala pro gly ala phe asp thr leu his ser leu ser thr leu asn ctc ttg gcc aat cct ttt aac tgt aac tgc tac ctg gct tgg ttg gga 2016 gag tgg ctg aga aag aag aga att gtc acg gga aat cct aga tgt caa 2064 glu trp leu arg lys lys arg ile val thr gly asn pro arg cys gln aaa cca tac ttc ctg aaa gaa ata ccc atc cag gat gtg gcc att cag 2112 lys pro tyr phe leu lys glu ile pro ile gln asp val ala ile gln gac ttc act tgt gat gac gga aat gat gac aat agt tgc tcc cca ctt 2160 tct cgc tgt cct act gaa tgt act tgc ttg gat aca gtc gtc cga tgt 2208 agc aac aag ggt ttg aag gtc ttg ccg aaa ggt att cca aga gat gtc 2256 aca gag ttg tat ctg gat gga aac caa ttt aca ctg gtt ccc aag gaa 2304 thr glu leu tyr leu asp gly asn gln phe thr leu val pro lys glu ctc tcc aac tac aaa cat tta aca ctt ata gac tta agt aac aac aga 2352 ata agc acg ctt tct aat cag agc ttc agc aac atg acc cag ctc ctc 2400 acc tta att ctt agt tac aac cgt ctg aga tgt att cct cct cgc acc 2448 ttt gat gga tta aag tct ctt cga tta ctt tct cta cat gga aat gac 2496 att tct gtt gtg cct gaa ggt gct ttc aat gat ctt tct gca tta tca 2544 ile ser val val pro glu gly ala phe asn asp leu ser ala leu ser cat cta gca att gga gcc aac cct ctt tac tgt gat tgt aac atg cag 2592 his leu ala ile gly ala asn pro leu tyr cys asp cys asn met gln tgg tta tcc gac tgg gtg aag tcg gaa tat aag gag cct gga att gct 2640 trp leu ser asp trp val lys ser glu tyr lys glu pro gly ile ala cgt tgt gct ggt cct gga gaa atg gca gat aaa ctt tta ctc aca act 2688 arg cys ala gly pro gly glu met ala asp lys leu leu leu thr thr ccc tcc aaa aaa ttt acc tgt caa ggt cct gtg gat gtc aat att cta 2736 pro ser lys lys phe thr cys gln gly pro val asp val asn ile leu gct aag tgt aac ccc tgc cta tca aat ccg tgt aaa aat gat ggc aca 2784 tgt aat agt gat cca gtt gac ttt tac cga tgc acc tgt cca tat ggt 2832 cys asn ser asp pro val asp phe tyr arg cys thr cys pro tyr gly ttc aag ggg cag gac tgt gat gtc cca att cat gcc tgc atc agt aac 2880 phe lys gly gln asp cys asp val pro ile his ala cys ile ser asn cca tgt aaa cat gga gga act tgc cac tta aag gaa gga gaa gaa gat 2928 gga ttc tgg tgt att tgt gct gat gga ttt gaa gga gaa aat tgt gaa 2976 gtc aac gtt gat gat tgt gaa gat aat gac tgt gaa aat aat tct aca 3024 tgt gtc gat ggc att aat aac tac aca tgc ctt tgc cca cct gag tat 3072 cys val asp gly ile asn asn tyr thr cys leu cys pro pro glu tyr aca ggt gag ttg tgt 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atc gcg gta gaa ctc tat cgg ggg cgt gtt cgt gcc agc tat gac 3648 his ile ala val glu leu tyr arg gly arg val arg ala ser tyr asp acc ggc tct cat cca gct tct gcc att tac agt gtg gag aca atc aat 3696 thr gly ser his pro ala ser ala ile tyr ser val glu thr ile asn gat gga aac ttc cac att gtg gaa cta ctt gcc ttg gat cag agt ctc 3744 asp gly asn phe his ile val glu leu leu ala leu asp gln ser leu tct ttg tcc gtg gat ggt ggg aac ccc aaa atc atc act aac ttg tca 3792 aag cag tcc act ctg aat ttt gac tct cca ctc tat gta gga ggc atg 3840 lys gln ser thr leu asn phe asp ser pro leu tyr val gly gly met cca ggg aag agt aac gtg gca tct ctg cgc cag gcc cct ggg cag aac 3888 gga acc agc ttc cac ggc tgc atc cgg aac ctt tac atc aac agt gag 3936 gly thr ser phe his gly cys ile arg asn leu tyr ile asn ser glu ctg cag gac ttc cag aag gtg ccg atg caa aca ggc att ttg cct ggc 3984 leu gln asp phe gln lys val pro met gln thr gly ile leu pro gly tgt gag cca tgc cac aag aag gtg tgt gcc cat ggc aca tgc cag ccc 4032 agc agc cag gca ggc ttc acc tgc gag tgc cag gaa gga tgg atg ggg 4080 ccc ctc tgt gac caa cgg acc aat gac cct tgc ctt gga aat aaa tgc 4128 gta cat ggc acc tgc ttg ccc atc aat gcg ttc tcc tac agc tgt aag 4176 val his gly thr cys leu pro ile asn ala phe ser tyr ser cys lys tgc ttg gag ggc cat gga ggt gtc ctc tgt gat gaa gag gag gat ctg 4224 ttt aac cca tgc cag gcg atc aag tgc aag cat ggg aag tgc agg ctt 4272 phe asn pro cys gln ala ile lys cys lys his gly lys cys arg leu tca ggt ctg ggg cag ccc tac tgt gaa tgc agc agt gga tac acg ggg 4320 gac agc tgt gat cga gaa atc tct tgt cga ggg gaa agg ata aga gat 4368 tat tac caa aag cag cag ggc tat gct gct tgc caa aca acc aag aag 4416 gtg tcc cga tta gag tgc aga ggt ggg tgt gca gga ggg cag tgc tgt 4464 gga ccg ctg agg agc aag cgg cgg aaa tac tct ttc gaa tgc act gac 4512 gly pro leu arg ser lys arg arg lys tyr ser phe glu cys thr asp ggc tcc tcc ttt gtg gac gag gtt gag aaa gtg gtg aag tgc ggc tgt 4560 acg agg tgt gtg tcc taaacacact cccggcagct 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val pro pro thr ile ala arg thr pro lys val pro lys gln gly gly met asn trp ala asp glu tyr asn ile ser val asp glu ser tyr asp gln glu met pro cys tyr ile ser gly pro leu val ser asp met asp thr asp ala pro glu asp leu glu ser ser val thr gly ser met ile asn gly trp gly ser ala ile lys ser pro thr ala gln ser lys thr gln leu glu val arg lys arg asp leu pro pro ala lys thr his leu ile gln glu asp ile gln ile val ala gln gly arg thr val thr phe pro cys glu thr lys gly asn pro gln pro ala val phe trp gln lys glu gly ser gln asn ser pro thr gly asp leu thr ile thr asn ile gln arg ser asp ala ala leu leu lys cys lys ala thr gly asp pro leu pro val ile ser trp leu lys glu gly phe thr phe pro gly arg asp pro arg ala thr ile gln glu gln gly thr leu gln ile lys asn leu arg ile ser asp trp ser ala val leu asp val thr glu ser gly ala thr ile ser lys thr asp val thr lys asn ser val thr leu ser trp gln pro gly thr pro gly thr leu pro ala ser ala tyr ile ile glu ala phe ser gln leu tyr thr val arg gly leu arg pro asn thr ile tyr leu phe met val arg ala ile asn pro lys val ser val thr gln xaa lys pro gln gln glu asn gly tyr asp ser asp ser trp cys pro pro leu pro val gln leu glu val arg pro val met val pro lys leu ala ser ile glu arg glu ala leu asp gly arg gln val thr asp leu arg thr asn pro