304 chitsulo chosapanga dzimbiri coil tubing mankhwala gawo, Kapangidwe ka SPCA6 ectodomain ali ndi osungidwa superfamily ya mapuloteni ogwirizana ndi gamete fusion.

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SPCA6 ndi puloteni yomwe imawonetsedwa ndi umuna yomwe imakhala yofunika kwambiri pakuphatikizana kwa gamete panthawi yoberekera.Ngakhale gawo lofunikirali, ntchito yeniyeni ya SPCA6 siyikumveka bwino.Timafotokozera mawonekedwe a kristalo a dera la extracellular la SPCA6 pa chigamulo cha 2.2 Å, kuwulula puloteni yamitundu iwiri yopangidwa ndi mtolo wazitsulo zinayi ndi masangweji a Ig-ngati β ophatikizidwa ndi ogwirizanitsa quasi-flexible.Kapangidwe kameneka kamafanana ndi IZUMO1, puloteni ina yogwirizana ndi kuphatikizika kwa gamete, kupangitsa SPCA6 ndi IZUMO1 kukhala mamembala a banja lapamwamba la mapuloteni okhudzana ndi umuna omwe amatchulidwa pano kuti IST superfamily.IST superfamily imatanthauzidwa mwadongosolo ndi mtolo wake wopotoka wa helix zinayi ndi ma CXXC motifs olumikizana ndi disulfide.Kufufuza kozikidwa kwa AlphaFold kwa proteome yamunthu kunazindikiritsa mamembala owonjezera a protein iyi;makamaka, ambiri mwa mapuloteniwa amatenga nawo gawo mu kuphatikizika kwa gamete.Mapangidwe a SPCA6 ndi ubale wake ndi mamembala ena a IST superfamily amapereka ulalo wosowa m'chidziwitso chathu cha kuphatikizika kwa nyama zam'mimba.
Moyo wa munthu aliyense umayamba ndi ma gamete awiri osiyana a haploid: umuna wa abambo ndi dzira la amayi.Ukalawu ndi umene umapambana posankha kwambiri pamene mamiliyoni ambiri a umuna amadutsa mu maliseche a mkazi, kugonjetsa zopinga zosiyanasiyana1 ndikukhala ndi capacitation, zomwe zimawonjezera kusuntha kwawo ndi machitidwe a pamwamba2,3,4.Ngakhale umuna ndi oocyte zipezana, ndondomekoyi siinathe.Oocyte imazunguliridwa ndi gulu la maselo a cumulus ndi chotchinga cha glycoprotein chotchedwa zona pellucida, chomwe umuna umayenera kudutsa kuti ulowe mu oocyte.Spermatozoa imagwiritsa ntchito kuphatikiza kwa mamolekyu omata pamwamba ndi ma enzyme okhudzana ndi nembanemba komanso obisika kuti athe kuthana ndi zopinga zomalizazi5.Mamolekyu ndi michere imeneyi makamaka amasungidwa mu nembanemba wamkati ndi matrix acrosomal ndipo amadziwikiratu pamene nembanemba wakunja wa umuna wa lysed pa acrosomal reaction6.Gawo lomaliza paulendo waukuluwu ndi chochitika cha kuphatikizika kwa umuna ndi dzira, momwe maselo awiriwa amalumikizana ndi nembanemba kuti akhale chamoyo chimodzi cha diploid7.Ngakhale kuti njirayi ndi yochititsa chidwi kwambiri pa kubereka kwa anthu, kugwirizana kofunikira kwa maselo sikumveka bwino.
Kuphatikiza pa umuna wa ma gametes, chemistry ya kuphatikizika kwa ma lipid bilayers awiri adawerengedwa mozama.Nthawi zambiri, kuphatikizika kwa membrane ndi njira yosasangalatsa yomwe imafuna kuti chothandizira puloteni chisinthe momwe zimakhalira zomwe zimapangitsa kuti nembanemba ziwiri ziyandikirane, ndikuphwanya kupitiliza kwake ndikupangitsa kuti fusion8,9.Zothandizira mapuloteniwa zimadziwika kuti fusogens ndipo zapezeka m'makina osawerengeka.Amafunika kuti ma virus alowe m'maselo omwe akulandira (mwachitsanzo, gp160 mu HIV-1, spike mu coronaviruses, hemagglutinin mu virus fuluwenza) 10,11,12 placental (syncytin) 13,14,15 ndi mapangidwe a gamete mu eukaryotes yotsika ( HAP2/GCS1 muzomera, ojambula ndi arthropods) 16,17,18,19.Mafusogens a gametes aumunthu sanapezekebe, ngakhale kuti mapuloteni angapo awonetsedwa kuti ndi ofunika kwambiri kuti ma gamete agwirizane ndi kuphatikizika.CD9 yowonetsedwa ndi oocyte, puloteni ya transmembrane yofunikira pakuphatikiza mbewa ndi ma gametes amunthu, inali yoyamba kupezeka 21,22,23.Ngakhale kuti ntchito yake yeniyeni imakhalabe yosadziwikiratu, gawo lomamatira, kapangidwe ka adhesion foci pa egg microvilli, ndi / kapena kutanthauzira kolondola kwa mapuloteni a oocyte pamwamba akuwoneka ngati 24,25,26.Mapuloteni awiri omwe ali ofunikira kwambiri pakuphatikizana kwa gamete ndi puloteni ya umuna IZUMO127 ndi puloteni ya oocyte JUNO28, ndipo kuyanjana kwawo ndi gawo lofunikira pakuzindikirika ndi kumamatira kwa gamete musanaphatikizidwe.Male Izumo1 knockout mbewa ndi wamkazi Juno knockout mbewa ndi wosabala, mu zitsanzo izi umuna kulowa perivitelline danga koma gametes samalumikizana.Mofananamo, kugwirizanitsa kunachepetsedwa pamene ma gametes amathandizidwa ndi anti-IZUMO1 kapena JUNO27,29 antibodies mu kuyesa kwaumunthu mu vitro feteleza.
Posachedwapa, gulu lomwe latulukira kumene la mapuloteni owonetsera umuna phenotypically ofanana ndi IZUMO1 ndi JUNO20,30,31,32,33,34,35.Mapuloteni a sperm acrosomal membrane-associated protein 6 (SPCAA6) adziwika kuti ndi ofunikira kuti apange umuna mu phunziro lalikulu la murine mutagenesis.Kuyika kwa transgene mu jini ya Spaca6 kumapanga spermatozoa yopanda mphamvu, ngakhale kuti spermatozoa imalowa mu malo a perivitelline 36.Kafukufuku wotsatira wogogoda mu mbewa adatsimikizira kuti Spaca6 ikufunika kuti gamete fusion 30,32 .SPCA6 imasonyezedwa pafupifupi m'ma testes ndipo ili ndi ndondomeko yokhazikika yofanana ndi ya IZUMO1, yomwe ili mkati mwa intima ya spermatozoa isanafike acrosomal reaction, ndiyeno imasamukira ku dera la equatorial pambuyo pa acrosomal reaction 30,32.Spaca6 homologues alipo mu mitundu yosiyanasiyana ya zinyama ndi ma eukaryotes 30 ndipo kufunika kwake kwa kuphatikizika kwa gamete yaumunthu kwasonyezedwa ndi kulepheretsa umuna waumunthu mu vitro mwa kukana SPCA6 30.Mosiyana ndi IZUMO1 ndi JUNO, tsatanetsatane wa kapangidwe kake, kuyanjana, ndi ntchito ya SPCA6 sizikudziwika.
Kuti timvetse bwino ndondomeko yoyambira kuphatikizika kwa umuna ndi mazira, zomwe zitithandiza kudziwa zomwe zidzachitike m'tsogolo pakulera ndi chithandizo cha chonde, tidachita maphunziro a SPCA6 okhudza kapangidwe kake ndi biochemical.Mapangidwe a kristalo a domain extracellular ya SPCA6 akuwonetsa mtolo wa helical anayi (4HB) ndi immunoglobulin-like (Ig-like) domain yolumikizidwa ndi zigawo zosinthika.Monga momwe zinanenedweratu m'maphunziro apitalo, 7,32,37 dongosolo la SPCA6 ndilofanana ndi la IZUMO1 laumunthu, ndipo mapuloteni awiriwa amagawana ndi zochitika zachilendo: 4HB yokhala ndi katatu ya helical pamwamba ndi ma disulfide-linked CXXC motifs.Tikulingalira kuti IZUMO1 ndi SPCA6 tsopano afotokoze za banja lalikulu, logwirizana kwambiri ndi mapuloteni okhudzana ndi kuphatikizika kwa gamete.Pogwiritsa ntchito mawonekedwe apadera a superfamily, tinafufuza mozama za AlphaFold structural human proteome, kuzindikiritsa mamembala owonjezera a banja lapamwambali, kuphatikiza mamembala angapo omwe akuchita nawo kuphatikizika kwa gamete ndi/kapena umuna.Tsopano zikuwoneka kuti pali gulu lodziwika bwino komanso lambiri la mapuloteni okhudzana ndi kuphatikizika kwa gamete, ndipo kapangidwe kathu kamapereka mapu a molekyulu a mbali yofunika iyi ya makina ophatikizika a gamete.
SPCA6 ndi puloteni ya transmembrane yodutsa imodzi yokhala ndi glycan imodzi yolumikizidwa ndi N ndi ma bond asanu ndi limodzi a putative disulfide (Zithunzi S1a ndi S2).Tinawonetsa dera la extracellular la SPCA6 yaumunthu (zotsalira 27-246) m'maselo a Drosophila S2 ndikuyeretsa mapuloteni pogwiritsa ntchito nickel affinity, cation exchange, ndi size exclusion chromatography (Fig. S1b).SPCA6 ectodomain yoyeretsedwa ndiyokhazikika komanso yofanana.Kusanthula pogwiritsa ntchito kukula kosaphatikiza chromatography pamodzi ndi kuwala kwa polygonal (SEC-MALS) kunavumbula nsonga imodzi yokhala ndi kulemera kwa maselo a 26.2 ± 0.5 kDa (mkuyu. S1c).Izi zikugwirizana ndi kukula kwa SPCA6 monomeric ectodomain, kusonyeza kuti oligomerization sinachitike pa kuyeretsedwa.Kuonjezera apo, masewero ozungulira a dichroism (CD) adavumbulutsa dongosolo losakanikirana la α / β ndi malo osungunuka a 51.3 ° C (Mkuyu S1d, e).Deconvolution ya CD spectra inavumbulutsa 38.6% α-helical ndi 15.8% β-stranded zinthu (Chithunzi S1d).
The SPCA6 ectodomain anali crystallized ntchito mwachisawawa masanjidwewo seeding38 chifukwa deta anapereka ndi kusamvana 2.2 Å (Table 1 ndi Chithunzi S3).Pogwiritsa ntchito kuphatikizika kwa ma cell-based fragment substitution and SAD phasesing data with bromide exposure for the structure (Table 1 ndi Figure S4), chomaliza choyeretsedwa chimakhala ndi zotsalira 27-246.Pa nthawi yomwe dongosololi linatsimikiziridwa, panalibe zoyesera kapena AlphaFold zomwe zilipo.SPCA6 ectodomain miyeso 20 Å × 20 Å × 85 Å, imakhala ndi ma helice asanu ndi awiri ndi ma β-strand asanu ndi anayi, ndipo ili ndi khola lapamwamba lokhazikika lokhazikika ndi zomangira zisanu ndi chimodzi za disulfide (Mkuyu 1a, b).Kuchuluka kwa ma elekitironi ofooka kumapeto kwa unyolo wa mbali ya Asn243 kumasonyeza kuti chotsalira ichi ndi N-linked glycosylation.Kapangidwe kamakhala madera awiri: ndi N-terminal anayi helix mtolo (4HB) ndi C-terminal Ig-ngati ankalamulira ndi wapakatikati hinge dera pakati pawo (mkuyu. 1c).
Mapangidwe a domain extracellular ya SPCA6.Chithunzi chojambula chamtundu wa extracellular wa SPCA6, mtundu wa unyolo kuchokera ku N kupita ku C-terminus kuchokera ku buluu wakuda mpaka kufiira kodera.Ma cysteines omwe amakhudzidwa ndi zomangira za disulfide amawonetsedwa mu magenta.b Topology ya domain extracellular ya SPCA6.Gwiritsani ntchito mtundu womwewo wa chithunzi 1a.c SPCA6 domain extracellular.Ma chart a 4HB, hinge, ndi ma Ig-like domain strip ali ndi utoto walalanje, wobiriwira, ndi wabuluu, motsatana.Zigawo sizimakokedwa pamlingo.
Dongosolo la 4HB la SPCA6 limaphatikizapo ma helice anayi (ma helice 1-4), omwe amakonzedwa mwa mawonekedwe a helical helix (mkuyu 2a), kusinthasintha pakati pa antiparallel ndi kugwirizana kofanana (mkuyu 2b).Helix yaing'ono yowonjezera imodzi (helix 1′) imayikidwa mozungulira pamtolo, ndikupanga makona atatu ndi helices 1 ndi 2. Makona atatuwa amapunduka pang'ono pakulongedza kopindika kwa helical kulongedza kwambiri kwa helices 3 ndi 4. Chithunzi 2a).
Chithunzi cha 4HB N-terminalb Mawonedwe apamwamba a mtolo wa ma helikisi anayi, helix iliyonse imawonekera mu buluu wakuda pa N-terminus ndi yofiira kwambiri pa C-terminus.c Chithunzi chozungulira pamwamba-pansi cha 4HB, chotsalira chilichonse chikuwonetsedwa ngati bwalo lolembedwa ndi code ya amino acid yokhala ndi chilembo chimodzi;ma amino acid anayi okha pamwamba pa gudumu amawerengedwa.Zotsalira zomwe si za polar zimakhala zachikasu, zotsalira za polar zosachangidwa zimakhala zobiriwira, zotsalira zowoneka bwino zimakhala za buluu wamitundu yosiyanasiyana, ndipo zotsalira zakuda zimakhala zofiira.d Nkhope za katatu za chigawo cha 4HB, chokhala ndi 4HBs mu lalanje ndi mahinji obiriwira.Zikhazikiko zonse zikuwonetsa ma bond a disulfide ngati ndodo.
4HB imakhazikika pakatikati pa hydrophobic core yopangidwa makamaka ndi zotsalira za aliphatic ndi zonunkhira (mkuyu 2c).Pakatikati pali mgwirizano wa disulfide pakati pa Cys41 ndi Cys55 womwe umalumikiza helices 1 ndi 2 palimodzi pamakona atatu apamwamba (mkuyu 2d).Zomangira ziwiri zowonjezera za disulfide zinapangidwa pakati pa CXXC motif mu Helix 1' ndi CXXC ina yopezeka pa nsonga ya β-hairpin m'dera la hinge (mkuyu 2d).Chotsalira cha arginine chokhazikika chokhala ndi ntchito yosadziwika (Arg37) chili mkati mwa katatu kakang'ono kopangidwa ndi helices 1′, 1, ndi 2. Aliphatic carbon maatomu Cβ, Cγ, ndi Cδ Arg37 amalumikizana ndi hydrophobic core, ndipo magulu ake a guanidine amayenda mozungulira. pakati pa helices 1' ndi 1 kudzera muzochita pakati pa msana wa Thr32 ndi unyolo wam'mbali (Mkuyu S5a, b).Tyr34 imafikira pabowo ndikusiya timipata tiwiri tating'ono momwe Arg37 imatha kulumikizana ndi zosungunulira.
Madomeni onga β-sangweji ndi gulu lalikulu la mapuloteni omwe amagawana zomwe zimafanana ndi ma β-sheets awiri kapena kuposerapo amphipathic amitundu yambiri omwe amalumikizana kudzera pa hydrophobic core 39. Malo a C-terminal Ig-like SPCA6 ali ndi mawonekedwe omwewo. ndipo imakhala ndi zigawo ziwiri (mkuyu S6a).Mapepala 1 ndi β-pepala la zingwe zinayi (zingwe D, F, H, ndi I) pamene zingwe F, H, ndi ine zimapanga dongosolo lotsutsana ndi kufanana, ndipo zingwe I ndi D zimagwirizana mofanana.Gulu 2 ndi pepala la beta laling'ono lolimbana ndi mizere iwiri (zingwe E ndi G).Chigwirizano chamkati cha disulfide chinawonedwa pakati pa C-terminus ya E chain ndi pakati pa H unyolo (Cys170-Cys226) (Mkuyu S6b).Chomangira cha disulfide ichi ndi chofanana ndi chomangira cha disulfide mu β-sandwich domain ya immunoglobulin40,41.
Mapepala anayi a β amapindika m'litali mwake, kupanga m'mphepete mwa asymmetrical omwe amasiyana mawonekedwe ndi ma electrostatics.Mphepete mwawoonda ndi malo athyathyathya a hydrophobic chilengedwe omwe amawonekera kwambiri poyerekeza ndi malo otsalira osagwirizana ndi ma electrostatically osiyanasiyana mu SPCA6 (mkuyu. S6b, c).Halo yamagulu a msana wa carbonyl/amino ndi maunyolo a polar akuzungulira hydrophobic pamwamba (mkuyu S6c).Mphepete mwambiri imaphimbidwa ndi kapu ya helical yomwe imatsekereza gawo la N-terminal la hydrophobic core ndikupanga ma hydrogen bond atatu ndi gulu lotseguka la polar la F chain backbone (mkuyu. S6d).Gawo la C-terminal la m'mphepete mwake limapanga thumba lalikulu lokhala ndi hydrophobic pachimake.Thumba lazunguliridwa ndi zolipiritsa zabwino chifukwa cha magawo atatu a zotsalira za arginine (Arg162-Arg221, Arg201-Arg205 ndi Arg212-Arg214) ndi histidine yapakati (His220) (Chithunzi S6e).
Dera la hinge ndi gawo laling'ono pakati pa chigawo cha helical ndi dera la Ig-like, lomwe lili ndi β-layer imodzi yotsutsana ndi katatu (nsonga A, B ndi C), helix yaing'ono ya 310, ndi zigawo zingapo zazitali zamtundu wautali.(mkuyu S7).Ma network a covalent and electrostatic contacts mu hinge region akuwoneka kuti akukhazikika pakati pa 4HB ndi domain ngati Ig.Network ikhoza kugawidwa m'magawo atatu.Gawo loyamba limaphatikizapo zigawo ziwiri za CXXC (27CXXC30 ndi 139CXXC142) zomwe zimapanga mgwirizano wa disulfide pakati pa β-hairpin mu hinge ndi 1' helix mu 4HB.Gawo lachiwiri limaphatikizapo kuyanjana kwa electrostatic pakati pa dera la Ig-ngati ndi hinge.Glu132 mu hinge imapanga mlatho wamchere wokhala ndi Arg233 m'malo ngati Ig ndi Arg135 mu hinge.Gawo lachitatu limaphatikizapo mgwirizano wogwirizana pakati pa dera la Ig-like ndi dera la hinge.Zomangira ziwiri za disulfide (Cys124-Cys147 ndi Cys128-Cys153) zimagwirizanitsa hinge loop ndi cholumikizira chomwe chimakhazikika ndi ma electrostatic interactions pakati pa Gln131 ndi gulu logwira ntchito la msana, kulola mwayi wopita ku dera loyamba la Ig.unyolo.
Mapangidwe a SPCA6 ectodomain ndi machitidwe aumwini a 4HB ndi ma Ig-like domains adagwiritsidwa ntchito kufufuza zolemba zofananira m'mapuloteni 42.Tidazindikira machesi okhala ndi zigoli zambiri za Dali Z, zopatuka zing'onozing'ono, ndi zigoli zazikulu za LALI (zomalizirazo zinali kuchuluka kwa zotsalira zofanana).Ngakhale kugunda kwa 10 koyambirira kuchokera pakusaka kwathunthu kwa ectodomain (Table S1) inali ndi Z-score yovomerezeka ya> 842, kufufuza kwa 4HB kapena Ig-like domain yokha kunasonyeza kuti zambiri mwazomwezi zimayenderana ndi masangweji a β okha.mpinda wopezeka paliponse m'mapuloteni ambiri.Kusaka konse kutatu ku Dali kwabweretsa chotsatira chimodzi chokha: IZUMO1.
Zakhala zikunenedwa kuti SPCA6 ndi IZUMO1 zimagawana zofanana7,32,37.Ngakhale ma ectodomains a mapuloteni awiriwa okhudzana ndi kuphatikizika kwa gamete amagawana 21% yokha yodziwika (Chithunzi S8a), umboni wovuta, kuphatikiza mawonekedwe otetezedwa a disulfide ndi malo onenedweratu a C-terminal Ig-ngati mu SPCA6, adalola kuyesa koyambirira kuti apange chitsanzo cha homology cha A a SPCA6 mbewa pogwiritsa ntchito IZUMO1 ngati template37.Kapangidwe kathu kakutsimikizira maulosi awa ndikuwonetsa kufanana kwenikweni.Ndipotu, mapangidwe a SPCA6 ndi IZUMO137,43,44 amagawana zomangamanga ziwiri (mkuyu S8b) ndi 4HB yofanana ndi β-sandwich domains yolumikizidwa ndi dera la hinge (Mkuyu S8c).
IZUMO1 ndi SPCA6 4HB ali ndi kusiyana kofanana ndi mitolo yozungulira yokhazikika.Ma 4HB odziwika bwino, monga omwe amapezeka mu mapuloteni a SNARE omwe amaphatikizidwa mu endosomal fusion 45,46, ali ndi ma helice osakanikirana omwe amasunga nthawi zonse kuzungulira pakati pa axis 47. Mosiyana ndi izi, madera a helical mu IZUMO1 ndi SPCA6 adasokonezedwa, ndi kupindika kosiyana komanso kulongedza katundu (Chithunzi S8d).Kupotoza, mwinamwake chifukwa cha katatu kopangidwa ndi helices 1', 1 ndi 2, kumasungidwa mu IZUMO1 ndi SPCA6 ndikukhazikika ndi CXXC yofanana pa helix 1'.Komabe, chomangira chowonjezera cha disulfide chopezeka mu SPCA6 (Cys41 ndi Cys55 cholumikiza ma helice 1 ndi 2 pamwambapa) chimapanga chokwera kwambiri pamakona atatu, ndikupangitsa SPCA6 kukhala yokhotakhota kwambiri kuposa IZUMO1, yokhala ndi makona atatu odziwika bwino.Kuphatikiza apo, IZUMO1 ilibe Arg37 yomwe imawonedwa pakatikati pabowo ili mu SPCA6.Mosiyana, IZUMO1 ili ndi hydrophobic core ya zotsalira za aliphatic ndi zonunkhira.
IZUMO1 ili ndi domain ngati Ig yokhala ndi β-sheet43 yazingwe ziwiri komanso zisanu.Chingwe chowonjezera mu IZUMO1 chimalowa m'malo mwa koyilo mu SPCA6, yomwe imalumikizana ndi F strand kuti ichepetse zomangira za hydrogen mu chingwe.Chosangalatsa chofanizira ndi kuchuluka komwe kunanenedweratu kwa magawo ngati Ig a mapuloteni awiriwa.Kumwamba kwa IZUMO1 kumakhala koyipa kwambiri kuposa malo a SPCA6.Chowonjezera china chili pafupi ndi C-terminus moyang'anizana ndi nembanemba ya umuna.Mu SPCA6, madera omwewo anali osalowerera ndale kapena abwino (Fig. S8e).Mwachitsanzo, malo a hydrophobic (m'mbali zoonda) ndi maenje otsekera bwino (m'mphepete mwake) mu SPCA6 amathiridwa moyipa mu IZUMO1.
Ngakhale kuti maubwenzi ndi makonzedwe achiwiri pakati pa IZUMO1 ndi SPCA6 amasungidwa bwino, kugwirizanitsa kwapangidwe kwa madera a Ig-ngati kunasonyeza kuti madera awiriwa amasiyana mosiyana ndi momwe amayendera (mkuyu S9).Mtolo wozungulira wa IZUMO1 ndi wopindika pafupi ndi sangweji ya β, ndikupanga mawonekedwe a "boomerang" omwe afotokozedwa kale pafupifupi 50 ° kuchokera pakati pa axis.Mosiyana ndi izi, mtengo wa helical mu SPCA6 udapendekeka pafupifupi 10 ° mbali ina.Kusiyanasiyana kwa machitidwewa kumakhala chifukwa cha kusiyana kwa dera la hinge.Pamlingo wotsatizana, IZUMO1 ndi SPCA6 zimagawana pang'ono kufanana kwa hinge, kupatula cysteine, glycine, ndi zotsalira za aspartic acid.Zotsatira zake, ma hydrogen bond ndi ma electrostatic network ndizosiyana kotheratu.β-mapepala achiwiri apangidwe amagawidwa ndi IZUMO1 ndi SPCA6, ngakhale maunyolo mu IZUMO1 ndi otalika kwambiri ndipo 310 helix (helix 5) ndi yapadera kwa SPCA6.Kusiyanaku kumabweretsa magawo osiyanasiyana amtundu wa mapuloteni awiri ofanana.
Kufufuza kwathu pa seva ya Dali kunavumbula kuti SPCA6 ndi IZUMO1 ndi ziwiri zokha zomwe zatsimikiziridwa moyesera zomwe zasungidwa munkhokwe ya mapuloteni omwe ali ndi 4HB fold iyi (Table S1).Posachedwapa, DeepMind (Alphabet/Google) yapanga AlphaFold, neural network based system yomwe imatha kulosera molondola mapangidwe a 3D a mapuloteni kuchokera kumagawo oyambira48.Titangotha ​​kuthetsa dongosolo la SPCA6, nkhokwe ya AlphaFold inatulutsidwa, ndikupereka zitsanzo zowonetseratu zomwe zimaphimba 98.5% ya mapuloteni onse mu proteome48,49 yaumunthu.Pogwiritsa ntchito dongosolo lathu la SPCA6 monga chitsanzo chofufuzira, kufufuza kwa homology kwachitsanzo mu AlphaFold human proteome kunapeza ofuna kufanana ndi SPCA6 ndi IZUMO1.Popeza zosaneneka kulondola kwa AlphaFold kulosera SPCA6 (mkuyu. S10a) -makamaka 1.1 Å rms ectodomain poyerekeza dongosolo wathu anatsimikiza (mkuyu. S10b) -tingakhale otsimikiza kuti anazindikira SPCA6 machesi zikuoneka kuti zolondola.
M'mbuyomu, PSI-BLAST idasakasaka gulu la IZUMO1 lokhala ndi mapuloteni ena atatu okhudzana ndi umuna: IZUMO2, IZUMO3, ndi IZUMO450.AlphaFold ananeneratu kuti mapuloteni a banja la IZUMO awa amapindika mu 4HB ndi njira yofanana ya disulfide ngati IZUMO1 (Zithunzi 3a ndi S11), ngakhale alibe gawo lofanana ndi Ig.Amaganiziridwa kuti IZUMO2 ndi IZUMO3 ndi maproteni a nembanemba a mbali imodzi ofanana ndi IZUMO1, pomwe IZUMO4 ikuwoneka kuti yatuluka.Ntchito za IZUMO 2, 3, ndi 4 mapuloteni mu kuphatikizika kwa gamete sizinadziwike.IZUMO3 imadziwika kuti imagwira ntchito mu acrosome biogenesis panthawi ya kukula kwa umuna51 ndipo mapuloteni a IZUMO apezeka kuti apanga ma complex50.Kusungidwa kwa mapuloteni a IZUMO mu nyama zoyamwitsa, zokwawa, ndi zamoyo zam'madzi kumasonyeza kuti ntchito yawo yomwe ingakhalepo ikugwirizana ndi mapuloteni ena odziwika okhudzana ndi gamete-fusion, monga DCST1/2, SOF1, ndi FIMP.
Chithunzi cha kamangidwe ka domain la IST superfamily, yokhala ndi 4HB, hinge, ndi madera ngati a Ig omwe adawonetsedwa mulalanje, zobiriwira, ndi buluu, motsatana.IZUMO4 ili ndi dera lapadera la C-terminal lomwe limawoneka lakuda.Zomangira zotsimikizika ndi putative disulfide zimawonetsedwa ndi mizere yolimba ndi madontho, motsatana.b IZUMO1 (PDB: 5F4E), SPCA6, IZUMO2 (AlphaFold DB: AF-Q6UXV1-F1), IZUMO3 (AlphaFold DB: AF-Q5VZ72-F1), IZUMO4 (AlphaFold DB: AF-Q1ZYL8-F95), ndi AF-Q1ZYL8-F95 DB: AF-Q1ZYL8-F1) : AF-Q1ZYL8-F1) : AF-Q3KNT9-F1) amawonetsedwa mumtundu womwewo monga gulu A. Zomangira za disulfide zikuwonetsedwa mu magenta.TMEM95, IZUMO2 ndi IZUMO3 transmembrane helices sawonetsedwa.
Mosiyana ndi mapuloteni a IZUMO, mapuloteni ena a SPCA (ie, SPCA1, SPCA3, SPCA4, SPCA5, ndi SPCA9) amaganiziridwa kuti ndi osiyana ndi SPCA6 (mkuyu S12).SPCA9 yokha yomwe ili ndi 4HB, koma sichikuyembekezeka kukhala ndi mawonekedwe ofanana ndi odana ndi kufanana kapena mgwirizano womwewo wa disulfide monga SPCA6.SPCA1 yokhayo ili ndi dera lofanana ndi Ig.AlphaFold amalosera kuti SPCA3, SPCA4 ndi SPCA5 ali ndi mawonekedwe osiyana kwambiri ndi SPCA6.Chosangalatsa ndichakuti, SPCA4 imadziwikanso kuti imagwira nawo umuna, koma mokulirapo kuposa SPCA6, m'malo mwake imathandizira kulumikizana pakati pa umuna ndi oocyte zona pellucida52.
Kusaka kwathu kwa AlphaFold kunapezanso zofananira za IZUMO1 ndi SPCA6 4HB, TMEM95.TMEM95, puloteni imodzi yokha ya umuna wa transmembrane, imapangitsa mbewa zamphongo kukhala zosabereka zikachotsedwa 32,33.Spermatozoa yosowa TMEM95 inali ndi morphology yabwinobwino, motility, komanso kuthekera kolowera kuzona pellucida ndikumanga ku nembanemba ya dzira, koma sinkatha kuphatikizira ndi nembanemba ya oocyte.Kafukufuku wam'mbuyomu awonetsa kuti TMEM95 imagawana zofananira ndi IZUMO133.Zoonadi, chitsanzo cha AlphaFold chinatsimikizira kuti TMEM95 ndi 4HB yokhala ndi zolemba zofanana za CXXC monga IZUMO1 ndi SPCA6 ndi mgwirizano womwewo wa disulfide pakati pa helices 1 ndi 2 wopezeka mu SPCA6 (Mkuyu 3a ndi S11).Ngakhale TMEM95 ilibe dera lofanana ndi Ig, ili ndi chigawo chokhala ndi disulfide bond pattern yofanana ndi madera a SPCA6 ndi IZUMO1 hinge (mkuyu 3b).Panthawi yosindikizidwa, seva yosindikiza inanena za kapangidwe ka TMEM95, kutsimikizira zotsatira za AlphaFold53.TMEM95 ndi yofanana kwambiri ndi SPCA6 ndi IZUMO1 ndipo imasungidwa kale mu amphibians (Mkuyu 4 ndi S13).
Kusaka kwa PSI-BLAST kudagwiritsa ntchito NCBI SPCA6, IZUMO1-4, TMEM95, DCST1, DCST2, FIMP, ndi SOF1 databases kuti adziwe malo omwe adatsatana mumtengo wamoyo.Kutalikirana pakati pa nsonga za nthambi sikuwonetsedwa kukula.
Kufanana kochititsa chidwi komwe kulipo pakati pa SPCA6 ndi IZUMO1 kukusonyeza kuti iwo ndi amene anayambitsa banja lapamwamba lotetezedwa lomwe limaphatikizapo mapuloteni a TMEM95 ndi IZUMO 2, 3, ndi 4.mamembala odziwika: IZUMO1, SPCA6 ndi TMEM95.Chifukwa ndi mamembala ochepa okha omwe ali ndi madera ngati a Ig, chizindikiro cha IST superfamily ndi 4HB domain, yomwe ili ndi mawonekedwe apadera omwe amafanana ndi mapuloteni onsewa: 1) 4HB yophimbidwa ndi ma helice okonzedwa mosinthana ndi anti-parallel/parallel (Mkuyu). 5a), 2) mtolowu uli ndi nkhope ya katatu yomwe imakhala ndi ma helice awiri mkati mwa mtolo ndi helix yachitatu yowongoka (Fig. key area (Fig. 5c) CXXC motif, yomwe imapezeka mu mapuloteni ngati thioredoxin, amadziwika kuti amagwira ntchito. monga sensa ya redox 54,55,56, pamene chithunzithunzi m'banja la IST chikhoza kugwirizanitsidwa ndi mapuloteni disulfide isomerases monga ERp57 mu fusion ya gamete.
Mamembala a IST superfamily amatanthauzidwa ndi mawonekedwe atatu a 4HB domain: ma helice anayi omwe amasinthana pakati pa ofanana ndi antiparallel orientation, ba-triangular helical bundle faces, ndi ca CXXC double motif yopangidwa pakati pa mamolekyu ang'onoang'ono.) N-terminal helixes (lalanje) ndi hinge dera β-hairpin (wobiriwira).
Poganizira kufanana pakati pa SPCA6 ndi IZUMO1, kuthekera kwa oyamba kumangiriza ku IZUMO1 kapena JUNO kunayesedwa.Biolayer interferometry (BLI) ndi njira yomangiriza ya kinetic yomwe yakhala ikugwiritsidwa ntchito poyesa kuyanjana pakati pa IZUMO1 ndi JUNO.Pambuyo pa kuyika kwa biotin-yotchedwa sensor yokhala ndi IZUMO1 ngati nyambo yokhala ndi chiwerengero chachikulu cha JUNO analyte, chizindikiro champhamvu chinapezeka (mkuyu S14a), kusonyeza kusintha komangiriza-kupangitsa kuti makulidwe a biomaterial agwirizane ndi nsonga ya sensa.Zizindikiro zofanana (ie, JUNO yophatikizidwa ndi sensa ngati nyambo yotsutsana ndi IZUMO1 analyte) (Mkuyu. S14b).Palibe chizindikiro chomwe chinadziwika pamene SPCA6 idagwiritsidwa ntchito ngati analyte motsutsana ndi IZUMO1 yomangidwa ndi sensa kapena JUNO (Chithunzi S14a,b).Kusowa kwa chizindikirochi kumasonyeza kuti dera la extracellular la SPCA6 siligwirizana ndi dera la extracellular la IZUMO1 kapena JUNO.
Chifukwa kuyesa kwa BLI kumachokera ku biotinylation ya zotsalira za lysine zaulere pa mapuloteni a nyambo, kusinthidwa kumeneku kungalepheretse kumangirira ngati zotsalira za lysine zikuphatikizidwa muzochita.Kuphatikiza apo, kuyang'ana komwe kumangiriza wachibale ku sensa kumatha kupangitsa zolepheretsa, kotero kuti zoyeserera zanthawi zonse zokokera pansi zidachitidwanso pa recombinant SPCA6, IZUMO1 ndi JUNO ectodomains.Ngakhale izi, SPCA6 sinayende bwino ndi IZUMO1 yomwe ili ndi zilembo Zake kapena JUNO (Fig. S14c, d), kusonyeza kuti palibe kuyanjana kogwirizana ndi zomwe zinawonetsedwa muzoyesera za BLI.Monga ulamuliro wabwino, tinatsimikizira kuyanjana kwa JUNO ndi dzina lake IZUMO1 (Zithunzi S14e ndi S15).
Ngakhale kufanana kwapangidwe pakati pa SPCA6 ndi IZUMO1, kulephera kwa SPCA6 kumanga JUNO sizodabwitsa.Pamwamba pa IZUMO1 yaumunthu ili ndi zotsalira zoposa 20 zomwe zimagwirizana ndi JUNO, kuphatikizapo zotsalira kuchokera kumadera onse atatu (ngakhale ambiri a iwo ali mu hinge dera) (Mkuyu S14f).Mwa zotsalira izi, imodzi yokha ndiyomwe imasungidwa mu SPCA6 (Glu70).Ngakhale kuti zotsalira zambiri zotsalira zinasungabe katundu wawo woyambirira wa biochemical, zotsalira za Arg160 zofunika mu IZUMO1 zidalowetsedwa m'malo ndi Asp148 yoyipa mu SPCA6;Kafukufuku wam'mbuyomu awonetsa kuti kusintha kwa Arg160Glu mu IZUMO1 pafupifupi kuthetseratu kumangirira ku JUNO43.Kuphatikiza apo, kusiyana kwa madera apakati pa IZUMO1 ndi SPCA6 kudakulitsa kwambiri malo a JUNO-binding site of the same area pa SPCA6 (Fig. S14g).
Ngakhale kufunikira kodziwika kwa SPCA6 kwa kuphatikizika kwa gamete komanso kufanana kwake ndi IZUMO1, SPCA6 sikuwoneka kuti ili ndi ntchito yomangirira yofanana ndi JUNO.Chifukwa chake, tayesetsa kuphatikiza deta yathu yamapangidwe ndi umboni wofunikira woperekedwa ndi chisinthiko cha biology.Kuyanjanitsa kwa ma homologues a SPCA6 kumawonetsa kusungidwa kwazomwe zimachitika kupitilira nyama zoyamwitsa.Mwachitsanzo, zotsalira za cysteine ​​​​zimapezeka ngakhale mu amphibians okhudzana kwambiri (mkuyu 6a).Pogwiritsa ntchito seva ya ConSurf, deta yosungirako katsatidwe kambiri kakutsatizana kwa 66 idajambulidwa pamtunda wa SPCA6.Kusanthula kotereku kungasonyeze kuti ndi zotsalira ziti zomwe zasungidwa panthawi ya kusintha kwa mapuloteni ndipo zingasonyeze kuti ndi zigawo ziti zomwe zimagwira ntchito.
kutsata kwa ma ectodomain a SPCA6 kuchokera ku mitundu 12 yosiyanasiyana yokonzedwa pogwiritsa ntchito CLUSTAL OMEGA.Malinga ndi kusanthula kwa ConSurf, malo osamala kwambiri amalembedwa buluu.Zotsalira za cysteine ​​​​zimawonetsedwa zofiira.Malire a madera ndi zigawo zachiwiri zimasonyezedwa pamwamba pa ndondomekoyi, pamene mivi imasonyeza β-strands ndi mafunde amasonyeza ma helice.NCBI Access Identifiers okhala ndi matsatidwewa ndi awa: anthu (Homo sapiens, NP_001303901), mandrill (Mandrilus leucophaeus, XP_011821277), capuchin nyani (Cebus mimic, XP_017359366), kavalo (Equus 002Or cabaci3), XP_017359366 23 XP_032_034).), nkhosa (Ovis aries, XP_014955560), njovu (Loxodonta africana, XP_010585293), galu (Canis lupus familyis, XP_025277208), mbewa (Mus musculus, NP_001156381), Tasmanian1156381), Tasmanian156381, Tasmanian156381 , 8) , 61_89 ndi Bullfrog (Bufo bufo, XP_040282113).Kuwerengera kumatengera dongosolo la anthu.b Kuyimira pamwamba kwa kapangidwe ka SPCA6 kokhala ndi 4HB pamwamba ndi domeni ngati Ig pansi, mitundu yotengera kuyerekezera kosungirako kuchokera pa seva ya ConSurf.Mbali zotetezedwa bwino kwambiri ndi za buluu, zotetezedwa bwino zimakhala zoyera, ndipo zochepa zomwe zimasungidwa ndi zachikasu.cysteine ​​wofiirira.Zigamba zitatu zapamwamba zosonyeza chitetezo chapamwamba zimasonyezedwa m’chimake cholembedwa zigamba 1, 2 ndi 3. Katuni ya 4HB yasonyezedwa m’chikhazikitso chakumanja pamwamba (chiwembu chamtundu womwewo).
Mapangidwe a SPCA6 ali ndi zigawo zitatu zotetezedwa kwambiri (mkuyu 6b).Patch 1 imapanga 4HB ndi dera la hinge ndipo ili ndi milatho iwiri yosungidwa ya CXXC disulfide, maukonde a hinge a Arg233-Glu132-Arg135-Ser144 (mkuyu.mkombero wotakata wa Ig-ngati ankalamulira (mkuyu. S6e), amene akuimira angapo zabwino mlandu zotsalira pa umuna pamwamba.Chosangalatsa ndichakuti chigambachi chili ndi antibody epitope yomwe idawonetsedwa kale kuti imasokoneza ntchito ya SPCA6 30.Dera la 3 limadutsa pa hinji ndi mbali imodzi ya dera lofanana ndi Ig;Derali lili ndi ma prolines otetezedwa (Pro126, Pro127, Pro150, Pro154) ndi zotsalira zoyang'ana kunja kwa polar/charged.Chodabwitsa n'chakuti, zotsalira zambiri zomwe zili pamwamba pa 4HB ndizosiyana kwambiri (mkuyu 6b), ngakhale kholalo limasungidwa mu SPCA6 homologue (monga momwe zimasonyezedwera ndi conservatism ya hydrophobic bundle core) komanso kupitirira IST superfamily.
Ngakhale kuti ili ndi dera laling'ono kwambiri mu SPCA6 lomwe lili ndi zinthu zochepa zomwe zimawoneka zachiwiri, zotsalira zambiri za hinge (kuphatikizapo dera la 3) zimasungidwa kwambiri pakati pa ma homologues a SPCA6, zomwe zingasonyeze kuti kuyang'ana kwa helical bundle ndi β-sandwich kumagwira ntchito.monga wodziletsa.Komabe, ngakhale maukonde a haidrojeni ambiri komanso maukonde a electrostatic m'chigawo cha hinge cha SPCA6 ndi IZUMO1, umboni wa kusinthasintha kwamkati utha kuwoneka polumikizana ndi kangapo kololedwa kwa IZUMO137,43,44.Kuyanjanitsa kwa madera amtunduwo kunadutsana bwino, koma mayendedwe a madera okhudzana ndi wina ndi mzake amasiyana kuchokera ku 50 ° mpaka 70 ° kuchokera kumtunda wapakati (mkuyu S16).Kuti mumvetsetse kusintha kwamphamvu kwa SPCA6 mu yankho, kuyesa kwa SAXS kunachitika (mkuyu S17a, b).Ab initio kukonzanso kwa SPCA6 ectodomain kumagwirizana ndi ndodo ya kristalo (mkuyu. S18), ngakhale kuti chiwembu cha Kratky chinasonyeza kusinthasintha (mkuyu. S17b).Kuphatikizikaku kumasiyana ndi IZUMO1, momwe puloteni yosamangidwa imatengera mawonekedwe a boomerang mu latisi ndi yankho43.
Kuti adziwe mwachindunji dera losinthika, hydrogen-deuterium exchange mass spectroscopy (H-DXMS) inachitidwa pa SPCA6 ndikuyerekeza ndi zomwe zinapezedwa kale pa IZUMO143 (Mkuyu 7a, b).SPCA6 imasinthasintha momveka bwino kuposa IZUMO1, monga zikuwonetseredwa ndi kusinthana kwapamwamba kwa deuterium mumpangidwe wonse pambuyo pa 100,000 s kusinthanitsa.M'mapangidwe onsewa, gawo la C-terminal la dera la hinge likuwonetsa kusinthana kwakukulu, komwe mwina kumalola kusinthasintha kochepa kwa madera a 4HB ndi ma Ig ngati ofanana.Chochititsa chidwi n'chakuti, gawo la C-terminal la hinge la SPCA6, lomwe lili ndi zotsalira za 147CDLPLDCP154, ndi dera lotetezedwa kwambiri 3 (mkuyu 6b), mwinamwake kusonyeza kuti kusinthasintha kwa interdomain ndi chinthu chosungidwa mwachisinthiko cha SPCAA6.Malingana ndi kusinthasintha kwa kusinthasintha, deta ya CD thermal melt inasonyeza kuti SPCA6 (Tm = 51.2 ° C) ndi yochepa kwambiri kuposa IZUMO1 (Tm = 62.9 ° C) (Fig. S1e ndi S19).
a H-DXMS zithunzi za SPCA6 ndi b IZUMO1.Maperesenti osinthanitsa a deuterium adadziwika pa nthawi zomwe zasonyezedwa.Kusinthanitsa kwa hydrogen-deuterium kumasonyezedwa ndi mtundu pamlingo wa gradient kuyambira buluu (10%) mpaka wofiira (90%).Mabokosi akuda amaimira madera osinthanitsa kwambiri.Malire a 4HB, hinge ndi ma Ig-like domain omwe amawonedwa mu mawonekedwe a kristalo akuwonetsedwa pamwamba pa mndandanda woyamba.Miyezo ya kusinthana kwa Deuterium pa 10 s, 1000 s, ndi 100,000 s inajambulidwa pa tchati cha mizere yojambulidwa pamwamba pa mamolekyu oonekera a SPCA6 ndi IZUMO1.Magawo azinthu zomwe zili ndi mulingo wosinthira wa deuterium pansi pa 50% ndi zoyera.Madera omwe ali pamwamba pa 50% H-DXMS kusinthana ndi utoto mu sikelo ya gradient.
Kugwiritsiridwa ntchito kwa CRISPR/Cas9 ndi njira za gene zogogoda za mbewa zapangitsa kuti pakhale kuzindikirika kwa zinthu zingapo zofunika pakumanga kwa umuna ndi dzira ndi kuphatikizika.Kupatula kuyanjana kodziwika bwino kwa IZUMO1-JUNO ndi CD9 kapangidwe kake, mapuloteni ambiri okhudzana ndi kuphatikizika kwa gamete amakhalabe mwadongosolo komanso mogwira ntchito modabwitsa.Maonekedwe a biophysical ndi kapangidwe ka SPCA6 ndi gawo lina la chithunzithunzi cha adhesion/fusion molecular pa nthawi ya umuna.
SPCA6 ndi mamembala ena a IST superfamily akuwoneka kuti ndi otetezedwa kwambiri mu nyama zoyamwitsa komanso mbalame payekha, zokwawa, ndi amphibians;kwenikweni, amaganiziridwa kuti SPCA6 imafunikanso umuna mu zebrafish 59. Kugawa kumeneku ndi kofanana ndi mapuloteni ena odziwika a gamete fusion-associated monga DCST134, DCST234, FIMP31, ndi SOF132, kutanthauza kuti zinthuzi ndizosowa HAP2 (komanso omwe amadziwika kuti GCS1) omwe amachititsa chidwi cha akatswiri ambiri ojambula., zomera, ndi arthropods.Mapuloteni ophatikizana ndi feteleza 60, 61. Ngakhale kuti pali kufanana kwakukulu kwapangidwe pakati pa SPCA6 ndi IZUMO1, kugogoda kwa majini omwe amaphatikizirapo imodzi mwa mapuloteniwa amachititsa kuti asabereke mbewa zamphongo, zomwe zimasonyeza kuti ntchito zawo mu kuphatikizika kwa gamete sizinabwerezedwe..Mochulukira, palibe mapuloteni odziwika a umuna omwe amafunikira gawo lophatikizika la maphatikizidwe omwe safunikira.
Funso lotseguka likadali lotseguka ngati SPCA6 (ndi mamembala ena a IST superfamily) amatenga nawo gawo pamagulu ophatikizika, kupanga ma intragametic network kuti apeze mapuloteni ofunikira kumalo ophatikizika, kapenanso kukhala ngati ma fusogens osawoneka.Maphunziro a co-immunoprecipitation m'maselo a HEK293T adawonetsa kuyanjana pakati pa kutalika kwa IZUMO1 ndi SPCA632.Komabe, ma ectodomain athu ophatikizananso sanagwirizane mu vitro, kutanthauza kuti kuyanjana komwe kumawoneka mu Noda et al.zonse zidafufutidwa pomanga (onani mchira wa cytoplasmic wa IZUMO1, womwe wasonyezedwa kuti ndi wosafunikira pa umuna62).Kapenanso, IZUMO1 ndi/kapena SPCA6 ingafunike malo omangirira omwe sitipanganso mu vitro, monga ma conformations okhudzana ndi thupi kapena mamolekyulu okhala ndi mapuloteni ena (odziwika kapena omwe sanapezekebe).Ngakhale IZUMO1 ectodomain imakhulupirira kuti imayimira kulumikizidwa kwa spermatozoa ku dzira mu perivitelline space, cholinga cha SPCA6 ectodomain sichikudziwika.
Mapangidwe a SPCA6 amawulula malo angapo otetezedwa omwe atha kukhala okhudzidwa ndi mapuloteni ndi mapuloteni.Gawo lotetezedwa la hinge dera lomwe lili pafupi ndi CXXC motif (lomwe latchulidwa Patch 1 pamwambapa) lili ndi zotsalira zingapo zowoneka kunja zomwe nthawi zambiri zimagwirizanitsidwa ndi kuyanjana kwa hydrophobic ndi π-stacking pakati pa ma biomolecules.Mbali zazikuluzikulu za dera la Ig-like (chigawo cha 2) zimapanga groove yabwino yokhala ndi Arg yosungidwa kwambiri ndi zotsalira Zake, ndipo ma antibodies olimbana ndi dera lino akhala akugwiritsidwa ntchito kale kuletsa gamete fusion 30.Antibody imazindikira mzere wa epitope 212RIRPAQLTHRGTFS225, yomwe ili ndi zotsalira zitatu mwa zisanu ndi chimodzi za arginine ndipo His220 yosungidwa kwambiri.Sizikudziwika ngati kusokonekeraku kumachitika chifukwa cha kutsekeka kwa zotsalira izi kapena dera lonselo.Malo omwe pali kusiyana kumeneku pafupi ndi C-terminus ya β-sandwich amasonyeza kugwirizana kwa cis ndi mapuloteni oyandikana nawo a umuna, koma osati ndi mapuloteni a oocyte.Komanso, kusungidwa kwa tangle yolemera kwambiri ya proline (malo 3) mkati mwa hinge ikhoza kukhala malo opangira mapuloteni-mapuloteni kapena, mwinamwake, amasonyeza kusungidwa kwa kusinthasintha pakati pa madera awiriwa.Jenda ndiyofunikira pa ntchito yosadziwika ya SPCA6.kuphatikiza kwa gametes.
SPCA6 ili ndi mphamvu zama protein adhesion intercellular, kuphatikiza masangweji a Ig ngati β.Mapuloteni ambiri omatira (monga ma cadherins, integrins, adhesins, ndi IZUMO1) ali ndi madera amodzi kapena angapo a β-sangweji omwe amakulitsa mapuloteni kuchokera ku cell membrane kupita ku zolinga zawo zachilengedwe63,64,65.Dera la Ig-like la SPCA6 lilinso ndi motif yomwe imapezeka mu β-sandwiches of adhesion and cohesion: double strand of parallel strand in the end of β-sandwiches, known as mechanical clamps66.Zimakhulupirira kuti izi zimawonjezera kukana kumeta ubweya wa ubweya, zomwe ndizofunikira kwa mapuloteni omwe amakhudzidwa ndi ma intercellular.Komabe, ngakhale izi zikufanana ndi zomatira, palibe umboni wosonyeza kuti SPCA6 imalumikizana ndi azungu a dzira.Ectodomain ya SPCA6 siyingathe kumangirira ku JUNO, ndi ma cell a HEK293T a SPCA6, monga momwe tawonetsera pano, samagwirizana kwambiri ndi ma oocyte opanda zona 32.Ngati SPCA6 ikhazikitsa ma intergametic bond, kuyanjana kumeneku kungafune kusinthidwa pambuyo pomasulira kapena kukhazikika ndi mapuloteni ena a umuna.Pothandizira malingaliro omaliza, spermatozoa yoperewera ya IZUMO1 imamangiriza ku oocyte, kusonyeza kuti mamolekyu ena osati IZUMO1 akuphatikizidwa mu sitepe ya 27 ya gamete.
Mapuloteni ambiri ophatikizika a ma virus, ma cell, komanso otukuka amakhala ndi zinthu zomwe zimalosera ntchito yawo ngati fusogens.Mwachitsanzo, ma viral fusion glycoproteins (makalasi I, II ndi III) amakhala ndi peptide ya hydrophobic fusion kapena loop kumapeto kwa puloteni yomwe imayikidwa mu nembanemba ya alendo.Mapu a hydrophilicity a IZUMO143 ndi kapangidwe kake (kutsimikiziridwa ndi kuneneratu) kwa IST superfamily sanasonyeze peptide yowoneka bwino ya hydrophobic fusion.Chifukwa chake, ngati mapuloteni aliwonse mu IST superfamily amagwira ntchito ngati fusogens, amatero mosiyana ndi zitsanzo zina zodziwika.
Pomaliza, ntchito za mamembala a IST superfamily of proteins okhudzana ndi gamete fusion akadali chinsinsi chodabwitsa.Molekyu yathu yodziwikanso ya SPCA6 komanso kapangidwe kake kokhazikika kadzapereka chidziwitso pa ubale womwe ulipo pakati pazigawo zomwe zimagawidwa komanso gawo lawo pakulumikizana ndi kuphatikizika kwa gamete.
Mayendedwe a DNA ofanana ndi omwe adanenedweratu a SPCA6 ectodomain (NCBI kulowa nambala NP_001303901.1; zotsalira 27-246) zidakonzedwa kuti ziwonetsedwe m'maselo a Drosophila melanogaster S2 ndikupangidwa ngati kadulidwe ka jini kotsatana ndi Geknofins Encoding Koza., chizindikiro cha katulutsidwe ka BiP ndi 5′ ndi 3′ yofananirayo imathera pakupanga kodziyimira pawokha kwa jini iyi kukhala vekitala ya mawu a pMT kutengera wolimbikitsa metallothionein wosinthidwa kuti asankhidwe ndi puromycin (pMT-puro).Vekita ya pMT-puro imatsekera malo ophwanyika a thrombin otsatiridwa ndi tagi ya 10x-His C-terminal tag (Chithunzi S2).
Kutumiza kokhazikika kwa SPCA6 pMT-puro vector mu maselo a D. melanogaster S2 (Gibco) anachitidwa mofanana ndi ndondomeko yogwiritsidwa ntchito pa IZUMO1 ndi JUNO43.Maselo a S2 adasungunuka ndikukula mu Schneider's sing'anga (Gibco) kuwonjezeredwa ndi ndende yomaliza ya 10% (v/v) yoletsa kutentha kwa fetal ng'ombe seramu (Gibco) ndi 1X antimycotic antibiotic (Gibco).Maselo oyambirira (3.0 x 106 maselo) anakutidwa mu zitsime payekha 6-chitsime mbale (Corning).Pambuyo pa maola 24 a incubation pa 27 ° C, maselo adasinthidwa ndi 2 mg ya SPCA6 pMT-puro vector ndi Effectene transfection reagent (Qiagen) malinga ndi ndondomeko ya wopanga.Ma cell opatsirana adakulungidwa kwa maola 72 ndiyeno amakololedwa ndi puromycin 6 mg/ml.Maselo adasiyanitsidwa ndi Schneider's wathunthu ndikuyikidwa mu seramu wopanda Insect-XPRESS medium (Lonza) kuti apange mapuloteni ambiri.Gulu la 1 L la chikhalidwe cha maselo a S2 linakula kukhala maselo a 8-10 × 106 ml-1 mu botolo la 2 L lokhala ndi mpweya wokwanira wa polypropylene Erlenmeyer ndipo kenako amawuzidwa ndi 500 µM CuSO4 (Millipore Sigma) ndi wosabala.kutengeka.Zikhalidwe zomwe zinapangitsidwa zidakulungidwa pa 27 ° C. pa 120 rpm kwa masiku anayi.
Sing'anga yokhala ndi SPCA6 idasiyanitsidwa ndi centrifugation pa 5660 × g pa 4 ° C ndikutsatiridwa ndi Centramate tangential flow filtration system (Pall Corp) yokhala ndi nembanemba ya 10 kDa MWCO.Ikani sing'anga yokhazikika yokhala ndi SPCA6 mpaka 2 ml Ni-NTA agarose resin (Qiagen) column.Utoto wa Ni-NTA udatsukidwa ndi ma voliyumu a 10 (CV) a bafa A ndiyeno 1 CV ya bafa A idawonjezedwa kuti ipereke ndende yomaliza ya imidazole ya 50 mm.SPCA6 idatulutsidwa ndi 10 ml ya buffer A yowonjezeredwa ndi imidazole mpaka ndende yomaliza ya 500 mm.Kuletsa kalasi ya thrombin (Millipore Sigma) inawonjezeredwa mwachindunji ku dialysis tubing (MWCO 12-14 kDa) pa 1 unit pa mg SPCAA6 vs. 1 L 10 mM Tris-HCl, pH 7.5 ndi 150 mM NaCl (buffer B) ya dialysis.) pa 4°C kwa maola 48.The thrombin-cleaved SPCA6 ndiye amachepetsedwa katatu kuti achepetse kuchuluka kwa mchere ndikuyikidwa pa 1 ml MonoS 5/50 GL cation exchange column (Cytiva/GE) yofanana ndi 10 mM Tris-HCl, pH 7.5.Chosinthira ma cation chinatsukidwa ndi 3 CV ya 10 mM Tris-HCl, pH 7.5, kenako SPCA6 idatulutsidwa ndi mzere wozungulira wa 0 mpaka 500 mM NaCl mu 10 mM Tris-HCl, pH 7.5 kwa 25 CV.Pambuyo pa ion exchange chromatography, SPCA6 idakhazikika ku 1 ml ndipo idasinthidwa isocratic kuchokera pagawo la ENrich SEC650 10 x 300 (BioRad) lolumikizana ndi buffer B. Malinga ndi chromatogram, dziwe ndi tizigawo tomwe tili ndi SPCA6.Kuyera kumayendetsedwa ndi Coomassie-stained electrophoresis pa 16% SDS-polyacrylamide gel.Kuchuluka kwa mapuloteni kunayesedwa ndi kuyamwa pa 280 nm pogwiritsa ntchito lamulo la Beer-Lambert ndi theoretical molar extinction coefficient.
SPCA6 yoyeretsedwa idasindikizidwa usiku wonse motsutsana ndi 10 mM sodium phosphate, pH 7.4 ndi 150 mM NaF ndikuchepetsedwa kukhala 0.16 mg/mL isanayambe kusanthula ndi CD spectroscopy.Kujambula kwapadera kwa ma CD okhala ndi kutalika kwa 185 mpaka 260 nm kunasonkhanitsidwa pa Jasco J-1500 spectropolarimeter pogwiritsa ntchito quartz cuvettes yokhala ndi 1 mm optical path length (Helma) pa 25 ° C pa mlingo wa 50 nm / min.Zowonera za CD zidakonzedwa moyambira, kupitilira kugulidwa kwa 10, ndikusinthidwa kukhala kutanthauza residual elliptical (θMRE) mu madigiri cm2/dmol:
kumene MW ndi kulemera kwa molekyulu ya chitsanzo chilichonse mu Da;N ndi chiwerengero cha amino zidulo;θ ndi elliptical mu millidegrees;d ikufanana ndi kutalika kwa njira ya kuwala mu masentimita;kuchuluka kwa mapuloteni mu mayunitsi.