304 bakin karfe nada tubing sinadaran sinadaran, Tsarin SPACA6 ectodomain ya ƙunshi babban iyali na sunadaran da ke da alaƙa da haɗin gamete.

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SPACA6 shine furotin da aka bayyana maniyyi wanda ke da mahimmanci don haɗuwa da gamete yayin haifuwar jima'i na mammalian.Duk da wannan muhimmiyar rawa, takamaiman aikin SPACA6 ba a fahimta sosai ba.Mun bayyana tsarin crystal na yankin extracellular na SPACA6 a ƙuduri na 2.2 Å, yana bayyana furotin yanki guda biyu wanda ya ƙunshi nau'i-nau'i huɗu da Ig-kamar β-sandwiches tare da masu haɗin gwiwa masu sassaucin ra'ayi.Wannan tsarin yayi kama da IZUMO1, wani furotin mai alaƙa da gamete fusion, wanda ke yin SPACA6 da IZUMO1 waɗanda suka kafa ƙungiyar superfamily na furotin masu alaƙa da hadi da ake magana a kai a cikin su azaman IST superfamily.IST superfamily an siffanta shi da tsari ta hanyar murɗaɗɗen gunkin helix guda huɗu da maƙallan CXXC masu alaƙa da disulfide.Binciken tushen tsarin AlphaFold na proteome na ɗan adam ya gano ƙarin membobi na furotin na wannan babban iyali;musamman, da yawa daga cikin waɗannan sunadaran suna da hannu wajen haɗakar da gamete.Tsarin SPACA6 da dangantakarsa da sauran membobin IST superfamily suna ba da hanyar haɗin da ta ɓace a cikin ilimin mu game da haɗuwar gamete mammalian.
Kowace rayuwar ɗan adam tana farawa da nau'in haploid gametes guda biyu: maniyyi uba da kwan uwa.Wannan maniyyi shi ne wanda ya yi nasara wajen zaɓe mai tsanani a lokacin da miliyoyin ƙwayoyin maniyyi suka ratsa cikin al'aurar mace, suka shawo kan matsaloli daban-daban1 kuma suna samun karfin aiki, wanda ke inganta motsin su da tsarin abubuwan da ke sama2,3,4.Ko da maniyyi da oocyte sun sami juna, tsarin bai ƙare ba tukuna.Oocyte yana kewaye da Layer na sel cumulus da shingen glycoprotein da ake kira zona pellucida, wanda dole ne maniyyi ya wuce don shiga oocyte.Spermatozoa yana amfani da haɗe-haɗe na ƙwayoyin mannewa saman ƙasa da haɗe-haɗe da enzymes masu alaƙa da ɓoye don shawo kan waɗannan shinge na ƙarshe5.Wadannan kwayoyin halitta da enzymes ana adana su ne a cikin membrane na ciki da kuma matrix na acrosomal kuma ana gano su lokacin da membrane na waje na maniyyi ya lysed a lokacin da ake ciki.Mataki na ƙarshe a cikin wannan ƙaƙƙarfan tafiya shine taron haɗakar maniyyi-kwai, wanda ƙwayoyin biyu suka haɗa membranes ɗinsu don zama kwayar diploid guda ɗaya7.Ko da yake wannan tsari yana da ƙarfi a cikin haifuwar ɗan adam, ba a fahimtar ma'amalar da ake buƙata ta ƙwayoyin cuta.
Baya ga hadi na gametes, an yi nazari sosai kan ilmin sinadarai na hadewar bilayers guda biyu.Gabaɗaya, haɗuwa da membrane tsari ne mara kuzari wanda ke buƙatar mai haɓaka furotin don fuskantar canjin tsarin tsari wanda ke kawo maɓalli guda biyu kusa da juna, yana karya ci gaba da haifar da fusion8,9.Wadannan furotin masu kara kuzari an san su da fusogens kuma an samo su a cikin tsarin fusion marasa adadi.Ana buƙatar su don shigarwar hoto ko bidiyo mai zagaya yanar gizo da sauri (misali, gp160 a cikin HIV-1, spike a coronaviruses, hemagglutinin a cikin ƙwayoyin mura) 10,11,12 placental (syncytin) 13,14,15 da gamete-forming fusions a cikin ƙananan eukaryotes ( HAP2/GCS1 a cikin shuke-shuke, protists da arthropods) 16,17,18,19.Fusogens ga gametes na ɗan adam har yanzu ba a gano su ba, kodayake wasu sunadaran sunadaran da aka nuna suna da mahimmanci ga haɗewar gamete da haɗuwa.CD9 da aka bayyana oocyte, furotin transmembrane da ake buƙata don haɗin linzamin kwamfuta da gametes na ɗan adam, shine farkon da aka gano 21,22,23.Kodayake ainihin aikinsa ya kasance ba a sani ba, rawar da ke cikin mannewa, tsarin mannewa foci akan microvilli kwai, da/ko daidaitaccen asalin sunadaran sunadaran saman oocyte yana da alama 24,25,26.Mafi yawan sunadaran sunadarai guda biyu waɗanda ke da mahimmanci don haɗakarwar gamete sune furotin IZUMO127 da furotin oocyte JUNO28, kuma haɗin gwiwar su shine muhimmin mataki na ganewar gamete da mannewa kafin haɗuwa.Namiji Izumo1 ƙwanƙwasa mice da mace Juno knockout mice gaba ɗaya bakararre ne, a cikin waɗannan samfuran maniyyi suna shiga sararin samaniya amma gametes ba sa fuse.Hakazalika, an rage haɗuwa lokacin da aka yi amfani da gametes da anti-IZUMO1 ko JUNO27,29 antibodies a cikin ɗan adam in vitro hadi gwaje-gwaje.
Kwanan nan, an gano wani sabon rukuni na sunadaran da aka bayyana maniyyi masu kama da IZUMO1 da JUNO20,30,31,32,33,34,35.Maniyyi acrosomal membrane-Associated protein 6 (SPACA6) an gano shi azaman mahimmanci don hadi a cikin babban binciken mutagenesis na murine.Shigar da transgene a cikin Spaca6 gene yana haifar da spermatozoa marasa amfani, kodayake waɗannan spermatozoa sun shiga cikin sararin samaniya 36.Binciken ƙwanƙwasa na gaba a cikin mice ya tabbatar da cewa ana buƙatar Spaca6 don haɗakarwar gamete 30,32.SPACA6 an bayyana kusan na musamman a cikin gwaje-gwajen kuma yana da yanayin wuri mai kama da na IZUMO1, wato a cikin intima na spermatozoa kafin maganin acrosomal, sa'an nan kuma ya yi hijira zuwa yankin equatorial bayan amsawar acrosomal 30,32.Spaca6 homologues sun kasance a cikin nau'o'in dabbobi masu shayarwa da sauran eukaryotes 30 kuma an nuna muhimmancinsa ga haɗuwa da gamete na mutum ta hanyar hana hadi na mutum a cikin vitro ta juriya ga SPACA6 30.Ba kamar IZUMO1 da JUNO ba, cikakkun bayanai na tsari, hulɗa, da aikin SPACA6 sun kasance ba a sani ba.
Don ƙarin fahimtar ainihin tsarin da ke tattare da haɗakar maniyyi da ƙwai, wanda zai ba mu damar sanar da abubuwan da ke faruwa a nan gaba a cikin tsarin iyali da kuma kula da haihuwa, mun gudanar da SPACA6 nazarin tsarin da kwayoyin halitta.Tsarin lu'ulu'u na yanki na SPACA6 yana nuna gungu mai ƙarfi huɗu (4HB) da yanki na immunoglobulin-kamar (Ig-like) wanda aka haɗa ta yankuna masu sassaucin ra'ayi.Kamar yadda aka annabta a cikin binciken da suka gabata, 7,32,37 tsarin yanki na SPACA6 yayi kama da na ɗan adam IZUMO1, kuma sunadaran sunadaran suna raba wani abu mai ban mamaki: 4HB tare da saman helical triangular da nau'i na CXXC masu alaƙa da disulfide.Muna ba da shawarar cewa IZUMO1 da SPACA6 yanzu suna ayyana babban dangi mai alaƙa da tsari na sunadaran da ke da alaƙa da haɗin gamete.Yin amfani da fasali na musamman ga babban iyali, mun gudanar da cikakken bincike don tsarin tsarin ɗan adam na AlphaFold, gano ƙarin membobi na wannan babban iyali, gami da mambobi da yawa da ke da hannu a haɗakar gamete da/ko hadi.Yanzu ya bayyana cewa akwai nau'in tsari na gama-gari da dangin sunadaran sunadaran da ke da alaƙa da haɗuwar gamete, kuma tsarin mu yana ba da taswirar kwayoyin halitta na wannan muhimmin al'amari na tsarin fusion na ɗan adam.
SPACA6 shine furotin transmembrane mai wucewa guda ɗaya tare da glycan mai alaƙa da N-da kuma haɗin haɗin disulfide guda shida (Figures S1a da S2).Mun bayyana yankin extracellular na ɗan adam SPACA6 (ragowar 27-246) a cikin ƙwayoyin Drosophila S2 kuma mun tsarkake furotin ta amfani da alaƙar nickel, musayar cation, da chromatography girma (Fig. S1b).SPACA6 ectodomain da aka tsarkake yana da ƙarfi sosai kuma yana kama da juna.Binciken ta yin amfani da chromatography mai girma da aka haɗe tare da watsawar haske na polygonal (SEC-MALS) ya bayyana kololu ɗaya tare da ƙididdige nauyin kwayoyin halitta na 26.2 ± 0.5 kDa (Fig. S1c).Wannan ya yi daidai da girman SPACA6 monomeric ectodomain, yana nuna cewa oligomerization bai faru ba yayin tsarkakewa.Bugu da ƙari, spectroscopy na madauwari dichroism (CD) ya bayyana tsarin α / β mai gauraye tare da ma'anar narkewa na 51.3 ° C (Fig. S1d, e).Deconvolution na sifofin CD sun bayyana 38.6% α-helical da 15.8% β-stranded abubuwa (Hoto S1d).
SPACA6 ectodomain an yi crystallized ta amfani da bazuwar matrix seeding38 wanda ya haifar da saitin bayanai tare da ƙuduri na 2.2 Å (Table 1 da Figure S3).Yin amfani da haɗe-haɗe-haɗe-haɗe-haɗe-haɗe-haɗe-haɗe da SAD phasing data tare da bayyanar bromide don ƙayyadaddun tsari (Table 1 da Figure S4), ƙirar mai ladabi ta ƙarshe ta ƙunshi ragowar 27-246.A lokacin da aka ƙayyade tsarin, babu wani gwaji ko tsarin AlphaFold da aka samu.SPACA6 ectodomain ma'auni na 20 Å × 20 Å × 85 Å, ya ƙunshi helices bakwai da tara β-strands, kuma yana da tsayin daka mai tsayi wanda aka daidaita ta hanyar haɗin disulfide shida (Fig. 1a, b).Rarraunan ƙarfin lantarki a ƙarshen sarkar gefen Asn243 yana nuna cewa wannan ragowar glycosylation ne mai alaƙa da N.Tsarin ya ƙunshi yanki guda biyu: N-terminal hudu-helix bundle (4HB) da wani yanki mai kama da C-terminal Ig tare da yanki mai tsaka-tsaki tsakanin su (Fig. 1c).
Tsarin yanki na extracellular na SPACA6.Zane mai zane na yankin extracellular na SPACA6, kalar sarkar daga N zuwa C-terminus daga shuɗi mai duhu zuwa ja mai duhu.Cysteine ​​​​da ke cikin haɗin gwiwar disulfide ana haskaka su a cikin magenta.b Topology na yankin extracellular na SPACA6.Yi amfani da tsarin launi iri ɗaya kamar yadda yake cikin Hoto 1a.c SPACA6 yanki na waje.4HB, hinge, da Ig-kamar taswirar yanki masu launin orange, kore, da shuɗi, bi da bi.Ba a zana yadudduka zuwa sikeli.
Yankin 4HB na SPACA6 ya haɗa da manyan helices guda huɗu (helices 1-4), waɗanda aka tsara su a cikin nau'in helix na helical (Fig. 2a), musanya tsakanin hulɗar antiparallel da layi daya (Fig. 2b).An dage farawa ƙaramin ƙaramin bi-da-biyu (helix 1′) daidai gwargwado ga dam ɗin, yana samar da triangle tare da helices 1 da 2. Wannan triangle yana ɗan lahani a cikin marufi-karkaɗɗen marufi na ingantacciyar marufi na helices 3 da 4 ( Hoto 2 a).
4HB N-Terminal tsiri ginshiƙi.b Babban ra'ayi na tarin helices guda huɗu, kowane helix mai haske da shuɗi mai duhu a N-terminus da ja mai duhu a C-terminus.c Tsarin dabaran karkace na sama na sama don 4HB, tare da kowane ragowar da aka nuna azaman da'irar da aka yiwa lakabi da lambar amino acid mai harafi ɗaya;amino acid guda hudu ne kawai a saman dabaran aka lissafta.Ragowar da ba na polar ba suna da launin rawaya, ragowar da ba a caje su ba suna da launin kore, ragowar da ba a caje su ba suna da launin shuɗi, sauran masu cajin da ba su da kyau suna da launin ja.d Fuskoki uku na yankin 4HB, tare da 4HBs a cikin lemu da hinges a kore.Dukansu insets suna nuna sandunan disulfide mai siffar sanda.
4HB yana mai da hankali akan ainihin hydrophobic na ciki wanda ya ƙunshi galibi na aliphatic da sauran abubuwan kamshi (Fig. 2c).Jigon ya ƙunshi haɗin disulfide tsakanin Cys41 da Cys55 wanda ke haɗa helices 1 da 2 tare a cikin maɗaukakin alwatika na sama (Fig. 2d).An samar da ƙarin haɗin disulfide guda biyu tsakanin motif na CXXC a cikin Helix 1 ′ da wani motsin CXXC da aka samu a ƙarshen β-hairpin a cikin yankin hinge (Fig. 2d).Ragowar arginine mai ra'ayin mazan jiya tare da aikin da ba a san shi ba (Arg37) yana cikin madaidaicin triangle da aka kafa ta helices 1′, 1, da 2. Aliphatic carbon atoms Cβ, Cγ, da Cδ Arg37 suna hulɗa tare da tushen hydrophobic, kuma ƙungiyoyin guanidine suna motsawa cikin sauri. tsakanin helices 1' da 1 ta hanyar hulɗar tsakanin kashin baya na Thr32 da sarkar gefe (Fig. S5a,b).Tyr34 ya shimfiɗa zuwa cikin rami yana barin ƙananan cavities guda biyu wanda Arg37 zai iya yin hulɗa tare da sauran ƙarfi.
Ig-kamar β-sandwich yanki babban dangi ne na sunadaran da ke raba fasalin gama gari na biyu ko fiye da yawa amphipathic β-sheets da ke hulɗa ta hanyar hydrophobic core 39. Yankin C-terminal Ig-like na SPACA6 yana da tsari iri ɗaya. kuma ya ƙunshi nau'i biyu (Fig. S6a).Sheet 1 shine β-sheet na madauri guda huɗu (strands D, F, H, da I) inda igiyoyi F, H, kuma na samar da tsari na gaba-gaba, kuma igiyoyi I da D suna yin hulɗar layi ɗaya.Tebu na 2 ƙaramin takardar beta ce mai madaidaicin-daidaitacce (matsalolin E da G).An lura da haɗin disulfide na ciki tsakanin C-terminus na sarkar E da tsakiyar sarkar H (Cys170-Cys226) (Fig. S6b).Wannan haɗin disulfide yana kama da haɗin disulfide a cikin yankin β-sandwich na immunoglobulin40,41.
Tabbataccen β-sheet ɗin guda huɗu yana jujjuya tare da tsayinsa duka, yana samar da gefuna na asymmetrical waɗanda suka bambanta da siffar da electrostatics.Ƙaƙwalwar bakin ciki shine shimfidar yanayi na hydrophobic mai lebur wanda ya fito waje idan aka kwatanta da sauran m da electrostatically bambance-bambancen saman a SPACA6 (Fig. S6b, c).Halo na ƙungiyoyin carbonyl/amino da aka fallasa da sarƙoƙi na gefen iyakacin duniya suna kewaye da saman hydrophobic (Fig. S6c).Faɗin gefe yana rufe da wani yanki mai lulluɓe wanda ke toshe ɓangaren N-terminal na hydrophobic core kuma ya samar da haɗin haɗin hydrogen guda uku tare da rukunin buɗewar iyakacin duniya na kashin bayan sarkar F (Fig. S6d).Ƙungiyar C-terminal na wannan gefen yana samar da babban aljihu tare da wani ɓangaren da aka fallasa ainihin hydrophobic.Aljihu yana kewaye da kyawawan zarge-zarge saboda saiti uku na ragowar arginine guda biyu (Arg162-Arg221, Arg201-Arg205 da Arg212-Arg214) da kuma tsakiyar histidine (His220) (Hoto S6e).
Yankin hinge ɗan gajeren yanki ne tsakanin yankin helical da yankin Ig-kamar yanki, wanda ya ƙunshi nau'ikan β-Layer guda uku masu kama da juna (strands A, B da C), ƙaramin helix na 310, da ɓangarorin helical masu tsayi da yawa.(Hoto S7).Cibiyar sadarwa na covalent da electrostatic lambobin sadarwa a cikin yankin hinge yana bayyana don daidaita daidaito tsakanin 4HB da yanki mai kama da Ig.Ana iya raba hanyar sadarwa zuwa sassa uku.Kashi na farko ya haɗa da motifs guda biyu na CXXC (27CXXC30 da 139CXXC142) waɗanda ke samar da haɗin haɗin disulfide guda biyu tsakanin β-hairpin a cikin hinge da 1′ helix a cikin 4HB.Kashi na biyu ya haɗa da hulɗar lantarki tsakanin yanki mai kama da Ig da hinge.Glu132 a cikin hinge yana samar da gadar gishiri tare da Arg233 a cikin yankin Ig-like da Arg135 a cikin hinge.Kashi na uku ya haɗa da haɗin kai tsakanin yanki mai kama da Ig da yankin hinge.Haɗin disulfide guda biyu (Cys124-Cys147 da Cys128-Cys153) suna haɗa madauki na hinge zuwa mai haɗin gwiwa wanda aka daidaita ta hanyar hulɗar lantarki tsakanin Gln131 da ƙungiyar aikin kashin baya, yana ba da damar shiga yankin Ig na farko.sarkar.
An yi amfani da tsarin SPACA6 ectodomain da tsarin kowane mutum na 4HB da Ig-like domains don nemo bayanan da aka tsara a cikin bayanan furotin 42.Mun gano matches tare da babban makin Dali Z, ƙananan madaidaitan sabani, da manyan makin LALI (na ƙarshe shine adadin ragowar tsarin da ya dace).Yayin da 10 na farko ya buge daga cikakken binciken ectodomain (Table S1) yana da ƙimar Z-score na> 842 mai karɓa, binciken 4HB ko Ig-like kawai ya nuna cewa yawancin waɗannan hits sun dace da β-sandwiches kawai.ninkin ko'ina da ake samu a cikin sunadarai da yawa.Duk bincike guda uku da aka yi a Dali sun dawo da sakamako daya kacal: IZUMO1.
An dade ana ba da shawarar cewa SPACA6 da IZUMO1 suna raba kamanni na tsari7,32,37.Kodayake ectodomains na waɗannan nau'ikan sunadaran haɗin gwiwar gamete guda biyu suna raba asalin 21% kawai (Hoto S8a), hadaddun shaida, gami da tsarin haɗin gwiwar disulfide da aka adana da kuma yanki mai kama da C-terminal Ig a cikin SPACA6, an ba da izinin yunƙurin farko na gina samfurin homology na linzamin kwamfuta na SPACA6 ta amfani da IZUMO1 azaman samfuri37.Tsarin mu yana tabbatar da waɗannan tsinkaya kuma yana nuna ainihin matakin kamance.A gaskiya ma, tsarin SPACA6 da IZUMO137,43,44 suna raba gine-ginen yanki guda biyu (Fig. S8b) tare da irin wannan yanki na 4HB da Ig-like β-sandwich da aka haɗa ta yankin hinge (Fig. S8c).
IZUMO1 da SPACA6 4HB suna da bambance-bambance na gama gari daga nau'ikan karkace na al'ada.Yawancin 4HBs, kamar waɗanda aka samo a cikin rukunin furotin na SNARE da ke cikin haɗin gwiwa na endosomal 45,46, suna da madaidaicin sararin samaniya da ke riƙe da kullun da ke kewaye da tsakiyar axis 47. Sabanin haka, yankunan helical a cikin IZUMO1 da SPACA6 sun kasance sun kasance sun karkace, tare da m curvature da canzawa. shiryarwa mara daidaituwa (Hoto S8d).Juyawa, mai yiwuwa ya haifar da alwatika da aka kafa ta helices 1', 1 da 2, ana kiyaye su a cikin IZUMO1 da SPACA6 kuma an daidaita su ta hanyar CXXC motif iri ɗaya akan helix 1′.Koyaya, ƙarin haɗin disulfide da aka samu a cikin SPACA6 (Cys41 da Cys55 haɗin gwiwar haɗin gwiwar helices 1 da 2 a sama) suna haifar da koli mai kaifi a kololuwar alwatika, yana sa SPACA6 ta fi karkacewa fiye da IZUMO1, tare da ƙarin madaidaicin madaidaicin ramuka.Bugu da ƙari, IZUMO1 ba shi da Arg37 da aka lura a tsakiyar wannan rami a cikin SPACA6.Sabanin haka, IZUMO1 yana da mafi yawan al'ada hydrophobic core na aliphatic da aromatic sharan gona.
IZUMO1 yana da yanki mai kama da Ig wanda ya ƙunshi madaidaicin madauri biyu da β-sheet43.Ƙarin madaidaicin a cikin IZUMO1 ya maye gurbin coil a cikin SPACA6, wanda ke hulɗa tare da F strand don iyakance kashin baya na hydrogen a cikin madaidaicin.Wani batu mai ban sha'awa na kwatanta shi ne ƙimar da aka annabta na yankunan Ig-kamar na sunadaran biyu.Filayen IZUMO1 yana da cajin da ba daidai ba fiye da saman SPACA6.Ana samun ƙarin cajin kusa da C-terminus da ke fuskantar maniyyi.A cikin SPACA6, yankuna iri ɗaya sun kasance masu tsaka tsaki ko ingantaccen caji (Fig. S8e).Misali, farfajiyar hydrophobic (gefukan bakin ciki) da ramuka masu fa'ida (fadi mai fadi) a cikin SPACA6 ana cajin su da kyau a cikin IZUMO1.
Kodayake dangantaka da abubuwa na biyu na tsarin tsakanin IZUMO1 da SPACA6 suna da kyau a kiyaye su, tsarin daidaitawa na yankunan Ig-like ya nuna cewa yankunan biyu sun bambanta a cikin yanayin gaba ɗaya dangane da juna (Fig. S9).Ƙungiyar karkace ta IZUMO1 tana lanƙwasa game da β-sandwich, ƙirƙirar siffar "boomerang" da aka kwatanta a baya a kusan 50 ° daga tsakiyar axis.Sabanin haka, an karkatar da igiyar helical a cikin SPACA6 kusan 10 ° a kishiyar shugabanci.Bambance-bambance a cikin waɗannan jagororin na iya yiwuwa saboda bambance-bambance a yankin hinge.A matakin matakin farko, IZUMO1 da SPACA6 suna raba ɗan kamanceceniya iri ɗaya a hinge, ban da cysteine, glycine, da ragowar aspartic acid.A sakamakon haka, hydrogen bond da electrostatic cibiyoyin sadarwa sun bambanta.β-sheet na biyu tsarin abubuwa suna raba ta IZUMO1 da SPACA6, kodayake sarƙoƙi a cikin IZUMO1 sun fi tsayi kuma 310 helix (helix 5) na musamman ga SPACA6.Waɗannan bambance-bambance suna haifar da daidaitawar yanki daban-daban don sunadaran gina jiki guda biyu in ba haka ba.
Binciken uwar garken Dali ɗin mu ya bayyana cewa SPACA6 da IZUMO1 sune kawai ƙayyadaddun sifofi biyu da aka ƙaddara a cikin ma'ajin furotin waɗanda ke da wannan nau'in 4HB na musamman (Table S1).Kwanan nan, DeepMind (Alphabet/Google) ya haɓaka AlphaFold, tsarin tushen hanyar sadarwa na jijiyoyi wanda zai iya yin hasashen daidai tsarin 3D na sunadaran daga jerin farko48.Ba da daɗewa ba bayan mun warware tsarin SPACA6, an fitar da bayanan AlphaFold, yana samar da ƙirar tsarin tsinkaya da ke rufe 98.5% na duk sunadaran da ke cikin proteome na ɗan adam48,49.Yin amfani da ƙayyadaddun tsarin SPACA6 ɗin mu azaman ƙirar bincike, binciken homology na tsari na ƙirar a cikin alfaFold proteome ɗan adam ya gano ƴan takara masu yuwuwar kamanni na tsari zuwa SPACA6 da IZUMO1.Idan aka ba da daidaito mai ban mamaki na AlphaFold a cikin tsinkayar SPACA6 (Fig. S10a) - musamman ma 1.1 Å rms ectodomain idan aka kwatanta da tsarin da aka tsara (Fig. S10b) - za mu iya zama da tabbaci cewa matakan SPACA6 da aka gano suna iya zama daidai.
A baya can, PSI-BLAST ya nemo gungu na IZUMO1 tare da wasu sunadaran da ke da alaƙa da maniyyi: IZUMO2, IZUMO3, da IZUMO450.AlphaFold ya annabta cewa waɗannan sunadaran dangin IZUMO suna ninka cikin yankin 4HB tare da tsarin haɗin kai iri ɗaya kamar IZUMO1 (Figures 3a da S11), kodayake basu da yanki mai kama da Ig.An yi hasashe cewa IZUMO2 da IZUMO3 sunadaran gina jiki mai gefe ɗaya kamar IZUMO1, yayin da IZUMO4 ya bayyana yana ɓoye.Ayyukan IZUMO 2, 3, da 4 sunadaran sunadaran a cikin fusion na gamete ba a ƙayyade ba.An san IZUMO3 yana taka rawa a cikin biogenesis na acrosome yayin haɓakar maniyyi51 kuma an gano furotin IZUMO don samar da hadaddun50.Kiyaye sunadaran IZUMO a cikin dabbobi masu rarrafe, dabbobi masu rarrafe, da masu amphibians suna nuna cewa yuwuwar aikinsu ya yi daidai da na sauran sanannun sunadaran da ke da alaƙa da gamete-fusion, irin su DCST1/2, SOF1, da FIMP.
Jadawalin tsarin gine-gine na babban iyali na IST, tare da 4HB, hinge, da yanki masu kama da Ig waɗanda aka haskaka cikin lemu, kore, da shuɗi, bi da bi.IZUMO4 yana da yanki na C-terminal na musamman wanda yayi kama da baki.An tabbatar da haɗin haɗin disulfide mai sanyawa ta hanyar tsayayyen layi da dige-dige, bi da bi.b IZUMO1 (PDB: 5F4E), SPACA6, IZUMO2 (AlphaFold DB: AF-Q6UXV1-F1), IZUMO3 (AlphaFold DB: AF-Q5VZ72-F1), IZUMO4 (AlphaFold DB: AF-Q1ZYL8-Fold): AF-Q1ZYL8-Fold DB: AF-Q1ZYL8-F1): AF-Q1ZYL8-F1): AF-Q3KNT9-F1) ana nuna su a cikin kewayon launi iri ɗaya kamar panel A. Ana nuna haɗin haɗin Disulfide a cikin magenta.TMEM95, IZUMO2 da IZUMO3 helices transmembrane ba a nuna su ba.
Ba kamar furotin IZUMO ba, sauran sunadaran SPACA (watau SPACA1, SPACA3, SPACA4, SPACA5, da SPACA9) ana tsammanin sun bambanta da tsarin SPACA6 (Fig. S12).SPACA9 kawai yana da 4HB, amma ba a sa ran samun daidaitattun daidaiton-anti-parallel orientation ko makamancin disulfide iri ɗaya kamar SPACA6.SPACA1 kawai yana da yanki mai kama da Ig.AlphaFold yayi annabta cewa SPACA3, SPACA4 da SPACA5 suna da tsari daban-daban fiye da SPACA6.Abin sha'awa shine, SPACA4 kuma an san yana taka rawa a cikin hadi, amma ya fi SPACA6, a maimakon haka yana sauƙaƙe hulɗar tsakanin maniyyi da oocyte zona pellucida52.
Binciken mu na AlphaFold ya sami wani wasa don IZUMO1 da SPACA6 4HB, TMEM95.TMEM95, ƙayyadaddun furotin transmembrane guda ɗaya na maniyyi, yana sa mice ba su da haihuwa lokacin da aka soke 32,33.Spermatozoa wanda ba shi da TMEM95 yana da ilimin halittar jiki na yau da kullun, motsi, da ikon shiga zona pellucida da ɗaure ga jikin kwai, amma ba zai iya haɗawa da membrane oocyte ba.Nazarin da suka gabata sun nuna cewa TMEM95 yana raba kamanceceniya na tsari tare da IZUMO133.Lalle ne, samfurin AlphaFold ya tabbatar da cewa TMEM95 shine 4HB tare da guda biyu na CXXC motifs kamar IZUMO1 da SPACA6 da kuma ƙarin disulfide bond tsakanin helices 1 da 2 da aka samu a SPACA6 (Fig. 3a da S11).Kodayake TMEM95 ba ta da yanki mai kama da Ig, yana da yanki tare da tsarin haɗin disulfide mai kama da SPACA6 da IZUMO1 hinge yankuna (Fig. 3b).A lokacin buga wannan rubutun, uwar garken da aka buga ya ba da rahoton tsarin TMEM95, yana mai tabbatar da sakamakon AlphaFold53.TMEM95 yayi kama da SPACA6 da IZUMO1 kuma an riga an kiyaye juyin halitta a cikin amphibians (Fig. 4 da S13).
Binciken PSI-BLAST yayi amfani da NCBI SPACA6, IZUMO1-4, TMEM95, DCST1, DCST2, FIMP, da SOF1 bayanai don sanin matsayin waɗannan jeri a cikin bishiyar rayuwa.Ba a nuna nisa tsakanin wuraren reshe zuwa sikeli ba.
Babban kamanni na tsarin gaba ɗaya tsakanin SPACA6 da IZUMO1 yana nuna cewa suna kafa mambobi ne na babban gida mai tsari wanda ya haɗa da TMEM95 da IZUMO 2, 3, da 4 sunadaran.sanannun membobin: IZUMO1, SPACA6 da TMEM95.Domin ƴan mambobi ne kawai ke da yankuna masu kama da Ig, alamar babban iyali na IST shine yankin 4HB, wanda ke da siffofi na musamman gama gari ga duk waɗannan sunadaran: 1) Rufe 4HB tare da helices da aka shirya a cikin juzu'i mai daidaitawa/daidaitacce (Figure). .5a), 2) daure yana da fuska mai siffar triangular wanda ya ƙunshi helices guda biyu a cikin damfara da kuma na uku a tsaye helix (Fig. key area (Fig. 5c) CXXC motif, samu a cikin thioredoxin-kamar sunadaran, an san yana aiki. a matsayin firikwensin redox 54,55,56, yayin da motif a cikin 'yan uwa na IST ana iya danganta shi da furotin disulfide isomerases kamar ERp57 a cikin fusion na gamete. Matsayin yana da alaƙa da 57,58.
Membobin IST superfamily an bayyana su ta hanyar sifofi guda uku na yanki na 4HB: helices guda huɗu masu musanya tsakanin layi ɗaya da daidaitawar gabaɗaya, ba-triangular helical bundle faces, da ca CXXC motif biyu da aka kafa tsakanin ƙananan ƙwayoyin cuta.) N-terminal helixes (orange) da yankin hinge β-hairpin (kore).
Ganin kamanceceniya tsakanin SPACA6 da IZUMO1, an gwada ƙarfin tsohon don ɗaure IZUMO1 ko JUNO.Biolayer interferometry (BLI) hanya ce ta ɗaure ta hanyar motsa jiki wacce aka yi amfani da ita a baya don ƙididdige hulɗar tsakanin IZUMO1 da JUNO.Bayan ƙaddamar da firikwensin mai lakabin biotin tare da IZUMO1 a matsayin koto tare da babban taro na JUNO analyte, an gano sigina mai ƙarfi (Fig. S14a), yana nuna canji mai ɗaure a cikin kauri na biomaterial da aka haɗe zuwa firikwensin firikwensin.Sigina iri ɗaya (watau JUNO haɗe da firikwensin a matsayin koto a kan analyte IZUMO1) (Fig. S14b).Ba a gano sigina ba lokacin da aka yi amfani da SPACA6 azaman nazari akan IZUMO1 da aka ɗaure firikwensin ko JUNO (Hoto S14a,b).Rashin wannan siginar yana nuna cewa yankin extracellular na SPACA6 baya hulɗa tare da yankin extracellular na IZUMO1 ko JUNO.
Saboda ƙididdigar BLI ta dogara ne akan biotinylation na ragowar lysine kyauta akan furotin bait, wannan gyare-gyare na iya hana ɗaurewa idan ragowar lysine ya shiga cikin hulɗar.Bugu da ƙari, daidaitawar dauri dangane da firikwensin na iya haifar da tsangwama, don haka an kuma yi gwaje-gwaje na yau da kullun akan SPACA6, IZUMO1 da JUNO ectodomains.Duk da haka, SPACA6 ba ta haɓaka ba tare da IZUMO1 mai alamar sa ko JUNO mai suna (Fig. S14c, d), yana nuna babu wata hulɗar da ta dace da abin da aka lura a cikin gwaje-gwajen BLI.A matsayin ingantaccen iko, mun tabbatar da hulɗar JUNO tare da lakabin IZUMO1 (Figures S14e da S15).
Duk da kamanceceniya tsakanin SPACA6 da IZUMO1, rashin iyawar SPACA6 don ɗaure JUNO ba abin mamaki bane.Fuskar ɗan adam IZUMO1 yana da fiye da ragowar 20 waɗanda ke hulɗa da JUNO, ciki har da ragowar kowane yanki na uku (ko da yake yawancin su suna cikin yankin hinge) (Fig. S14f).Daga cikin waɗannan ragowar, ɗaya ne kawai aka adana a cikin SPACA6 (Glu70).Yayin da yawancin sauran sauran abubuwan da suka rage sun ci gaba da riƙe ainihin kaddarorin sinadarai na halitta, ragowar Arg160 mai mahimmanci a cikin IZUMO1 an maye gurbinsu da Asp148 da aka caje a SPACA6;Nazarin da suka gabata sun nuna cewa maye gurbi na Arg160Glu a cikin IZUMO1 kusan ya kawar da ɗaurin JUNO43.Bugu da ƙari, bambance-bambance a cikin daidaitawar yanki tsakanin IZUMO1 da SPACA6 ya haɓaka girman filin JUNO-dauri na yankin daidai akan SPACA6 (Fig. S14g).
Duk da sanannen buƙatar SPACA6 don haɗakarwar gamete da kamanceceniya da IZUMO1, SPACA6 ba ya bayyana yana da aikin ɗaurin JUNO daidai.Don haka, mun nemi haɗa bayanan tsarin mu tare da shaidar mahimmancin da ilimin juyin halitta ya bayar.Jeri jeri na SPACA6 homologues yana nuna kiyaye tsarin gama gari fiye da dabbobi masu shayarwa.Misali, ragowar cysteine ​​suna nan har ma a cikin amphibians masu alaƙa (Fig. 6a).Yin amfani da uwar garken ConSurf, bayanan riƙon jeri da yawa na jeri 66 an tsara su zuwa saman SPACA6.Irin wannan bincike na iya nuna waɗanne ragowar da aka adana a lokacin juyin halittar furotin kuma yana iya nuna waɗanne yankuna na saman ke taka rawa wajen aiki.
Jeri jeri na SPACA6 ectodomains daga nau'ikan nau'ikan nau'ikan nau'ikan 12 da aka shirya ta amfani da CLUSTAL OMEGA.Bisa ga binciken ConSurf, mafi yawan matsayi masu ra'ayin mazan jiya suna da alamar shuɗi.Cysteine ​​​​sauran ana haskaka su cikin ja.Ana nuna iyakokin yanki da abubuwa na biyu na tsarin a saman jeri, inda kibau ke nuna β-strands da raƙuman ruwa suna nuna helices.Abubuwan Gano Samun damar NCBI waɗanda ke ɗauke da jerin abubuwa sune: ɗan adam (Homo sapiens, NP_001303901), mandrill (Mandrilus leucophaeus, XP_011821277), biri capuchin (Cebus mimic, XP_017359366), doki (Equus01355) 23 XP_032_034).), tumaki (Ovis aries, XP_014955560), giwa (Loxodonta africana, XP_010585293), kare ( Canis lupus familyis, XP_025277208), linzamin kwamfuta (Mus musculus, NP_001156381), Tasmanian devil , 8) , 61_89 da Bullfrog (Bufo bufo, XP_040282113).Ƙididdigar ta dogara ne akan tsarin ɗan adam.b Wakilin saman tsarin SPACA6 tare da 4HB a sama da yanki mai kama da Ig a ƙasa, launuka dangane da kimar kiyayewa daga uwar garken ConSurf.Mafi kyawun sassan da aka kiyaye su suna cikin shuɗi, sassan da aka kiyaye su a matsakaici suna cikin fari, kuma mafi ƙarancin kiyayewa suna cikin rawaya.purple cysteine.Ana nuna facin saman sama guda uku waɗanda ke nuna babban matakin kariya a cikin saitin da aka yiwa lakabin faci 1, 2 da 3. Ana nuna zane mai ban dariya 4HB a cikin sa a saman dama (tsarin launi iri ɗaya).
Tsarin SPACA6 yana da yankuna uku da aka kiyaye sosai (Fig. 6b).Patch 1 yana ɗaukar 4HB da yankin hinge kuma ya ƙunshi gada biyu na CXXC disulfide da aka adana, hanyar sadarwa ta Arg233-Glu132-Arg135-Ser144 (Fig. S7), da sauran abubuwan ƙamshi guda uku da aka adana (Phe31, Tyr73, Phe137)).babban gefen yanki mai kama da Ig (Fig. S6e), wanda ke wakiltar ragowar abubuwan da aka caje da yawa a saman maniyyi.Abin sha'awa shine, wannan facin ya ƙunshi epitope na antibody wanda aka nuna a baya don tsoma baki tare da aikin SPACA6 30.Yanki na 3 ya mamaye hinge da gefe ɗaya na yankin Ig-kamar;wannan yanki yana ƙunshe da ƙayyadaddun prolines (Pro126, Pro127, Pro150, Pro154) da sauran abubuwan da ke fuskantar waje.Abin mamaki shine, yawancin ragowar da ke saman 4HB suna da matukar canzawa (Fig. 6b), ko da yake an adana ninki a cikin SPACA6 homologue (kamar yadda aka nuna ta conservatism na hydrophobic bundle core) kuma bayan IST superfamily.
Kodayake wannan shine yanki mafi ƙanƙanta a cikin SPACA6 tare da ƙananan abubuwan da aka gano na biyu na tsarin, yawancin ragowar yanki (ciki har da yanki 3) ana kiyaye su sosai a tsakanin SPACA6 homologues, wanda zai iya nuna cewa daidaitawa na gunkin helical da β-sandwich suna taka rawa.a matsayin mai ra'ayin mazan jiya.Duk da haka, duk da yawan haɗin haɗin gwiwar hydrogen da hanyoyin sadarwa na lantarki a cikin yankin hinge na SPACA6 da IZUMO1, ana iya ganin shaidar sassaucin ra'ayi a cikin daidaitawar tsarin da aka yarda da yawa na IZUMO137,43,44.Daidaitawa na kowane yanki ya mamaye da kyau, amma daidaitawar yankunan dangane da juna ya bambanta daga 50 ° zuwa 70 ° daga tsakiyar axis (Fig. S16).Don fahimtar abubuwan da suka dace na SPACA6 a cikin bayani, an yi gwajin SAXS (Fig. S17a,b).Ab initio sake ginawa na SPACA6 ectodomain ya dace da tsarin kristal na sanda (Fig. S18), kodayake makircin Kratky ya nuna wani mataki na sassauci (Fig. S17b).Wannan daidaituwa ya bambanta da IZUMO1, wanda furotin da ba a ɗaure shi yana ɗaukar siffar boomerang duka a cikin lattice da kuma a cikin bayani43.
Don gano takamaiman yanki mai sassauƙa, hydrogen-deuterium musayar spectroscopy (H-DXMS) an yi shi akan SPACA6 kuma idan aka kwatanta da bayanan da aka samu a baya akan IZUMO143 (Fig. 7a,b).SPACA6 a fili ya fi sauƙi fiye da IZUMO1, kamar yadda aka nuna ta hanyar musayar deuterium mafi girma a cikin tsarin bayan 100,000 s na musayar.A cikin sassan biyu, ɓangaren C-terminal na yankin hinge yana nuna babban matakin musayar, wanda mai yiwuwa ya ba da damar iyakance iyaka na 4HB da Ig-kamar yanki dangane da juna.Abin sha'awa shine, ɓangaren C-terminal na SPACA6 hinge, wanda ya ƙunshi ragowar 147CDLPLDCP154, yanki ne mai girma 3 (Fig. 6b), mai yiwuwa yana nuna cewa sassaucin ra'ayi shine fasalin juyin halitta na SPACA6.Bisa ga ƙididdigar sassaucin ra'ayi, bayanan narke na CD na CD ya nuna cewa SPACA6 (Tm = 51.2 ° C) ba shi da kwanciyar hankali fiye da IZUMO1 (Tm = 62.9 ° C) (Fig. S1e da S19).
Hotunan H-DXMS na SPACA6 da b IZUMO1.An ƙayyade yawan musayar deuterium a wuraren da aka nuna.Matakan musayar hydrogen-deuterium ana nuna su ta launi akan sikelin gradient daga shuɗi (10%) zuwa ja (90%).Baƙaƙen kwalaye suna wakiltar wuraren babban musayar.Iyakoki na 4HB, hinge da Ig-kamar yanki da aka lura a cikin tsarin crystal ana nuna su sama da jerin farko.Matakan musayar Deuterium a 10 s, 1000 s, da 100,000 s an ƙirƙira su akan ginshiƙi mai ɗorewa a kan madaidaicin filayen kwayoyin halitta na SPACA6 da IZUMO1.Sassan tsarin tare da matakin musayar deuterium a ƙasa da 50% fari ne masu launi.Wuraren da ke sama da 50% H-DXMS musayar suna da launi a cikin sikelin gradient.
Amfani da CRISPR/Cas9 da linzamin kwamfuta na linzamin kwamfuta dabaru sun haifar da gano abubuwa da yawa masu mahimmanci ga maniyyi da ɗaure kwai da haɗuwa.Baya ga kyakkyawar ma'amala ta IZUMO1-JUNO da tsarin CD9, yawancin sunadaran da ke da alaƙa da haɗin gamete suna kasancewa cikin tsari da aiki mai ban mamaki.Siffar yanayin halitta da sifa na SPACA6 wani yanki ne na mannewa/fusion wasan wuyar warwarewa na ƙwayoyin cuta yayin hadi.
SPACA6 da sauran membobin IST superfamily sun bayyana cewa ana kiyaye su sosai a cikin dabbobi masu shayarwa da tsuntsayen tsuntsaye, dabbobi masu rarrafe, da masu amphibians;a gaskiya ma, ana tunanin cewa SPACA6 har ma ana buƙata don hadi a cikin zebrafish 59. Wannan rarraba yana kama da sauran sanannun sunadaran sunadaran da suka hada da gamete fusion irin su DCST134, DCST234, FIMP31, da SOF132, suna nuna cewa waɗannan abubuwan suna da kasawa HAP2 (har ila yau. da aka sani da GCS1) sunadaran da ke da alhakin ayyukan haɓakar protists da yawa., shuke-shuke, da arthropods.Hakika fusion proteins 60, 61. Duk da ƙaƙƙarfan kamanceceniya tsakanin SPACA6 da IZUMO1, ƙwanƙwasa ƙwayoyin halittar da ke ɓoye ɗayan waɗannan sunadaran sun haifar da rashin haihuwa a cikin berayen maza, yana nuna cewa ayyukansu a cikin fusion na gamete ba su kwafi..Fiye da yawa, babu ɗayan sanannun sunadaran sunadaran maniyyi da ake buƙata don lokacin mannewa na fusion ɗin da ya yi yawa.
Ya kasance buɗaɗɗen tambaya ko SPACA6 (da sauran membobin IST superfamily) suna shiga tsaka-tsakin tsaka-tsakin tsaka-tsakin, samar da hanyoyin sadarwa na intragametic don ɗaukar mahimman furotin zuwa wuraren haɗakarwa, ko wataƙila ma suna aiki azaman fusogens.Nazarin haɗin gwiwar rigakafin rigakafi a cikin sel HEK293T ya bayyana hulɗar tsakanin cikakken tsayin IZUMO1 da SPACA632.Koyaya, ectodomains ɗin mu na sake haɗawa ba su yi hulɗa a cikin vitro ba, yana nuna cewa hulɗar da aka gani a Noda et al.an share su duka a cikin ginin (lura wutsiyar cytoplasmic na IZUMO1, wanda aka nuna ba dole ba ne don hadi62).Madadin haka, IZUMO1 da/ko SPACA6 na iya buƙatar takamaiman mahalli masu ɗaure waɗanda ba mu haifuwa a cikin vitro ba, kamar ƙayyadaddun ƙayyadaddun physiologically ko rukunin kwayoyin halitta waɗanda ke ɗauke da wasu sunadaran (an sani ko ba a gano su ba tukuna).Ko da yake IZUMO1 ectodomain an yi imanin yana daidaita haɗin maniyyi zuwa kwai a cikin sararin perivitelline, manufar SPACA6 ectodomain ba ta da tabbas.
Tsarin SPACA6 yana bayyana wurare da yawa da aka adana waɗanda zasu iya shiga cikin hulɗar furotin-gina jiki.Bangaren da aka adana na yankin hinge nan da nan kusa da manufar CXXC (wanda aka zana Patch 1 a sama) yana da ragowar kamshi da yawa masu fuskantar waje waɗanda galibi ana danganta su da ma'amalar hydrophobic da π-stacking tsakanin biomolecules.Faɗin ɓangarorin Ig-kamar yanki (yankin 2) suna samar da tsagi mai inganci tare da kiyayewa sosai da Arg da ragowarsa, kuma an yi amfani da ƙwayoyin rigakafi a kan wannan yanki a baya don toshe fusion na gamete 30.Maganin rigakafi ya gane madaidaicin epitope 212RIRPAQLTHRGTFS225, wanda ke da uku daga cikin ragowar arginine shida kuma His220 ya kiyaye sosai.Ba a bayyana ko tabarbarewar ta samo asali ne sakamakon toshewar waɗannan takamaiman abubuwan da suka rage ko kuma yankin gaba ɗaya.Wurin wannan rata a kusa da C-terminus na β-sandwich yana nuna hulɗar cis tare da sunadaran maniyyi makwabta, amma ba tare da sunadaran oocyte ba.Bugu da ƙari kuma, riƙewar tangle mai arziƙi mai sassaucin ra'ayi (shafin 3) a cikin hinge na iya zama wurin haɗin gwiwar furotin-protein ko kuma, mafi mahimmanci, yana nuna riƙe da sassauci tsakanin sassan biyu.Jinsi yana da mahimmanci don rawar da ba a sani ba na SPACA6.Fusion na gametes.
SPACA6 yana da kaddarorin sunadaran mannewa tsakanin salula, gami da Ig-kamar β-sandwiches.Yawancin sunadaran mannewa (misali, cadherins, integrins, adhesins, da IZUMO1) suna da yanki ɗaya ko fiye da β-sandwich waɗanda ke shimfiɗa sunadaran daga jikin tantanin halitta zuwa ga mahallin muhalli63,64,65.Yankin Ig-kamar na SPACA6 kuma ya ƙunshi motif da aka saba samu a cikin β-sandwiches na mannewa da haɗin kai: ninki biyu na madauri mai kama da juna a ƙarshen β-sandwiches, wanda aka sani da maƙallan inji66.An yi imani da cewa wannan motif yana ƙara juriya ga ƙarfin ƙarfi, wanda ke da mahimmanci ga sunadaran da ke cikin hulɗar intercellular.Duk da haka, duk da wannan kamance da adhesins, a halin yanzu babu wata shaida da ke nuna cewa SPACA6 yana hulɗa tare da fararen kwai.SPACA6 ectodomain ba zai iya ɗaure zuwa JUNO ba, kuma SPACA6-bayyana ƙwayoyin HEK293T, kamar yadda aka nuna a nan, da wuya yin hulɗa tare da oocytes marasa zona 32.Idan SPACA6 ta kafa haɗin gwiwar juna, waɗannan hulɗar na iya buƙatar gyare-gyaren bayan fassarar ko kuma a daidaita su ta wasu sunadaran maniyyi.A cikin goyan bayan ra'ayi na ƙarshe, IZUMO1-rashin spermatozoa yana ɗaure zuwa oocytes, yana nuna cewa kwayoyin da ba IZUMO1 ba suna shiga cikin mataki na 27 gamete adhesion.
Yawancin ƙwayoyin cuta, salon salula, da sunadaran haɗin haɓaka suna da kaddarorin da ke hasashen aikin su azaman fusogens.Misali, glycoproteins fusion na hoto ko bidiyo mai zagaya yanar gizo da sauri (azuzuwan I, II da III) suna da peptide fusion na hydrophobic ko madauki a ƙarshen furotin da aka saka a cikin membrane mai watsa shiri.Taswirar hydrophilicity na IZUMO143 da tsarin (ƙaddara da annabta) na IST superfamily ya nuna babu wani bayyanar peptide hydrophobic fusion.Don haka, idan kowane sunadaran da ke cikin IST superfamily suna aiki azaman fusogens, suna yin haka ta hanyar da ta bambanta da sauran sanannun misalan.
A ƙarshe, ayyukan membobin ƙungiyar IST superfamily na sunadaran da ke da alaƙa da haɗuwar gamete sun kasance wani asiri mai ban mamaki.SPACA6 ɗinmu na sake haɗawa da ƙayyadaddun tsarinsa zai ba da haske game da alaƙar da ke tsakanin waɗannan sifofin da aka raba da kuma rawar da suke takawa a cikin haɗe-haɗe da haɗuwa da gamete.
Jerin DNA wanda ya dace da ɗan adam SPACA6 ectodomain da aka annabta (lambar shiga NCBI NP_001303901.1; saura 27-246) an inganta shi don magana a cikin sel Drosophila melanogaster S2 kuma an haɗa su azaman rarrabuwar kwayoyin halitta tare da tsarin shigar da Kozaknom (Eurofin)., Siginar ɓoyewar BiP da madaidaicin 5' da 3' ya ƙare don cloning mai zaman kansa na wannan kwayar halitta a cikin sigar magana ta pMT dangane da mai tallata metallothionein wanda aka gyara don zaɓi tare da puromycin (pMT-puro).Vector pMT-puro yana ɓoye wurin tsagewar thrombin tare da alamar C-Terminal 10x-Sa (Hoto S2).
Canjin kwanciyar hankali na SPACA6 pMT-puro vector zuwa cikin sel D. melanogaster S2 (Gibco) an yi daidai da ka'idar da aka yi amfani da ita don IZUMO1 da JUNO43.Kwayoyin S2 sun narke kuma sun girma a cikin matsakaicin Schneider (Gibco) an ƙara su tare da ƙaddamarwa na ƙarshe na 10% (v / v) zafi mara amfani da ƙwayar maraƙi (Gibco) da 1X maganin rigakafi na antimycotic (Gibco).Kwayoyin wucewa na farko (kwayoyin 3.0 x 106) an yi su a cikin rijiyoyin guda ɗaya na faranti 6-rijiya (Corning).Bayan sa'o'i 24 na shiryawa a 27 ° C, an canza sel tare da cakuda 2 MG na SPACA6 pMT-puro vector da Effectene transfection reagent (Qiagen) bisa ga ka'idar masana'anta.An yi amfani da ƙwayoyin da aka canza zuwa 72 hours sannan kuma an girbe su tare da 6 mg / ml puromycin.An keɓe ƙwayoyin sel daga cikakkiyar matsakaicin matsakaicin Schneider kuma an sanya su cikin matsakaicin ƙwayar cuta-XPRESS (Lonza) kyauta don samar da furotin mai girma.A 1 L batch na S2 cell al'ada aka girma zuwa 8-10 × 106 ml-1 Kwayoyin a cikin 2 L ventilated lebur-kasa polypropylene Erlenmeyer flask sa'an nan haifuwa tare da karshe taro na 500 µM CuSO4 (Millipore Sigma) da kuma bakararre tace.jawo.An ƙaddamar da al'adun da aka haifar a 27 ° C. a 120 rpm na kwanaki hudu.
Matsakaicin matsakaici mai ɗauke da SPACA6 an keɓe shi ta hanyar centrifugation a 5660×g a 4°C sannan kuma tsarin tacewa na Centramate tangential (Pall Corp) tare da membrane 10 kDa MWCO.Aiwatar da matsakaitan matsakaita mai ɗauke da SPACA6 zuwa ginshiƙi na Ni-NTA agarose resin (Qiagen) 2 ml.An wanke resin Ni-NTA tare da kundin ginshiƙi 10 (CV) na buffer A sannan an ƙara 1 CV na buffer A don ba da ƙaddamarwar imidazole na ƙarshe na 50 mM.An cire SPACA6 tare da 10 ml na buffer A wanda aka haɓaka tare da imidazole zuwa ƙaddamarwar ƙarshe na 500 mM.Ƙuntataccen thrombin (Millipore Sigma) an ƙara kai tsaye zuwa bututun dialysis (MWCO 12-14 kDa) a 1 raka'a ta MG SPACA6 vs. 1 L 10 mM Tris-HCl, pH 7.5 da 150 mM NaCl (buffer B) don dialysis.a 4 ° C na awanni 48.SPACA6 mai thrombin-cleaved an diluted sau uku don rage yawan gishiri kuma an ɗora shi a kan 1 ml MonoS 5/50 GL cation musayar ginshiƙi (Cytiva/GE) daidai da 10 mM Tris-HCl, pH 7.5.An wanke mai musayar cation ɗin tare da 3 CV na 10 mM Tris-HCl, pH 7.5, sannan SPACA6 ya ƙare tare da madaidaiciyar madaidaiciyar 0 zuwa 500 mM NaCl a cikin 10 mM Tris-HCl, pH 7.5 don 25 CV.Bayan musayar chromatography na ion, SPACA6 an mayar da hankali ga 1 ml kuma an cire shi ta hanyar keɓancewa daga ginshiƙi na ENrich SEC650 10 x 300 (BioRad) wanda aka daidaita tare da buffer B. Bisa ga chromatogram, tafkin da tattara ɓangarorin da ke ɗauke da SPACA6.Ana sarrafa tsabta ta hanyar Coomassie-stained electrophoresis akan 16% SDS-polyacrylamide gel.An ƙididdige tattarawar sunadaran ta hanyar sha a 280 nm ta amfani da dokar Beer-Lambert da ƙayyadaddun ƙayyadaddun ƙayyadaddun molar.
An yi wa SPACA6 mai tsafta da daddare akan 10 mM sodium phosphate, pH 7.4 da 150 mM NaF kuma an diluted zuwa 0.16 mg/mL kafin bincike ta CD spectroscopy.An tattara sikanin sikanin CD ɗin tare da tsawon 185 zuwa 260 nm akan Jasco J-1500 spectropolarimeter ta amfani da ma'aunin quartz cuvettes tare da tsayin hanya na gani na 1 mm (Helma) a 25 ° C akan ƙimar 50 nm/min.An gyara siginar CD na asali, an ƙididdige sama da saye 10, kuma an canza su zuwa ma'anar saura ellipticity (θMRE) a cikin digiri cm2/dmol:
inda MW shine nauyin kwayoyin kowane samfurin a cikin Da;N shine adadin amino acid;θ shine elpticity a cikin millidegrees;d yayi daidai da tsawon hanyar gani a cikin cm;furotin maida hankali a cikin raka'a.