304 Stainless simbi coil tubing kemikari chikamu, Chimiro cheSPCA6 ectodomain ine yakachengetedzwa superfamily yemapuroteni ane chekuita negamete fusion.

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SPCA6 iprotein-inotaridzirwa pamusoro pesperm iyo yakakosha pakuita gamete fusion panguva yekubereka kwepabonde.Kunyangwe iri basa rakakosha, iro chairo basa reSPCA6 harinzwisisike.Isu tinotsanangudza chimiro chekristaro cheiyo extracellular domain yeSPCA6 pakugadziriswa kwe2.2 Å, ichiburitsa maviri-domain protein inoumbwa neine-stranded bundle uye Ig-like β-sandwiches akabatanidzwa ne quasi-flexible linkers.Chimiro ichi chakafanana neIZUMO1, imwezve mapuroteni ane gamete fusion-associated, zvichiita kuti SPCA6 neIZUMO1 vave nhengo dzemhuri huru yemapuroteni ane hukama nefotereza anonzi pano seIST superfamily.Iyo IST superfamily inotsanangurwa zvine mutsindo neyakamonyoroka ina-helix bundle uye peya ye disulfide-yakabatana CXXC motifs.Kutsvaga-kwakavakirwa AlphaFold kutsvaga kweproteome yemunhu yakaratidza mamwe mapuroteni nhengo dzeiyi superfamily;kunyanya, mazhinji emapuroteni aya anobatanidzwa mugamete fusion.Iyo SPCA6 chimiro uye hukama hwayo kune dzimwe nhengo dzeIST superfamily inopa chinongedzo chisipo muruzivo rwedu rwemammalian gamete fusion.
Hupenyu hwese hwemunhu hunotanga nemagete maviri ehaploid akasiyana: urume hwababa uye zai raamai.Urume uhu ndihwo hunokunda pakusarudza kwakasimba panguva iyo mamiriyoni emaseru embeu anopfuura nepanhengo yemukadzi, achikunda zvipingaidzo zvakasiyana-siyana1 uye anoita capacitation, izvo zvinosimudzira kufamba kwavo uye maitiro ezvikamu zvepamusoro2,3,4.Kunyange kana mbeu neoocyte zvikawanana, maitiro haasati apera.Iyo oocyte yakakomberedzwa nekamuchuru maseru uye glycoprotein barrier inonzi zona pellucida, iyo urume hunofanira kupfuura kuti hupinde muoocyte.Spermatozoa inoshandisa musanganiswa wepamusoro adhesion molecules uye membrane-inobatanidzwa uye yakavanzwa enzymes kukunda izvi zvekupedzisira zvipingamupinyi5.Aya mamorekuru nema enzymes anonyanya kuchengetwa mumukati me membrane uye acrosomal matrix uye anoonekwa kana yekunze membrane yembeu inonyungudika panguva yeacrosomal reaction6.Danho rekupedzisira murwendo rwakadzama urwu chiitiko chekusanganiswa kwesperm-zai, umo masero maviri anosanganisa ma membranes kuti ave chipenyu chimwe chediploid7.Kunyange zvazvo kuita uku kuri kunetsa mukuberekana kwevanhu, kupindirana kwema molecular kunodiwa hakuna kunzwisiswa.
Pamusoro pekusangana kwemagametes, chemistry yekusanganiswa kwema lipid bilayers yakadzidzwa zvakanyanya.Kazhinji, membrane fusion isimba risingafadzi hurongwa hunoda protein catalyst kuti iite shanduko yechimiro inounza mamembrane maviri pedyo pamwe chete, kutyora kuenderera kwawo uye kukonzera fusion8,9.Aya maprotein catalysts anozivikanwa sefusogens uye akawanikwa mune zvisingaverengeki fusion masisitimu.Izvo zvinodiwa kuti hutachiona hupinde mumasero anotambira (semuenzaniso, gp160 muHIV-1, spike muconaviruses, hemagglutinin muhutachiona hwefuruwenza) 10,11,12 placental (syncytin) 13,14,15 uye gamete-inogadzira fusions mune yakaderera eukaryotes ( HAP2/GCS1 muzvirimwa, mapuroti uye arthropods) 16,17,18,19.Mafusogens emagametes emunhu haasati awanikwa, kunyangwe mapuroteni akati wandei akaratidzwa kuve akakosha pakubatanidzwa kwegamete uye fusion.Iyoocyte-yakaratidza CD9, transmembrane protein inodiwa pakusanganisa mbeva nemagametes evanhu, ndiyo yakatanga kuwanikwa 21,22,23.Kunyange zvazvo basa rayo chairo rinoramba risina kujeka, basa rekunamatira, chimiro chekunamatira foci pazai microvilli, uye / kana nzvimbo yakarurama yeoocyte pamusoro pemapuroteni inoita se24,25,26.Mapuroteni maviri anonyanya kukosha akakosha pakusanganiswa kwegamete iprotein yesperm IZUMO127 neoocyte protein JUNO28, uye kushamwaridzana kwavo idanho rakakosha mukuzivikanwa kwegamete uye kunamatira kusati kwasanganiswa.Male Izumo1 knockout mbeva uye hadzi Juno knockout mbeva hadzina kubereka zvachose, mumhando idzi urume hunopinda munzvimbo yeperivitelline asi magametes haasanganise.Saizvozvo, confluence yakaderedzwa apo ma gametes akabatwa ne anti-IZUMO1 kana JUNO27,29 antibodies mumunhu in vitro fertilization miedzo.
Munguva pfupi yapfuura, boka richangobva kuwanikwa remapuroteni anoratidzwa nembeu phenotypically yakafanana ne IZUMO1 uye JUNO20,30,31,32,33,34,35 yakawanikwa.Sperm acrosomal membrane-associated protein 6 (SPACA6) yakaonekwa seyakakosha pakugadzirisa mune yakakura-scale murine mutagenesis kudzidza.Kuiswa kwe transgene muSpaca6 gene kunogadzira nonfusible spermatozoa, kunyange zvazvo idzi spermatozoa inopinda mu perivitelline space 36.Zvidzidzo zvinotevera zvekugogodza mumakonzo zvakasimbisa kuti Spaca6 inodiwa kuti gamete fusion 30,32.SPCA6 inotaridzwa kanenge kari muma testes uye ine maitiro enzvimbo yakafanana neiyo IZUMO1, iyo mukati me intima ye spermatozoa isati yaita acrosomal reaction, uye yobva yatamira kunharaunda ye equatorial mushure mekuita kwe acrosomal 30,32.Spaca6 homologues iripo mumhando dzakasiyana-siyana dzemhuka uye dzimwe eukaryotes 30 uye kukosha kwayo kumunhu gamete fusion kwakaratidzwa nekudziviswa kwekusangana kwevanhu mu vitro nekupikisa SPCA6 30.Kusiyana neIZUMO1 neJUNO, iwo maumbirwo, kudyidzana, uye basa reSPCA6 harina kujeka.
Kuti tinzwisise zviri nani maitiro akakosha ari pasi pekusanganiswa kwembeu nemazai evanhu, izvo zvinozotigonesa kuzivisa zvichazoitika mukuronga mhuri uye kurapwa kwembereko, takaita SPCA6 zvidzidzo zvemaumbirwo uye zvebiochemical.Iyo crystal chimiro cheiyo extracellular domain yeSPCA6 inoratidza ina-helical bundle (4HB) uye immunoglobulin-yakafanana (Ig-yakafanana) domain yakabatana neiyo quasi-flexible regions.Sezvakafanotaurwa mune zvidzidzo zvekare, 7,32,37 iyo domain structure yeSPCA6 yakafanana neyemunhu IZUMO1, uye mapuroteni maviri anogovera maitiro asina kujairika: 4HB ine triangular helical surface uye peya ye disulfide-yakabatana CXXC motifs.Isu tinokurudzira kuti IZUMO1 neSPCA6 zvino vatsanangure mhuri huru, ine hukama hwepamusoro yemapuroteni ane chekuita negamete fusion.Tichishandisa maficha akasarudzika kumhuri yepamusoro, takaita tsvakiridzo yakazara yeAlphaFold structural human proteome, tichizivisa dzimwe nhengo dzemhuri iyi yepamusoro, kusanganisira nhengo dzakati wandei dzakabatanidzwa mukusanganiswa kwegamete uye/kana fetiraiza.Ikozvino zvave kuita sekunge kune yakajairika dhizaini kupeta uye superfamily yemapuroteni ane hukama negamete fusion, uye chimiro chedu chinopa mepu yemamorekuru yeiyi yakakosha chikamu chemunhu gamete fusion mechanism.
SPCA6 is a single-pass transmembrane protein ine imwe N-yakabatana glycan uye matanhatu putative disulfide bond (Figures S1a uye S2).Takaratidza extracellular domain yevanhu SPCA6 (masara 27-246) muDrosophila S2 masero uye takanatsa puroteni tichishandisa nickel affinity, cation exchange, uye size exclusion chromatography (Fig. S1b).Iyo yakacheneswa SPCA6 ectodomain yakagadzikana uye ine homogeneous.Ongororo ichishandisa saizi yekusarudzika chromatography yakasanganiswa nepolygonal light scattering (SEC-MALS) yakaratidza imwe peak ine akaverengerwa molecular huremu hwe26.2 ± 0.5 kDa (Fig. S1c).Izvi zvinoenderana nehukuru hweSPCA6 monomeric ectodomain, zvichiratidza kuti oligomerization haina kuitika panguva yekucheneswa.Mukuwedzera, denderedzwa dichroism (CD) spectroscopy yakaratidza yakasanganiswa α / β chimiro nechinonyunguduka che 51.3 ° C (Fig. S1d, e).Deconvolution yeCD spectra yakaratidza 38.6% α-helical uye 15.8% β-stranded elements (Mufananidzo S1d).
The SPCA6 ectodomain was crystallized using random matrix seeding38 zvichikonzera data set ine resolution ye 2.2 Å (Table 1 uye Figure S3).Kushandisa musanganiswa wechidimbu-based molecular substitution uye SAD phasing data ine bromide exposure yekugadzirisa chimiro (Table 1 uye Figure S4), yekupedzisira yakanatswa modhi ine masara 27-246.Panguva iyo chimiro chakatemwa, pakanga pasina kuyedza kana AlphaFold zvimiro zviripo.Iyo SPCA6 ectodomain inoyera 20 Å × 20 Å × 85 Å, ine helice nomwe nemapfumbamwe β-strand, uye ine elongated tertiary kupetwa yakadzikamiswa nematanhatu disulfide bond (Fig. 1a, b).Iyo isina simba maerekitironi density pakupera kweAsn243 side chain inoratidza kuti iyi yasara iN-yakabatana glycosylation.Chimiro chacho chine madomasi maviri: N-terminal mana-helix bundle (4HB) uye C-terminal Ig-like domain ine yepakati hinge nharaunda pakati pavo (Fig. 1c).
Chimiro cheiyo extracellular domain yeSPCA6.Mutsara dhiyagiramu weiyo extracellular domain yeSPCA6, iwo ruvara rwecheni kubva kuN kuenda kuC-terminus kubva kune yakasviba bhuruu kusvika kune yakasviba tsvuku.Cysteines inobatanidzwa mune disulfide zvisungo zvinoratidzwa mumagenta.b Topology ye extracellular domain ye SPCA6.Shandisa ruvara rumwechete semufananidzo 1a.c SPCA6 extracellular domain.Iyo 4HB, hinge, uye Ig-senge domain strip machati ane mavara orenji, girini, uye bhuruu, zvichiteerana.Iwo akaturikidzana haana kukweverwa pachiyero.
Iyo 4HB domain yeSPCA6 inosanganisira mana makuru helices (helices 1-4), iyo yakarongedzwa nenzira yehelical helix (Fig. 2a), kushandurana pakati peantiparallel uye parallel kupindirana (Fig. 2b).Imwe diki yekuwedzera imwe-turn helix (helix 1′) yakaiswa perpendicular kune sumbu, ichiita katatu ine helices 1 uye 2. Iyi katatu yakakanganiswa zvishoma muhelical-yakamonyoroka packing yeine dense kurongedza kwehelices 3 uye 4 ( Mufananidzo 2a).
4HB N-terminal mitsetse chati.b Maonero epamusoro ebhurugwa remahelikisi mana, imwe neimwe helix yakamisikidzwa nerima rebhuruu paN-terminus uye dema dema paC-terminus.c Pamusoro-pasi spiral vhiri dhiyagiramu ye4HB, ine chisaririra chega chega chinoratidzwa sedenderedzwa rakanyorwa nekodhi yebhii-amino acid;maamino acids mana chete pamusoro pevhiri anoverengwa.Non-polar residues dzine ruvara rweyero, polar uncharged residues dzine ruvara rwegirinhi, zvinochajiswa zvakanaka zvisaririra zvine ruvara rwebhuruu, uye zvinochajiswa zvisina kunaka zvine ruvara rutsvuku.d Zviso zvetrianngular zve 4HB domain, ine 4HBs muorenji uye mahinji mugirini.Maseseti ese ari maviri anoratidza mabhondi akaita setsvimbo disulfide.
4HB inonangidzirwa pane yemukati hydrophobic core inoumbwa zvakanyanya nealiphatic uye inonhuhwirira zvisaririra (Fig. 2c).Nheyo yacho ine disulfide bond pakati peCys41 neCys55 inobatanidza helices 1 ne2 pamwe chete mugonyo rakasimudzwa repamusoro (Fig. 2d).Zvisungo zviviri zve disulfide zvakaumbwa pakati peCXXC motif muHelix 1 'uye imwe CXXC motif inowanikwa pamucheto we-β-hairpin munharaunda yehinge (Fig. 2d).A conservative arginine residue ine basa risingazivikanwe (Arg37) inowanikwa mukati megonyo retatu rinoumbwa nehelices 1′, 1, uye 2. Aliphatic carbon atoms Cβ, Cγ, uye Cδ Arg37 inodyidzana nehydrophobic core, uye mapoka ayo eguanidine anotenderera pakati pehelices 1' uye 1 kuburikidza nekubatana pakati peThr32 musana uye necheni yemativi (Fig. S5a, b).Tyr34 inoenda mugomba ichisiya maviri madiki maburi ayo Arg37 anogona kupindirana neanonyungudutsa.
Ig-like β-sandwich domains imhuri huru yemapuroteni ayo anogovana zvakajairika maviri kana anopfuura akawanda ane stranded amphipathic β-sheets achidyidzana kuburikidza nehydrophobic core 39. C-terminal Ig-like domain yeSPCAA6 ine maitiro akafanana. uye ine zvidimbu zviviri (Fig. S6a).Shepa 1 i beta-pepa retambo ina (tambo D, F, H, uye I) apo tambo F, H, uye ini ndinoumba anti-parallel gadziriro, uye tambo I naD dzinotora kupindirana kwakafanana.Tafura 2 idiki anti-parallel kaviri-yakasungwa beta sheet (tambo E uye G).Chisungo chemukati che disulfide chakaonekwa pakati peC-terminus yeketani yeE uye pakati peH chain (Cys170-Cys226) (Fig. S6b).Iyi disulfide bond inofananidzwa ne disulfide bond mu β-sandwich domain ye immunoglobulin40,41.
Iwo mana-strand β-sheet inomonereka nehurefu hwayo hwese, ichigadzira asymmetrical edges ayo akasiyana muchimiro uye electrostatics.Mupendero wakatetepa inzvimbo yakati sandara ye hydrophobic yemhoteredzo inomira kunze kana ichienzaniswa neyakasara isina kuenzana uye yemagetsi akasiyana siyana muSPCA6 (Fig. S6b, c).Halo yemusana wakafumurwa kabhoni/amino mapoka uye polar side cheni inotenderedza hydrophobic surface (Fig. S6c).Iyo yakafara margin yakafukidzwa necapped helical segment inovhara N-terminal chikamu cheiyo hydrophobic core uye inoumba matatu ehydrogen zvisungo neakavhurika polar boka reF chain backbone (Fig. S6d).Iyo C-terminal chikamu chemupendero uyu inoumba homwe hombe ine chikamu chakafumurwa hydrophobic core.Homwe yakakomberedzwa nemachaji akanaka nekuda kwemaseti matatu eviri arginine zvakasara (Arg162-Arg221, Arg201-Arg205 uye Arg212-Arg214) uye yepakati histidine (His220) (Mufananidzo S6e).
Nharaunda yehinge chikamu chipfupi pakati peiyo helical domain uye Ig-like domain, ine imwe antiparallel ine tambo nhatu β-layer (strand A, B uye C), diki 310 helix, uye akati wandei marefu asina kurongeka helical segments.(Mufananidzo S7).Iyo network ye covalent uye electrostatic contacts munharaunda yehinge inoita seinodzikamisa kutaridzika pakati pe4HB neIg-senge domain.Iyo network inogona kukamurwa kuita zvikamu zvitatu.Chikamu chekutanga chinosanganisira maviri CXXC motifs (27CXXC30 uye 139CXXC142) iyo inoumba peya ye disulfide zvisungo pakati pe β-hairpin mu hinge uye 1′ helix mu4HB.Chikamu chechipiri chinosanganisira kupindirana kwemagetsi pakati peIg-like domain uye hinge.Glu132 muhinge inoumba bhiriji remunyu neArg233 muIg-like domain uye Arg135 muhinge.Chikamu chechitatu chinosanganisira covalent chisungo pakati peIg-like domain uye hinge dunhu.Zvisungo zviviri zve disulfide (Cys124-Cys147 uye Cys128-Cys153) inobatanidza hinge loop kune chinongedzo chinogadziriswa ne electrostatic interactions pakati peGln131 nemusana unoshanda boka, zvichibvumira kupinda kune yekutanga Ig-like domain.cheni.
Chimiro cheSPCA6 ectodomain uye zvivako zvega zve 4HB uye Ig-like domains zvakashandiswa kutsvaga zvinyorwa zvakagadziriswa zvakafanana mumapuroteni databases 42.Isu takaona machisi ane akakwira Dali Z zvibodzwa, madiki akajairwa akatsauka, uye makuru eLALI zvibodzwa (iyo yekupedzisira iri iyo nhamba yezvimiro zvakaenzana zvakasara).Kunyange zvazvo yekutanga 10 hits kubva pakutsvaga ectodomain yakazara (Table S1) yakanga ine Z-score inogamuchirwa ye> 842, kutsvaga kwe4HB kana Ig-like domain chete kwakaratidza kuti zvizhinji zvezvipembenene izvi zvakaenderana ne-β-sandwiches chete.kupeta kwose kwose kunowanikwa mumapuroteni akawanda.Tsvakiridzo dzese dziri nhatu muDali dzakaburitsa chinhu chimwe chete: IZUMO1.
Zvakagara zvichitaurwa kuti SPCA6 neIZUMO1 vanogovana zvimiro zvakafanana7,32,37.Kunyange zvazvo ectodomains yeaya mapuroteni maviri e-gamete fusion-akabatanidzwa anogovana chete 21% sequence identity (Mufananidzo S8a), humbowo hwakaoma, kusanganisira yakachengetedzwa disulfide bond pattern uye yakafanotaurwa C-terminal Ig-like domain muSPCA6, yakabvumira kuedza kwekutanga kuvaka homology modhi yeA a SPCA6 mouse inoshandisa IZUMO1 setemplate37.Chimiro chedu chinosimbisa fungidziro idzi uye chinoratidza chiyero chechokwadi chekufanana.Muchokwadi, iyo SPCA6 uye IZUMO137,43,44 zvimiro zvinogovana zvakafanana-domain architecture (Fig. S8b) yakafanana 4HB uye Ig-like β-sandwich domains yakabatana nehinge region (Fig. S8c).
IZUMO1 uye SPCA6 4HB vane misiyano yakajairwa kubva kune yakajairwa spiral bundles.Yakajairika 4HBs, yakafanana neaya anowanikwa muSNARE protein complexes inobatanidzwa mu endosomal fusion 45,46, ine yakaenzana nzvimbo helices kuchengetedza nguva dzose curvature kumativi epakati axis 47. Kusiyana neizvi, helical domains mune zvose IZUMO1 uye SPCAA6 yakakanganiswa, ine variable curvature uye kusaenzana kurongedza (Mufananidzo S8d).The twist, zvichida inokonzerwa nekatatu yakaumbwa nehelices 1′, 1 uye 2, inochengetwa muIZUMO1 uye SPCA6 uye yakasimbiswa neiyo CXXC motif pahelix 1′.Nekudaro, iyo yekuwedzera disulfide bond inowanikwa muSPCA6 (Cys41 neCys55 inobatanidza zvine hushamwari helices 1 ne2 pamusoro) inogadzira yakapinza apex pakona yekona, zvichiita kuti SPCA6 iwedzere kumonyoroka kupfuura IZUMO1, ine mamwe magonyeti matatu akataurwa.Pamusoro pezvo, IZUMO1 inoshaya Arg37 inocherechedzwa pakati pegomba iri muSPCA6.Mukupesana, IZUMO1 ine yakajairika hydrophobic musimboti wealiphatic uye anonhuwirira masara.
IZUMO1 ine Ig-yakafanana domain ine ine tambo-kaviri uye shanu-tambo β-sheet43.Iyo yakawedzera tambo muIZUMO1 inotsiva coil muSPCA6, iyo inodyidzana neF strand kudzikamisa musana wehydrogen bond mutambo.Chinhu chinonakidza chekuenzanisa ndiyo yakafanotaurwa pamusoro pechaji yeIg-senge madomasi emapuroteni maviri.Iyo IZUMO1 inochajiswa zvakashata pane iyo SPCA6.Imwezve kuchaja inowanikwa pedyo neC-terminus yakatarisana nesperm membrane.Mu SPCA6, nzvimbo dzakafanana dzakanga dzisina kwazvakarerekera kana kuti dzakapomerwa zvakanaka (Fig. S8e).Semuenzaniso, iyo hydrophobic surface (micheto mitete) uye makomba ane chaji (yakafara micheto) muSPCAA6 anochajwa zvisina kunaka muIZUMO1.
Kunyange zvazvo hukama uye chechipiri chekugadzirisa zvinhu pakati peIZUMO1 uye SPCA6 zvakachengetedzwa zvakanaka, kurongeka kwemaitiro eIg-like domains kwakaratidza kuti nzvimbo mbiri dzinosiyana muhutano hwavo hwose maererano nemumwe (Fig. S9).Iyo spiral bundle ye IZUMO1 yakakomberedzwa nezve β-sandwich, ichigadzira iyo yakambotsanangurwa "boomerang" chimiro pa50 ° kubva pakati peaxis.Kusiyana neizvi, helical beam muSPCA6 yaive yakarereka ingangoita 10 ° kune rumwe rutivi.Misiyano yemaitiro aya ingangove nekuda kwekusiyana munharaunda yehinge.Padanho rekutanga rekutevedzana, IZUMO1 uye SPCA6 vanogovana zvishoma kutevedzana kufanana pahinge, kunze kwecysteine, glycine, uye aspartic acid zvisaririra.Nekuda kweizvozvo, hydrogen zvisungo uye electrostatic network zvakasiyana zvachose.β-sheet secondary structure zvinhu zvakagovaniswa ne IZUMO1 ne SPCA6, kunyange zvazvo maketani ari mu IZUMO1 akareba uye 310 helix (helix 5) yakasiyana neSPCA6.Misiyano iyi inokonzeresa kutarisisa kwakasiyana kwedomasi kune maviri mamwe mapuroteni akafanana.
Kutsvaga kwedu kweDali server kwakaratidza kuti SPCA6 neIZUMO1 ndidzo chete zvimiro zviviri zvakatemerwa zvakachengetwa mudura reprotein zvine iyi 4HB kupeta (Table S1).Munguva pfupi yapfuura, DeepMind (Alphabet/Google) yakagadzira AlphaFold, neural network yakavakirwa sisitimu inogona kufanotaura nemazvo 3D zvimiro zveprotein kubva kukutanga sequences48.Nguva pfupi mushure mekunge tagadzirisa iyo SPCA6 chimiro, iyo AlphaFold dhatabhesi yakaburitswa, ichipa fungidziro yemhando yemhando inovhara 98.5% yemapuroteni ese ari mumunhu proteome48,49.Tichishandisa chimiro chedu cheSPCA6 chakagadziriswa semuenzaniso wekutsvagisa, tsvakiridzo yehomology yemuenzaniso muAlphaFold human proteome yakaratidza vamiriri vane zvimwe zvakafanana zvechimiro kune SPCA6 neIZUMO1.Tichifunga nezve incredible kururama kweAlphaFold mukufanotaura SPCA6 (Fig. S10a) -kunyanya 1.1 Å rms ectodomain kana ichienzaniswa neyakagadziriswa chimiro chedu (Fig. S10b) -tinogona kuva nechivimbo chokuti yakaonekwa SPCA6 machisi angangove akarurama.
Kare, PSI-BLAST yaitsvaga IZUMO1 cluster ine mamwe matatu maprotein akabatana nesperm: IZUMO2, IZUMO3, neIZUMO450.AlphaFold yakafanotaura kuti mapuroteni emhuri eIZUMO aya anopetana mu4HB ane disulfide bond patani seIZUMO1 (Figure 3a neS11), kunyange zvazvo asina nzvimbo yakaita seIg.Zvinofungidzirwa kuti IZUMO2 neIZUMO3 maprotein emu membrane ane divi rimwe chete akafanana neIZUMO1, ukuwo IZUMO4 ichiita seyakavharika.Mabasa eIZUMO 2, 3, uye 4 mapuroteni mugamete fusion haana kutsanangurwa.IZUMO3 inozivikanwa kuti inobata basa muacrosome biogenesis panguva yekukura kwembeu51 uye protein yeIZUMO yakaonekwa ichigadzira complex50.Kuchengetedzwa kweIZUMO mapuroteni mumhuka dzinoyamwisa, zvinokambaira, uye mhuka dzinogara mumvura inonaya mvura inopfungaira inoratidza kuti basa razvo rinogoneka rinoenderana nemamwe mapuroteni anozivikanwa ane gamete-fusion-associated, akadai seDCST1/2, SOF1, uye FIMP.
Dhiagiramu yedhizaini yedhizaini yeIST superfamily, ine 4HB, hinge, uye Ig-senge madomasi akasimbiswa mune orenji, girinhi, uye bhuruu, zvichiteerana.IZUMO4 ine yakasarudzika C-terminal dunhu rinotaridzika dema.Yakasimbiswa uye inoisa disulfide zvisungo zvinoratidzwa nemitsara yakasimba uye ine doti, zvichiteerana.b IZUMO1 (PDB: 5F4E), SPCA6, IZUMO2 (AlphaFold DB: AF-Q6UXV1-F1), IZUMO3 (AlphaFold DB: AF-Q5VZ72-F1), IZUMO4 (AlphaFold DB: AF-EMQ1ZYL8-F95), (AlphaFold TM) DB: AF-Q1ZYL8-F1) : AF-Q1ZYL8-F1) : AF-Q3KNT9-F1) inoratidzwa mumhando imwechete yeruvara sepanera A. Disulfide bonds inoratidzwa mumagenta.TMEM95, IZUMO2 uye IZUMO3 transmembrane helices haina kuratidzwa.
Kusiyana neprotein yeIZUMO, mamwe mapuroteni eSPCA (kureva, SPCA1, SPCA3, SPCA4, SPCA5, uye SPCA9) anofungidzirwa kuti akasiyana neSPCA6 (Fig. S12).SPCA9 chete ine 4HB, asi haitarisirwe kuve neyakafanana-inopokana-inotarisana neyakafanana kana imwechete disulfide bond seSPCA6.Chete SPCA1 ine yakafanana Ig-senge domain.AlphaFold inofanotaura kuti SPCA3, SPCA4 uye SPCA5 ine chimiro chakasiyana zvachose pane SPCA6.Sezvineiwo, SPCA4 inozivikanwa zvakare kuita basa mukusangana, asi kusvika pamwero mukuru kupfuura SPCA6, pachinzvimbo ichifambisa kudyidzana pakati pesperm ne oocyte zona pellucida52.
Kutsvaga kwedu kweAlphaFold kwakawana mumwe mutambo weIZUMO1 uye SPCA6 4HB, TMEM95.TMEM95, imwe sperm-specific transmembrane protein, inoita kuti mbeva dzechirume dzishaye mbereko kana dzabviswa 32,33.Spermatozoa yaishaya TMEM95 yaive neyakajairika morphology, motility, uye kugona kupinda muzona pellucida nekusunga kune membrane yezai, asi yaisagona kusanganisa neoocyte membrane.Zvidzidzo zvakapfuura zvakaratidza kuti TMEM95 inogovera kufanana kwechimiro neIZUMO133.Zvechokwadi, mufananidzo weAlphaFold wakasimbisa kuti TMEM95 i4HB ine mbiri yakafanana yeCXXC motifs seIZUMO1 uye SPCA6 uye imwechete yekuwedzera disulfide bond pakati pehelices 1 uye 2 inowanikwa muSPCA6 (Fig. 3a uye S11).Kunyange zvazvo TMEM95 isina nzvimbo yeIg-like, ine nharaunda ine disulfide bond pattern yakafanana neSPCA6 uye IZUMO1 hinge regions (Fig. 3b).Panguva yekuburitswa kwechinyorwa ichi, preprint server yakashuma chimiro cheTMEM95, ichisimbisa iyo AlphaFold53 mhedzisiro.TMEM95 yakafanana zvikuru neSPCA6 neIZUMO1 uye inochengetedzwa neshanduko kare mumhuka dzinonzi amphibians (Fig. 4 neS13).
Tsvakiridzo yePSI-BLAST yakashandisa NCBI SPCA6, IZUMO1-4, TMEM95, DCST1, DCST2, FIMP, neSOF1 dhatabhesi kuti ione nzvimbo yenhevedzano iyi mumuti wehupenyu.Nzvimbo dziri pakati pemapazi emazita hazviratidzirwe kuyera.
Kufanana kunokatyamadza kwemaitiro pakati peSPCA6 neIZUMO1 kunoratidza kuti ivo ndivo vanovamba nhengo dzemhuri yakachengetedzwa yakasarudzika inosanganisira TMEM95 neIZUMO 2, 3, uye 4 mapuroteni.nhengo dzinozivikanwa: IZUMO1, SPCA6 uye TMEM95.Nekuti nhengo shoma chete dzine maIg-like domains, chiratidzo cheIST superfamily ndiyo 4HB domain, ine yakasarudzika maficha akajairika kune ese mapuroteni aya: 1) Yakavharidzirwa 4HB ine helices yakarongwa mune anti-parallel/parallel alternation (Fig. 5a), 2) bundle rine chiso chetatu chinosanganisira helice mbiri mukati memusumbu uye yechitatu vertical helix (Fig. key area (Fig. 5c) CXXC motif, inowanikwa mu thioredoxin-like proteins, inozivikanwa kushanda. se redox sensor 54,55,56 , nepo motif mu IST nhengo dzemhuri inogona kusanganiswa neprotein disulfide isomerases yakadai seERp57 mu gamete fusion.
Nhengo dzeIST superfamily dzinotsanangurwa nehunhu hutatu hwenzvimbo ye4HB: mahelikisi mana anochinjana pakati peparallel uye antiparallel orientation, ba-triangular helical bundle faces, uye ca CXXC kaviri motif yakaumbwa pakati pemamorekuru madiki.) N-terminal helixes (orenji) uye hinge dunhu β-hairpin (girinhi).
Tichifunga nezvekufanana pakati peSPCA6 neIZUMO1, kugona kweiyo yekutanga kusunga kuIZUMO1 kana JUNO kwakaedzwa.Biolayer interferometry (BLI) ndeye kinetic-based binding method yakashandiswa kare kuyera kuwirirana pakati peIZUMO1 neJUNO.Mushure mokuvharidzirwa kwebiotin-yakanyorwa sensor ne IZUMO1 sechiredzo chine huwandu hwepamusoro hweJUNO analyte, chiratidzo chakasimba chakaonekwa (Fig. S14a), zvichiratidza kusungirirwa-kushanduka kwekuchinja kwehuwandu hwebiomaterial yakasungirirwa kune imwe pfungwa.Zviratidzo zvakafanana (kureva, JUNO yakabatanidzwa kune sensor sechiredzo chinopesana neIZUMO1 analyte) (Fig. S14b).Hapana chiratidzo chakaonekwa apo SPCA6 yakashandiswa seanalyte inopesana ne sensor-bound IZUMO1 kana sensor-bound JUNO (Mufananidzo S14a,b).Kusavapo kwechiratidzo ichi kunoratidza kuti extracellular domain yeSPCA6 haipindirani neiyo extracellular domain ye IZUMO1 kana JUNO.
Nekuti BLI assay yakavakirwa pabiotinylation yemahara lysine residues pane chirauro protein, ichi chigadziriso chinogona kudzivirira kusunga kana lysine masara achibatanidzwa mukudyidzana.Pamusoro pezvo, kutariswa kwechisungo chehukama kune sensor kunogona kugadzira zvipingamupinyi zvesteric, saka zvakajairwa kudonhedza-pasi assays zvakaitwawo pane recombinant SPCA6, IZUMO1 uye JUNO ectodomains.Pasinei neizvi, SPCA6 haina kukurumidza neYake-tagged IZUMO1 kana Yake-tagged JUNO (Fig. S14c, d), ichiratidza kuti hapana kupindirana kunoenderana neyakaonekwa mukuedza kweBLI.Sekutonga kwakanaka, takasimbisa kudyidzana kweJUNO ine yakanyorwa Yake IZUMO1 (Figure S14e uye S15).
Pasinei nekufanana kwechimiro pakati peSPCA6 neIZUMO1, kusakwanisa kweSPCA6 kusunga JUNO hazvishamisi.Pamusoro pemunhu IZUMO1 ine zvinodarika makumi maviri zvakasara zvinodyidzana neJUNO, kusanganisira zvakasara kubva kune imwe neimwe yematunhu matatu (kunyangwe mazhinji acho ari munharaunda yehinge) (Fig. S14f).Pazvisaririra izvi, imwe chete ndiyo inochengetedzwa muSPCA6 (Glu70).Nepo zvakawanda zvinotsiva zvisaririra zvakachengeta hunhu hwazvo hwepakutanga hwebiochemical, zvakakosha Arg160 zvakasara muIZUMO1 zvakatsiviwa neAsp148 yakachajiswa zvisina kunaka muSPCAA6;zvidzidzo zvekare zvakaratidza kuti Arg160Glu mutation muIZUMO1 inopotsa yabvisa zvachose kusunga kuJUNO43.Pamusoro pezvo, mutsauko wekutenderera kwedomasi pakati peIZUMO1 neSPCA6 wakawedzera zvakanyanya nzvimbo yeJUNO-inosunga saiti yenzvimbo yakaenzana paSPCA6 (Fig. S14g).
Zvisinei nekudiwa kunozivikanwa kweSPCA6 yegamete fusion uye kufanana kwayo neIZUMO1, SPCA6 hairatidzike kunge ine basa rekusunga rakafanana neJUNO.Naizvozvo, takatsvaga kubatanidza data redu rezvimiro nehumbowo hwekukosha hunopihwa neiyo evolutionary biology.Sequence alignment yeSPCA6 homologues inoratidza kuchengetedza kwechimiro chakajairwa kupfuura mhuka dzinoyamwisa.Semuenzaniso, cysteine ​​​​residues iripo kunyange mune amphibians ane hukama kure (Fig. 6a).Uchishandisa sevha yeConSurf, yakawanda kutevedzana kwekuchengetedza data ye66 sequences yakamepurwa kune SPCA6 pamusoro.Rudzi urwu rwekuongorora runogona kuratidza kuti ndeapi masara akachengetedzwa panguva yekushanduka kweprotein uye anogona kuratidza kuti ndeapi nzvimbo dzepamusoro dzinoita basa.
a Sequence alignment yeSPCA6 ectodomains kubva kumarudzi gumi nemaviri akasiyana akagadzirirwa uchishandisa CLUSTAL OMEGA.Zvinoenderana nekuongorora kweConSurf, nzvimbo dzakanyanya kuchengetedza dzakaiswa mubhuruu.Cysteine ​​​​zvisaririra zvinoratidzwa mutsvuku.Domain miganhu uye chechipiri chimiro zvinhu zvinoratidzwa pamusoro pekugadzirisa, apo miseve inoratidza β-strands uye masaisai anoratidza helices.Iyo NCBI Access Identifiers ine zvakatevedzana ndeiyi: vanhu (Homo sapiens, NP_001303901), mandrill (Mandrilus leucophaeus, XP_011821277), capuchin monkey (Cebus mimic, XP_017359366), bhiza (Equus 0002 Orchea3), XP_017359366 23 XP_032_034) .), hwai (Ovis aries, XP_014955560), nzou (Loxodonta africana, XP_010585293), imbwa (Canis lupus familyis, XP_025277208), mbeva (Mus musculus, NP_001156381), Tasmanian africana_1, XP_XP_38 dhiyabhorosi 3 , 8) , 61_89 neBullfrog (Bufo bufo, XP_040282113).Kuverengera kunoenderana nekurongeka kwevanhu.b Kumiririrwa kwepamusoro kweiyo SPCA6 chimiro chine 4HB kumusoro uye Ig-sedomasi pazasi, mavara anoenderana nekuchengetedza fungidziro kubva kuConSurf server.Zvikamu zvakachengetedzwa zvakanakisisa zviri mubhuruu, zvikamu zvakachengetwa zvine mwero zviri muchena, uye zvishoma zvakachengetwa zviri muyero.yepepuru cysteine.Mapeche matatu epamusoro anoratidza kudzivirira kwepamusoro anoratidzwa muchikamu chakanyorwa zvigamba 1, 2 uye 3. Katuni ye4HB inoratidzwa muchikamu chiri kumusoro kurudyi (same color scheme).
Iyo SPCA6 chimiro chine matatu akachengetedzwa zvakanyanya pamusoro penzvimbo (Fig. 6b).Patch 1 inotenderera 4HB uye nharaunda yehinge uye ine maviri akachengetedzwa CXXC disulfide mabhiriji, Arg233-Glu132-Arg135-Ser144 hinge network (Fig. S7), uye matatu akachengetedzwa ekunze anonhuhwirira masara (Phe31, Tyr73, Phe137).rim rakafara reIg-like domain (Fig. S6e), iyo inomiririra akati wandei anochajiswa masara paurume hwepamusoro.Sezvineiwo, chigamba ichi chine antibody epitope yakamboratidzwa kuvhiringa SPCA6 30 basa.Dunhu 3 rinotenderera hinge uye rimwe divi reIg-senge domain;nharaunda iyi ine prolines akachengetedzwa (Pro126, Pro127, Pro150, Pro154) uye kunze-yakatarisana nepolar/charge residues.Zvinotoshamisa ndezvokuti, zvizhinji zvezvisara zviri pamusoro pe4HB zvakanyanya kushanduka (Fig. 6b), kunyange zvazvo kupeta kwakachengetedzwa mukati meSPCA6 homologue (sezvinoratidzwa nekuchengetedza kweiyo hydrophobic bundle core) uye kunze kweIST superfamily.
Kunyangwe iyi iri dunhu diki muSPCA6 ine mashoma anoonekwa echipiri maumbirwo emhando, akawanda hinge nharaunda yakasara (kusanganisira dunhu 3) inochengetedzwa zvakanyanya pakati peSPCA6 homologues, izvo zvinogona kuratidza kuti kutaridzika kwehelical bundle uye β-sandwich inoita basa.seanochengetedza.Zvakadaro, zvisinei nekuwanda kwehydrogen bonding uye maelectrostatic network munzvimbo yehinge yeSPCA6 neIZUMO1, humbowo hwekuchinjika kwemukati hunogona kuonekwa mukurongeka kwezvimiro zvinotenderwa zvakawanda zveIZUMO137,43,44.Kurongeka kwemadomasi ega ega aipindirana zvakanaka, asi kutariswa kwemadomasi kune mumwe nemumwe kwakasiyana kubva ku50 ° kusvika 70 ° kubva pakati peaxis (Fig. S16).Kuti unzwisise conformational dynamics ye SPCA6 mune mhinduro, SAXS miedzo yakaitwa (Fig. S17a, b).Ab initio reconstruction of the SPCA6 ectodomain inopindirana nerod crystal structure (Fig. S18), kunyange zvazvo Kratky plot yakaratidza imwe dhigirii yekushandura (Fig. S17b).Kuwirirana uku kunosiyana neIZUMO1, umo protein isina kusungwa inotora chimiro cheboomerang zvese mulatisi uye mumushonga43.
Kunyatsoona nzvimbo inoshanduka, hydrogen-deuterium exchange mass spectroscopy (H-DXMS) yakaitwa paSPCA6 uye ichienzaniswa nedheta rakambowanikwa paIZUMO143 (Fig. 7a,b).SPCA6 inonyatso kuchinjika kupfuura IZUMO1, sezvinoratidzwa nepamusoro deuterium exchange mukati mechimiro mushure me100,000 s yekuchinjana.Mune zvese zviri zviviri zvimiro, iyo C-terminal chikamu chehinge dunhu rinoratidza mwero wepamusoro wekutsinhana, izvo zvingangobvumira kutenderera kudiki kwe4HB uye Ig-senge madomasi ane hukama kune mumwe nemumwe.Sezvineiwo, C-terminal chikamu cheSPCA6 hinge, inosanganisira 147CDLPLDCP154 yakasara, inzvimbo yakachengetedzwa zvikuru 3 (Fig. 6b), zvichida ichiratidza kuti interdomain flexibility chinhu chakachengetedzwa chakachengetedzwa cheSPCA6.Maererano nekuongorora kushanduka, CD thermal melt data yakaratidza kuti SPCA6 (Tm = 51.2 ° C) haina kugadzikana kupfuura IZUMO1 (Tm = 62.9 ° C) (Fig. S1e uye S19).
a H-DXMS mifananidzo yeSPCA6 uye b IZUMO1.Iyo percentage deuterium exchange yakatemerwa panguva dzakatarwa nguva.Mwero yehydrogen-deuterium exchange inoratidzwa neruvara pachiyero che gradient kubva kubhuruu (10%) kusvika kune tsvuku (90%).Mabhokisi madema anomiririra nzvimbo dzekutsinhana kwepamusoro.Iyo miganhu ye4HB, hinge uye Ig-senge domain inocherechedzwa mune yekristaro chimiro inoratidzwa pamusoro pekutanga kutevedzana.Deuterium exchange level at 10 s, 1000 s, and 100,000 s akarongerwa pastrip chart akaiswa pamusoro pepachena mamorekuru epaSPCA6 neIZUMO1.Zvikamu zvezvivakwa zvine deuterium exchange level pazasi 50% zvine mavara machena.Nzvimbo dziri pamusoro pe50% H-DXMS kuchinjanisa dzine mavara muchiyero chegradient.
Kushandiswa kweCRISPR/Cas9 uye mbeva gene kugogodza geneti nzira kwatungamira mukuzivikanwa kwezvinhu zvakati wandei zvakakosha kune urume uye zai kusunga uye fusion.Kunze kwekubatana kwakanaka kweIZUMO1-JUNO uye CD9 chimiro, mapuroteni akawanda ane chokuita negamete fusion anoramba ari maitiro uye anoshanda enigmatic.Iyo biophysical uye chimiro chechimiro cheSPCA6 chimwe chidimbu cheadhesion/fusion molecular puzzle panguva yekusangana.
SPCA6 nedzimwe nhengo dzeIST superfamily dzinoita sedzinochengetedzwa zvakanyanya mumhuka dzinoyamwisa pamwe chete neshiri imwe neimwe, zvinokambaira, uye mhuka dzemufibhi;kutaura zvazviri, zvinofungidzirwa kuti SPCA6 inotodiwa pakusangana kwezebrafish 59. Kugoverwa uku kwakafanana nemamwe mapuroteni anozivikanwa egamete fusion-associated akadai DCST134, DCST234, FIMP31, uye SOF132, zvichiratidza kuti zvinhu izvi HAP2-inoshaya (zvakare. inozivikanwa seGCS1) mapuroteni ane mutoro wekuita kwekuita kwevazhinji maprotist., zvinomera, uye arthropods.Fertilized fusion proteins 60, 61. Pasinei nekufanana kwakasimba kwechimiro pakati peSPCA6 neIZUMO1, kugogodza kwemajini encoding imwe yeiyi mapuroteni kwakaguma nekushaya mbereko mumakonzo emurume, zvichiratidza kuti mabasa avo mugamete fusion haana kudhindwa..Zvakawanda zvakanyanyisa, hapana yeanozivikanwa mapuroteni esperm anodiwa kune adhesion chikamu chefusion akawandisa.
Inoramba iri mubvunzo wakavhurika kana SPCA6 (nedzimwe nhengo dzeIST superfamily) dzichitora chikamu mumijaho yeintergametic, inogadzira intragametic network yekutora mapuroteni akakosha kune fusion point, kana pamwe kutoita seasinganzwisisike fusogens.Co-immunoprecipitation zvidzidzo muHEK293T masero zvakaratidza kudyidzana pakati pehurefu hwakazara IZUMO1 uye SPCA632.Nekudaro, yedu recombinant ectodomains haina kupindirana mu vitro, zvichiratidza kuti kudyidzana kunoonekwa muNoda et al.zvose zvakadzimwa mukuvaka (ona iyo cytoplasmic muswe weIZUMO1, iyo yakaratidzwa kuva isingakodzeri pakuberekesa62).Neimwe nzira, IZUMO1 uye/kana SPCA6 ingangoda nharaunda dzakasungirirwa dzatisingaburitse mu vitro, senge physiologically conformations kana mamolecular complexes ane mamwe mapuroteni (anozivikanwa kana asati awanikwa).Kunyangwe iyo IZUMO1 ectodomain ichitendwa kuti inoyananisa kusungirirwa kwespermatozoa kune zai riri munzvimbo yeperivitelline, chinangwa cheSPCA6 ectodomain hachina kujeka.
Mamiriro eSPCA6 anoburitsa akati wandei akachengeterwa nzvimbo angangove akabatanidzwa mukudyidzana kweprotein-protein.Iyo yakachengetedzwa chikamu chehinge dunhu chiri padyo neCXXC motif (yakasarudzwa Patch 1 pamusoro) ine akati wandei akatarisana nekunze anonhuhwirira zvisaririra izvo zvinowanzobatanidzwa nehydrophobic uye π-stacking kudyidzana pakati pebiomolecules.Mativi akafara eIg-like domain (region 2) anoumba groove ine positive charged ine Arg yakachengetedzwa zvakanyanya uye masara ake, uye masoja ekudzivirira chirwere anorwisa dunhu iri akamboshandiswa kuvhara gamete fusion 30.Antibody inoziva mutsara epitope 212RIRPAQLTHRGTFS225, iyo ine matatu pamatanhatu arginine asara uye akachengetedzwa zvakanyanya His220.Hazvina kujeka kana kusashanda kuri kukonzerwa nekuvharika kwezvisaririra izvi kana dunhu rese.Nzvimbo yepakati iri padhuze neC-terminus ye β-sandwich inoratidza cis-kudyidzana nemapuroteni eurume akavakidzana, asi kwete neoocyte mapuroteni.Uyezve, kuchengetwa kweproline-rich tangle yakanyanyisa (nzvimbo 3) mukati mehinge inogona kunge iri nzvimbo yeprotein-protein interaction kana, zvichida, inoratidza kuchengetwa kwekugadzirisa pakati pezvikamu zviviri.Gender yakakosha pabasa risingazivikanwe reSPCA6.kusanganiswa kwe gametes.
SPCA6 ine zvimiro zve intercellular adhesion proteins, kusanganisira Ig-like β-sandwiches.Mapuroteni akawanda anonamira (eg, cadherins, integrins, adhesins, uye IZUMO1) ane imwe kana kupfuura β-sandwich domains inotambanudza mapuroteni kubva musero membrane kuenda kune yavo yezvakatipoteredza zvinangwa63,64,65.Ig-like domain yeSPCA6 inewo motif inowanzowanikwa mu β-sandwiches yekunamatira uye kubatana: mapeji maviri emakumbo akafanana pamagumo e-β-sandwiches, anozivikanwa se mechanical clamps66.Zvinotendwa kuti iyi motif inowedzera kuramba kune shear forces, iyo inokosha kune mapuroteni anobatanidzwa mukubatana kwe intercellular.Nekudaro, zvisinei nekufanana uku kune adhesin, parizvino hapana humbowo hwekuti SPCA6 inodyidzana nevachena zai.Iyo SPCA6 ectodomain haikwanisi kusunga kuJUNO, uye SPCA6-inotaura HEK293T masero, sezvaratidzwa pano, haatombodyidzana nemaoocyte asina iyo 32.Kana SPCA6 ikamisa zvisungo zveintergametic, kudyidzana uku kungangoda kugadziridzwa mushure mekushandura kana kudzikamiswa nemamwe mapuroteni embeu.Mukutsigira kwekupedzisira hypothesis, IZUMO1-inoshaya spermatozoa inosunga maoocytes, ichiratidza kuti mamorekuru kunze kweIZUMO1 anobatanidzwa mumutambo wekunamatira danho 27.
Mazhinji hutachiona, masero, uye ekusimudzira fusion mapuroteni ane zvivakwa zvinofanotaura basa ravo sefusogens.Semuenzaniso, viral fusion glycoproteins (makirasi I, II uye III) ane hydrophobic fusion peptide kana loop pamagumo eprotein inopinzwa mu membrane yevaenzi.Mepu yehydrophilicity yeIZUMO143 uye chimiro (chakatemerwa uye chakafanotaurwa) cheIST superfamily chakaratidza hapana inooneka hydrophobic fusion peptide.Saka, kana chero mapuroteni ari muIST superfamily anoshanda sefusogens, vanozviita nenzira yakasiyana nemimwe mienzaniso inozivikanwa.
Mukupedzisa, mashandiro enhengo dzeIST superfamily yemapuroteni ane hukama negamete fusion anoramba ari chakavanzika chinokatyamadza.Yedu inozivikanwa SPCA6 recombinant molecule uye chimiro chayo chakagadziriswa chinopa nzwisiso muhukama pakati peizvi zvakagovaniswa zvimiro uye basa ravo mukunamatira gamete uye fusion.
Kutevedzana kweDNA kunoenderana neyakafanotaurwa yemunhu SPCA6 ectodomain (NCBI accession number NP_001303901.1; residues 27–246) yakagadziridzwa codon-optimized for expression in Drosophila melanogaster S2 cells and synthesized as a gene fragment with the sequence encoding GeknomicEncoding Koza., chiratidzo cheBiP chekuvhara uye chinopindirana 5′ uye 3′ inogumira pakubatanidzwa kwakazvimirira kwejini iyi kuita pMT expression vector zvichibva papromota yemetallothionein yakagadziridzwa kuti isarudzwe nepuromycin (pMT-puro).Iyo pMT-puro vector inoisa thrombin cleavage saiti ichiteverwa ne10x-Yake C-terminal tag (Mufananidzo S2).
Kuchinja kwakagadzikana kweSPCA6 pMT-puro vector muD. melanogaster S2 (Gibco) masero akaitwa zvakafanana neprotocol inoshandiswa IZUMO1 uye JUNO43.Masero eS2 akanyungudutswa uye akakura muSchneider's medium (Gibco) akawedzerwa nechikamu chekupedzisira che10% (v/v) kupisa-inactivated fetal mhuru serum (Gibco) uye 1X antimycotic antibiotic (Gibco).Masero ekutanga (3.0 x 106 masero) akaputirwa mumatsime ega e6-tsime mahwendefa (Corning).Mushure memaawa makumi maviri nemana ekukuchidzira pa 27°C, maseru akachinjwa nemusanganiswa we 2 mg ye SPCA6 pMT-puro vector uye Effectene transfection reagent (Qiagen) maererano neprotocol yemugadziri.Masero akachinjwa akaiswa kwemaawa makumi manomwe nemaviri ndokuzokohwewa ne 6 mg/ml puromycin.Masero akabva aparadzaniswa nepakati peSchneider's akazara uye akaiswa muserum isina Insect-XPRESS yepakati (Lonza) yekugadzira mapuroteni makuru.A 1 L batch yeS2 cell tsika yakakura kusvika 8-10 × 106 ml-1 maseru mu2 L inofefetedza flat-bottomed polypropylene Erlenmeyer flask uye ndokuzoitwa sterilized nekupedzisira kuungana kwe500 µM CuSO4 (Millipore Sigma) uye isina kupepetwa.induced.Tsika dzakakonzerwa dzakaiswa pa 27 ° C. pa 120 rpm kwemazuva mana.
Conditioned medium ine SPCA6 yakaparadzaniswa necentrifugation pa 5660 × g pa 4 ° C ichiteverwa neCentramate tangential flow filtration system (Pall Corp) ine 10 kDa MWCO membrane.Isa yakadzika midzi ine SPCA6 kune 2 ml Ni-NTA agarose resin (Qiagen) column.Iyo Ni-NTA resin yakagezwa ne10 column volumes (CV) yebuffer A uyezve 1 CV yebuffer A yakawedzerwa kupa midazole yekupedzisira ye50 mM.SPCA6 yakaburitswa ne 10 ml yebuffer A yakawedzerwa nemidazole kusvika kuchikamu chekupedzisira che500 mM.Restriction class thrombin (Millipore Sigma) yakawedzerwa zvakananga ku dialysis tubing (MWCO 12-14 kDa) pa 1 unit per mg SPCAA6 vs. 1 L 10 mM Tris-HCl, pH 7.5 uye 150 mM NaCl (buffer B) ye dialysis.) pa4°C kwemaawa makumi mana nemasere.The thrombin-cleaved SPCA6 yakabva yadirwa katatu kuti ideredze kuwedzera kwemunyu uye kutakurwa pa1 ml MonoS 5/50 GL cation exchange column (Cytiva/GE) yakaenzana ne10 mM Tris-HCl, pH 7.5.Iyo cation exchanger yakagezwa ne3 CV ye10 mM Tris-HCl, pH 7.5, ipapo SPCA6 yakaburitswa ine linear gradient ye0 kusvika 500 mM NaCl mu10 mM Tris-HCl, pH 7.5 ye25 CV.Mushure meion exchange chromatography, SPCA6 yakanamirwa ku 1 ml uye yakaburitswa isocratically kubva kuENrich SEC650 10 x 300 column (BioRad) yakaenzana nebuffer B. Maererano nechromatogram, dziva uye concentrate zvikamu zvine SPCAA6.Kuchena kwaidzorwa neCoomassie-stained electrophoresis pane 16% SDS-polyacrylamide gel.Protein concentration yakaverengerwa nekunyura pa280 nm uchishandisa mutemo weBeer-Lambert uye theoretical molar extinction coefficient.
Yakanatswa SPCA6 yakaitwa dialyzed usiku hwose inopesana ne10 mM sodium phosphate, pH 7.4 uye 150 mM NaF uye yakaderedzwa kusvika 0.16 mg/mL isati yaongororwa neCD spectroscopy.Spectral scanning yeCDs ine wavelength ye185 kusvika 260 nm yakaunganidzwa paJasco J-1500 spectropolarimeter uchishandisa quartz cuvettes ine 1 mm optical nzira urefu (Helma) pa25 ° C pamwero we50 nm/min.Iwo maCD spectra aive ekutanga akagadziridzwa, akaenzana pamusoro pe10 kutora, uye akashandurwa kureva residual elliptical (θMRE) mumadhigirii cm2/dmol:
uko MW ndiyo huremu hwemamorekuru emuenzaniso wega wega muDa;N nhamba yeamino acids;θ ndiyo ellipcity mumamillidegrees;d inopindirana nehurefu hwenzira yemaziso mu cm;protein concentration muzvikamu.