I-304 yensimbi engenastainless coil tubing chemical component, Ulwakhiwo lwe-SPCAA6 ectodomain luqulathe usapho olukhulu olugciniweyo lweeproteni ezinxulumene nokudityaniswa kwegamete.

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I-SPCAA6 yiprotein yomphezulu evezwe ngamadlozi ebaluleke kakhulu ekudibaneni kwegamete ngexesha lokuzala kwezilwanyana ezincancisayo.Ngaphandle kwale ndima ingundoqo, umsebenzi othile we-SPCAA6 awuqondwa kakuhle.Sicacisa isakhiwo se-crystal ye-domain ye-extracellular ye-SPCAA6 kwisisombululo se-2.2 Å, ityhila iprotheni ye-domain-domain ehlanganiswe ne-bundle ezine-stranded kunye ne-Ig-like β-sandwiches ezidityaniswe yi-quasi-flexible linkers.Esi sakhiwo sifana ne-IZUMO1, enye iprotein ehambelana ne-gamete fusion-associated, eyenza i-SPCAA6 kunye ne-IZUMO1 ibe ngamalungu aseka i-superfamily yeeproteni ezinxulumene nokuchumisa ekubhekiswa kuzo apha njenge-IST superfamily.I-IST superfamily ichazwa ngokwesakhiwo ngokujija kwayo i-helix ezine-helix kunye ne-disulfide-linked CXXC motifs.Uphando lwe-AlphaFold olusekwe kwisakhiwo seproteome yomntu ichonge amalungu eprotheyini eyongezelelweyo yale superfamily;ngokuphawulekayo, ezininzi zezi proteni zibandakanyeka kwi-gamete fusion.Ulwakhiwo lwe-SPACA6 kunye nobudlelwane bayo namanye amalungu e-IST superfamily ibonelela ngekhonkco elilahlekileyo kulwazi lwethu lwe-mammalian gamete fusion.
Bonke ubomi bomntu buqala ngeegamete ezimbini ezahlukeneyo ze-haploid: idlozi likabawo kunye neqanda likamama.Le sperm iphumelele inkqubo yokhetho ebukhali apho izigidi zeeseli zesidoda zidlula kwilungu lobufazi, zoyise imiqobo eyahlukeneyo1 kwaye zingene kwi-capacitation, ephucula ukuhamba kwazo kunye nenkqubo yamalungu omphezulu2,3,4.Nangona i-sperm kunye ne-oocyte bafumana enye kwenye, inkqubo ayikapheli.I-oocyte ijikelezwe ngumaleko weeseli ze-cumulus kunye nomqobo we-glycoprotein obizwa ngokuba yi-zona pellucida, ekufuneka idlozi lidlule kuyo ukuze lingene kwi-oocyte.I-Spermatozoa isebenzisa indibaniselwano yeemolekyuli zokubambelela kumphezulu kunye ne-membrane edibeneyo kunye ne-enzyme efihliweyo ukuze inqobe le miqobo yokugqibela5.Ezi molekyuli kunye nee-enzyme zigcinwa ikakhulu kwi-membrane yangaphakathi kunye ne-acrosomal matrix kwaye zichongwa xa i-membrane yangaphandle yesidoda i-lysed ngexesha le-acrosomal reaction6.Inyathelo lokugqibela kolu hambo lunzima sisiganeko sokudityaniswa kwedlozi-iqanda, apho iiseli ezimbini zidibanisa inwebu yazo ukuze zibe yi-diploid organism7.Nangona le nkqubo isisiseko ekuzalweni kwabantu, unxibelelwano oluyimfuneko lwemolekyuli aluqondwa kakuhle.
Ukongeza kwi-fertilition ye-gametes, i-chemistry ye-fusion ye-lipid bilayers ifundwe ngokubanzi.Ngokuqhelekileyo, i-membrane fusion yinkqubo engathandekiyo enamandla efuna iprotheni ye-protein ye-protein ye-protein ye-conformation ye-structural conformation eyenza i-membrane ezimbini zisondelelane, ziphule ukuqhubeka kwazo kwaye zibangele i-fusion8,9.Ezi zixhobo zeprotheni zaziwa njenge-fusogens kwaye zifunyenwe kwiinkqubo ezininzi zokudibanisa.Ziyafuneka ekungeneni kwentsholongwane kwiiseli zomkhosi (umzekelo, i-gp160 kwi-HIV-1, i-spike kwi-coronaviruses, i-hemagglutinin kwintsholongwane yomkhuhlane) 10,11,12 placental (syncytin) 13,14,15 kunye ne-gamete-forming fusions kwi-eukaryotes esezantsi ( I-HAP2 / GCS1 kwizityalo, iiprotisti kunye ne-arthropods) 16,17,18,19.IiFusogens zeegametes zabantu azikafunyaniswa, nangona iiproteni ezininzi zibonakalisiwe zibalulekile kuncamathiselo lwe-gamete kunye nokudibanisa.I-CD9 ye-oocyte-expressed, iprotein ye-transmembrane efunekayo ukuze kudityaniswe i-mouse kunye ne-gametes yabantu, yayiyeyokuqala ukufunyanwa i-21,22,23.Nangona umsebenzi wayo ochanekileyo uhlala ungacacanga, indima ekunamatheleni, isakhiwo se-adhesion foci kwi-egg microvilli, kunye / okanye indawo echanekileyo ye-oocyte surface proteins ibonakala ngathi i-24,25,26.Iiprotheni ezimbini eziqhelekileyo ezibaluleke kakhulu kwi-gamete fusion yiprotheni yesidoda i-IZUMO127 kunye neprotheni ye-oocyte JUNO28, kunye nobudlelwane babo bokubambisana linyathelo elibalulekileyo ekuqapheliseni i-gamete kunye nokubambelela ngaphambi kokudibanisa.Iimpuku eziyindoda ze-Izumo1 zokunkqonkqoza kunye neempuku zeJuno ezibhinqileyo azinyumba kwaphela, kule mifuziselo isidoda singena kwindawo yeperivitelline kodwa iigeyithi azidibanisi.Ngokufanayo, i-confluence yancitshiswa xa i-gametes iphathwa nge-anti-IZUMO1 okanye i-JUNO27,29 antibodies kwi-experimenti yabantu kwi-vitro fertilization.
Kutshanje, iqela elisandula ukufunyanwa leeprotheyini ze-sperm-expressed phenotypically ezifana ne-IZUMO1 kunye ne-JUNO20,30,31,32,33,34,35 ifunyenwe.I-sperm acrosomal membrane-enxulumene neprotheyini ye-6 (SPACA6) ichongiwe njengento ebalulekileyo ekuchumeni kwisifundo esikhulu se-murine mutagenesis.Ukufakwa kwe-transgene kwi-Spaca6 gene kuvelisa i-spermatozoa engabonakaliyo, nangona le spermatozoa ingena kwi-perivitelline space 36.Izifundo ezilandelelweyo zokunkqonkqoza kwiigundane ziqinisekisile ukuba i-Spaca6 iyadingeka kwi-gamete fusion 30,32.I-SPCAA6 ibonakaliswe phantse ngokukodwa kwii-testes kwaye inepateni yendawo efana ne-IZUMO1, eyile ngaphakathi kwe-intima ye-spermatozoa phambi kwe-acrosomal reaction, kwaye emva koko ifudukela kummandla we-equatorial emva kwe-acrosomal reaction 30,32.I-Spaca6 homologues ikhona kwiindidi zezilwanyana ezanyisayo kunye nezinye i-eukaryotes 30 kwaye ukubaluleka kwayo kwi-gamete fusion yabantu ibonakaliswe ngokuthintela ukuchumisa komntu kwi-vitro ngokuchasana ne-SPCAA6 30.Ngokungafani ne-IZUMO1 kunye ne-JUNO, iinkcukacha zesakhiwo, ukusebenzisana, kunye nomsebenzi we-SPCAA6 zihlala zingacacanga.
Ukuqonda ngcono inkqubo esisiseko esisiseko sokudityaniswa kwedlozi lomntu kunye namaqanda, okuya kusenza sikwazi ukwazisa uphuhliso lwexesha elizayo kucwangciso-ntsapho kunye nonyango lokuchuma, siye saqhuba izifundo ze-SPCA6 zesakhiwo kunye ne-biochemical.Isakhiwo se-crystal ye-domain ye-extracellular ye-SPCAA6 ibonisa i-helical bundle ezine (4HB) kunye ne-immunoglobulin-like (i-Ig-like) domain exhunywe yimimandla e-quasi-flexible.Njengoko kuxelwe kwangaphambili kwizifundo zangaphambili, i-7,32,37 i-domain structure ye-SPCA6 ifana ne-IZUMO1 yabantu, kwaye iiprotheni ezimbini zabelana nge-motif engavamile: i-4HB ene-triangular helical surface kunye ne-disulfide-linked CXXC motifs.Sicebisa ukuba i-IZUMO1 kunye ne-SPCAA6 ngoku zichaze usapho olukhulu, olunxulumene nolwakhiwo lweprotheyini enxulumene nokudityaniswa kwegeyithi.Sisebenzisa iimpawu ezizodwa kwi-superfamily, senze uphando olunzulu lwe-AlphaFold structural human proteome, ukuchonga amalungu ongezelelweyo ale superfamily, kubandakanywa amalungu amaninzi abandakanyekayo kwi-gamete fusion kunye / okanye ukuchumisa.Ngoku kubonakala ngathi kukho ulwakhiwo oluqhelekileyo kunye ne-superfamily yeeprotheyini ezinxulumene ne-gamete fusion, kwaye ulwakhiwo lwethu lubonelela ngemephu yemolekyuli yalo mba ubalulekileyo wendlela yokudityaniswa kwegeyithi yomntu.
I-SPACA6 yiprotheyini ye-transmembrane ye-single-pass kunye ne-N-linked glycan enye kunye ne-six putative disulfide bond (Amanani S1a kunye ne-S2).Sibonise i-domain ye-extracellular ye-SPACA6 yabantu (iintsalela ze-27-246) kwiiseli ze-Drosophila S2 kwaye sihlambulule iprotheni usebenzisa i-nickel affinity, i-cation exchange, kunye nobukhulu be-chromatography yokukhutshwa (Fig. S1b).I-ectodomain ye-SPCA6 ecocekileyo izinzile kwaye i-homogeneous.Uhlalutyo usebenzisa i-chromatography yokukhutshwa kobukhulu edityaniswe ne-polygonal light scattering (SEC-MALS) ibonise incopho enye enobunzima obubaliweyo be-molecular 26.2 ± 0.5 kDa (Fig. S1c).Oku kuhambelana nobukhulu be-SPCA6 monomeric ectodomain, ebonisa ukuba i-oligomerization ayizange yenzeke ngexesha lokuhlanjululwa.Ukongezelela, i-circular dichroism (CD) i-spectroscopy ibonakalise isakhiwo esixubileyo se-α / β kunye ne-melting point ye-51.3 ° C (Fig. S1d, e).I-Deconvolution ye-CD spectra ibonakalise i-38.6% α-helical kunye ne-15.8% ye-β-stranded elements (Figure S1d).
I-ectodomain ye-SPCAA6 yenziwe ngekristali kusetyenziswa i-random matrix seeding38 ibangele isethi yedatha kunye nesisombululo se-2.2 Å (Itheyibhile 1 kunye ne-Figure S3).Ukusebenzisa inhlanganisela ye-fragment-based substitution ye-molecular kunye ne-SAD yedatha yesigaba kunye ne-bromide exposure yokuzimisela kwesakhiwo (Itheyibhile 1 kunye ne-Figure S4), imodeli ecocekileyo yokugqibela iqukethe iintsalela ze-27-246.Ngexesha lokumiselwa kwesakhiwo, kwakungekho zakhiwo zovavanyo okanye ze-AlphaFold ezikhoyo.I-SPCAA6 imilinganiselo ye-ectodomain 20 Å × 20 Å × 85 Å, iqulethe iiheliksi ezisixhenxe kunye neentambo ezilithoba ze-β, kwaye inomgoqo ophakamileyo ophakamileyo ozinzisiweyo ngamabhondi amathandathu e-disulfide (Umfanekiso 1a, b).Ubuninzi be-electron obubuthathaka ekupheleni kwekhonkco ye-Asn243 ibonisa ukuba le ntsalela yi-N-linked glycosylation.Isakhiwo siquka imimandla emibini: i-N-terminal ezine-helix bundle (4HB) kunye ne-C-terminal Ig-like domain kunye nommandla we-hinge ophakathi phakathi kwabo (umzobo 1c).
Ubume besizinda se-extracellular ye-SPCA6.Umzobo we-Strip we-domain ye-extracellular ye-SPACA6, umbala wekhonkco ukusuka kwi-N ukuya kwi-C-terminus ukusuka kumnyama omnyama ukuya kubomvu obumnyama.I-Cysteines ebandakanyekayo kwiibhondi ze-disulfide zigxininiswe kwi-magenta.b I-Topology ye-domain ye-extracellular ye-SPCA6.Sebenzisa umbala ofanayo weskimu njengoko kuMfanekiso 1a.c SPACA6 idomeyini ye-extracellular.I-4HB, ihinge, kunye neetshathi zesizinda se-Ig-ezifana ne-Ig zinemibala e-orenji, eluhlaza, kunye nebhlowu, ngokulandelelanayo.Iileya azitsalwanga kwisikali.
I-domain ye-4HB ye-SPACA6 ibandakanya ii-helices ezine eziphambili (i-helices 1-4), ezihlelwe ngendlela ye-helical helix (Umfanekiso 2a), i-alternating phakathi kwe-antiparallel kunye ne-parallel interactions (Umfanekiso 2b).I-helix encinci eyongezelelweyo ye-single-turn (i-helix 1′) ibekwe i-perpendicular kwi-bundle, yenza unxantathu kunye ne-helices 1 kunye ne-2. Lo nxantathu uphazamisekile kancinci kwi-helical-twisted packing of the packing dense of helices 3 kunye ne-4. Isazobe 2a).
4HB N-terminal strip itshati.b Umbono ophezulu wenqwaba yeeheliksi ezine, iheliksi nganye igqanyiswe ngombala oluhlaza okwesibhakabhaka kwi-N-terminus kunye nobomvu obumnyama kwi-C-terminus.c Umzobo wevili elijiyileyo eliPhezulu-phantsi le-4HB, kunye nentsalela nganye eboniswa njengesangqa esinombhalo wekhowudi ye-amino acid enonobumba omnye;kuphela ezine amino acids kumphezulu wevili zibaliwe.Iintsalela ezingezizo ezipolar zinemibala etyheli, iintsalela ezingatshajwanga zinombala oluhlaza, iintsalela ezinecala elincomekayo zinombala ozuba, kwaye iintsalela ezinecala elibi zinombala obomvu.d Ubuso obunxantathu besizinda se-4HB, kunye ne-4HBs e-orenji kunye neehenjisi eziluhlaza.Zombini iziseti zibonisa iibhondi ze-disulfide ezimile okwentonga.
I-4HB igxile kumbindi we-hydrophobic ongaphakathi owenziwe ikakhulu ziintsalela ze-aliphatic kunye nevumba elimnandi (Umfanekiso 2c).Ingundoqo iqulethe i-disulfide bond phakathi kwe-Cys41 kunye ne-Cys55 edibanisa i-helices 1 kunye ne-2 kunye nonxantathu ophakanyisiweyo ophezulu (umzobo 2d).Izibophelelo ezimbini ezongezelelweyo ze-disulfide zenziwe phakathi kwe-CXXC motif kwi-Helix 1 kunye nenye i-CXXC i-motif efunyenwe kwi-tip ye-β-hairpin kwingingqi ye-hinge (Umfanekiso 2d).Intsalela ye-arginine ye-conservative kunye nomsebenzi ongaziwayo (Arg37) ifumaneka ngaphakathi kunxantathu ongenanto eyenziwe ngama-helices 1′, 1, kunye no-2. I-aliphatic carbon athom i-Cβ, i-Cγ, kunye ne-Cδ i-Arg37 isebenzisana ne-hydrophobic core, kunye namaqela ayo e-guanidine ahamba ngebhayisikile. phakathi kwe-helices 1 kunye ne-1 ngokusebenzisana phakathi kwe-Thr32 backbone kunye ne-side chain (Fig. S5a, b).I-Tyr34 iyonabela kumngxuma ishiya imingxuma emibini emincinci apho iArg37 inokunxibelelana nesinyibilikisi.
I-Ig-like β-sandwich domains yi-superfamily enkulu yeeprotheyini ezabelana ngeempawu eziqhelekileyo ezimbini okanye ngaphezulu ze-amphipathic β-sheets ezininzi ezixutywe nge-hydrophobic core 39. I-C-terminal Ig-like domain ye-SPCAA6 inomzekelo ofanayo. kwaye iqulathe iileya ezimbini (Fig. S6a).Iphepha le-1 liyi-β-ishidi lemicu emine (iintambo D, F, H, kunye ne-I) apho iintambo F, H, kunye ndenza ilungiselelo elichasene nokulinganisa, kunye neentambo I kunye no-D zithatha ukusebenzisana okufanayo.Itheyibhile 2 yiphepha elincinci le-beta elichasene ne-parallel elinemisonto ephindwe kabini (imisonto E kunye no-G).Ibhondi yangaphakathi ye-disulfide yabonwa phakathi kwe-C-terminus ye-chain ye-E kunye neziko le-H chain (Cys170-Cys226) (Fig. S6b).Le bhondi ye-disulfide ifana ne-disulfide bond kwi-β-sandwich domain ye-immunoglobulin40,41.
I-beta-sheet enemisonto emine ijija ngobude bayo bonke, yenze imiphetho engalinganiyo neyahlukileyo ngokwemilo kunye ne-electrostatics.Umphetho obhityileyo ngumphezulu wokusingqongileyo osicaba we-hydrophobic ome ngaphandle xa kuthelekiswa neendawo eziseleyo ezingalinganiyo kunye nezahlukeneyo zombane kwi-SPCAA6 (Fig. S6b,c).I-halo ye-backbone ye-carbonyl / amaqela e-amino kunye ne-polar side chain zijikeleze umphezulu we-hydrophobic (Fig. S6c).Umda obanzi ugutyungelwe yinxalenye ye-helical ene-capped evimba inxalenye ye-N-terminal ye-hydrophobic core kwaye yenza iibhondi ezintathu ze-hydrogen kunye neqela elivulekileyo le-polar le-F chain backbone (Fig. S6d).Inxalenye ye-C-terminal yalo mphetho yenza ipokotho enkulu ene-hydrophobic core evezwe ngokuyinxenye.Ipokotho ijikelezwe ziintlawulo ezilungileyo ngenxa yeeseti ezintathu zeentsalela ze-arginine (Arg162-Arg221, Arg201-Arg205 kunye neArg212-Arg214) kunye ne-histidine ephakathi (His220) (Umfanekiso S6e).
Ummandla we-hinge uyingxenye emfutshane phakathi kwesizinda se-helical kunye ne-domain efana ne-Ig, equka enye i-antiparallel enemisonto emithathu ye-β-layer (imicu A, B kunye ne-C), i-310 helix encinci, kunye namacandelo amaninzi amade e-helical.(Umfanekiso S7).Uthungelwano lwabafowunelwa be-covalent kunye ne-electrostatic kummandla wehinge lubonakala luzinzisa ukuqhelaniswa phakathi kwe-4HB kunye nesizinda esifana ne-Ig.Inethiwekhi inokwahlulwa ibe ngamacandelo amathathu.Inxalenye yokuqala ibandakanya ezimbini ze-CXXC motifs (27CXXC30 kunye ne-139CXXC142) eyenza idibanti ye-disulfide phakathi kwe-β-hairpin kwi-hinge kunye ne-1'helix kwi-4HB.Inxalenye yesibini ibandakanya ukusebenzisana kwe-electrostatic phakathi kwesizinda esifana ne-Ig kunye nehinge.I-Glu132 kwihinge yenza ibhulorho yetyuwa ene-Arg233 kwi-domain efana ne-Ig kunye ne-Arg135 kwihinge.Inxalenye yesithathu ibandakanya i-covalent bond phakathi kwe-domain efana ne-Ig kunye nommandla we-hinge.Iibhondi ezimbini ze-disulfide (i-Cys124-Cys147 kunye ne-Cys128-Cys153) zidibanisa i-hinge loop kwi-linker eqiniswe ngokubambisana kwe-electrostatic phakathi kwe-Gln131 kunye neqela elisebenzayo le-backbone, elivumela ukufikelela kwi-domain yokuqala efana ne-Ig.ikhonkco.
Ubume be-ectodomain ye-SPCA6 kunye nezakhiwo zomntu ngamnye ze-4HB kunye ne-Ig-like domains zisetyenziselwa ukukhangela iirekhodi ezifanayo ngokwesakhiwo kwiiprotheyini ze-42.Sichonge iimidlalo ezinamanqaku aphezulu eDali Z, ukutenxa kwimigangatho emincinci, kunye namanqaku amakhulu e-LALI (eli lokugqibela linani leentsalela ezilinganayo ngokwesakhiwo).Ngelixa i-10 yokuqala ibetha kwi-ectodomain epheleleyo yokukhangela (Itheyibhile S1) ine-Z-score eyamkelekileyo> 842, ukukhangela i-4HB okanye i-Ig-like domain yodwa ibonise ukuba ininzi yezi hits zihambelana ne-β-sandwiches kuphela.i-fold ubiquitous efumaneka kwiiprotheni ezininzi.Zontathu izikhangelo eDali zibuyise isiphumo esinye kuphela: IZUMO1.
Kudala kucetyiswa ukuba i-SPCA6 kunye ne-IZUMO1 babelane ngokufana kwesakhiwo7,32,37.Nangona i-ectodomains yezi zimbini ze-gamete fusion-associated proteins zabelana kuphela nge-21% ye-sequence identity (Figure S8a), ubungqina obuntsonkothileyo, kubandakanywa ipateni egciniweyo ye-disulfide bond kunye ne-C-terminal Ig-like domain eqikelelweyo kwi-SPACA6, ivumele imizamo yokuqala yokwakha imodeli yehomology ye-A a SPCA6 mouse usebenzisa i-IZUMO1 njenge template37.Ulwakhiwo lwethu luqinisekisa ezi ngqikelelo kwaye lubonisa inqanaba lokwenyani lokufana.Enyanisweni, i-SPCA6 kunye ne-IZUMO137,43,44 izakhiwo zabelana nge-architecture ye-domain-domain efanayo (Umfanekiso we-S8b) kunye ne-4HB efanayo kunye ne-Ig-like β-sandwich domains exhunywe ngummandla we-hinge (Fig. S8c).
I-IZUMO1 kunye ne-SPCA6 4HB zineeyantlukwano ezifanayo kwimiqulu eqhelekileyo ejiyileyo.I-4HBs eqhelekileyo, njengalezo zifunyenwe kwiiprotheni ze-SNARE ezibandakanyekayo kwi-endosomal fusion 45,46, zineendawo ezilinganayo ze-helices ezigcina i-curvature ejikelezayo kwi-axis ephakathi 47. Ngokwahlukileyo, imimandla ye-helical kwi-IZUMO1 kunye ne-SPACA6 yaphazamiseka, kunye ne-curvature eguquguqukayo ukupakisha ngokungalingani (Figure S8d).I-twist, mhlawumbi ibangelwa ngunxantathu owenziwe ngama-helices 1, 1 kunye ne-2, igcinwe kwi-IZUMO1 kunye ne-SPACA6 kwaye iqiniswe yi-motif ye-CXXC efanayo kwi-helix 1'.Nangona kunjalo, ibhondi eyongezelelweyo ye-disulfide efunyenwe kwi-SPACA6 (i-Cys41 kunye ne-Cys55 edibanisa ngokudibeneyo i-helices 1 kunye ne-2 apha ngasentla) yenza i-pex ebukhali kwi-apex ye-triangle, isenza i-SPACA6 ijijeke ngakumbi kune-IZUMO1, kunye nonxantathu we-cavity ocacileyo.Ukongeza, i-IZUMO1 ayinayo i-Arg37 ejongwe embindini walo mgodi kwi-SPCAA6.Ngokwahlukileyo, i-IZUMO1 inondoqo we-hydrophobic oqhelekileyo we-aliphatic kunye neentsalela ezinevumba elimnandi.
I-IZUMO1 ine-domain efana ne-Ig equka umsonto ophindwe kabini kunye nentlanu β-sheet43.I-strand eyongezelelweyo kwi-IZUMO1 ithatha indawo yekhoyili kwi-SPACA6, edibanisa ne-F strand ukukhawulela i-backbone bonds ye-hydrogen kwi-strand.Inqaku elinomdla lokuthelekisa yintlawulo eqikelelweyo yendawo ye-Ig-like domains yeeprotheni ezimbini.Umphezulu we-IZUMO1 uhlawuliswe kakubi kakhulu kunomphezulu we-SPCA6.Intlawulo eyongezelelweyo ifumaneka kufuphi neC-terminus ejongene nenwebu yesidoda.Kwi-SPACA6, imimandla efanayo yayingathathi hlangothi okanye ihlawuliswe kakuhle (Umfanekiso we-S8e).Ngokomzekelo, i-hydrophobic surface (imida encinci) kunye nemigodi ehlanjululwe kakuhle (imida ebanzi) kwi-SPCAA6 ihlawuliswa kakubi kwi-IZUMO1.
Nangona ubudlelwane kunye nezinto zesakhiwo sesibini phakathi kwe-IZUMO1 kunye ne-SPCAA6 zigcinwe kakuhle, ukulungelelaniswa kwesakhiwo se-Ig-like domains kubonise ukuba imimandla emibini ihluke kwi-orientation yabo ngokubanzi ngokumalunga nomnye (Fig. S9).I-Spiral bundle ye-IZUMO1 igophe malunga ne-β-sandwich, idala ubume obuchazwe ngaphambili "boomerang" malunga ne-50 ° ukusuka kwi-axis esembindini.Ngokwahlukileyo, i-helical beam kwi-SPCAA6 yayithambekele malunga ne-10 ° kwicala elichaseneyo.Umahluko kolu lungelelwaniso kungenzeka ngenxa yeyantlukwano kwingingqi yehinge.Kwinqanaba eliphambili lokulandelelana, i-IZUMO1 kunye ne-SPACA6 yabelana ngokulandelelana okuncinci ukufana kwihinge, ngaphandle kwe-cysteine, i-glycine, kunye neentsalela ze-aspartic acid.Ngenxa yoko, iibhondi ze-hydrogen kunye neenethiwekhi ze-electrostatic zahluke ngokupheleleyo.Iimpawu ze-β-sheet zesibini zesakhiwo zabelwana ngazo yi-IZUMO1 kunye ne-SPACA6, nangona amatyathanga e-IZUMO1 ade kakhulu kwaye i-310 helix (i-helix 5) iyingqayizivele kwi-SPACA6.Lo mahluko ubangela ukuqhelaniswa kwesizinda okwahlukileyo kwiiprotheni ezimbini ezifanayo.
Ukukhangela kwethu iseva ye-Dali kubonise ukuba i-SPACA6 kunye ne-IZUMO1 zizona zakhiwo ezibini kuphela ezimiselwe ngokovavanyo ezigcinwe kuvimba weenkcukacha zeprotheyini ezinale fold 4HB (Itheyibhile S1).Ngoku kutshanje, i-DeepMind (i-Alphabet / Google) iphuhlise i-AlphaFold, i-neural network based system enokuthi iqikelele ngokuchanekileyo izakhiwo ze-3D zeeprotheni ezivela kwii-primary sequences48.Kungekudala emva kokuba sisombulule isakhiwo se-SPACA6, i-database ye-AlphaFold yakhululwa, ibonelela ngeemodeli zesakhiwo esiqikelelwayo ezigubungela i-98.5% yazo zonke iiprotheni kwiproteome48,49 yabantu.Ukusebenzisa ulwakhiwo lwethu lwe-SPCAA6 olusonjululweyo njengomzekelo wokukhangela, ukukhangela kwe-homology yesakhiwo kwimodeli kwi-AlphaFold human proteome ichonge abaviwa abanokufana okunokwenzeka kwesakhiwo kwi-SPCAA6 kunye ne-IZUMO1.Ngenxa yokuchaneka okungakholelekiyo kwe-AlphaFold ekuqikeleleni i-SPACA6 (Fig. S10a)-ingakumbi i-1.1 Å rms ectodomain xa kuthelekiswa nolwakhiwo lwethu olusonjululweyo (Fig. S10b)-sinokuqiniseka ukuba iimatshisi ze-SPCAA6 ezichongiweyo kusenokwenzeka ukuba zichanekile.
Ngaphambili, i-PSI-BLAST ibikhangela iqela le-IZUMO1 kunye nezinye iiproteni ezintathu ezinxulumene namadlozi: IZUMO2, IZUMO3, kunye ne-IZUMO450.I-AlphaFold iqikelele ukuba ezi proteni zosapho ze-IZUMO zigoqeka kwi-domain ye-4HB kunye nepateni efanayo ye-disulfide bond njenge-IZUMO1 (Amanani 3a kunye ne-S11), nangona zingenayo i-domain efana ne-Ig.Kucingelwa ukuba i-IZUMO2 kunye ne-IZUMO3 ziiprotheyini zenwebu ezicalanye ezifanayo ne-IZUMO1, ngelixa i-IZUMO4 ibonakala ngathi ikhutshiwe.Imisebenzi ye-IZUMO 2, 3, kunye ne-4 yeeprotheni kwi-gamete fusion ayikamiselwa.I-IZUMO3 yaziwa ngokudlala indima kwi-acrosome biogenesis ngexesha lophuhliso lwamadlozi51 kwaye iprotein ye-IZUMO kufunyaniswe ukuba yenze i-complex50.Ukugcinwa kweeprotheyini ze-IZUMO kwizilwanyana ezincancisayo, izilwanyana ezirhubuluzayo, kunye nezilwanyana eziphila emhlabeni nasemanzini zibonisa ukuba umsebenzi wabo onokwenzeka uhambelana nenye eyaziwa ngokuba yi-gamete-fusion-associated proteins, njenge-DCST1/2, SOF1, kunye ne-FIMP.
Umzobo we-domain architecture ye-IST superfamily, ene-4HB, i-hinge, kunye ne-Ig-like domains egxininiswe kwi-orenji, eluhlaza, kunye ne-blue, ngokulandelanayo.I-IZUMO4 inommandla owodwa we-C-terminal obonakala umnyama.Iibhondi ze-disulfide eziqinisekisiweyo kunye ne-puative ziboniswa ngemigca eqinileyo kunye namachaphaza, ngokulandelanayo.b IZUMO1 (PDB: 5F4E), SPCA6, IZUMO2 (AlphaFold DB: AF-Q6UXV1-F1), IZUMO3 (AlphaFold DB: AF-Q5VZ72-F1), IZUMO4 (AlphaFold DB: AF-Q1ZYL8-FEM95), kunye ne-Alpha DB: AF-Q1ZYL8-F1) : AF-Q1ZYL8-F1) : AF-Q3KNT9-F1) ziboniswa kumbala ofanayo wombala njengephaneli A. Iibhondi ze-Disulfide ziboniswa kwi-magenta.I-TMEM95, IZUMO2 kunye ne-IZUMO3 i-helices ye-transmembrane ayiboniswa.
Ngokungafaniyo neprotheyini ye-IZUMO, ezinye iiprotheni ze-SPCA (oko kukuthi, i-SPACA1, i-SPACA3, i-SPACA4, i-SPACA5, kunye ne-SPACA9) zicingelwa ukuba zihluke ngokwesakhiwo kwi-SPACA6 (Fig. S12).Kuphela i-SPCAA9 ene-4HB, kodwa akulindelekanga ukuba ibe ne-parallel-anti-parallel orientation efanayo okanye i-disulfide bond efanayo ne-SPCAA6.Kuphela i-SPCA1 ene-domain efana ne-Ig efanayo.I-AlphaFold iqikelela ukuba i-SPACA3, i-SPACA4 kunye ne-SPCA5 inesakhiwo esahluke ngokupheleleyo kune-SPACA6.Okubangela umdla kukuba, i-SPACA4 yaziwa ngokudlala indima ekuchumiseni, kodwa ukuya kuthi ga kwinqanaba le-SPACA6, endaweni yoko iququzelele intsebenziswano phakathi kwedlozi kunye ne-oocyte zona pellucida52.
Uphendlo lwethu lwe-AlphaFold lufumene omnye umdlalo we-IZUMO1 kunye ne-SPACA6 4HB, TMEM95.I-TMEM95, iprotheni ye-sperm-specific transmembrane protein, yenza iigundane zamadoda zingabi nabantwana xa zichithwa i-32,33.I-Spermatozoa eswele i-TMEM95 yayine-morphology eqhelekileyo, i-motility, kunye nokukwazi ukungena kwi-zona pellucida kwaye ibophe kwi-membrane yeqanda, kodwa ayikwazi ukudibanisa ne-oocyte membrane.Uphononongo lwangaphambili lubonise ukuba i-TMEM95 yabelana ngokufana kwesakhiwo kunye ne-IZUMO133.Enyanisweni, imodeli ye-AlphaFold iqinisekisile ukuba i-TMEM95 yi-4HB kunye nesibini esifanayo se-CXXC ye-motifs njenge-IZUMO1 kunye ne-SPACA6 kunye ne-disulfide bond eyongezelelweyo phakathi kwee-helices 1 kunye ne-2 efunyenwe kwi-SPCA6 (Umfanekiso 3a kunye ne-S11).Nangona i-TMEM95 ingenayo i-domain efana ne-Ig, inommandla kunye nepatheni yebhondi ye-disulfide efana ne-SPACA6 kunye ne-IZUMO1 imimandla yeehinge (Umfanekiso 3b).Ngexesha lokupapashwa kwalo mbhalo-ngqangi, iseva yokuprinta yaxela ubume be-TMEM95, iqinisekisa isiphumo se-AlphaFold53.I-TMEM95 ifana kakhulu ne-SPCA6 kunye ne-IZUMO1 kwaye igcinwe ngokuguquguqukayo sele igcinwe kwii-amphibians (Umfanekiso 4 kunye ne-S13).
Uphando lwe-PSI-BLAST lusebenzise i-NCBI SPCA6, IZUMO1-4, TMEM95, DCST1, DCST2, FIMP, kunye ne-SOF1 yogcino-lwazi ukumisela indawo yolu landelelwano kumthi wobomi.Umgama phakathi kwamanqaku esebe awuboniswanga kwisikali.
Ukufana okumangalisayo kwesakhiwo kukonke phakathi kwe-SPACA6 kunye ne-IZUMO1 kuphakamisa ukuba bangamalungu aseka i-superfamily yesakhiwo egciniweyo equka i-TMEM95 kunye ne-IZUMO 2, 3, kunye neeprotheni ezi-4.amalungu aziwayo: IZUMO1, SPCA6 kunye TMEM95.Ngenxa yokuba ngamalungu ambalwa kuphela anemimandla efana ne-Ig, uphawu lwe-IST superfamily yi-domain ye-4HB, eneempawu ezizodwa eziqhelekileyo kuzo zonke ezi proteni: 1) I-4HB ehlanganisiweyo kunye ne-helices ecwangciswe kwi-anti-parallel / parallel alternation (Fig 5a), i-2) i-bundle inobuso obunxantathu obuqulethwe kwii-helices ezimbini ngaphakathi kwe-bundle kunye ne-helix ye-vertical vertical (Fig. Indawo engundoqo (umzobo 5c) I-motif ye-CXXC, efumaneka kwiiprotheni ezifana ne-thioredoxin, iyaziwa ukuba isebenze. njengenzwa ye-redox 54,55,56 , ngelixa i-motif kumalungu osapho lwe-IST inokudibaniswa ne-protein disulfide isomerases ezifana ne-ERp57 kwi-gamete fusion.Iindima zihambelana ne-57,58.
Amalungu e-IST superfamily achazwa ngeempawu ezintathu ze-domain ye-4HB: ii-helice ezine ezitshintshanayo phakathi kwe-parallel kunye ne-antiparallel orientation, ubuso be-ba-triangular helical bundle, kunye ne-ca CXXC i-motif ephindwe kabini eyenziwe phakathi kwama-molecule amancinci.) I-N-terminal helixes (i-orange) kunye ne-hinge region β-hairpin (eluhlaza).
Ngenxa yokufana phakathi kwe-SPACA6 kunye ne-IZUMO1, ukukwazi kwabokuqala ukubophelela kwi-IZUMO1 okanye i-JUNO kwavavanywa.I-Biolayer interferometry (BLI) yindlela yokubopha i-kinetic esetyenziswe ngaphambili ukulinganisa ukusebenzisana phakathi kwe-IZUMO1 kunye neJUNO.Emva kokufakwa kwe-sensor ebhalwe nge-biotin ene-IZUMO1 njenge-bait ene-concentration ephezulu ye-JUNO analyte, isibonakaliso esinamandla sifunyenwe (umfanekiso we-S14a), ebonisa utshintsho olubophezelayo kwi-thickness of biomaterial efakwe kwi-tip sensor.Imiqondiso efanayo (okt, JUNO idityaniswe kwinzwa njengesithiyelo ngokuchasene ne-IZUMO1 analyte) (Fig. S14b).Akukho mqondiso ufunyenwe xa i-SPCAA6 isetyenziswe njengohlalutyi ngokuchasene ne-sensor-bound IZUMO1 okanye i-sensor-bound JUNO (Figure S14a,b).Ukungabikho kwesi siginali kubonisa ukuba i-domain ye-extracellular ye-SPACA6 ayisebenzi kunye ne-extracellular domain ye-IZUMO1 okanye i-JUNO.
Ngenxa yokuba uvavanyo lwe-BLI lusekelwe kwi-biotinylation yee-residues ze-lysine zamahhala kwiprotheni ye-bait, oku kuguqulwa kunokuthintela ukubopha ukuba i-lysine i-residues ibandakanyeka ekusebenzisaneni.Ukongeza, ukuqhelaniswa kwesalamane esibophezelayo kwinzwa kunokudala izithintelo zesteric, ngoko ke iimvavanyo eziqhelekileyo zokutsalwa phantsi nazo zenziwa kwi-recombinant SPCA6, IZUMO1 kunye ne-JUNO ectodomains.Ngaphandle koku, i-SPCAA6 ayizange iqhube nge-IZUMO1 ephawulwe Yakhe okanye i-JUNO ephawulwe Yakhe (Umfanekiso we-S14c, d), ebonisa ukuba akukho ntsebenziswano ehambelana noko kubonwa kwiimvavanyo ze-BLI.Njengolawulo oluhle, siqinisekisile ukusebenzisana kweJUNO ebhalwe i-IZUMO1 Yakhe (Amanani S14e kunye ne-S15).
Ngaphandle kokufana kwesakhiwo phakathi kwe-SPCAA6 kunye ne-IZUMO1, ukungakwazi kwe-SPCAA6 ukubopha i-JUNO akumangalisi.Umphezulu we-IZUMO1 yomntu uneentsalela ezingaphezulu kwama-20 ezisebenzisana neJUNO, kubandakanywa iintsalela ezivela kummandla ngamnye kule mimandla mithathu (nangona uninzi lwayo lukwingingqi yehinge) (Fig. S14f).Kwezi ntsalela, inye kuphela egciniweyo kwi-SPACA6 (Glu70).Ngelixa ukutshintshwa kwentsalela okuninzi kugcine iipropati zoqobo zebhayokhemikhali, intsalela ye-Arg160 eyimfuneko kwi-IZUMO1 yathatyathelwa indawo yi-Asp148 ehlawuliswe kakubi kwi-SPCAA6;Uphononongo lwangaphambili lubonise ukuba ukuguqulwa kwe-Arg160Glu kwi-IZUMO1 phantse kuphelise ngokupheleleyo ukubophelela kwi-JUNO43.Ukongeza, umahluko kwi-domain orientation phakathi kwe-IZUMO1 kunye ne-SPACA6 yandisa kakhulu indawo yendawo ye-JUNO-binding site yommandla olinganayo kwi-SPACA6 (Fig. S14g).
Ngaphandle kwesidingo esaziwayo se-SPACA6 yokudibanisa i-gamete kunye nokufana kwayo ne-IZUMO1, i-SPACA6 ayibonakali inomsebenzi olinganayo wokubopha we-JUNO.Ke ngoko, siye safuna ukudibanisa idatha yethu yolwakhiwo kunye nobungqina bokubaluleka obubonelelwa yi-evolutionary biology.Ulungelelwaniso lolandelelwano lweehomologue ze-SPCAA6 lubonisa ulondolozo lwesakhiwo esiqhelekileyo ngaphaya kwezilwanyana ezanyisayo.Umzekelo, iintsalela ze-cysteine ​​​​zikhona nakwii-amphibians ezinxulumene kude (umzobo 6a).Ukusebenzisa i-server ye-ConSurf, idatha yokugcinwa kolandelelwano olulandelelanayo lwe-66 elandelelanayo yenziwe imephu kumphezulu we-SPCAA6.Olu hlobo lokuhlalutya lunokubonisa ukuba zeziphi iintsalela eziye zagcinwa ngexesha lokuziphendukela kweprotheyini kwaye zingabonisa ukuba yeyiphi imimandla ephezulu edlala indima emsebenzini.
Ulungelelwaniso lolandelelwano lwe-SPCA6 ectodomains ukusuka kwiintlobo ezili-12 ezahlukeneyo ezilungiselelwe kusetyenziswa i-CLUSTAL OMEGA.Ngokohlalutyo lwe-ConSurf, ezona zikhundla zolondolozo ziphawulwe ngombala oluhlaza okwesibhakabhaka.Iintsalela ze-cysteine ​​​​ziphawulwe ngombala obomvu.Imida ye-Domain kunye nezinto zesakhiwo sesibini ziboniswa phezulu kwi-alignment, apho iintolo zibonisa i-β-strands kunye namaza abonisa i-helices.I-NCBI Access Identifiers equlathe ulandelelwano zezi: human (Homo sapiens, NP_001303901), mandrill (Mandrilus leucophaeus, XP_011821277), capuchin monkey (Cebus mimic, XP_017359366), ihashe (Equus 1002 Orchea3) 23 XP_032_034) .), iigusha (Ovis aries, XP_014955560), elephant (Loxodonta africana, XP_010585293), inja (Canis lupus familyis, XP_025277208), impuku (Mus musculus, NP_001156381), Tasmanian XP, 3Plarisi3 XPPlarisi1 , 8) , 61_89 kunye neBullfrog (Bufo bufo, XP_040282113).Amanani asekelwe kulandelelwano lomntu.b Ukumelwa komphezulu wesakhiwo se-SPACA6 kunye ne-4HB phezulu kunye ne-Ig-like domain ezantsi, imibala esekelwe kuqikelelo lolondolozo olusuka kwi-ConSurf server.Ezona ndawo zigcinwe kakuhle zinombala oluhlaza okwesibhakabhaka, iindawo ezilondolozwe ngokuphakathi zimhlophe, kwaye ezona ndawo zincinci zigcinwe zityheli.icysteine ​​emfusa.Iziqwenga ezintathu zomphezulu ezibonisa umgangatho ophezulu wokhuseleko zibonisiwe kwindawo ebhalwe iziqwenga 1, 2 kunye no-3. Ikhathuni ye-4HB iboniswe kwi-inset phezulu ngasekunene (umbala ofanayo wombala).
Ulwakhiwo lwe-SPCA6 lunemimandla emithathu egcinwe phezulu egcinwe phezulu (Umfanekiso 6b).Isiqwenga se-1 sidibanisa i-4HB kunye nommandla we-hinge kwaye iqulethe iibhulorho ezimbini ezigciniweyo ze-CXXC ze-disulfide, i-Arg233-Glu132-Arg135-Ser144 network hinge (Fig. S7), kunye neentsalela ezilondolozwe zangaphandle ezinevumba elimnandi (Phe31, Tyr73, Phe137).ungqameko olubanzi lwe-domain efana ne-Ig (Fig. S6e), emele iintsalela ezininzi ezihlawuliswa ngokufanelekileyo kumphezulu wamadlozi.Okubangel 'umdla kukuba, le patch iqulethe i-antibody epitope eboniswe ngaphambili ukuphazamisa umsebenzi we-SPCAA6 30.Ummandla we-3 udibanisa ihenjisi kunye necala elinye le-domain efana ne-Ig;lo mmandla uqulethe iiprolines ezigciniweyo (Pro126, Pro127, Pro150, Pro154) kunye neentsalela ezijongene ne-polar / ezihlawuliweyo.Okumangalisa kukuba, ininzi ye-residus ebusweni be-4HB iguquguquka kakhulu (Umfanekiso we-6b), nangona i-fold fold igcinwe kwi-homologue ye-SPACA6 (njengoko kuboniswe yi-conservatism ye-hydrophobic bundle core) nangaphaya kwe-IST superfamily.
Nangona lo ngowona mmandla uncinci kwi-SPCAA6 kunye nezona zinto zimbalwa zibonwayo zesakhiwo sesibini, iintsalela ezininzi ze-hinge (kubandakanywa nommandla we-3) zigcinwe kakhulu phakathi kwe-SPCAA6 homologues, enokuthi ibonise ukuba ukuqhelaniswa kwe-helical bundle kunye ne-β-sandwich idlala indima.njengomntu ogcinayo.Nangona kunjalo, nangona i-hydrogen bonding ebanzi kunye nothungelwano lwe-electrostatic kummandla wehinge we-SPCA6 kunye ne-IZUMO1, ubungqina bokuguquguquka kwangaphakathi bunokubonwa ekulungelelaneni kwezakhiwo ezininzi ezivunyelweyo ze-IZUMO137,43,44.Ukulungelelaniswa kweendawo zomntu ngamnye zigqithise kakuhle, kodwa ukuqhelaniswa kwemimandla ehambelana nomnye kwahluka ukusuka kwi-50 ° ukuya kwi-70 ° ukusuka kwi-axis ephakathi (Fig. S16).Ukuqonda i-conformational dynamics ye-SPCA6 kwisisombululo, iimvavanyo ze-SAXS zenziwa (Umfanekiso we-S17a, b).Ukwakhiwa kwakhona kwe-Ab initio ye-SPCA6 ectodomain ihambelana nesakhiwo sekristal yentonga (Umfanekiso we-S18), nangona isicwangciso seKratky sibonise iqondo elithile lokuguquguquka (Fig. S17b).Oku kuhambelana ne-IZUMO1, apho iprotheyini engabonakaliyo ithatha i-boomerang shape zombini kwi-lattice kunye nesisombululo43.
Ukuchonga ngokuthe ngqo ummandla oguquguqukayo, i-hydrogen-deuterium exchange mass spectroscopy (H-DXMS) yenziwa kwi-SPCAA6 kwaye xa kuthelekiswa nedatha efunyenwe ngaphambili kwi-IZUMO143 (Umfanekiso 7a, b).I-SPCAA6 ngokucacileyo iguquguquka ngakumbi kune-IZUMO1, njengoko ibonakaliswe yi-deuterium ephezulu yotshintshiselwano kwisakhiwo sonke emva kwe-100,000 ye-exchange exchange.Kuzo zombini izakhiwo, i-C-terminal inxalenye yendawo ye-hinge ibonisa izinga eliphezulu lokutshintshiselana, mhlawumbi livumela ukujikeleza okulinganiselwe kwe-4HB kunye ne-Ig-like domains ngokubhekiselele komnye nomnye.Okubangel 'umdla kukuba, inxalenye ye-C-terminal yehinge ye-SPCAA6, equka i-147CDLPLDCP154 i-residu, yindawo egcinwe kakhulu ye-3 (umzobo 6b), mhlawumbi ebonisa ukuba ukuguquguquka kwe-interdomain yinto egcinwe ngokuguquguqukayo ye-SPACA6.Ngokutsho kohlalutyo lokuguquguquka, idatha ye-CD thermal melt ibonise ukuba i-SPACA6 (Tm = 51.2 ° C) ayizinzile ngaphantsi kwe-IZUMO1 (Tm = 62.9 ° C) (Fig. S1e kunye ne-S19).
a H-DXMS imifanekiso SPCA6 kunye b IZUMO1.Ipesenti yokutshintshiselana kwe-deuterium yamiselwa kwiindawo ezibonisiweyo zexesha.Amanqanaba okutshintshana kwe-hydrogen-deuterium aboniswa ngombala kwi-gradient scale ukusuka eluhlaza okwesibhakabhaka (10%) ukuya kubomvu (90%).Iibhokisi ezimnyama zibonisa iindawo zokutshintshiselana okuphezulu.Imida ye-4HB, i-hinge kunye ne-Ig-efana ne-domain ebonwe kwisakhiwo se-crystal iboniswe ngasentla kokulandelelana okuphambili.Amanqanaba otshintshiselwano lwe-Deuterium kwi-10 s, 1000 s, kunye ne-100,000 s aye acwangciswa kwitshathi yestrip ebekwe ngaphezulu kumphezulu wemolekyuli ocacileyo we-SPACA6 kunye ne-IZUMO1.Iinxalenye zezakhiwo ezinezinga lotshintshiselwano lwe-deuterium ngaphantsi kwe-50% zinemibala emhlophe.Iindawo ezingaphezulu kwe-50% ye-H-DXMS yotshintshiselwano zinemibala kwi-gradient scale.
Ukusetyenziswa kwe-CRISPR/Cas9 kunye ne-mouse gene knockout gene qhinga liye lakhokelela ekuchongweni kwezinto ezininzi ezibalulekileyo kwi-sperm kunye neqanda lokubopha kunye nokudibanisa.Ngaphandle kwentsebenziswano ecacileyo ye-IZUMO1-JUNO kunye nesakhiwo se-CD9, ezininzi iiprotheni ezinxulumene ne-gamete fusion zihlala zihlelekile kwaye zisebenza ngokufihlakeleyo.I-biophysical kunye ne-structural characterization ye-SPCAA6 sesinye isiqwenga sepuzzle ye-adhesion/fusion yemolekyuli ngexesha lokuchumisa.
I-SPCAA6 kunye namanye amalungu e-IST superfamily abonakala egcinwe kakhulu kwizilwanyana ezanyisayo kunye neentaka ezizimeleyo, ezirhubuluzayo, kunye nezilwanyana ezihlala emanzini;eneneni, kucingelwa ukuba i-SPCAA6 iyafuneka nasekuchumiseni kwi-zebrafish 59. Olu lwabiwo luyafana nezinye iiproteni ezaziwayo ze-gamete fusion-associated ezifana ne-DCST134, DCST234, FIMP31, kunye ne-SOF132, ebonisa ukuba ezi zinto zi-HAP2-zinqongophele (kwakhona. eyaziwa ngokuba yi-GCS1) iiprotheyini ezinoxanduva lomsebenzi we-catalytic wamaprotisti amaninzi., izityalo, kunye ne-arthropods.Iiprotheyini ezidityanisiweyo ezidityanisiweyo 60, 61. Ngaphandle kokufana okuqinileyo kwesakhiwo phakathi kwe-SPACA6 kunye ne-IZUMO1, i-knockout ye-gene e-encoding enye yezi proteni ibangele ubudlolo kwiimpuku zamadoda, ebonisa ukuba imisebenzi yabo kwi-gamete fusion ayiphindanga..Ngokubanzi ngakumbi, akukho nanye kwiiproteni zesidoda ezaziwayo ezifunekayo kwisigaba sokuncamathela sokudityaniswa kuninzi.
Kuhlala kungumbuzo ovulekileyo ukuba ngaba i-SPACA6 (kunye namanye amalungu e-IST superfamily) athatha inxaxheba kwi-intergametic junctions, yenza i-intragametic networks ukufumana iiprotheni ezibalulekileyo kwiindawo zokudityaniswa, okanye mhlawumbi zisebenze njenge-fusogens engabonakaliyo.Uphononongo lwe-Co-immunoprecipitation kwiiseli ze-HEK293T luveze intsebenziswano phakathi kobude obupheleleyo be-IZUMO1 kunye ne-SPCA632.Nangona kunjalo, i-ectodomains yethu edibeneyo ayizange isebenze kwi-vitro, iphakamisa ukuba intsebenziswano ebonwe kwi-Noda et al.zaye zacinywa zombini ekwakhiweni (qaphela umsila we-cytoplasmic we-IZUMO1, oye waboniswa ukuba awuyomfuneko kwi-fertilition62).Kungenjalo, i-IZUMO1 kunye/okanye i-SPACA6 inokufuna iimeko ezisibophelelayo ezikhethekileyo esingazivelisiyo kwi-vitro, ezifana neengqamaniso ezithile ze-physiologically okanye ii-molecular complexes ezinezinye iiproteni (ezaziwa okanye ezingekafunyanwa).Nangona i-IZUMO1 ectodomain kukholelwa ukuba ilamla uncamathiselo lwe-spermatozoa kwiqanda kwindawo ye-perivitelline, injongo ye-SPCA6 ectodomain ayicacanga.
Ulwakhiwo lwe-SPACA6 lubonisa iindawo ezininzi ezigciniweyo ezinokubandakanyeka kwintsebenziswano yeprotheni-protein.Inxalenye egciniweyo yommandla wehinge ekufuphi ngokukhawuleza ne-CXXC motif (eyiyo iPatch 1 ngasentla) ineentsalela ezinevumba elimnandi ezijonge ngaphandle ezihlala zinxulunyaniswa ne-hydrophobic kunye ne-π-stacking interactions phakathi kwe-biomolecules.Amacala abanzi e-Ig-like domain (ingingqi ye-2) enza i-groove ene-positive charged groove ene-Arg egcinwe kakhulu kunye neentsalela Zakhe, kunye ne-antibodies echasene nalo mmandla ngaphambili isetyenziswe ukuvimba i-gamete fusion 30.I-antibody iyayibona i-epitope engumgca 212RIRPAQLTHRGTFS225, eneentsalela ze-arginine ezintandathu kwaye igcinwe kakhulu i-His220.Akucaci ukuba ukungasebenzi kubangelwa ukuvaleka kwezi ntsalela ezithile okanye ummandla wonke.Indawo yalo msantsa kufuphi ne-C-terminus ye-β-sandwich ibonisa i-cis-intsebenziswano kunye neeprotheni zesidoda ezingabamelwane, kodwa kungekhona ngeeprotheni ze-oocyte.Ngaphezu koko, ukugcinwa kwe-proline-rich tangle eguquguqukayo kakhulu (indawo ye-3) ngaphakathi kwe-hinge ingaba yindawo ye-protein-protein interaction okanye, mhlawumbi, ibonisa ukugcinwa kokuguquguquka phakathi kweendawo ezimbini.Isini sibalulekile kwindima engaziwayo ye-SPCA6.ukudibanisa kwe-gametes.
I-SPCAA6 ineempawu ze-intercellular adhesion proteins, kuquka i-Ig-like β-sandwiches.Iiprotheyini ezininzi ezinamathele (umzekelo, i-cadherins, i-integrins, i-adhesin, kunye ne-IZUMO1) zinendawo enye okanye ngaphezulu ye-β-sandwich eyandisa iiprotheni ukusuka kwi-cell membrane ukuya kwiithagethi zazo zokusingqongileyo63,64,65.I-domain efana ne-Ig ye-SPCAA6 iqulethe i-motif efumaneka ngokuqhelekileyo kwi-β-sandwiches yokubambelela kunye nokudibanisa: ukuphindaphinda kwemicu ehambelanayo ekupheleni kwe-β-sandwiches, eyaziwa ngokuba yi-clamps mechanical66.Kukholelwa ukuba le motif yandisa ukuchasana nemikhosi yokucheba, exabisekileyo kwiiprotheni ezibandakanyekayo kwintsebenziswano ye-intercellular.Nangona kunjalo, ngaphandle koku kufana nokunamathela, okwangoku akukho bungqina bokuba i-SPCAA6 isebenzisana nabamhlophe beqanda.I-SPCA6 ectodomain ayikwazi ukubophelela kwi-JUNO, kunye ne-SPCA6-ebonisa iiseli ze-HEK293T, njengoko kubonisiwe apha, kunzima ukusebenzisana ne-oocyte eswele indawo ye-32.Ukuba i-SPCAA6 iseka iibhondi ze-intergametic, olu nxibelelwano lunokufuna ukuguqulwa emva koguqulelo okanye luzinziswe ngezinye iiprotheni zesidoda.Ukuxhasa i-hypothesis yokugqibela, i-IZUMO1-i-spermatozoa elahlekileyo ibophelela kwi-oocytes, ebonisa ukuba iamolekyu ngaphandle kwe-IZUMO1 zibandakanyeka kwi-gamete adhesion step 27.
Iiprotheni ezininzi zentsholongwane, iiseli, kunye nophuhliso lwe-fusion zineempawu eziqikelela umsebenzi wazo njenge-fusogens.Ngokomzekelo, i-viral fusion glycoproteins (iiklasi I, II kunye ne-III) zine-peptide ye-hydrophobic fusion okanye i-loop ekupheleni kweprotheni efakwe kwi-membrane yomkhosi.Imephu ye-hydrophilicity ye-IZUMO143 kunye nesakhiwo (esimiselweyo kwaye siqikelelwe) se-IST superfamily ibonise ukuba akukho peptide ye-hydrophobic fusion icacileyo.Ke, ukuba naziphi na iiproteni ezikwi-IST superfamily zisebenza njenge-fusogens, zikwenza oko ngendlela eyahlukileyo kweminye imizekelo eyaziwayo.
Ukuqukumbela, imisebenzi yamalungu e-IST superfamily yeeproteni ezinxulumene ne-gamete fusion ihlala iyimfihlakalo ekhangayo.I-molecule yethu ye-SPCA6 ye-recombinant kunye nesakhiwo sayo esisonjululwe siya kunika ingqiqo kubudlelwane phakathi kwezi zakhiwo ekwabelwana ngazo kunye nendima yazo kwi-gamete attachment kunye nokudibanisa.
Ulandelelwano lwe-DNA oluhambelana ne-ectodomain yabantu eqikelelweyo ye-SPCA6 (inombolo yokufikelela kwi-NCBI NP_001303901.1; iintsalela ze-27-246) zenziwe i-codon-optimized ukuze ibonakaliswe kwiiseli ze-Drosophila melanogaster S2 kwaye zenziwe njengeqhekeza lemfuza kunye ne-encoding ye-Encoding ye-Koza, umqondiso wokukhuselwa kwe-BiP kunye neziphelo ezi-5′ kunye ne-3′ ehambelanayo yokwenziwa kwekloni ezimeleyo yalo jini ibeyivektha yokubonakalisa ye-pMT esekwe kumxhasi wemetallothionein eguqulelwe kukhetho ngepuromycin (pMT-puro).Ivektha ye-pMT-puro ifaka iikhowudi kwindawo yethrombin yoqhekeko elandelwa yi-10x-Ithegi Yakhe ye-C-terminal (Figure S2).
Ukudluliselwa okuzinzile kwe-SPCA6 pMT-puro vector kwiiseli ze-D. melanogaster S2 (Gibco) zenziwe ngokufanayo neprotocol esetyenziselwa i-IZUMO1 kunye ne-JUNO43.Iiseli ze-S2 zanyibilika kwaye zakhuliswa kwi-Schneider's medium (Gibco) zongezwa ngokugxininiswa kokugqibela kwe-10% (v/v) ubushushu obungasebenziyo kwi-fetal ithole serum (Gibco) kunye ne-1X antimycotic antibiotic (Gibco).Iiseli zokuhamba zangaphambili (i-3.0 x 106 iiseli) zifakwe kumaqula ahlukeneyo e-6-well plates (Corning).Emva kweeyure ezingama-24 zokufukanyelwa kwi-27°C, iiseli zatshintshwa ngomxube we-2 mg ye-SPCA6 pMT-puro vector kunye ne-Effectene transfection reagent (Qiagen) ngokweprothokholi yomenzi.Iiseli ezosulelayo ziye zafukanywa iiyure ezingama-72 kwaye emva koko zavunwa nge-6 mg/ml puromycin.Iiseli ke zaye zabekwa zodwa kwiSchneider's medium epheleleyo kwaye zafakwa kwiserum engenazinambuzane-XPRESS medium (Lonza) ukwenzela ukuveliswa kweprotheyini enkulu.Ibhetshi ye-1 L yenkcubeko yeseli ye-S2 yakhuliswa ukuya kwi-8-10 × 106 ml-1 iiseli kwi-2 L ye-flask ene-flat-bottomed polypropylene Erlenmeyer iflask kwaye emva koko yazaliswa ngoxinaniso lokugqibela lwe-500 µM CuSO4 (Millipore Sigma) kunye ne-sterile yahluzwa.enziwe.Iinkcubeko ezibangelwayo zafukanywa kwi-27 ° C. kwi-120 rpm iintsuku ezine.
I-conditioned medium equkethe i-SPCAA6 yahlulwa yi-centrifugation kwi-5660 × g kwi-4 ° C ilandelwa yi-Centramate tangential filtration system (Pall Corp) ene-membrane ye-10 kDa MWCO.Faka i-concentrated medium equlethe i-SPCA6 ukuya kwikholamu ye-2 ml ye-Ni-NTA agarose resin (Qiagen).I-resin ye-Ni-NTA yahlanjwa kunye nemiqulu ye-10 yekholomu (CV) ye-buffer A kunye ne-1 CV ye-buffer A yongezwa ukunika ugxininiso lokugqibela lwe-imidazole lwe-50 mM.I-SPCAA6 yahlanjululwa nge-10 ml ye-buffer A eyongezelelweyo imidazole ukuya kugxininiso lokugqibela lwe-500 mM.I-thrombin yeklasi yokukhawulela (i-Millipore Sigma) yongezwa ngokuthe ngqo kwi-dialysis tubing (MWCO 12-14 kDa) kwiyunithi ye-1 nge-mg SPCA6 vs. 1 L 10 mM Tris-HCl, pH 7.5 kunye ne-150 mM NaCl (buffer B) ye-dialysis.) kwi-4°C iiyure ezingama-48.I-thrombin-cleaved SPCA6 yabe ihlanjululwe kathathu ukuze inciphise ityuwa kwaye ilayishwe kwi-1 ml ye-MonoS 5 / 50 ikholomu yokutshintshiselana kwe-GL cation (Cytiva / GE) ehambelana ne-10 mM Tris-HCl, pH 7.5.Umtshintshi wombane wahlanjwa nge-3 CV ye-10 mM Tris-HCl, i-pH 7.5, emva koko i-SPACA6 yahluthwa nge-linear gradient ye-0 ukuya ku-500 mM NaCl kwi-10 mM Tris-HCl, pH 7.5 ye-25 CV.Emva kwe-ion exchange chromatography, i-SPCAA6 yagxininiswa kwi-1 ml kwaye yahluthwa ngokwe-isocratically ukusuka kwi-ENrich SEC650 10 x 300 ikholamu (BioRad) ehambelana ne-buffer B. Ngokwechromatogram, i-pool kunye ne-concentrate fractions equkethe i-SPCAA6.Ubunyulu bulawulwa yi-Coomassie-stained electrophoresis kwi-16% ye-SDS-polyacrylamide gel.Uxinaniso lweprotheyini luqikelelwa ngokutsalwa kwi-280 nm usebenzisa umthetho we-Beer-Lambert kunye ne-coefficient ye-molar extinction coefficient.
I-SPACA6 ecocekileyo yahlanjululwa ngobusuku malunga ne-10 mM i-sodium phosphate, i-pH 7.4 kunye ne-150 mM NaF kwaye ihlanjululwe kwi-0.16 mg / mL ngaphambi kokuhlalutya nge-CD spectroscopy.I-Spectral scanning ye-CD ene-wavelength ye-185 ukuya kwi-260 nm iqokelelwe kwi-spectropolarimeter ye-Jasco J-1500 usebenzisa i-quartz cuvettes ene-1 mm ubude bendlela ye-optical (Helma) kwi-25 ° C ngesantya se-50 nm / min.I-CD spectra yalungiswa ngokwesiseko, i-avareji ngaphezulu kwe-10 yokufunyanwa, kwaye yaguqulelwa kuthetha i-elliptical residual (θMRE) kwiidigri ze-cm2/dmol:
apho i-MW bubunzima bemolekyuli yesampulu nganye kwi-Da;N linani le-amino acids;θ bubude obuyimilidigremu;d ihambelana nobude bendlela ye-optical in cm;Ugxininiso lweprotheyini kwiiyunithi.