304 ibyuma bitagira umuyonga coing tubing chimique, Imiterere ya ectodomain ya SPACA6 irimo superfamily yabitswe na proteine ​​zijyanye no guhuza gamete.

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SPACA6 ni intungamubiri zigaragaza intungamubiri zifite akamaro kanini muguhuza gamete mugihe cyimyororokere yinyamabere.Nubwo uru ruhare rwibanze, imikorere yihariye ya SPACA6 ntabwo yunvikana neza.Turasobanura neza imiterere ya kristu ya domaine idasanzwe ya SPACA6 ku cyemezo cya 2.2 Å, tugaragaza proteine ​​ebyiri-igizwe na bundle igizwe n'imirongo ine hamwe na Ig-nka β-sandwiches ihujwe na quasi-flexible.Iyi miterere isa na IZUMO1, indi proteine ​​ifitanye isano na gamete, bigatuma SPACA6 na IZUMO1 bashinze abanyamuryango ba superfamily ya poroteyine zijyanye no gusama bavugwa hano nka IST birenze urugero.IST mu buryo budasobanutse isobanurwa muburyo bwahinduwe na bine-helix bundle hamwe na disulfide ihuza CXXC.Ubushakashatsi bushingiye kuri AlphaFold ubushakashatsi bwa proteome yumuntu bwerekanye izindi poroteyine zigize iyi superfamily;biragaragara, inyinshi muri izo poroteyine zigira uruhare mu guhuza gamete.Imiterere ya SPACA6 nubusabane bwayo nabandi banyamuryango ba IST birenze urugero bitanga umurongo wabuze mubumenyi bwacu bwinyamanswa yinyamanswa.
Ubuzima bwa buri muntu butangirira kumikino ibiri itandukanye ya haploid: intanga za se nintanga ya nyina.Iyi ntanga niyo yatsindiye uburyo bukomeye bwo gutoranya aho amamiriyoni yintangangabo zinyura mumyanya ndangagitsina yumugore, zigatsinda inzitizi zitandukanye1 kandi zikagira ubushobozi, ibyo bikaba byongera imbaraga zabo hamwe nibikorwa byubutaka2,3,4.Nubwo intanga na oocyte zibonana, inzira ntirarangira.Oocyte ikikijwe nigice cya selile cumulus na bariyeri ya glycoproteine ​​yitwa zona pellucida, intanga zigomba kunyuramo kugirango zinjire muri oocyte.Spermatozoa koresha uruvange rwa molekules ya adhesion hamwe na membrane ifitanye isano na enzymes zasohotse kugirango utsinde izo nzitizi zanyuma5.Izi molekile na enzymes zibikwa cyane muri membrane y'imbere na matrix ya acrosomal kandi bikamenyekana mugihe ururenda rwinyuma rwintanga rwatewe mugihe cya acrosomal reaction6.Intambwe yanyuma mururwo rugendo rukomeye nigikorwa cyo guhuza intanga-amagi, aho selile zombi zihuza ururenda kugirango zibe ikinyabuzima kimwe cya diploid7.Nubwo iyi nzira ari intangiriro mu myororokere yabantu, imikoranire ya molekile ikenewe ntisobanutse neza.
Usibye gusama kwa gametes, chimie yo guhuza ibice bibiri bya lipid bilayers yarizwe cyane.Muri rusange, guhuza franse ni inzira idakenewe cyane isaba intungamubiri za poroteyine kugira ngo ihindure imiterere ihuza ibice bibiri byegeranye, bisenya gukomeza kandi bitera guhuza 8,9.Izi poroteyine zizwi nka fusogène kandi zabonetse muri sisitemu zitabarika.Barasabwa kwinjirira virusi muri selile zakira (urugero, gp160 muri VIH-1, spike muri coronavirus, hemagglutinin muri virusi yibicurane) 10,11,12 insimburangingo (syncytin) 13,14,15 hamwe nudukino twa gamete muri eukaryote yo hepfo ( HAP2 / GCS1 mu bimera, abigaragambyaga na arthropodes) 16,17,18,19.Fusogène kumikino yabantu ntiravumburwa, nubwo proteine ​​nyinshi zerekanwe ko ari ingenzi mu guhuza imikino no guhuza.CD9 ya oocyte yagaragajwe, poroteyine ya transembrane isabwa mu guhuza imbeba n'imikino y'abantu, ni yo ya mbere yavumbuwe 21,22,23.Nubwo imikorere yacyo idasobanutse neza, uruhare mukwizirika, imiterere ya adhesion yibanze kuri microvilli yamagi, hamwe na / cyangwa aho bikwiye bya poroteyine zo mu bwoko bwa oocyte bisa nkaho ari 24,25,26.Poroteyine ebyiri zisanzwe zifite akamaro kanini mu guhuza gamete ni intanga ngabo ya IZUMO127 na proteine ​​oocyte JUNO28, kandi guhuza kwabo ni intambwe ikomeye mu kumenyekanisha imikino no kuyifata mbere yo guhuza.Imbeba zumugabo Izumo1 nimbeba za Juno knockout imbeba ni sterile rwose, murubu buryo intanga ngabo zinjira mumwanya wa perivitelline ariko gametes ntizihuza.Mu buryo nk'ubwo, ihuriro ryaragabanutse igihe imikino yakoreshwaga na antibodiyide zirwanya IZUMO1 cyangwa JUNO27,29 mu bantu mu bushakashatsi bwo gufumbira vitro.
Vuba aha, havumbuwe itsinda rishya rya poroteyine zigaragaza intanga ngabo zisa na IZUMO1 na JUNO20,30,31,32,33,34,35.Intanga ngabo ya acrosomal membrane ifitanye isano na poroteyine 6 (SPACA6) byagaragaye ko ari ngombwa mu gusama mu bushakashatsi bunini bwa murine mutagenezi.Kwinjiza transgene muri gen ya Spaca6 itanga spermatozoa idashoboka, nubwo izo spermatozoya zinjira mumwanya wa perivitelline 36.Ubushakashatsi bwakomotse kuri knockout mu mbeba bwemeje ko Spaca6 isabwa kugirango gamete fusion 30,32.SPACA6 igaragazwa cyane cyane mubizamini kandi ifite uburyo bwaho busa nubwa IZUMO1, cyane cyane muri intima ya spermatozoa mbere ya reaction ya acrosomal, hanyuma ikimukira mukarere ka ecatori nyuma ya acrosomal reaction 30,32.Spaca6 homologues ibaho mubinyamabere zinyuranye nizindi eukaryote 30 kandi akamaro kayo muguhuza imikino yabantu byagaragajwe no kubuza ifumbire mvaruganda muri vitro kurwanya SPACA6 30.Bitandukanye na IZUMO1 na JUNO, ibisobanuro birambuye kumiterere, imikoranire, n'imikorere ya SPACA6 ntibisobanutse neza.
Kugira ngo twumve neza inzira y'ibanze ishingiye ku guhuza intanga ngabo n'amagi, bizadufasha kumenyesha iterambere rizaza mu kuboneza urubyaro no kuvura uburumbuke, twakoze ubushakashatsi ku miterere n'ibinyabuzima bya SPACA6.Imiterere ya kristu ya domaine idasanzwe ya SPACA6 yerekana bundle enye (4HB) hamwe na immunoglobuline imeze (Ig-imeze) ihuza uturere tworoshye.Nkuko byari byarahanuwe mubushakashatsi bwabanje, 7.32,37 imiterere ya domaine ya SPACA6 isa niy'umuntu IZUMO1, kandi poroteyine zombi zisangiye motif idasanzwe: 4HB ifite ubuso butatu bwa mpandeshatu hamwe na moteri ya CXXC ihuza disulfide.Turasaba ko IZUMO1 na SPACA6 ubu bisobanura binini, bifitanye isano na superfamily ya proteine ​​zijyanye no guhuza gamete.Twifashishije ibintu byihariye bidasanzwe, twakoze ubushakashatsi bwimbitse kuri proteome yumuntu ya AlphaFold, tumenyekanisha abandi banyamuryango biyi superfamily, harimo nabanyamuryango benshi bagize uruhare mumikino yo guhuza no / cyangwa gusama.Ubu biragaragara ko hariho imiterere ihuriweho hamwe na superfamily ya proteine ​​zijyanye no guhuza gamete, kandi imiterere yacu itanga ikarita ya molekile yiyi ngingo yingenzi yuburyo bwimikorere yabantu.
SPACA6 ni poroteyine imwe-imwe ya poroteyine hamwe na glycan imwe ihuza N hamwe na batandatu ya disulfide ihuza (Ishusho S1a na S2).Twerekanye indangagitsina zidasanzwe za muntu SPACA6 (ibisigara 27-2246) muri selire ya Drosophila S2 hanyuma dusukura poroteyine dukoresheje nikel, guhanahana cation, hamwe no gutandukanya chromatografiya (Ishusho S1b).SPACA6 isukuye ectodomain irahagaze neza kandi ni umwe.Isesengura ukoresheje ubunini bwo gutandukanya chromatografiya hamwe no gukwirakwiza urumuri rwa polygonal (SEC-MALS) byagaragaje impinga imwe ifite uburemere bwa molekile yabazwe bwa 26.2 ± 0.5 kDa (Ishusho S1c).Ibi bihuye nubunini bwa SPACA6 monomeric ectodomain, byerekana ko oligomerisation itabayeho mugihe cyo kwezwa.Byongeye kandi, dichroism izenguruka (CD) spekitroscopi yerekanye imiterere ivanze α / β ifite aho ishonga ya 51.3 ° C (Ishusho S1d, e).Deconvolution ya CD yerekanwe yerekanaga 38,6% α-ihindagurika na 15.8% β -ibintu (Ishusho S1d).
Ectodomain ya SPACA6 yashizwemo hifashishijwe imbuto ya matrix idasanzwe 38 bivamo amakuru yashyizweho hamwe na 2.2 Å (Imbonerahamwe 1 nishusho S3).Ukoresheje uruvangitirane rushingiye ku gusimbuza ibice bya molekuline hamwe na SAD ikurikirana ibyiciro hamwe na bromide yerekanwe kugirango igaragaze imiterere (Imbonerahamwe 1 nishusho S4), moderi yanyuma yatunganijwe igizwe nibisigara 27-224.Mugihe imiterere yagenwe, ntabigeragezo cyangwa AlphaFold yubatswe yaboneka.SPACA6 ectodomain ipima 20 Å × 20 Å × 85 Å, igizwe na helices zirindwi na cyenda β-imirongo, kandi ifite urwego rurerure rurerure rwahagaritswe n’imigozi itandatu ya disulfide (Ishusho 1a, b).Ubucucike bwa electron nkeya kumpera yumunyururu wa Asn243 byerekana ko iki gisigara ari glycosylation ya N ihuza.Imiterere igizwe na domaine ebyiri: N-terminal enye-helix bundle (4HB) hamwe na C-terminal Ig-imeze nka domeni ifite akarere kegeranye hagati yabo (Ishusho 1c).
Imiterere ya domaine idasanzwe ya SPACA6.Igishushanyo mbonera cya domaine idasanzwe ya SPACA6, ibara ryumunyururu kuva N kugeza C-terminus kuva mubururu bwijimye kugeza umutuku wijimye.Cysteine ​​igira uruhare muri disulfide iragaragazwa muri magenta.b Topologiya ya domaine idasanzwe ya SPACA6.Koresha ibara rimwe nkuko bigaragara mu gishushanyo 1a.c Indanganturo ya SPACA6.4HB, hinge, na Ig-bisa nkibishushanyo mbonera byerekana ibara rya orange, icyatsi, nubururu.Ibice ntibishushanyije ku munzani.
Indangantego ya 4HB ya SPACA6 ikubiyemo ibice bine by'ingenzi (helices 1-4), bitunganijwe muburyo bwa helix helix (Ishusho 2a), bisimburana hagati yimikoranire itandukanye kandi ibangikanye (Ishusho 2b).Agace gato kiyongereyeho icyerekezo kimwe (helix 1 ′) gishyirwa kuri perpendikulari kuri bundle, kigakora inyabutatu ifite helices 1 na 2. Iyi mpandeshatu yahinduwe gato mubipfunyika byahinduwe byapakiye ugereranije nibipfunyika bya tekinike ya 3 na 4 ( Igishushanyo 2a).
4HB N-imbonerahamwe yerekana umurongo.b Hejuru yo hejuru ya bundle ya helices enye, buri helix yamuritse mubururu bwijimye kuri N-terminus n'umutuku wijimye kuri C-terminus.c Igishushanyo mbonera cyo hejuru hejuru ya 4HB, hamwe na buri gisigara cyerekanwe nkuruziga rwanditseho inyuguti imwe ya kode ya amino acide;acide enye gusa aminide hejuru yiziga irabaze.Ibisigazwa bitari inkingi bifite ibara ry'umuhondo, ibisigazwa bitarimo polar bisigara bifite ibara ry'icyatsi, ibisigara byuzuye neza bifite ibara ry'ubururu, naho ibisigazwa byuzuye nabi bifite ibara ry'umutuku.d Isura ya mpandeshatu ya domaine ya 4HB, hamwe na 4HB muri orange na hinges mubyatsi.Inets zombi zerekana inkoni imeze nka disulfide.
4HB yibanze kuri hydrophobique y'imbere igizwe ahanini n'ibisigazwa bya alifatique na aromatiya (Ishusho 2c).Intangiriro ikubiyemo isano ya disulfide hagati ya Cys41 na Cys55 ihuza helices 1 na 2 hamwe muri mpandeshatu yazamuye hejuru (Ishusho 2d).Ubundi buryo bubiri bwa disulfide bwakozwe hagati ya motif ya CXXC muri Helix 1 ′ nindi motif ya CXXC iboneka ku isonga rya hair-umusatsi mu karere ka hinge (Ishusho 2d).Ibisigisigi bya arginine bisigara bifite ibikorwa bitazwi (Arg37) biherereye imbere muri mpandeshatu itagira ingano ikozwe na helices 1 ′, 1, na 2. Atipi ya karubone ya Alifatique Cβ, Cγ, na Cδ Arg37 ikorana na hydrophobique, kandi amatsinda yayo ya guanidine agenda azunguruka. hagati ya helices 1 ′ na 1 binyuze mumikoranire hagati yumugongo wa Thr32 numurongo wuruhande (Ishusho S5a, b).Tyr34 igera mu cyuho hasigara imyanya ibiri ntoya Arg37 ishobora gukorana na solve.
Ig-nka β-sandwich domaine nini nini ya superfamily ya proteine ​​isangiye ibintu bisanzwe biranga amphipathic ebyiri cyangwa nyinshi zirenze urugero β -impapuro zikorana binyuze muri hydrophobique 39. C-terminal Ig-imeze nka domeni ya SPACA6 ifite icyitegererezo kimwe kandi igizwe n'ibice bibiri (Ishusho S6a).Urupapuro 1 ni β -urupapuro rw'imirongo ine (imirongo D, F, H, na I) aho imirongo F, H, nanjye nkora gahunda irwanya parallel, naho imirongo I na D ifata imikoranire ibangikanye.Imbonerahamwe 2 ni ntoya irwanya-parallel kabiri-urupapuro rwa beta (imirongo E na G).Imbere ya disulfide y'imbere yagaragaye hagati ya C-terminus ya E na rwagati rwumunyururu H (Cys170-Cys226) (Ishusho S6b).Iyi nkunga ya disulfide irasa nubusabane bwa disulfide muri domain-sandwich domaine ya immunoglobulin40,41.
Imirongo ine-urupapuro ruzunguruka mu burebure bwarwo bwose, rukora impande zidasanzwe zidafite imiterere na electrostatike.Uruhande ruto cyane ni hydrophobique yubuso bwibidukikije igaragara neza ugereranije nubuso busigaye butaringaniye kandi butandukanye butandukanye muri SPACA6 (Ishusho S6b, c).Halo yerekana imigongo ya karubone / amatsinda hamwe nuruhererekane rwuruhande ruzengurutse hydrophobique (Ishusho S6c).Imipaka yagutse itwikiriwe nigice cyafashwe gihagaritse igice cya N-terminal cya hydrophobique kandi kigakora imigozi itatu ya hydrogène hamwe nitsinda rya polar rifunguye ryurunigi rwa F (Ishusho S6d).Igice cya C-terminal cyuruhande kigize umufuka munini hamwe na hydrophobique yibanze igice.Umufuka uzengurutswe nuburyo bwiza bitewe nuburyo butatu bwibisigisigi bya arginine ebyiri (Arg162-Arg221, Arg201-Arg205 na Arg212-Arg214) hamwe na histidine yo hagati (His220) (Igicapo S6e).
Agace ka hinge nigice kigufi hagati ya domaine ihanamye hamwe na Ig-imeze nka domeni, igizwe na antiparallel imwe igizwe n'imirongo itatu β-layer (imirongo A, B na C), agace gato 310, hamwe nibice byinshi birebire bidasanzwe.(Isanamu S7).Umuyoboro wa covalent na electrostatike ihuza mukarere ka hinge bigaragara ko ihindura icyerekezo hagati ya 4HB na Ig-imeze nka domeni.Umuyoboro urashobora kugabanywamo ibice bitatu.Igice cya mbere kirimo ibishushanyo bibiri bya CXXC (27CXXC30 na 139CXXC142) bigizwe nubusabane bwa disulfide hagati ya β-umusatsi uri muri hinge na 1 ′ helix muri 4HB.Igice cya kabiri kirimo imikoranire ya electrostatike hagati ya Ig-nka domaine na hinge.Glu132 muri hinge ikora ikiraro cyumunyu hamwe na Arg233 murwego rwa Ig-na Arg135 muri hinge.Igice cya gatatu kirimo uburinganire hagati ya Ig-imeze nkakarere ka hinge.Imiyoboro ibiri ya disulfide (Cys124-Cys147 na Cys128-Cys153) ihuza hinge loop ihuza umuhuza uhagarikwa n’imikoranire ya electrostatike hagati ya Gln131 nitsinda ryimikorere ryumugongo, ryemerera kugera kumurongo wambere wa Ig.urunigi.
Imiterere ya ectodomain ya SPACA6 hamwe nuburyo bwihariye bwa 4HB na Ig-bisa na domeni byakoreshejwe mugushakisha inyandiko zisa muburyo bwububiko bwa protein 42.Twabonye imipira ifite amanota menshi ya Dali Z, gutandukana guto, hamwe n amanota manini ya LALI (icya nyuma ni umubare wibisigisigi byubatswe).Mugihe ibitaramo 10 byambere bivuye mubushakashatsi bwuzuye bwa ectodomain (Imbonerahamwe S1) byari bifite Z-amanota yemewe ya> 842, ubushakashatsi bwakozwe kuri 4HB cyangwa Ig-imeze nka Ig bwonyine bwerekanye ko ibyinshi muri ibyo bitaramo byari bihuye na sand-sandwiches gusa.ububiko bugaragara hose buboneka muri poroteyine nyinshi.Ishakisha uko ari itatu muri Dali ryagaruye igisubizo kimwe gusa: IZUMO1.
Kuva kera byavuzwe ko SPACA6 na IZUMO1 basangiye imiterere 7,32,37.Nubwo ectodomaine yizo poroteyine zombi zifitanye isano na gamete zisangiye 21% gusa zikurikirana (Igicapo S8a), ibimenyetso bigoye, harimo uburyo bwo kubika disulfide yabitswe hamwe na C-terminal Ig-imeze nka domeni muri SPACA6, yemeye kugerageza hakiri kare kubaka a icyitegererezo cya homology yimbeba ya SPACA6 ukoresheje IZUMO1 nkicyitegererezo37.Imiterere yacu yemeza ibyo bihanura kandi yerekana urwego nyarwo rusa.Mubyukuri, inzego za SPACA6 na IZUMO137,43,44 zisangiye imyubakire imwe-yububiko bubiri (Igicapo S8b) hamwe na 4HB hamwe na Ig-nka β-sandwich domaine ihujwe nakarere ka hinge (Ishusho S8c).
IZUMO1 na SPACA6 4HB bifite aho bihurira nibisanzwe bizunguruka.Ubusanzwe 4HBs, kimwe nizisangwa muri SNARE proteine ​​complexe zigira uruhare muri fonction endosomal 45,46, zagiye ziringaniza helices zigumya guhora zihindagurika hafi yumurongo wo hagati 47. Ibinyuranye, domaine ya helical muri IZUMO1 na SPACA6 yagoretse, hamwe na curvature ihinduka kandi gupakira kutaringaniye (Igicapo S8d).Impinduramatwara, birashoboka ko yatewe na mpandeshatu yakozwe na helices 1 ′, 1 na 2, igumana muri IZUMO1 na SPACA6 kandi igahagarikwa na motif imwe ya CXXC kuri helix 1 ′.Nyamara, inkwano yinyongera ya disulfide iboneka muri SPACA6 (Cys41 na Cys55 ihuza guhuza helices 1 na 2 hejuru) itera hejuru cyane kuri mpandeshatu ya mpandeshatu, bigatuma SPACA6 ihindagurika kurusha IZUMO1, hamwe na mpandeshatu za cavity.Mubyongeyeho, IZUMO1 ibura Arg37 igaragara hagati yu mwobo muri SPACA6.Ibinyuranye, IZUMO1 ifite hydrophobique yibanze yibisigisigi bya alifatique na aromatic.
IZUMO1 ifite Ig-imeze nka domeni igizwe ninshuro ebyiri-eshanu β -urupapuro43.Umugozi winyongera muri IZUMO1 usimbuza coil muri SPACA6, ihuza umurongo wa F kugirango ugabanye imigozi ya hydrogène yumugongo.Ingingo ishimishije yo kugereranya nuburyo buteganijwe hejuru yubuso bwa Ig-nka domaine za poroteyine zombi.Ubuso bwa IZUMO1 bwishyuzwa nabi kuruta ubuso bwa SPACA6.Amafaranga yinyongera aherereye hafi ya C-terminus ireba intanga ngabo.Muri SPACA6, uturere tumwe twarushijeho kutabogama cyangwa kwishyurwa neza (Ishusho S8e).Kurugero, hejuru ya hydrophobique (impande zoroheje) hamwe nibyobo byuzuye neza (impande nini) muri SPACA6 byishyuzwa nabi muri IZUMO1.
Nubwo umubano nibintu byubatswe hagati ya IZUMO1 na SPACA6 bibitswe neza, guhuza imiterere ya Ig-bisa na domaine byerekanaga ko domaine zombi zitandukanye mubyerekezo rusange ugereranije nizindi (Ishusho S9).Umuzenguruko uzunguruka wa IZUMO1 uhetamye hafi ya sand-sandwich, urema imiterere ya "boomerang" yavuzwe mbere kuri 50 ° uhereye kumurongo wo hagati.Ibinyuranyo, urumuri ruhebuje muri SPACA6 rwegamye nka 10 ° muburyo butandukanye.Itandukaniro muribi byerekezo rishobora guterwa no gutandukana mukarere ka hinge.Kurwego rwibanze, IZUMO1 na SPACA6 basangiye bike bikurikiranye kuri hinge, usibye sisitemu, glycine, nibisigisigi bya acide.Nkigisubizo, imiyoboro ya hydrogène hamwe numuyoboro wa electrostatike biratandukanye rwose.et -urupapuro rwimiterere yinyongera rusangiwe na IZUMO1 na SPACA6, nubwo iminyururu yo muri IZUMO1 ari ndende cyane kandi 310 helix (helix 5) yihariye SPACA6.Ibinyuranyo bivamo ibyerekezo bitandukanye kuri poroteyine ebyiri zisa.
Ishakisha rya Dali ryerekanye ko SPACA6 na IZUMO1 aribwo buryo bubiri bwonyine bwageragejwe bubitswe mububiko bwa poroteyine bufite ubu bubiko bwa 4HB (Imbonerahamwe S1).Vuba aha, DeepMind (Alphabet / Google) yateje imbere AlphaFold, sisitemu ishingiye ku mitsi ishobora guhanura neza imiterere ya 3D ya poroteyine uhereye ku cyiciro cya mbere48.Nyuma gato yo gukemura imiterere ya SPACA6, ububiko bwa AlphaFold bwasohotse, butanga imiterere yuburyo buteganijwe bukubiyemo 98.5% bya poroteyine zose ziri muri proteome zabantu 48,49.Twifashishije imiterere yacu ya SPACA6 nkicyitegererezo cyo gushakisha, gushakisha imiterere ya homologiya kuri moderi muri proteome ya AlphaFold yerekanaga abakandida bafite imiterere isa na SPACA6 na IZUMO1.Urebye ubunyangamugayo budasanzwe bwa AlphaFold mu guhanura SPACA6 (Igicap.
Mbere, PSI-BLAST yashakishije cluster ya IZUMO1 hamwe nizindi poroteyine eshatu zifitanye isano nintanga: IZUMO2, IZUMO3, na IZUMO450.AlphaFold yahanuye ko izo poroteyine z'umuryango wa IZUMO zinjira muri domaine ya 4HB hamwe na disulfide ihuza urugero nka IZUMO1 (Ishusho 3a na S11), nubwo zidafite domaine isa na Ig.Biravugwa ko IZUMO2 na IZUMO3 ari poroteyine imwe yo mu bwoko bwa membrane isa na IZUMO1, mu gihe IZUMO4 isa nkaho yasohotse.Imikorere ya IZUMO 2, 3, na 4 za poroteyine muguhuza gamete ntizamenyekanye.IZUMO3 izwiho kugira uruhare muri acrosome biogenezi mugihe cyo gukura kwintanga51 kandi proteine ​​ya IZUMO yasanze ikora complexe50.Kubungabunga poroteyine za IZUMO mu nyamaswa z’inyamabere, ibikururuka hasi, na amphibian byerekana ko imikorere yabo ishobora guhuza n’izindi poroteyine zizwi cyane zifitanye isano na gamete-fusion, nka DCST1 / 2, SOF1, na FIMP.
Igishushanyo mbonera cyububiko bwa IST birenze urugero, hamwe na 4HB, hinge, na Ig-bisa na domaine byerekanwe muri orange, icyatsi, nubururu.IZUMO4 ifite akarere kadasanzwe C-terminal gasa n'umukara.Impapuro zemejwe kandi zishyirwaho disulfide zerekanwa numurongo ukomeye kandi utudomo.b IZUMO1 (PDB: 5F4E), SPACA6, IZUMO2 (AlphaFold DB: AF-Q6UXV1-F1), IZUMO3 (AlphaFold DB: AF-Q5VZ72-F1), IZUMO4 (AlphaFold DB: AF-Q1ZYL8-F1), DB.TMEM95, IZUMO2 na IZUMO3 transembrane helices ntabwo bigaragara.
Bitandukanye na poroteyine ya IZUMO, izindi poroteyine za SPACA (ni ukuvuga SPACA1, SPACA3, SPACA4, SPACA5, na SPACA9) zitekereza ko zitandukanye mu buryo butandukanye na SPACA6 (Ishusho S12).Gusa SPACA9 ifite 4HB, ariko ntabwo byitezwe ko igira icyerekezo kimwe-cyo kurwanya-icyerekezo kimwe cyangwa isano ya disulfide imwe na SPACA6.Gusa SPACA1 ifite domeni isa na Ig.AlphaFold ihanura ko SPACA3, SPACA4 na SPACA5 bifite imiterere itandukanye rwose na SPACA6.Igishimishije, SPACA4 nayo izwiho kugira uruhare mu gusama, ariko ku rugero runini kuruta SPACA6, aho koroshya imikoranire hagati yintanga na oocyte zona pellucida52.
Ishakisha ryacu rya AlphaFold ryabonye undi mukino wa IZUMO1 na SPACA6 4HB, TMEM95.TMEM95, intungamubiri imwe yihariye ya poroteyine ya transembrane, ituma imbeba z'abagabo zitabyara iyo zivuyeho 32,33.Spermatozoa idafite TMEM95 yari ifite morphologie isanzwe, kugenda, hamwe nubushobozi bwo kwinjira muri zona pellucida no guhambira mu magi, ariko ntishobora guhuza na oocyte membrane.Ubushakashatsi bwibanze bwerekanye ko TMEM95 isangiye imiterere na IZUMO133.Mubyukuri, moderi ya AlphaFold yemeje ko TMEM95 ari 4HB ifite moteri imwe ya CXXC nka IZUMO1 na SPACA6 hamwe nubundi buryo bwa disulfide bwiyongera hagati ya helices 1 na 2 buboneka muri SPACA6 (Ishusho 3a na S11).Nubwo TMEM95 idafite indangarubuga isa na Ig, ifite akarere gafite imiterere ya disulfide isa na SPACA6 na IZUMO1 hinge (Ishusho 3b).Mugihe cyo gutangaza iyi nyandiko yandikishijwe intoki, seriveri yabanje kwerekana imiterere ya TMEM95, yemeza ibisubizo bya AlphaFold53.TMEM95 isa cyane na SPACA6 na IZUMO1 kandi ibitswe mubwihindurize isanzwe muri amphibian (Ishusho 4 na S13).
Ishakisha rya PSI-BLAST ryakoresheje NCBI SPACA6, IZUMO1-4, TMEM95, DCST1, DCST2, FIMP, na SOF1 ububiko bwamakuru kugirango hamenyekane umwanya wurwo rutonde mubiti byubuzima.Intera iri hagati yingingo zishami ntizerekanwa kurwego.
Ibintu bigaragara muri rusange bihuza imiterere hagati ya SPACA6 na IZUMO1 byerekana ko bashinze abanyamuryango babungabunzwe muburyo budasanzwe burimo TMEM95 na IZUMO 2, 3, na 4 za poroteyine.abanyamuryango bazwi: IZUMO1, SPACA6 na TMEM95.Kuberako abanyamuryango bake gusa bafite domaine isa na Ig, ikiranga IST superfamily ni domaine ya 4HB, ifite ibintu byihariye bihuriweho na poroteyine zose: 1) Bishyizwe hamwe 4HB hamwe na helices zitunganijwe muburyo butandukanye bwo kurwanya (parallel) . 5a), 2) bundle ifite isura ya mpandeshatu igizwe na helices ebyiri muri bundle hamwe na veritike ya gatatu ihagaritse (Igishushanyo cy'ingenzi (Ishusho 5c) nka sensor ya redox 54,55,56, mugihe motif yo mumuryango wa IST irashobora guhuzwa na protein disulfide isomerases nka ERp57 muguhuza gamete.Uruhare rufitanye isano 57.58.
Abagize IST birenze urugero basobanurwa nibintu bitatu biranga indangarubuga ya 4HB: ibice bine bisimburana hagati yicyerekezo kibangikanye na antiparallel icyerekezo, ba-triangular helical bundle mumaso, hamwe na ca CXXC motif ikorwa hagati ya molekile nto.) N-terminal helixes (orange) hamwe na hinge region β-umusatsi (icyatsi).
Urebye isano iri hagati ya SPACA6 na IZUMO1, harageragejwe ubushobozi bwambere bwo guhuza IZUMO1 cyangwa JUNO.Biolayer interferometrie (BLI) nuburyo bwa kinetic bushingiye kuburyo bukoreshwa mbere mukugereranya imikoranire hagati ya IZUMO1 na JUNO.Nyuma yubushakashatsi bwa biotin yanditseho sensor hamwe na IZUMO1 nkigisambo gifite intungamubiri nyinshi za analyte ya JUNO, habonetse ikimenyetso gikomeye (Igicapo S14a), cyerekana impinduka zatewe no guhuza umubyimba wa biomaterial wometse kumutwe wa sensor.Ibimenyetso bisa (ni ukuvuga, JUNO ihujwe na sensor nk'icyambo cyo kurwanya analyte ya IZUMO1) (Ishusho S14b).Nta kimenyetso cyagaragaye igihe SPACA6 yakoreshwaga nka analyse irwanya sensor ya IZUMO1 cyangwa JUNO ihuza sensor (Ishusho S14a, b).Kubura kw'iki kimenyetso byerekana ko indangagihe idasanzwe ya SPACA6 idakorana na domaine idasanzwe ya IZUMO1 cyangwa JUNO.
Kuberako isuzuma rya BLI rishingiye kuri biotinylation yibisigazwa bya lysine yubusa kuri proteine ​​ya bait, iri hinduka rirashobora gukumira guhuza niba ibisigazwa bya lysine bigira uruhare mubikorwa.Byongeye kandi, icyerekezo cyo guhuza ugereranije na sensor kirashobora gutera imbogamizi zidasanzwe, bityo rero ibisanzwe byo gukuramo hasi byakozwe no kuri recombinant SPACA6, IZUMO1 na JUNO ectodomain.Nubwo bimeze gurtyo, SPACA6 ntiyaguye hamwe na IZUMO1 cyangwa Yashizweho na JUNO (Igicapo S14c, d), byerekana ko nta mikoranire ijyanye niyagaragaye mubushakashatsi bwa BLI.Nkigenzura ryiza, twemeje imikoranire ya JUNO yanditseho IZUMO1 (Ishusho S14e na S15).
Nubwo imiterere ihuza SPACA6 na IZUMO1, kuba SPACA6 idashobora guhuza JUNO ntabwo bitangaje.Ubuso bwabantu IZUMO1 bufite ibisigazwa birenga 20 bikorana na JUNO, harimo ibisigisigi biva muri buri turere dutatu (nubwo ibyinshi muri byo biherereye mukarere ka hinge) (Ishusho S14f).Muri ibyo bisigazwa, kimwe gusa kibitswe muri SPACA6 (Glu70).Mugihe ibyinshi byasimbuwe byagumanye imiterere yumwimerere ya biohimiki, ibisigisigi byingenzi bya Arg160 muri IZUMO1 byasimbuwe na Asp148 yishyuwe nabi muri SPACA6;ubushakashatsi bwibanze bwerekanye ko ihinduka rya Arg160Glu muri IZUMO1 hafi gukuraho burundu guhuza JUNO43.Mubyongeyeho, itandukaniro mubyerekezo byerekeranye na domeni hagati ya IZUMO1 na SPACA6 byongereye cyane ubuso bwubuso bwa JUNO-buhuza akarere kangana kuri SPACA6 (Ishusho S14g).
Nubwo bizwi ko hakenewe SPACA6 yo guhuza gamete no guhuza na IZUMO1, SPACA6 ntabwo bigaragara ko ifite imikorere ihwanye na JUNO.Kubwibyo, twashatse guhuza amakuru yimiterere yacu nibimenyetso byingirakamaro bitangwa na biyolojiya.Guhuza urutonde rwa homologues ya SPACA6 byerekana kubungabunga imiterere rusange irenze inyamaswa z’inyamabere.Kurugero, ibisigazwa bya sisitemu birahari no muri amphibian bifitanye isano ya kure (Ishusho 6a).Ukoresheje seriveri ya ConSurf, amakuru menshi yo kugumya guhuza amakuru yo kubika 66 yakurikiranwe ku buso bwa SPACA6.Ubu bwoko bwisesengura burashobora kwerekana ibisigisigi byabitswe mugihe cyubwihindurize bwa poroteyine kandi birashobora kwerekana uturere two hejuru tugira uruhare mumikorere.
guhuza Urutonde rwa ectodomaine ya SPACA6 kuva mubwoko 12 butandukanye bwateguwe ukoresheje CLUSTAL OMEGA.Ukurikije isesengura rya ConSurf, imyanya myinshi yibumbira hamwe irangwa mubururu.Ibisigisigi bya sisitemu byerekanwe mumutuku.Imipaka ya domeni hamwe nuburyo bwa kabiri ibintu byerekanwe hejuru hejuru yo guhuza, aho imyambi yerekana β -imirongo n'imiraba byerekana helices.Indangamuntu ya NCBI ikubiyemo urutonde ni: abantu (Homo sapiens, NP_001303901), mandrill (Mandrilus leucophaeus, XP_011821277), inkende ya capuchin (Cebus mimic, XP_017359366), ifarashi (Equus caballus, XP_02350610 34).), intama (Ovis aries, XP_014955560), inzovu (Loxodonta africana, XP_010585293), imbwa (Canis lupus familyis, XP_025277208), imbeba (Mus musculus, NP_001156381), shitani Tasmanian (Sarcophilus X_03 , 61_89 na Bullfrog (Bufo bufo, XP_040282113).Umubare ushingiye kuri gahunda yabantu.b Ubuso bwerekana imiterere ya SPACA6 hamwe na 4HB hejuru na Ig-imeze nka domaine hepfo, amabara ashingiye kubigereranyo byo kubungabunga uhereye kuri seriveri ya ConSurf.Ibice byabitswe neza biri mubururu, ibice bibitswe neza biri mweru, naho bike bibitswe biri mumuhondo.cysteine.Ibice bitatu byubuso byerekana urwego rwo hejuru rwuburinzi byerekanwe muri inset yanditseho ibipapuro 1, 2 na 3. Ikarito ya 4HB irerekanwa muri inset hejuru iburyo (ibara rimwe).
Imiterere ya SPACA6 ifite uturere dutatu twabitswe cyane (Ishusho 6b).Igice cya 1 kigizwe na 4HB n'akarere ka hinge kandi kirimo ibiraro bibiri byabitswe bya CXXC disulfide, Arg233-Glu132-Arg135-Ser144 umuyoboro wa hinge (Igicapo S7), hamwe n'ibisigarira bitatu byabitswe neza (Phe31, Tyr73, Phe137).umugari mugari wa Ig-imeze (Igishusho S6e), igereranya ibisigisigi byinshi byashizwe hejuru hejuru yintanga.Igishimishije, iyi patch irimo antibody epitope yerekanwe mbere kubangamira imikorere ya SPACA6 30.Intara 3 izenguruka hinge nuruhande rumwe rwa Ig-imeze;kano karere karimo protine zabitswe (Pro126, Pro127, Pro150, Pro154) hamwe n’ibisigazwa bya polar / byashizwe hanze.Igitangaje, ibyinshi mubisigara hejuru yubuso bwa 4HB birahinduka cyane (Igishusho 6b), nubwo ububiko bwabitswe muri homologue ya SPACA6 (nkuko bigaragazwa na conservatism ya hydrophobic bundle core) ndetse no hejuru ya IST birenze urugero.
Nubwo aka ari agace gato muri SPACA6 hamwe nibintu bike byamenyekanye byubatswe, ibisigisigi byinshi byo mukarere ka hinge (harimo n'akarere ka 3) bibitswe cyane mubantu bahuje ibitsina ba SPACA6, bishobora kwerekana ko icyerekezo cya bundle na β-sandwich gifite uruhare.nk'umuntu uharanira inyungu.Nubwo, nubwo hydrogène ihuza byinshi hamwe numuyoboro wa electrostatike mukarere ka hinge ka SPACA6 na IZUMO1, ibimenyetso byerekana imiterere ihinduka irashobora kugaragara muguhuza inzego nyinshi zemewe za IZUMO137,43,44.Guhuza domaine kugiti cye byuzuzanya neza, ariko icyerekezo cya domaine ugereranije nizindi zitandukanye kuva kuri 50 ° kugeza 70 ° uhereye kumurongo wo hagati (Ishusho S16).Kugira ngo dusobanukirwe ningaruka zijyanye na SPACA6 mugisubizo, hakozwe ubushakashatsi bwa SAXS (Ishusho S17a, b).Ab initio kwiyubaka kwa ectodomain ya SPACA6 yahujwe nuburyo bwo korohereza inkoni (Igicap. S18), nubwo umugambi wa Kratky werekanye urwego runaka rwo guhinduka (Ishusho S17b).Ihinduka ritandukanye na IZUMO1, aho poroteyine idahuza ifata imiterere ya bomerang haba muri kasike no mubisubizo43.
Kugirango umenye neza akarere gahinduka, hydrogen-deuterium yo guhanahana ibintu rusange (H-DXMS) yakorewe kuri SPACA6 kandi ugereranije namakuru yabonetse kuri IZUMO143 (Ishusho 7a, b).SPACA6 biragaragara ko ihindagurika kurusha IZUMO1, nkuko bigaragazwa no guhana kwa deuterium murwego rwose nyuma ya 100.000 s yo kuvunja.Muri ubwo buryo bwombi, C-terminal igice cyakarere ka hinge yerekana urwego rwo hejuru rwo guhanahana amakuru, birashoboka ko yemerera kuzenguruka kugarukira kwa 4HB na Ig-nka domeni ugereranije nundi.Igishimishije, igice cya C-igice cya SPACA6 hinge, kigizwe n’ibisigisigi 147CDLPLDCP154, ni akarere gakunzwe cyane 3 (Igishusho 6b), birashoboka ko byerekana ko guhuza imiterere hagati y’imiterere ari ikintu cyabitswe mu bwihindurize bwa SPACA6.Ukurikije isesengura ryoroshye, amakuru ya CD yumuriro wa CD yerekanaga ko SPACA6 (Tm = 51.2 ° C) idahagaze neza kurusha IZUMO1 (Tm = 62.9 ° C) (Ishusho S1e na S19).
amashusho ya H-DXMS ya SPACA6 na b IZUMO1.Ijanisha rya deuterium ryagenwe kumwanya wagenwe.Urwego rwo guhana hydrogène-deuterium rwerekanwa namabara kurwego rwa gradient kuva mubururu (10%) kugeza umutuku (90%).Agasanduku k'umukara kagaragaza uduce two guhanahana amakuru.Imipaka ya 4HB, hinge na Ig-isa na domaine igaragara muburyo bwa kristu irerekanwa hejuru yurwego rwibanze.Urwego rwo guhana Deuterium kuri 10 s, 1000 s, na 100.000 s byateguwe ku mbonerahamwe yerekana umurongo hejuru ya molekile igaragara ya SPACA6 na IZUMO1.Ibice byimiterere hamwe na deuterium urwego rwo munsi ya 50% bifite ibara ryera.Uturere turi hejuru ya 50% H-DXMS guhanahana amabara murwego rwo hejuru.
Gukoresha CRISPR / Cas9 hamwe nimbeba gene knockout ingamba za genetike byatumye hamenyekana ibintu byinshi byingenzi byintanga ngabo hamwe no guhuza amagi no guhuza.Usibye imikoranire irangwa neza ya IZUMO1-JUNO na CD9, proteine ​​nyinshi zijyanye no guhuza gamete ziguma zubatswe kandi zikora neza.Ibinyabuzima biranga ibinyabuzima na SPACA6 ni ikindi gice cya puzzle ya adhesion / fusion mugihe cyo gusama.
SPACA6 hamwe nabandi banyamuryango ba IST birenze urugero bigaragara ko bibitswe cyane mubinyamabere kimwe ninyoni, ibikururuka hasi, na amphibian;mubyukuri, biratekerezwa ko SPACA6 isabwa no gusama muri zebrafish 59. Iri gabana risa nizindi poroteyine zizwi zifitanye isano na gamete fusion nka DCST134, DCST234, FIMP31, na SOF132, byerekana ko ibyo bintu ari bike HAP2 (nayo izwi nka GCS1) proteyine zishinzwe ibikorwa bya catalitiki yabigaragambyaga benshi., ibimera, na arthropods.Poroteyine zifumbire mvaruganda 60, 61. Nuburyo bukomeye bwubatswe hagati ya SPACA6 na IZUMO1, knockout ya genes ikubiyemo imwe muri izo poroteyine byaviriyemo kutabyara imbeba zabagabo, byerekana ko imikorere yabo muguhuza imikino idakopororwa..Muri rusange, ntanimwe muri poroteyine zizwi zikenewe kugirango icyiciro cya fonction fusion ntikirenze.
Biracyari ikibazo gifunguye niba SPACA6 (nabandi banyamuryango ba IST birenze urugero) bitabira guhuza imiyoboro, gushiraho imiyoboro ihuza abantu kugirango bashakishe poroteyine zingenzi aho zihurira, cyangwa wenda zikora nka fusogène zoroshye.Ubushakashatsi bwakozwe na immunoprecipitation muri selile HEK293T bwerekanye imikoranire hagati yuburebure bwuzuye IZUMO1 na SPACA632.Ariko, ectodomain yacu ya recombinant ntabwo yigeze ikora muri vitro, byerekana ko imikoranire igaragara muri Noda nabandi.byombi byasibwe mubwubatsi (andika umurizo wa cytoplasmeque wa IZUMO1, byagaragaye ko bidakenewe mu gusama 62).Ubundi, IZUMO1 na / cyangwa SPACA6 birashobora gusaba ibidukikije bihuza bidasanzwe tutabyara muri vitro, nkibintu byihariye bihuza umubiri cyangwa ibice bya molekile birimo izindi poroteyine (bizwi cyangwa bitaravumburwa).Nubwo ectodomain ya IZUMO1 yizera ko ihuza guhuza intanga ngabo ku magi mu mwanya wa perivitelline, intego ya ectodomain ya SPACA6 ntisobanutse.
Imiterere ya SPACA6 igaragaza ubuso butandukanye bwabitswe bushobora kugira uruhare mubikorwa bya poroteyine-proteyine.Igice cyabitswe mukarere ka hinge gihita kegeranye na motif ya CXXC (yagenwe Patch 1 hejuru) ifite ibisigazwa byinshi byimbere byimbere byamavuta bikunze kuba bifitanye isano na hydrophobique na π -kubika hagati ya biomolecules.Impande nini za domaine isa na Ig (akarere 2) ikora igikonjo cyuzuye neza hamwe na Arg hamwe nibisigisigi bye byabitswe cyane, kandi antibodiyite zirwanya kariya karere zagiye zikoreshwa muguhagarika umukino wa 30.Antibody imenya epitope y'umurongo 212RIRPAQLTHRGTFS225, ifite bitatu muri bitandatu bisigaye bya arginine kandi ibitse cyane220.Ntabwo byumvikana niba imikorere mibi iterwa no guhagarika ibyo bisigazwa byihariye cyangwa akarere kose.Ahantu h'iki cyuho hafi ya C-terminus ya sand-sandwich yerekana cis-imikoranire na poroteyine zituranye, ariko ntabwo ari proteine ​​za oocyte.Ikigeretse kuri ibyo, kugumana ibintu byoroshye cyane bya porine ikungahaye cyane (urubuga 3) muri hinge birashobora kuba urubuga rwimikoranire ya poroteyine-proteyine cyangwa, cyane cyane, byerekana kugumana guhinduka hagati yinzego zombi.Uburinganire ni ngombwa ku ruhare rutazwi rwa SPACA6.guhuza imikino.
SPACA6 ifite imiterere ya proteine ​​intercellular adhesion, harimo Ig-nka β-sandwiches.Poroteyine nyinshi zifata (urugero, cadherine, integin, adhesine, na IZUMO1) zifite domeni imwe cyangwa nyinshi β-sandwich yongerera poroteyine kuva mu ngirabuzimafatizo kugera ku ntego z’ibidukikije 63,64,65.Ig-imeze nka domeni ya SPACA6 nayo irimo motif ikunze kuboneka muri β-sandwiches yo gufatana hamwe no guhuriza hamwe: inshuro ebyiri zumugozi ugereranije kumpera ya sand-sandwiches, izwi nka clamps ya mashini66.Byizerwa ko iyi motif yongerera imbaraga imbaraga zo gukata, zifite agaciro kuri poroteyine zigira uruhare mu mikoranire.Nubwo, nubwo ibyo bisa na adhesine, kuri ubu nta kimenyetso cyerekana ko SPACA6 ikorana n’abazungu b'igi.Ectodomain ya SPACA6 ntishobora guhuza na JUNO, hamwe na selile ya HEK293T ya SPACA6, nkuko bigaragara hano, ntibishobora gukorana na oocytes idafite zona 32.Niba SPACA6 ishyiraho isano ihuza imikoranire, iyi mikoranire irashobora gusaba nyuma yo guhindurwa cyangwa guhindurwa nizindi poroteyine.Mu rwego rwo gushyigikira hypothesis ya nyuma, spermatozoya ibuze IZUMO1 ihuza na oocytes, byerekana ko molekile zitari IZUMO1 zigira uruhare mu gukina umukino wa 27.
Intungamubiri nyinshi za virusi, selile, niterambere rya fusion proteine ​​zifite ibintu byerekana imikorere yazo nka fusogène.Kurugero, virusi ya virusi glycoproteine ​​(icyiciro cya I, II na III) ifite hydrophobique fusion peptide cyangwa loop kumpera ya poroteyine yinjizwa muri membrane yakira.Ikarita ya hydrophilicity ya IZUMO143 nuburyo (byagenwe kandi byahanuwe) bya IST birenze urugero byerekanaga nta hydrophobique fusion peptide igaragara.Rero, niba poroteyine zose muri IST zidasanzwe zikora nka fusogène, babikora muburyo butandukanye nizindi ngero zizwi.
Mu gusoza, imikorere yabanyamuryango ba IST birenze urugero bya poroteyine zijyanye no guhuza gamete bikomeje kuba amayobera.Ibiranga SPACA6 recombinant molekile hamwe nuburyo byakemuwe bizatanga ubushishozi kumubano uri hagati yizi nzego zisangiwe nuruhare rwazo muguhuza imikino no guhuza.
Urutonde rwa ADN ruhuye na ectodomain ya SPACA6 yahanuwe (numero yinjira ya NCBI nimero NP_001303901.1; ibisigara 27-2246) yashizwemo codon kugirango igaragare mu tugari twa Drosophila melanogaster S2 kandi ihindurwamo agace ka gene hamwe na kodegisi ya Kozak (Eurofins Genomics)., ikimenyetso cyo gusohora kwa BiP hamwe na 5 ′ na 3 ′ birangira kugirango clone-yigenga ikoronijwe niyi gene mumagambo ya pMT ashingiye kuri porotokoro ya metallothionein yahinduwe kugirango ahitemo puromycine (pMT-puro).Imashini ya pMT-puro ikubiyemo urubuga rwa trombin rukurikirwa na 10x-Ikirangantego cye C (Terminal S2).
Ihererekanyabubasha rya virusi ya SPACA6 pMT-puro muri selile D. melanogaster S2 (Gibco) byakozwe kimwe na protocole yakoreshejwe kuri IZUMO1 na JUNO43.Ingirabuzimafatizo za S2 zashwanyagujwe kandi zikura mu buryo bwa Schneider (Gibco) hiyongeraho 10% (v / v) inyana y’inyana y’inda y’inyana (Gibco) na antibiyotike ya 1X (Gibco).Utugingo ngengabuzima twa mbere (3.0 x 106 selile) washyizwe mu mariba ku giti cye cy'amasahani 6 (Corning).Nyuma yamasaha 24 yubushakashatsi kuri 27 ° C, selile zandujwe nuruvange rwa mg 2 za vector ya SPACA6 pMT-puro na reagent ya Effectene (Qiagen) nkuko protocole yabayikoze.Ingirabuzimafatizo zahinduwe zashyizwemo amasaha 72 hanyuma zisarurwa na mg / ml 6 puromycine.Ingirabuzimafatizo noneho zitandukanijwe nuburyo bwuzuye bwa Schneider hanyuma zishyirwa muri serumu yubusa Udukoko-XPRESS (Lonza) kugirango habeho umusaruro munini wa poroteyine.Icyiciro cya 1 L cyumuco w'akagari S2 cyakuze kigera kuri 8-10 × 106 ml-1 muri selile ya 2 L ihumeka neza ya polypropilene Erlenmeyer hanyuma igahagarikwa hamwe na 500 µM CuSO4 (Millipore Sigma) hanyuma ikayungurura sterile.gushukwa.Imico yatewe yashizwemo kuri 27 ° C. saa 120 rpm muminsi ine.
Ikigereranyo gikubiyemo SPACA6 cyatandukanijwe na centrifugation kuri 5660 × g kuri 4 ° C ikurikirwa na sisitemu ya Centramate tangential flow filtration (Pall Corp) hamwe na 10 kDa MWCO.Koresha uburyo bwibanze burimo SPACA6 kuri ml 2 Ni-NTA agarose resin (Qiagen).Isuku ya Ni-NTA yogejwe hamwe nubunini 10 bwinkingi (CV) ya buffer A hanyuma hiyongeraho 1 CV ya buffer A kugirango itange imidazole yanyuma ya mM 50.SPACA6 yashizwemo na ml 10 ya buffer A yongerewe na imidazole kugeza kuri mM 500.Icyiciro cyo kubuza trombine (Millipore Sigma) yongewemo muburyo butaziguye kuri dialyse tubing (MWCO 12-14 kDa) kuri 1 kuri mg SPACA6 na 1 L 10 mM Tris-HCl, pH 7.5 na 150 mM NaCl (buffer B) kuri dialyse.) kuri 4 ° C mu masaha 48.SPACA6 yashizwemo na trombine noneho yongerewe inshuro eshatu kugirango igabanye umunyu hanyuma ishyirwa kuri ml 1 MonoS 5/50 GL yo guhanahana cation (Cytiva / GE) ihwanye na mM 10 Tris-HCl, pH 7.5.Guhindura cation byogejwe hamwe na CV 3 ya 10 mM Tris-HCl, pH 7.5, hanyuma SPACA6 isubizwa hamwe na gradient ya metero 0 kugeza 500 mM NaCl muri 10 mM Tris-HCl, pH 7.5 kuri 25 CV.Nyuma yo guhanahana chromatografiya, SPACA6 yibanze kuri ml 1 hanyuma ivanwa muburyo bwa demokarasi bivuye muri ENrich SEC650 10 x 300 inkingi (BioRad) ihwanye na buffer B. Dukurikije chromatogramu, pisine hamwe nuduce twinshi turimo SPACA6.Isuku yagenzurwaga na Coomassie yandujwe na electrophoreis kuri geli ya 16% SDS-polyacrylamide.Intungamubiri za poroteyine zapimwe no kwinjizwa kuri 280 nm ukoresheje amategeko ya Beer-Lambert hamwe na coefficente de l'oritifike.
SPACA6 yasukuwe yaraye ikoresheje ijoro ryose irwanya mM 10 ya sodium fosifate, pH 7.4 na mM 150 NaF hanyuma ikavangwa kuri 0.16 mg / mL mbere yo gusesengura na CD spectroscopy.Gusikana kuri CD bifite uburebure bwa 185 kugeza 260 nm byakusanyirijwe kuri Jasco J-1500 spectropolarimeter ikoresheje quartz cuvettes ifite uburebure bwa mm 1 ya optique (Helma) kuri 25 ° C ku gipimo cya 50 nm / min.CD yerekanwe yakosowe, ikigereranyo kirenga 10 yaguzwe, hanyuma ihindurwa bisobanura elliptike isigaye (θMRE) muri dogere cm2 / dmol:
aho MW nuburemere bwa molekuline ya buri sample muri Da;N ni umubare wa aside amine;θ ni elliptike muri milidegree;d ihuye n'uburebure bw'inzira nziza muri cm;intungamubiri za poroteyine mu bice.