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317 Mea hoʻolako mai i nā mea hoʻolako i nā paipu wili wili hao

Pākaukau hoʻohui kemika o ke kila kuhiliʻole

ʻO ka SPACA6 kahi protein i hōʻike ʻia i ka sperm i mea koʻikoʻi no ka hui ʻana o ka gamete i ka wā o ka hoʻohua ʻana i ka moekolohe.ʻOiai ʻo kēia kuleana koʻikoʻi, ʻaʻole maopopo ka hana kikoʻī o SPACA6.Hōʻike mākou i ke ʻano aniani o ka domain extracellular o SPACA6 ma kahi hoʻonā o 2.2 Å, e hōʻike ana i kahi protein ʻelua-domain i haku ʻia me kahi pūʻulu ʻehā a me Ig-like β-sandwiches i hui pū ʻia e nā mea hoʻopili quasi-flexible.Ua like kēia ʻano me IZUMO1, kekahi pūmua pili i ka gamete fusion, e hoʻokumu ana i ka SPACA6 a me IZUMO1 i mau lālā o ka ʻohana nui o nā protein pili i ka fertilization i kapa ʻia ʻo ka IST superfamily.Ua wehewehe ʻia ka IST superfamily e kāna pūʻolo ʻehā-helix wili a me nā kumu kumu CXXC i hoʻopili ʻia disulfide.Ua ʻike ʻia kahi hulina AlphaFold i hoʻokumu ʻia o ka proteome kanaka i nā lālā protein hou o kēia ʻohana super;ʻO ka mea nui, ʻo ka hapa nui o kēia mau protein i komo i ka gamete fusion.ʻO ka ʻōnaehana SPACA6 a me kona pilina me nā lālā ʻē aʻe o ka superfamily IST e hāʻawi i ka loulou nalo i kā mākou ʻike o ka mammalian gamete fusion.
Hoʻomaka kēlā me kēia ola kanaka me ʻelua haploid gametes kaʻawale: ka sperm o ka makua kāne a me ka hua o ka makuahine.ʻO kēia sperm ka lanakila o ke kaʻina koho ikaika i ka wā e hele ai nā miliona o nā sperm cell i loko o ka ʻāpana wahine, lanakila i nā pilikia like ʻole1 a hoʻokō i ka capacitation, kahi e hoʻonui ai i ko lākou motility a me ke kaʻina hana o nā mea ʻili2,3,4.ʻOiai inā loaʻa ka sperm a me ka oocyte kekahi i kekahi, ʻaʻole i pau ke kaʻina hana.Hoʻopuni ʻia ka oocyte e kahi papa o nā cell cumulus a me kahi pale glycoprotein i kapa ʻia ʻo zona pellucida, kahi e hele ai ka sperm e komo i ka oocyte.Hoʻohana ʻo Spermatozoa i ka hui pū ʻana o nā molekole pili i ka ʻili a me nā enzyme pili me ka membrane a huna ʻia e lanakila ai i kēia mau pale hope5.Mālama ʻia kēia mau molekele a me nā enzyme i loko o ka membrane i loko a me ka matrix acrosomal a ʻike ʻia ke hoʻoheheʻe ʻia ka membrane waho o ka sperm i ka wā o ka hopena acrosomal6.ʻO ka hana hope loa i kēia huakaʻi ikaika, ʻo ia ka hanana hui ʻana o ka hua manu-sperm, kahi e hoʻohui ai nā ʻelua i ko lākou mau membrane e lilo i mea ola diploid hoʻokahi.ʻOiai ʻo kēia kaʻina hana i hoʻokumu ʻia i ka hana hou ʻana o ke kanaka, ʻaʻole maopopo ka pono o ka pilina molekala.
Ma waho aʻe o ka fertilization o gametes, ua aʻo nui ʻia ka kemika o ka hui ʻana o ʻelua lipid bilayers.Ma ka laulā, ʻo ka hui ʻana o ka membrane he kaʻina hana maikaʻi ʻole e koi ai i kahi catalyst protein e hana i kahi hoʻololi conformation structural e hoʻopili kokoke ai i ʻelua mau membrane, e uhaʻi i ko lākou hoʻomau ʻana a hoʻoulu i ka fusion8,9.Ua ʻike ʻia kēia mau mea hoʻoheheʻe protein he fusogens a ua loaʻa i nā ʻōnaehana fusion lehulehu.Pono lākou no ke komo ʻana o ka maʻi i loko o nā cell host (eg, gp160 i ka HIV-1, spike i nā coronaviruses, hemagglutinin i nā maʻi maʻi influenza) 10,11,12 placental (syncytin)13,14,15 a me nā fusions hana gamete i nā eukaryotes haʻahaʻa ( HAP2/GCS1 i na mea kanu, protist a me arthropods) 16,17,18,19.ʻAʻole i ʻike ʻia nā Fusogens no nā gametes kanaka, ʻoiai ua hōʻike ʻia kekahi mau protein i mea koʻikoʻi no ka hoʻopili ʻana a me ka hui ʻana.ʻO ka CD9 i hōʻike ʻia i ka oocyte, he protein transmembrane i koi ʻia no ka hui ʻana o ka ʻiole a me nā gametes kanaka, ʻo ia ka mea mua i ʻike ʻia 21,22,23.ʻOiai ʻaʻole maopopo kāna hana pololei, he kuleana i ka adhesion, ke ʻano o ka adhesion foci ma ka microvilli hua manu, a / a i ʻole ka localization kūpono o nā protein oocyte surface me he 24,25,26.ʻO nā protein maʻamau ʻelua i koʻikoʻi no ka fusion gamete ʻo ia ka sperm protein IZUMO127 a me ka oocyte protein JUNO28, a ʻo kā lākou hui ʻana he hana koʻikoʻi i ka ʻike gamete a me ka hoʻopili ʻana ma mua o ka hui ʻana.Kane Izumo1 knockout iole a me ka wahine Juno knockout iole ua piha sterile, i loko o kēia mau kŘkohu sperm komo i ka perivitelline ākea akā, gametes ole fuse.Pēlā nō, hoʻemi ʻia ka hui ʻana i ka wā i mālama ʻia ai nā gametes me nā anti-IZUMO1 a i ʻole JUNO27,29 antibodies i nā hoʻokolohua fertilization in vitro kanaka.
I kēia mau lā, ua ʻike ʻia kahi pūʻulu hou o ka sperm-expressed proteins phenotypically like me IZUMO1 a me JUNO20,30,31,32,33,34,35.Ua ʻike ʻia ʻo Sperm acrosomal membrane-associated protein 6 (SPACA6) he mea pono no ka hoʻoulu ʻana i kahi noiʻi mutagenesis murine nui.ʻO ka hoʻokomo ʻana o ka transgene i loko o ka gene Spaca6 e hoʻopuka i ka spermatozoa nonfusible, ʻoiai ua komo kēia mau spermatozoa i ka perivitelline space 36.Ua hōʻoia ʻia nā haʻawina knockout ma hope mai e koi ʻia ʻo Spaca6 no ka gamete fusion 30,32.Hōʻike ʻia ʻo SPACA6 ma kahi o nā testes a loaʻa kahi ʻano localization e like me ka IZUMO1, ʻo ia hoʻi i loko o ka intima o ka spermatozoa ma mua o ka hopena acrosomal, a laila neʻe i ka ʻāpana equatorial ma hope o ka hopena acrosomal 30,32.Aia nā homologues Spaca6 i loko o nā ʻano mammals a me nā eukaryotes ʻē aʻe 30 a ua hōʻike ʻia kona koʻikoʻi no ka hui ʻana o ke kanaka ma ke kāohi ʻana i ka fertilization kanaka in vitro ma ke kūʻē ʻana iā SPACA6 30.ʻAʻole like me IZUMO1 a me JUNO, ʻaʻole maopopo nā kikoʻī o ke ʻano, ka pilina, a me ka hana o SPACA6.
No ka hoʻomaopopo maikaʻi ʻana i ke kaʻina hana ma lalo o ka hui ʻana o ka sperm a me nā hua manu, e hiki ai iā mākou ke hoʻomaopopo i nā hanana e hiki mai ana i ka hoʻolālā ʻohana a me ka mālama ʻana i ka momona, ua alakaʻi mākou i nā haʻawina hoʻolālā a me nā biochemical SPACA6.Hōʻike ka ʻano aniani o ka domain extracellular o SPACA6 i kahi pūpū helika ʻehā (4HB) a me kahi domain immunoglobulin-like (Ig-like) i hoʻopili ʻia e nā ʻāpana quasi-flexible.E like me ka wānana i nā haʻawina mua, ʻo 7,32,37 ka hoʻolālā domain o SPACA6 e like me ke kanaka IZUMO1, a ʻo nā protein ʻelua e hoʻokaʻawale i kahi kumu maʻamau: 4HB me kahi triangular helical surface a me kahi pālua o disulfide-linked CXXC motifs.Manaʻo mākou e wehewehe ʻo IZUMO1 a me SPACA6 i kahi superfamily e pili ana i ke ʻano o nā protein e pili ana me ka gamete fusion.Me ka hoʻohana ʻana i nā hiʻohiʻona kūʻokoʻa i ka superfamily, ua hana mākou i kahi hulina piha no ka AlphaFold structural human proteome, e ʻike ana i nā lālā hou o kēia superfamily, me kekahi mau lālā i komo i ka gamete fusion a / a i ʻole fertilization.ʻIke ʻia i kēia manawa aia kahi papa hana maʻamau a me ka ʻohana nui o nā protein e pili ana me ka gamete fusion, a hāʻawi kā mākou hale i kahi palapala molekala o kēia ʻano koʻikoʻi o ka mīkini hui gamete kanaka.
ʻO SPACA6 kahi protein transmembrane hoʻokahi-pass me hoʻokahi N-linked glycan a me ʻeono putative disulfide bond (Figures S1a a me S2).Ua hōʻike mākou i ke kikowaena extracellular o SPACA6 kanaka (koe 27-246) i loko o nā pūnaewele Drosophila S2 a hoʻomaʻemaʻe i ka protein me ka hoʻohana ʻana i ka nickel affinity, cation exchange, a me ka nui exclusion chromatography (Fig. S1b).ʻO ka SPACA6 ectodomain i hoʻomaʻemaʻe ʻia he kūpaʻa a homogeneous.Hōʻike ʻia ka hoʻohana ʻana i ka chromatography exclusion size i hui pū ʻia me ka polygonal light scattering (SEC-MALS) i hoʻokahi piko me ke kaumaha molekala helu o 26.2 ± 0.5 kDa (Fig. S1c).Kūlike kēia me ka nui o ka SPACA6 monomeric ectodomain, e hōʻike ana ʻaʻole i loaʻa ka oligomerization i ka wā hoʻomaʻemaʻe.Eia hou, ua hōʻike ka circular dichroism (CD) spectroscopy i kahi hui α/β me kahi helu heheʻe o 51.3 °C (Fig. S1d,e).Ua hōʻike ʻia ka deconvolution o ka spectra CD he 38.6% α-helical a me 15.8% β-stranded element (Figure S1d).
ʻO ka SPACA6 ectodomain i crystallized me ka hoʻohana 'ana i ka matrix seeding38 ma muli o kahi hoʻonohonohoʻikepili me ka hoʻonā o 2.2 Å (Table 1 and Figure S3).Ke hoʻohana nei i ka hui pū ʻana o ka molecular substitution-based a me SAD phasing data me ka bromide exposure no ka hoʻoholo ʻana i ka hoʻolālā (Papa 1 a me ke Kiʻi S4), ʻo ke kumu hoʻohālike hope loa he mau koena 27-246.I ka manawa i hoʻoholo ʻia ai ka hale, ʻaʻohe mea hoʻokolohua a i ʻole AlphaFold i loaʻa.ʻO ka SPACA6 ectodomain ana he 20 Å × 20 Å × 85 Å, he ʻehiku helices a he ʻeiwa β-strands, a he ʻāpana tertiary elongated i hoʻopaʻa ʻia e nā paʻa disulfide ʻeono (Fig. 1a, b).ʻO ka mānoanoa electron nāwaliwali ma ka hope o ke kaulahao ʻaoʻao Asn243 e hōʻike ana he glycosylation pili i kēia koena.Aia i loko o ka hale he ʻelua mau kāʻei kapu: he pūʻulu ʻehā-helix N-terminal (4HB) a me kahi kikowaena C-terminal Ig-like me kahi ʻāpana hinge waena ma waena o lākou (Fig. 1c).
ʻO kahi hoʻolālā o ka pūnaewele extracellular o SPACA6.E wehe i ke kiʻikuhi o ke kikowaena extracellular o SPACA6, ke kala o ke kaulahao mai N a i C-terminus mai ka uliuli ʻeleʻele a i ka ʻulaʻula ʻeleʻele.Hōʻike ʻia nā Cysteine ​​i loko o nā paʻa disulfide i ka magenta.b Topology o ka extracellular domain o SPACA6.E hoʻohana i ka hoʻolālā kala like me ka Figure 1a.c SPACA6 domain extracellular.ʻO ka 4HB, hinge, a me Ig-like domain strip charts he ʻalani, ʻōmaʻomaʻo, a me ka uliuli.ʻAʻole huki ʻia nā papa i ka nui.
Aia ka 4HB domain o SPACA6 i nā helices nui ʻehā (helices 1-4), i hoʻonohonoho ʻia ma ke ʻano o ka helical helix (Fig. 2a), e hoʻololi ana ma waena o nā pilina antiparallel a me nā mea like (Fig. 2b).Ua waiho ʻia kahi huinakolu liʻiliʻi liʻiliʻi hoʻololi hoʻokahi (helix 1′) e kū pono ana i ka pūʻolo, e hana ana i kahi huinakolu me nā helices 1 a me 2. Ua hoʻololi iki ʻia kēia huinakolu i loko o ka hoʻopili ʻana i ka helical-twisted o ka hoʻopili paʻa o nā helices 3 a me 4 ( Fig. 2a).
4HB N-terminal strip pakuhi.b Nānā kiʻekiʻe o ka pūʻolo o nā ʻehā helices, ua hōʻike ʻia kēlā me kēia helix i ka uliuli ʻeleʻele ma ka N-terminus a me ka ʻulaʻula ʻeleʻele ma ka C-terminus.c Kiʻikuhi huila huila luna-lalo no 4HB, me kēlā me kēia koena i hōʻike ʻia ma ke ʻano he pōʻai i hoʻopaʻa inoa ʻia me ka helu helu amino acid hoʻokahi;ʻo nā ʻakika amino ʻehā wale nō ma luna o ka huila i helu ʻia.He melemele nā ​​koena non-polar, ʻōmaʻomaʻo nā koena i hoʻopaʻa ʻole ʻia, ʻulaʻula nā koena i hoʻopiʻi maikaʻi ʻia.d He mau helehelena huikolu o ka domain 4HB, me 4HBs i ka alani a me na hinges i ka omaomao.Hōʻike nā mea hoʻokomo ʻelua i nā mea paʻa disulfide koʻikoʻi.
Hoʻopili ʻia ʻo 4HB ma kahi kumu hydrophobic i loko i haku ʻia me nā koena aliphatic a me nā mea ʻala (Fig. 2c).Aia i loko o ke kumu kahi paʻa disulfide ma waena o Cys41 a me Cys55 e hoʻopili ai i nā helices 1 a me 2 i kahi huinakolu kiʻekiʻe (Fig. 2d).Ua hoʻokumu ʻia ʻelua mau paʻa disulfide hou ma waena o ke kumu CXXC ma Helix 1 'a me kekahi kumu CXXC i loaʻa ma ka piko o ka β-hairpin ma ka ʻāpana hinge (Fig. 2d).Aia kekahi koena arginine conservative me kahi hana ike ole (Arg37) i loko o ka huinakolu puha i hanaia e na helices 1′, 1, a me 2. Atoma kalapona Aliphatic Cβ, Cγ, a me Cδ Arg37 e pili ana me ke kumu hydrophobic, a ne'e cyclically kona mau hui guanidine. ma waena o nā helices 1' a me 1 ma o ka pilina ma waena o ka iwi kuamoʻo Thr32 a me ke kaulahao ʻaoʻao (Fig. S5a,b).Hoʻonui ʻo Tyr34 i loko o ka lua e waiho ana i ʻelua mau lua liʻiliʻi e hiki ai iā Arg37 ke launa pū me ka mea hoʻoheheʻe.
ʻO nā kāʻei kapu β-sandwich like Ig he ʻohana nui loa o nā protein e kaʻana like i ka hiʻohiʻona maʻamau o ʻelua a ʻoi aku paha nā ʻāpana amphipathic β-sheet e launa pū ana ma o ka hydrophobic core 39. He ʻano like ka C-terminal Ig-like domain o SPACA6 a he ʻelua papa (Fig. S6a).ʻO ka Pepa 1 he β-pepa o nā kaula ʻehā (nā kaula D, F, H, a me I) kahi e hana ai nā kaula F, H, a me I i hoʻonohonoho kūʻokoʻa, a hui like nā kaula I a me D.ʻO ka papa 2 he pepa beta liʻiliʻi kūʻokoʻa-parallel kaulua (mau kaula E a me G).Ua ʻike ʻia kahi paʻa disulfide kūloko ma waena o ka C-terminus o ke kaulahao E a me ke kikowaena o ke kaulahao H (Cys170-Cys226) (Fig. S6b).Hoʻohālikelike kēia paʻa disulfide me ka paʻa disulfide i loko o ka β-sandwich domain o immunoglobulin40,41.
Huli ka lau β ʻehā ma kona lōʻihi holoʻokoʻa, e hana ana i nā kihi asymmetrical i ʻokoʻa i ke ʻano a me nā electrostatics.ʻO ka ʻaoʻao ʻoi aku ka lahilahi he ʻili honua hydrophobic pālahalaha e kū nei i waho i ka hoʻohālikelike ʻia me nā ʻāpana like ʻole a electrostatically ʻokoʻa i loko o SPACA6 (Fig. S6b,c).He halo o ka iwi kuamoʻo hui carbonyl/amino a me nā kaulahao ʻaoʻao polar e hoʻopuni ana i ka ʻili hydrophobic (Fig. S6c).Ua uhi ʻia ka ʻaoʻao ākea e kahi ʻāpana helical i hoʻopaʻa ʻia e ālai ana i ka ʻāpana N-terminal o ke kumu hydrophobic a hana i ʻekolu mau paʻa hydrogen me ka pūʻulu polar hāmama o ka iwi hope F (Fig. S6d).ʻO ka ʻāpana C-terminal o kēia ʻaoʻao he ʻeke nui me kahi ʻāpana hydrophobic i ʻike ʻia.Hoʻopuni ʻia ka ʻeke e nā koina kūpono ma muli o ʻekolu mau ʻāpana o nā koena arginine pālua (Arg162-Arg221, Arg201-Arg205 a me Arg212-Arg214) a me kahi histidine waena (His220) (Figure S6e).
He māhele pōkole ka ʻāpana hinge ma waena o ka domain helical a me ka domain like Ig, me hoʻokahi antiparallel three-stranded β-layer (nā kaula A, B a me C), he 310 helix liʻiliʻi, a me kekahi mau ʻāpana helical lōʻihi.(Fig. S7).ʻO kahi pūnaewele o nā pilina covalent a me electrostatic ma ka ʻāpana hinge e ʻike ʻia e hoʻopaʻa i ka hoʻonohonoho ʻana ma waena o 4HB a me ka domain like Ig.Hiki ke hoʻokaʻawale ʻia ka pūnaewele i ʻekolu ʻāpana.ʻO ka hapa mua he ʻelua mau kumu CXXC (27CXXC30 a me 139CXXC142) i hana i nā paʻa disulfide ma waena o ka β-hairpin i ka hinge a me ka 1′ helix ma 4HB.ʻO ka ʻāpana ʻelua e pili ana i nā pilina electrostatic ma waena o ka domain like Ig a me ka hinge.Hoʻokumu ʻo Glu132 i ka hinge i alahaka paʻakai me Arg233 ma ka ʻaoʻao Ig-like a me Arg135 i ka hinge.Aia ka ʻāpana ʻekolu i kahi paʻa covalent ma waena o ka domain like Ig a me ka ʻāpana hinge.ʻElua mau paʻa disulfide (Cys124-Cys147 a me Cys128-Cys153) e hoʻopili i ka loop hinge i kahi loulou i hoʻopaʻa ʻia e nā pilina electrostatic ma waena o Gln131 a me ka hui hana iwi kuamoʻo, e ʻae ana i ke komo ʻana i ka domain like Ig mua.kaulahao.
Ua hoʻohanaʻia keʻano o ka SPACA6 ectodomain a me nā hale hoʻokahi o 4HB a me Ig-like domain no ka huliʻana i nā moʻolelo likeʻole i loko o nā pūnaewele protein 42.Ua ʻike mākou i nā pāʻani me nā helu Dali Z kiʻekiʻe, nā ʻokoʻa maʻamau liʻiliʻi, a me nā helu LALI nui (ʻo ia ka helu o nā koena like structurally).ʻOiai ʻo ka 10 hits mua loa mai ka hulina ectodomain piha (Pable S1) loaʻa ka Z-score o >842, ʻo ka huli ʻana no 4HB a i ʻole Ig-like domain wale nō i hōʻike i ka hapa nui o kēia mau hits i pili i nā β-sandwich wale nō.he paʻi ma nā wahi a pau i loaʻa i nā protein he nui.Hoʻokahi wale nō hopena o nā hulina ʻekolu ma Dali: IZUMO1.
Ua manaʻo lōʻihi ʻo SPACA6 a me IZUMO1 e kaʻana like i nā ʻano like7,32,37.ʻOiai ʻo nā ectodomains o kēia mau protein pili i ka gamete fusion-pili i ka 21% kaʻina kikoʻī wale nō (Figure S8a), nā hōʻike paʻakikī, me kahi kumu hoʻopaʻa disulfide i mālama ʻia a me kahi kikowaena C-terminal Ig-like domain i SPACA6, ʻae ʻia nā hoʻāʻo mua e kūkulu i kahi homology model o A a SPACA6 iole me IZUMO1 ma ke ano he template37.Hōʻoia kā mākou hale i kēia mau wanana a hōʻike i ke kiʻekiʻe maoli o ka like.ʻO kaʻoiaʻiʻo,ʻo nā hale SPACA6 a me IZUMO137,43,44 kaʻana like i ka hoʻolālā hoʻolālāʻelua (Fig. S8b) me nā kikowaena like 4HB a me Ig-like β-sandwich i hoʻohuiʻia e kahi'āpana hinge (Fig. S8c).
Loaʻa iā IZUMO1 a me SPACA6 4HB nā ʻokoʻa maʻamau mai nā puʻupuʻu spiral maʻamau.ʻO nā 4HB maʻamau, e like me nā mea i loaʻa i loko o ka SNARE protein complexes i komo i ka endosomal fusion 45,46, ua hoʻokaʻawale ʻia nā helices e mālama ana i ka curvature mau a puni kahi kikowaena axis 47. ʻO ka mea like ʻole, ua hoʻololi ʻia nā kikowaena helical ma IZUMO1 a me SPACA6, me ka curvature loli a hoʻopili ʻole ʻia (Figure S8d).ʻO ka wili, ma muli paha o ka huinakolu i hana ʻia e nā helices 1′, 1 a me 2, ua mālama ʻia ma IZUMO1 a me SPACA6 a hoʻopaʻa ʻia e ke kumu like CXXC ma ka helix 1′.Eia naʻe, ʻo ka paʻa disulfide hou aʻe i loaʻa ma SPACA6 (Cys41 a me Cys55 e hoʻopili ana i nā helices 1 a me 2 ma luna) e hana i kahi piko ʻoi aʻe ma ka piko triangle, e hoʻololi ai i ka SPACA6 i ʻoi aku ka wili ma mua o IZUMO1, me nā huinakolu lua i ʻōlelo ʻia.Eia kekahi, nele ʻo IZUMO1 iā Arg37 i ʻike ʻia i waenakonu o kēia lua ma SPACA6.ʻO ka ʻokoʻa, loaʻa iā IZUMO1 kahi kumu hydrophobic maʻamau o nā koena aliphatic a me nā mea ʻala.
Loaʻa iā IZUMO1 kahi kikowaena Ig-like me kahi β-sheet43 ʻelua-stranded a me ʻelima.Hoʻololi ke kaula hou ma IZUMO1 i ka wili ma SPACA6, e hui pū me ke kaula F e kaupalena i nā paʻa hydrogen iwi kuamoʻo i ke kaula.ʻO kahi mea hoihoi o ka hoʻohālikelike ʻana, ʻo ia ka hoʻopiʻi ʻana o ka ʻili o nā kāʻei like Ig o nā protein ʻelua.ʻOi aku ka maikaʻi o ka IZUMO1 ma mua o ka SPACA6 surface.Aia kahi uku hou ma kahi kokoke i ka C-terminus e kū pono ana i ka membrane sperm.Ma SPACA6, ʻoi aku ka kūʻokoʻa a i ʻole ka hoʻopiʻi maikaʻi ʻana o nā ʻāpana like (Fig. S8e).No ka laʻana, ʻo ka ʻili hydrophobic (nā ʻaoʻao ʻoi aku ka lahilahi) a me nā lua i hoʻopiʻi maikaʻi ʻia (nā ʻaoʻao ākea) ma SPACA6 i hoʻopiʻi ʻino ʻia ma IZUMO1.
ʻOiai ua mālama maikaʻi ʻia ka pilina a me nā mea hana lua ma waena o IZUMO1 a me SPACA6, ua hōʻike ʻia ka hoʻonohonoho ʻana o nā kāʻei like Ig i ka ʻokoʻa o nā kikowaena ʻelua i ko lākou ʻano nui e pili ana i kekahi i kekahi (Fig. S9).Hoʻopili ʻia ka puʻupuʻu spiral o IZUMO1 e pili ana i ka β-sandwich, e hana ana i ke ʻano "boomerang" i wehewehe mua ʻia ma kahi o 50° mai ke koʻi waena.ʻO ka ʻokoʻa, ua hili ʻia ka beam helical ma SPACA6 ma kahi o 10 ° ma ka ʻaoʻao ʻē aʻe.ʻO nā ʻokoʻa o kēia mau kuhikuhi ma muli paha o nā ʻokoʻa o ka ʻāpana hinge.Ma ka pae kaʻina mua, IZUMO1 a me SPACA6 kaʻana like liʻiliʻi o ke kaʻina like ma ka hinge, koe naʻe nā koena cysteine, glycine, a me aspartic acid.ʻO ka hopena, ʻokoʻa loa nā paʻa hydrogen a me nā pūnaewele electrostatic.Hoʻokaʻawale ʻia nā mea hana kiʻekiʻe β-sheet e IZUMO1 a me SPACA6, ʻoiai ʻoi aku ka lōʻihi o nā kaulahao ma IZUMO1 a ʻokoʻa ka 310 helix (helix 5) iā SPACA6.ʻO kēia mau ʻokoʻa ka hopena i nā ʻano kikowaena ʻokoʻa no ʻelua mau protein like ʻole.
Ua hōʻike ʻia kā mākou ʻimi kikowaena Dali ʻo SPACA6 a me IZUMO1 wale nō ʻelua mau hale hoʻokolohua i hoʻopaʻa ʻia i mālama ʻia i loko o ka waihona protein i loaʻa kēia 4HB fold (Table S1).I kēia mau lā, ua hoʻomohala ʻo DeepMind (Alphabet/Google) iā AlphaFold, kahi ʻōnaehana neural network e hiki ke wānana pololei i nā hana 3D o nā protein mai nā kaʻina kumu mua48.Ma hope koke iho o kā mākou hoʻoponopono ʻana i ka ʻōnaehana SPACA6, ua hoʻokuʻu ʻia ka waihona AlphaFold, e hāʻawi ana i nā hiʻohiʻona wānana e uhi ana i ka 98.5% o nā protein āpau i ka proteome48,49 kanaka.Ke hoʻohana nei i kā mākou hoʻolālā SPACA6 i hoʻoholo ʻia ma ke ʻano he ʻimi hulina, kahi hulina homology structural no ke kumu hoʻohālike i ka AlphaFold human proteome i ʻike i nā moho me nā ʻano like like me SPACA6 a me IZUMO1.Hāʻawi ʻia i ka pololei kupaianaha o AlphaFold i ka wānana ʻana i ka SPACA6 (Fig. S10a) - ʻoi loa ka 1.1 Å rms ectodomain i hoʻohālikelike ʻia me kā mākou hoʻolālā hoʻoholo (Fig. S10b) - hiki iā mākou ke hilinaʻi i ka pololei o nā pāʻani SPACA6 i ʻike ʻia.
Ma mua, ua ʻimi ʻo PSI-BLAST i ka puʻupuʻu IZUMO1 me ʻekolu mau protein pili i ka sperm: IZUMO2, IZUMO3, a me IZUMO450.Ua wānana ʻo AlphaFold e hoʻopili ʻia kēia mau protein ʻohana IZUMO i ka domain 4HB me ke ʻano hoʻopaʻa disulfide like me IZUMO1 (Nā Kiʻi 3a a me S11), ʻoiai ʻaʻole lākou i kahi kikowaena Ig-like.Ua manaʻo ʻia ʻo IZUMO2 a me IZUMO3 he mau protein membrane ʻaoʻao hoʻokahi e like me IZUMO1, ʻoiai ʻo IZUMO4 ka mea huna.ʻAʻole i hoʻoholo ʻia nā hana o IZUMO 2, 3, a me 4 protein i ka gamete fusion.ʻIke ʻia ʻo IZUMO3 e pāʻani i ka biogenesis acrosome i ka wā o ka ulu ʻana o spermatozoa51, a ʻo ka protein IZUMO e hana i kahi complex50.ʻO ka mālama ʻana i nā proteins IZUMO i loko o nā mammals, reptile, a me nā amphibians e hōʻike ana e kūlike kā lākou hana me nā protein pili gamete-fusion ʻē aʻe, e like me DCST1/2, SOF1, a me FIMP.
Ke kiʻikuhi o ka papa kuhikuhi kahua o ka IST superfamily, me 4HB, hinge, a me Ig-like domain i hōʻike ʻia i ka ʻalani, ʻōmaʻomaʻo, a me ka uliuli.Loaʻa iā IZUMO4 kahi ʻāpana C-terminal kūʻokoʻa e nānā ʻeleʻele.Hōʻike ʻia nā paʻa disulfide i hōʻoia ʻia a me ka putative e nā laina paʻa a me nā kiko kiko.b IZUMO1 (PDB: 5F4E), SPACA6, IZUMO2 (AlphaFold DB: AF-Q6UXV1-F1), IZUMO3 (AlphaFold DB: AF-Q5VZ72-F1), IZUMO4 (AlphaFold DB: AF-Q1ZYL8-FEMF95), a me DB: AF-Q1ZYL8-F1) : AF-Q1ZYL8-F1): AF-Q3KNT9-F1) hōʻike ʻia ma ka laulā kala like me ka panel A. Hōʻike ʻia nā paʻa disulfide i ka magenta.ʻAʻole hōʻike ʻia nā helices transmembrane TMEM95, IZUMO2 a me IZUMO3.
ʻAʻole like me ka protein IZUMO, ua manaʻo ʻia nā protein SPACA ʻē aʻe (ʻo ia hoʻi, SPACA1, SPACA3, SPACA4, SPACA5, a me SPACA9) he ʻokoʻa ke ʻano mai ka SPACA6 (Fig. S12).ʻO SPACA9 wale nō ka 4HB, akā, ʻaʻole i manaʻo ʻia e loaʻa ka hoʻohālikelike like ʻole-anti-parallel a i ʻole ka pilina disulfide like me SPACA6.ʻO SPACA1 wale nō ka ʻāpana like Ig.Wahi a AlphaFold he ʻano ʻokoʻa loa ko SPACA3, SPACA4 a me SPACA5 ma mua o SPACA6.ʻO ka mea e mahalo ai, ua ʻike ʻia ʻo SPACA4 e pāʻani i ka fertilization, akā ʻoi aku ka nui ma mua o SPACA6, ma kahi o ka hoʻomaʻamaʻa ʻana i ka pilina ma waena o ka sperm a me ka oocyte zona pellucida52.
Ua loaʻa i kā mākou hulina AlphaFold kekahi pāʻani no IZUMO1 a me SPACA6 4HB, TMEM95.ʻO TMEM95, kahi protein transmembrane kikoʻī sperm hoʻokahi, e hoʻolilo i nā ʻiole kāne male ʻole ke hoʻopau ʻia 32,33.ʻO Spermatozoa nele i ka TMEM95 he morphology maʻamau, motility, a me ka hiki ke komo i ka zona pellucida a hoʻopaʻa i ka membrane hua manu, akā ʻaʻole hiki ke hoʻohui me ka membrane oocyte.Ua hōʻike ʻia nā haʻawina mua e kaʻana like ana ʻo TMEM95 me IZUMO133.ʻOiaʻiʻo, ua hōʻoia ka AlphaFold model i ka TMEM95 he 4HB me nā kumu like CXXC e like me IZUMO1 a me SPACA6 a me ka pilina disulfide hou ma waena o nā helices 1 a me 2 i loaʻa ma SPACA6 (Fig. 3a a me S11).ʻOiai ʻaʻohe o TMEM95 i kahi kikowaena Ig-like, loaʻa iā ia kahi ʻāpana me kahi kumu hoʻopaʻa disulfide e like me nā wahi hinge SPACA6 a me IZUMO1 (Fig. 3b).I ka manawa o ka paʻi ʻia ʻana o kēia manuscript, ua hōʻike ka server preprint i ke ʻano o TMEM95, e hōʻoia ana i ka hopena AlphaFold53.Ua like loa ka TMEM95 me SPACA6 a me IZUMO1 a ua mālama ʻia i loko o nā amphibians (Fig. 4 a me S13).
Ua hoʻohana ka hulina PSI-BLAST i nā ʻikepili NCBI SPACA6, IZUMO1-4, TMEM95, DCST1, DCST2, FIMP, a me SOF1 e hoʻoholo i ke kūlana o kēia mau kaʻina i ka lāʻau o ke ola.ʻAʻole i hōʻike ʻia nā mamao ma waena o nā kiko lālā i ka nui.
ʻO ke ʻano like ʻole o ka hoʻolālā holoʻokoʻa ma waena o SPACA6 a me IZUMO1 e hōʻike ana e hoʻokumu ana lākou i nā lālā o kahi superfamily i mālama ʻia e komo pū ana me nā protein TMEM95 a me IZUMO 2, 3, a me 4.ʻike ʻia nā lālā: IZUMO1, SPACA6 a me TMEM95.No ka mea he liʻiliʻi wale nā ​​lālā i loaʻa i nā kāʻei like Ig, ʻo ka hōʻailona o ka superfamily IST ka domain 4HB, nona nā hiʻohiʻona kūʻokoʻa i maʻamau i kēia mau protein a pau: 1) Coiled 4HB me nā helices i hoʻonohonoho ʻia i kahi hoʻololi anti-parallel/parallel (Fig). . 5a), 2) he alo triangular ka pūʻolo i loaʻa i nā helices ʻelua i loko o ka pūʻolo a me ke kolu o ka helix vertical (Fig. kī wahi (Fig. 5c). ʻO ke kumu CXXC, i loaʻa i nā protein like thioredoxin, ua ʻike ʻia e hana. ma ke ʻano he redox sensor 54,55,56, ʻoiai ke kumu o ka ʻohana IST hiki ke hoʻopili ʻia me nā isomerases disulfide protein e like me ERp57 i ka gamete fusion. Ua pili nā kuleana 57,58.
Ua wehewehe ʻia nā lālā o ka IST superfamily e ʻekolu mau hiʻohiʻona o ka domain 4HB: ʻehā helices e hoʻololi ana ma waena o ka hoʻohālikelike a me ka hoʻohālikelike antiparallel, nā alo puʻupuʻu helical ba-triangular, a me kahi kumu pālua CXXC i hoʻokumu ʻia ma waena o nā mole liʻiliʻi.) N-terminal helixes (ʻalani) a me ka ʻāpana hinge β-hairpin ('ōmaʻomaʻo).
Hāʻawi i ka like ma waena o SPACA6 a me IZUMO1, ua hoʻāʻo ʻia ka hiki o ka mea mua e hoʻopaʻa iā IZUMO1 a i ʻole JUNO.ʻO ka biolayer interferometry (BLI) kahi hana hoʻopaʻa kinetic-based i hoʻohana mua ʻia e helu i ka pilina ma waena o IZUMO1 a me JUNO.Ma hope o ka incubation o ka biotin-labeled sensor me IZUMO1 ma ke ʻano he maunu me kahi kiʻekiʻe o ka JUNO analyte, ua ʻike ʻia kahi hōʻailona ikaika (Fig. S14a), e hōʻike ana i kahi hoʻololi hoʻopaʻa paʻa i ka mānoanoa o ka biomaterial i hoʻopili ʻia i ka piko sensor.Nā hōʻailona like (ʻo ia hoʻi, hui pū ʻia ʻo JUNO me ka mea ʻike ma ke ʻano he maunu kūʻē iā IZUMO1 analyte) (Fig. S14b).ʻAʻohe hōʻailona i ʻike ʻia ke hoʻohana ʻia ʻo SPACA6 ma ke ʻano he analyte e kūʻē i ka IZUMO1 paʻa i ka sensor a i ʻole JUNO paʻa i ka sensor (Figure S14a,b).ʻO ka loaʻa ʻole o kēia hōʻailona e hōʻike ana ʻaʻole pili ka domain extracellular o SPACA6 me ka domain extracellular o IZUMO1 a i ʻole JUNO.
Ma muli o ka BLI assay e pili ana i ka biotinylation o nā koena lysine manuahi ma ka protein maunu, hiki i kēia hoʻololi ke pale i ka hoʻopaʻa ʻana inā pili nā koena lysine i ka pilina.Eia kekahi, hiki i ka orientation o ka hoʻopaʻa ʻana e pili ana i ka sensor ke hana i nā hindrances steric, no laila ua hana pū ʻia nā hoʻokolohua huki-iho maʻamau i ka recombinant SPACA6, IZUMO1 a me JUNO ectodomains.ʻOiai naʻe, ʻaʻole i hoʻokahe ʻia ʻo SPACA6 me kāna IZUMO1 i hōʻailona ʻia a i ʻole JUNO (Fig. S14c, d), e hōʻike ana ʻaʻohe pilina pili me ka mea i ʻike ʻia ma nā hoʻokolohua BLI.Ma keʻano he mana maikaʻi, ua hōʻoia mākou i ka pilina o JUNO me ka inoaʻo kāna IZUMO1 (Figures S14e a me S15).
ʻOiai ke ʻano like o ke ʻano ma waena o SPACA6 a me IZUMO1, ʻaʻole he mea kupanaha ka hiki ʻole o SPACA6 ke hoʻopaʻa iā JUNO.ʻO ka ʻili o ke kanaka IZUMO1 he ʻoi aku ma mua o 20 mau koena e launa pū me JUNO, me nā koena mai kēlā me kēia o nā ʻāpana ʻekolu (ʻoiai ʻo ka hapa nui o lākou aia ma ka ʻāpana hinge) (Fig. S14f).ʻO kēia mau koena, hoʻokahi wale nō i mālama ʻia ma SPACA6 (Glu70).ʻOiai ka nui o nā mea hoʻololi i hoʻopaʻa ʻia i ko lākou mau waiwai biochemical maoli, ua hoʻololi ʻia ke koena Arg160 koʻikoʻi ma IZUMO1 e ka Asp148 i hoʻopiʻi maikaʻi ʻia ma SPACA6;ua hōʻike mua nā haʻawina i ka hoʻololi ʻana o Arg160Glu ma IZUMO1 kokoke loa e hoʻopau i ka hoʻopaʻa ʻana iā JUNO43.Eia kekahi, ʻo ka ʻokoʻa o ka hoʻonohonoho ʻāina ma waena o IZUMO1 a me SPACA6 i hoʻonui nui i ka ʻili o ka JUNO-binding site o ka ʻāpana like ma SPACA6 (Fig. S14g).
ʻOiai ka pono i ʻike ʻia no SPACA6 no ka hui ʻana o ka gamete a me kona ʻano like me IZUMO1, ʻaʻole ʻike ʻia ka SPACA6 i kahi hana hoʻopaʻa JUNO like.No laila, ua ʻimi mākou e hoʻohui i kā mākou ʻikepili kūkulu me nā hōʻike koʻikoʻi i hāʻawi ʻia e ka biology evolutionary.Hōʻike ka hoʻonohonoho ʻana o nā homologues SPACA6 i ka mālama ʻana i ka hale maʻamau ma mua o nā mammals.No ka laʻana, aia nā koena cysteine ​​i loko o nā amphibians pili loa (Fig. 6a).Ke hoʻohana nei i ka kikowaena ConSurf, ua paʻi ʻia nā ʻikepili hoʻopaʻa ʻana i nā kaʻina hana he 66 i ka ʻili SPACA6.Hiki i kēia ʻano loiloi ke hōʻike i nā koena i mālama ʻia i ka wā o ka ulu ʻana o ka protein a hiki ke hōʻike i ke ʻano o nā ʻāpana ili i ka hana.
kahi hoʻonohonoho ʻana o nā ectodomains SPACA6 mai 12 mau ʻano ʻano like ʻole i hoʻomākaukau ʻia me ka CLUSTAL OMEGA.Wahi a ka loiloi ConSurf, ua kaha ʻia nā kūlana conservative i ka uliuli.Hōʻike ʻia nā koena Cysteine ​​​​i ka ʻulaʻula.Hōʻike ʻia nā palena ʻāina a me nā mea hana lua ma luna o ka hoʻopololei, kahi e hōʻike ai nā pua i nā kaula β a me nā nalu e hōʻike i nā helices.ʻO ka NCBI Access Identifiers e loaʻa ana nā kaʻina: kanaka (Homo sapiens, NP_001303901), mandrill (Mandrilus leucophaeus, XP_011821277), ka monkey capuchin (Cebus mimic, XP_017359366), lio (Equus caballus, XP_061_02rca3), ka lio (Equus caballus o XP_02_02rca3, XP_061821277). 3 XP_032_034) .), hipa (Ovis aries, XP_014955560), ʻelepani (Loxodonta africana, XP_010585293), ʻīlio (Canis lupus familyis, XP_025277208), ʻiole (Mus musculus, NP_001156381) ypus, 8) , 61_89 a me Bullfrog (Bufo bufo, XP_040282113).Hoʻokumu ʻia ka helu ʻana i ka hoʻonohonoho kanaka.b Hōʻike i ka ʻili o ka hoʻolālā SPACA6 me 4HB ma luna a me Ig-like domain ma lalo, nā waihoʻoluʻu e pili ana i nā koho mālama mai ka kikowaena ConSurf.ʻO nā ʻāpana mālama maikaʻi loa i ka uliuli, ʻo nā ʻāpana i mālama ʻia ma ke keʻokeʻo, a ʻo ka mea liʻiliʻi i mālama ʻia i ka melemele.ʻōniʻoniʻo poni.Hōʻike ʻia ʻekolu mau ʻāpana ʻili e hōʻike ana i kahi pae kiʻekiʻe o ka pale ma ka hoʻokomo ʻia ʻana i nā ʻāpana 1, 2 a me 3. Hōʻike ʻia kahi kiʻi kiʻi 4HB ma ka hoʻokomo ʻana ma ka ʻaoʻao ʻākau o luna (ka hoʻolālā kala like).
ʻEkolu mau ʻāpana ili i mālama nui ʻia ka hale SPACA6 (Fig. 6b).Hoʻopili ʻo Patch 1 i ka 4HB a me ka ʻāpana hinge a loaʻa i ʻelua mau alahaka disulfide CXXC i mālama ʻia, kahi pūnaewele hinge Arg233-Glu132-Arg135-Ser144 (Fig. S7), a me ʻekolu mau koena ʻono o waho i mālama ʻia (Phe31, Tyr73, Phe137)).he rim akea o ka Ig-like domain (Fig. S6e), e hoike ana i kekahi mau koena i hookauia maikai ma ka sperm surface.ʻO ka mea e mahalo ai, aia kēia ʻāpana i kahi epitope antibody i hōʻike mua ʻia e hoʻopilikia i ka hana SPACA6 30.Māhele ʻo 3 i ka hinge a me kekahi ʻaoʻao o ka domain like Ig;Aia i loko o kēia māhele nā ​​proline i mālama ʻia (Pro126, Pro127, Pro150, Pro154) a me nā koena polar/kau i waho.ʻO ka mea kupanaha, ʻo ka hapa nui o nā koena ma ka ʻili o ka 4HB he loli nui (Fig. 6b), ʻoiai ua mālama ʻia ka pā i loko o ka homologue SPACA6 (e like me ka mea i hōʻike ʻia e ka conservatism o ka pūpū hydrophobic core) a ma waho o ka IST superfamily.
ʻOiai ʻo kēia ka ʻāpana liʻiliʻi loa ma SPACA6 me nā mea liʻiliʻi loa i ʻike ʻia, ʻoi aku ka nui o nā koena ʻāpana hinge (me ka ʻāpana 3) i mālama nui ʻia ma waena o nā homologues SPACA6, e hōʻike ana i ka hoʻonohonoho ʻana o ka pūpū helical a me ka β-sandwich.ma ke ano he conservative.Eia nō naʻe, ʻoiai ka nui o ka hoʻopaʻa ʻana i ka hydrogen a me nā ʻenehana electrostatic ma ka ʻāpana hinge o SPACA6 a me IZUMO1, hiki ke ʻike ʻia nā hōʻike o ka hiki ke hoʻololi ʻia i ka alignment o nā hale i ʻae ʻia o IZUMO137,43,44.ʻO ka alignment o nā kāʻei hoʻokahi i hoʻopili maikaʻi ʻia, akā ʻo ka hoʻonohonoho ʻana o nā kikowaena pili i kekahi i kekahi mai 50 ° a 70 ° mai ke axis waena (Fig. S16).No ka hoʻomaopopo ʻana i ka conformational dynamics o SPACA6 i ka hopena, ua hana ʻia nā hoʻokolohua SAXS (Fig. S17a, b).Ab initio reconstruction o ka SPACA6 ectodomain kulike me ke koʻokoʻo crystal structure (Fig. S18), oiai ka Kratky plot i hoike mai i kekahi degere o ka maʻalahi (Fig. S17b).ʻOkoʻa kēia conformation me IZUMO1, kahi i manaʻo ai ka protein unbound i kahi ʻano boomerang ma ka lattice a me ka solution43.
No ka hoʻomaopopo pono ʻana i ka ʻāina maʻalahi, ua hana ʻia ka hydrogen-deuterium exchange mass spectroscopy (H-DXMS) ma SPACA6 a hoʻohālikelike ʻia me ka ʻikepili i loaʻa mua ma IZUMO143 (Fig. 7a,b).ʻOi aku ka maʻalahi o ka SPACA6 ma mua o IZUMO1, e like me ka mea i hōʻike ʻia e kahi hoʻololi deuterium kiʻekiʻe i loko o ka hale ma hope o 100,000 s o ka hoʻololi.Ma nā hale ʻelua, ʻo ka ʻāpana C-terminal o ka ʻāpana hinge e hōʻike ana i kahi kiʻekiʻe o ka hoʻololi ʻana, hiki paha ke ʻae i ka hoʻololi ʻana o ka 4HB a me Ig-like domain e pili ana i kekahi.ʻO ka mea e mahalo ai, ʻo ka ʻāpana C-terminal o ka hinge SPACA6, ʻo ia hoʻi ka 147CDLPLDCP154 koena, he ʻāina i mālama nui ʻia 3 (Fig. 6b), e hōʻike ana paha i ka hoʻololi ʻana o interdomain he hiʻohiʻona evolutionarily conserved o SPACA6.E like me ka hoʻololi ʻana, hōʻike ʻia ka CD thermal melt data i ka SPACA6 (Tm = 51.2 ° C) ʻoi aku ka paʻa ma mua o IZUMO1 (Tm = 62.9 ° C) (Fig. S1e a me S19).
he kiʻi H-DXMS o SPACA6 a me b IZUMO1.Hoʻoholo ʻia ka pākēneka o ka hoʻololi deuterium ma nā wahi manawa i hōʻike ʻia.Hōʻike ʻia nā pae o ka hoʻololi ʻana i ka hydrogen-deuterium e ka waihoʻoluʻu ma kahi pālākiō gradient mai ka uliuli (10%) a i ka ʻulaʻula (90%).Hōʻike nā pahu ʻeleʻele i nā wahi o ka hoʻololi kiʻekiʻe.Hōʻike ʻia nā palena o 4HB, hinge a me Ig-like domain i ʻike ʻia ma ke ʻano aniani ma luna o ke kaʻina mua.Ua hoʻolālā ʻia nā pae hoʻololi Deuterium ma 10 s, 1000 s, a me 100,000 s ma ka palapala kiʻi i hoʻopaʻa ʻia ma luna o nā ʻili molekele alohilohi o SPACA6 a me IZUMO1.ʻO nā ʻāpana o nā hale me kahi pae hoʻololi deuterium ma lalo o 50% he keʻokeʻo keʻokeʻo.ʻO nā wahi ma luna o 50% H-DXMS hoʻololi i waihoʻoluʻu ʻia i kahi pālākiō gradient.
ʻO ka hoʻohana ʻana i ka CRISPR/Cas9 a me ka ʻiole gene knockout genetic strategies ua alakaʻi i ka ʻike ʻana i nā mea koʻikoʻi no ka sperm a me ka paʻa hua manu a me ka hui ʻana.Ma waho aʻe o ka launa pū ʻana o IZUMO1-JUNO a me ka hoʻolālā CD9, ʻo ka hapa nui o nā protein e pili ana me ka gamete fusion e hoʻomau i ka structurally and functionally enigmatic.ʻO ka hiʻohiʻona biophysical a me ka hoʻolālā o SPACA6 kahi ʻāpana ʻē aʻe o ka puʻupuʻu adhesion/fusion molecular i ka wā fertilization.
ʻO SPACA6 a me nā lālā ʻē aʻe o ka IST superfamily i mālama nui ʻia i nā mammals a me nā manu pākahi, nā mea kolo, a me nā amphibians;ʻoiaʻiʻo, ua manaʻo ʻia he koi ʻia ʻo SPACA6 no ka hoʻoulu ʻana i ka iʻa zebrafish 59. Ua like kēia māhele ʻana me nā proteins pili i ka gamete fusion i ʻike ʻia e like me DCST134, DCST234, FIMP31, a me SOF132, e hōʻike ana he HAP2-deficient kēia mau mea (ʻo ia hoʻi. ʻike ʻia ʻo GCS1) nā protein nona ke kuleana no ka hana catalytic o nā protist he nui., nā mea kanu, a me nā ʻāʻī.Fertilized fusion proteins 60, 61. ʻOiai ke ʻano like o ke ʻano ma waena o SPACA6 a me IZUMO1, ʻo ka knockout o nā genes e hoʻopili ana i kekahi o kēia mau protein i loaʻa i ka infertility i nā ʻiole kāne, e hōʻike ana ʻaʻole i hoʻohālikelike ʻia kā lākou mau hana i ka gamete fusion..ʻOi aku ka laulā, ʻaʻohe o nā protein sperm i ʻike ʻia e koi ʻia no ka pae adhesion o ka hui ʻana.
He nīnau wehe ia inā komo ʻo SPACA6 (a me nā lālā ʻē aʻe o ka superfamily IST) i nā hui intergametic, e hana i nā pūnaewele intragametic e kiʻi i nā protein koʻikoʻi i nā wahi fusion, a i ʻole e hana like me nā fusogens paʻakikī.Ua hōʻike ʻia nā haʻawina co-immunoprecipitation ma HEK293T i kahi pilina ma waena o IZUMO1 lōʻihi a me SPACA632.Eia naʻe, ʻaʻole i launa pū kā mākou ectodomains recombinant in vitro, e hōʻike ana i nā pilina i ʻike ʻia ma Noda et al.ua holoi ʻia nā mea ʻelua i ka hana (e nānā i ka huelo cytoplasmic o IZUMO1, i hōʻike ʻia ʻaʻole pono no ka fertilization62).ʻO kahi ʻē aʻe, makemake paha ʻo IZUMO1 a/a i ʻole SPACA6 i nā kaiapuni paʻa kikoʻī ʻaʻole mākou e hana hou i loko o ka vitro, e like me nā conformations kikoʻī physiologically a i ʻole nā ​​molecular complex i loaʻa nā protein ʻē aʻe (ʻike ʻia a ʻike ʻole ʻia paha).ʻOiai ua manaʻo ʻia ka IZUMO1 ectodomain e hoʻopili i ka hoʻopili ʻana o ka spermatozoa i ka hua manu ma ka perivitelline space, ʻaʻole maopopo ke kumu o ka SPACA6 ectodomain.
Hōʻike ke ʻano o SPACA6 i kekahi mau wahi i mālama ʻia e pili ana i ka pilina protein-protein.ʻO ka ʻāpana i mālama ʻia o ka ʻāpana hinge e pili kokoke ana i ke kumu CXXC (i koho ʻia ʻo Patch 1 ma luna) he mau koena ʻala i waho e pili pinepine ana me ka pilina hydrophobic a me π-stacking ma waena o nā biomolecules.ʻO nā ʻaoʻao ākea o ka ʻāpana Ig-like ('āpana 2) i hoʻokumu ʻia i kahi awāwa i hoʻopiʻi maikaʻi ʻia me Arg a me kāna mau koena i mālama nui ʻia, a ua hoʻohana mua ʻia nā antibodies e kūʻē i kēia māhele e pale i ka hui ʻana o ka gamete 30.Hoʻomaopopo ka antibody i ka epitope linear 212RIRPAQLTHRGTFS225, nona ʻekolu o nā koena arginine ʻeono a mālama nui ʻia ʻo His220.ʻAʻole maopopo i ka hana ʻana ma muli o ka paʻa ʻana o kēia mau koena kikoʻī a i ʻole ka ʻāina holoʻokoʻa.ʻO kahi o kēia āpau kokoke i ka C-terminus o ka β-sandwich e hōʻike ana i ka cis-interactions me nā protein sperm kokoke, akā ʻaʻole me nā protein oocyte.Eia kekahi, ʻo ka paʻa ʻana o kahi kuʻekuʻe waiwai proline maʻalahi loa (pae 3) i loko o ka hinge, ʻo ia paha ke kahua o kahi pilina protein-protein a i ʻole, e hōʻike ana i ka paʻa ʻana o ka maʻalahi ma waena o nā kikowaena ʻelua.He mea koʻikoʻi ke kāne no ka hana ʻike ʻole o SPACA6.hui ʻana o nā gametes.
Loaʻa iā SPACA6 nā waiwai o nā protein adhesion intercellular, me ka Ig-like β-sandwiches.Nui nā protein adhesive (eg, cadherins, integrins, adhesins, a me IZUMO1) loaʻa i hoʻokahi a ʻoi aku paha nā kāʻei β-sandwich e hoʻolōʻihi i nā protein mai ka membrane cell i ko lākou mau pahuhopu kaiapuni63,64,65.Aia i loko o ka ʻaoʻao Ig-like o SPACA6 kekahi kumu i loaʻa maʻamau i loko o nā β-sandwiches o ka hoʻopili a me ka cohesion: nā papalua o nā kaula like ʻole ma nā wēlau o nā β-sandwiches, i kapa ʻia ʻo mechanical clamps66.Manaʻo ʻia ʻo kēia kumu e hoʻonui ai i ke kūʻē ʻana i nā ikaika shear, he mea waiwai ia no nā protein i komo i loko o nā pilina intercellular.Eia naʻe, ʻoiai kēia like me nā adhesins, ʻaʻohe mea hōʻike i kēia manawa e pili ana ka SPACA6 me nā hua keʻokeʻo.ʻAʻole hiki i ka SPACA6 ectodomain ke hoʻopaʻa iā JUNO, a me ka SPACA6-expressing HEK293T cell, e like me ia i hōʻike ʻia ma ʻaneʻi, paʻakikī ke launa pū me nā oocytes i nele i ka zona 32.Inā hoʻokumu ʻo SPACA6 i nā paʻa intergametic, pono paha kēia mau pilina i ka hoʻololi ʻana ma hope o ka unuhi ʻana a i ʻole ka hoʻopaʻa ʻana e nā protein sperm ʻē aʻe.I ke kākoʻo ʻana i ke kuhiakau hope, hoʻopaʻa ʻia ka IZUMO1-deficient spermatozoa i nā oocytes, e hōʻike ana i nā molekala ʻē aʻe ma mua o IZUMO1 e pili ana i ka hana adhesion gamete 27.
He nui nā proteins viral, cellular, a me developmental fusion protein i nā waiwai e wānana i kā lākou hana ma ke ʻano he fusogens.No ka laʻana, loaʻa i nā glycoproteins fusion viral (papa I, II a me III) kahi peptide fusion hydrophobic a i ʻole loop ma ka hope o ka protein i hoʻokomo ʻia i loko o ka membrane host.ʻO ka palapala ʻāina hydrophilicity o IZUMO143 a me ke ʻano (hoʻoholo ʻia a wānana ʻia) o ka superfamily IST i hōʻike ʻole i kahi peptide fusion hydrophobic.No laila, inā hana kekahi mau protein i loko o ka superfamily IST ma ke ʻano he fusogens, hana lākou pēlā ma ke ʻano ʻokoʻa mai nā hiʻohiʻona ʻē aʻe.
I ka hopena, ʻo nā hana a nā lālā o ka IST superfamily o nā protein e pili ana me ka gamete fusion e noho mau i kahi mea pohihihi.ʻO kā mākou SPACA6 recombinant mole a me kāna ʻano i hoʻoholo ʻia e hāʻawi i ka ʻike i ka pilina ma waena o kēia mau hale kaʻana like a me kā lākou kuleana i ka hoʻopili gamete a me ka fusion.
ʻO ke kaʻina DNA e pili ana i ka ectodomain SPACA6 kanaka i wānana ʻia (helu helu helu NCBI NP_001303901.1; koena 27-246) i codon-optimized no ka hōʻike ʻana i nā pūnae Drosophila melanogaster S2 a synthesized ma ke ʻano he ʻāpana gene me ke kaʻina hoʻopulapula Kozak (Eurofins Genomics)., ka hōʻailona huna BiP a me ka 5 'a me 3' hopena no ka ligation-kūʻokoʻa cloning o kēia gene i loko o ka pMT expression vector ma muli o kahi mea hoʻolaha metallothionein i hoʻololi ʻia no ke koho ʻana me puromycin (pMT-puro).Hoʻopili ka pMT-puro vector i kahi pae thrombin cleavage a ukali ʻia e kahi 10x-His C-terminal tag (Figure S2).
ʻO ka hoʻololi paʻa o ka SPACA6 pMT-puro vector i D. melanogaster S2 (Gibco) pūnaewele i hana like me ka protocol i hoʻohanaʻia no IZUMO1 a me JUNO43.Ua hoʻoheheʻe ʻia nā pūnaewele S2 a hoʻonui ʻia i loko o Schneider's medium (Gibco) i hoʻohui ʻia me ka hopena hope o 10% (v/v) heat-inactivated fetal calf serum (Gibco) a me 1X antimycotic antibiotic (Gibco).Ua uhi ʻia nā pūnāwai mua (3.0 x 106 cell) i nā pūnāwai pākahi o nā pā 6-well (Corning).Ma hope o 24 mau hola o ka incubation ma 27 ° C, ua hoʻololi ʻia nā cell me ka hui ʻana o 2 mg o ka SPACA6 pMT-puro vector a me Effectene transfection reagent (Qiagen) e like me ka protocol o ka mea hana.Hoʻopiliʻia nā pūnaewele i hoʻololiʻia no nā hola 72 a laila eʻohiʻia me 6 mg / ml puromycin.A laila hoʻokaʻawale ʻia nā kelepona mai ke kikowaena Schneider piha a waiho ʻia i loko o ka serum free Insect-XPRESS medium (Lonza) no ka hana ʻana i ka protein nui.Hoʻonui ʻia kahi pūʻulu 1 L o ka moʻomeheu cell S2 i 8-10 × 106 ml-1 mau keena i loko o ka 2 L ventilated flat-bottomed polypropylene Erlenmeyer flask a laila sterilized me ka hopena hope o 500 µM CuSO4 (Millipore Sigma) a kānana sterile.hoʻokomo ʻia.Hoʻopiliʻia nā moʻomeheu i hoʻokomoʻia ma 27 ° C. ma 120 rpm no nā lāʻehā.
Ua hoʻokaʻawale ʻia ke ʻano mea i loaʻa i ka SPACA6 e ka centrifugation ma 5660 × g ma 4 ° C a ukali ʻia e kahi ʻōnaehana hoʻoheheʻe ʻia ʻo Centramate tangential (Pall Corp) me kahi membrane 10 kDa MWCO.E hoʻopili i ka mea kikowaena i loaʻa ka SPACA6 i kahi kolamu 2 ml Ni-NTA agarose resin (Qiagen).Ua holoi ʻia ka resin Ni-NTA me 10 mau kolamu kolamu (CV) o ka buffer A a laila ua hoʻohui ʻia 1 CV o ka buffer A e hāʻawi i ka manaʻo imidazole hope loa o 50 mM.Ua hoʻokuʻu ʻia ʻo SPACA6 me 10 ml o ka buffer A i hoʻohui ʻia me ka imidazole i ka hopena hope o 500 mM.Hoʻopili pololei ʻia ka thrombin papa kapu (Millipore Sigma) i ka paipu dialysis (MWCO 12-14 kDa) ma 1 ʻāpana no ka mg SPACA6 vs. 1 L 10 mM Tris-HCl, pH 7.5 a me 150 mM NaCl (buffer B) no ka dialysis.) ma 4°C no 48 mau hola.Ua hoʻoheheʻe ʻia ka SPACA6 i hoʻokaʻawale ʻia i ka thrombin e hoʻemi i ka paʻakai paʻakai a hoʻouka ʻia ma kahi kolamu hoʻololi cation 1 ml MonoS 5/50 GL (Cytiva/GE) i hoʻohālikelike ʻia me 10 mM Tris-HCl, pH 7.5.Ua holoi ʻia ka mea hoʻololi cation me 3 OK 10 mM Tris-HCl, pH 7.5, a laila hoʻokaʻawale ʻia ʻo SPACA6 me kahi gradient linear mai 0 a 500 mm NaCl i 10 mm Tris-HCl, pH 7.5 no 25 OK.Ma hope o ka chromatography hoʻololi ion, ua hoʻopaʻa ʻia ʻo SPACA6 i ka 1 ml a ua hoʻokaʻawale ʻia mai kahi kolamu ENrich SEC650 10 x 300 (BioRad) i hoʻohālikelike ʻia me ka buffer B. E like me ka chromatogram, pool and concentrate hakina i loaʻa iā SPACA6.Ua mālama ʻia ka maʻemaʻe e Coomassie-stained electrophoresis ma kahi 16% SDS-polyacrylamide gel.Ua helu ʻia ka manaʻo o ka protein e ka absorbance ma 280 nm me ka hoʻohana ʻana i ke kānāwai Beer-Lambert a me ka coefficient molar extinction coefficient.
Hoʻomaʻemaʻe ʻia ʻo SPACA6 i ka pō e kūʻē i ka 10 mM sodium phosphate, pH 7.4 a me 150 mM NaF a hoʻoheheʻe ʻia i ka 0.16 mg/mL ma mua o ka nānā ʻana e CD spectroscopy.Ua hōʻiliʻili ʻia ka nānā ʻana i nā CD me ka lōʻihi o ka nalu o 185 a i ka 260 nm ma kahi spectropolarimeter Jasco J-1500 me ka hoʻohana ʻana i nā cuvettes quartz me kahi 1 mm ka lōʻihi o ke ala optical (Helma) ma 25 ° C ma ka wikiwiki o 50 nm / min.Ua hoʻoponopono ʻia ka kikoʻī CD, ua hoʻololi ʻia i ke koena ellipticity (θMRE) ma nā degere cm2/dmol:
kahi MW ke kaumaha molekala o kēlā me kēia hāpana ma Da;ʻO N ka helu o nā ʻakika amino;ʻO θ ka ellipticity i nā miledegrees;d pili i ka lōʻihi o ke ala ʻike ma ke knm;ka nui o ka protein ma nā ʻāpana.