304 igwe anaghị agba nchara tubing kemịkalụ kemịkalụ, Ọdịdị nke SPACA6 ectodomain nwere nnukwu ezinụlọ echekwara nke protein jikọtara ya na ngwakọta gamete.

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SPACA6 bụ protein n'elu sperm gosipụtara nke dị oke mkpa maka njikọta gamete n'oge mmeputakwa mmekọahụ.N'agbanyeghị ọrụ a dị mkpa, a na-aghọtachaghị ọrụ SPACA6.Anyị na-akọwapụta usoro kristal nke mpaghara extracellular nke SPACA6 na mkpebi nke 2.2 Å, na-ekpughe protein ngalaba abụọ mejupụtara ngwugwu anọ na Ig-like β-sandwiches jikọtara ya na quasi-flexible linkers.Ihe owuwu a yiri IZUMO1, protein ọzọ jikọrọ gamete fusion, na-eme SPACA6 na IZUMO1 ndị mebere ndị otu nnukwu ezinụlọ nke protein ndị metụtara fatịlaịza na-ezo aka na nke a dị ka ezinụlọ IST superfamily.A kọwapụtara ezinụlọ superfamily IST site na ngwugwu helix anọ gbagọrọ agbagọ na otu ụzọ CXXC nwere njikọ disulfide.Nchọgharị AlphaFold dabere na nhazi nke proteome mmadụ chọpụtara ndị otu protein agbakwunyere na nnukwu ezinụlọ a;nke ọma, ọtụtụ n'ime protein ndị a na-etinye aka na ngwakọta gamete.Usoro SPACA6 na mmekọrịta ya na ndị ọzọ so na ezinụlọ superfamily IST na-enye njikọ na-efu efu na ihe ọmụma anyị gbasara ngwakọta gamete mammalian.
Ndụ mmadụ ọ bụla na-amalite site na haploid gametes abụọ dị iche iche: spam nna na akwa nne.Nke a spam bụ onye mmeri nke oké nhọrọ usoro n'oge nke ọtụtụ nde sperm cell na-agafe na nwanyi genital tract, merie dị iche iche mgbochi1 na-enweta capacitation, nke enhances ha motility na usoro nke elu components2,3,4.Ọ bụrụgodị na spam na oocyte chọtara ibe ha, usoro ahụ agwụbeghị.Okirikiri mkpụrụ ndụ cumulus na ihe mgbochi glycoprotein a na-akpọ zona pellucida gbara oocyte gburugburu, bụ nke spam ga-esi na ya gafere iji banye n'ime oocyte.Spermatozoa na-eji ngwakọta nke ụmụ irighiri ihe nkedo elu na membran metụtara na enzymes zoro ezo iji merie ihe mgbochi ikpeazụ ndị a5.A na-echekwa ụmụ irighiri ihe ndị a na enzymes n'ime akpụkpọ ahụ dị n'ime na matrix acrosomal ma chọpụta mgbe akpụkpọ ahụ dị n'èzí nke spam na-agbaze n'oge mmeghachi omume acrosomal6.Nzọụkwụ ikpeazụ n'ime njem a siri ike bụ mmemme njikọta spam na akwa, nke mkpụrụ ndụ abụọ ahụ na-ejikọta akpụkpọ ahụ ha ka ọ bụrụ otu diploid organism7.Ọ bụ ezie na usoro a na-agbawa agbawa n'ime ọmụmụ mmadụ, a ghọtaghị mmekọrịta dị mkpa nke mkpụrụ ndụ ihe nketa.
Na mgbakwunye na fatịlaịza nke gametes, a mụọla kemịkalụ nke ngwakọta nke abụọ lipid bilayers.N'ozuzu, njikọta akpụkpọ ahụ bụ usoro na-adịghị mma nke na-achọ ka onye na-emepụta protein na-eme mgbanwe mgbanwe nhazi nke na-eme ka akpụkpọ ahụ abụọ dịkwuo nso, na-emebi ọganihu ha na-akpata fusion8,9.A maara ihe na-eme ka protein ndị a dị ka fusogens ma hụ na a na-ahụ ya n'ime usoro mgbagwoju anya.A na-achọrọ ha maka ịbanye n'ime sel ndị ọbịa (dịka, gp160 na HIV-1, spike na coronaviruses, hemagglutinin na nje influenza) 10,11,12 placental (syncytin) 13,14,15 na gamete-forming fusions na ala eukaryotes. HAP2/GCS1 na osisi, protists na arthropods) 16,17,18,19.Fusogens maka gametes ụmụ mmadụ ka achọpụtabeghị, ọ bụ ezie na egosila ọtụtụ protein dị oke mkpa maka njikọta gamete na ngwakọta.CD9 nke egosipụtara oocyte, protein transmembrane chọrọ maka njikọ nke òké na gametes mmadụ, bụ nke mbụ achọpụtara 21,22,23.Ọ bụ ezie na ọrụ ya ka na-edoghị anya, ọrụ dị na adhesion, nhazi nke adhesion foci na egg microvilli, na / ma ọ bụ nhazi ziri ezi nke proteins elu oocyte yiri 24,25,26.Protein abụọ a na-ahụkarị nke dị oke mkpa maka ngwakọta gamete bụ protein sperm IZUMO127 na protein oocyte JUNO28, na mkpakọrịta ha na-emekọrịta ihe bụ nzọụkwụ dị mkpa na njirimara gamete na adhesion tupu njikọ.Nwoke Izumo1 knockout ụmụ oke na nwanyi Juno knockout ụmụ oke bụ kpamkpam akpali mmasị, na ụdị spam na-abanye na perivitelline ohere ma gametes adịghị fuse.N'otu aka ahụ, e belatara mkparịta ụka mgbe a na-eji ọgwụ mgbochi IZUMO1 ma ọ bụ JUNO27,29 agwọ ọrịa gametes n'ime mmadụ in vitro fatịlaịza nnwale.
N'oge na-adịbeghị anya, a chọpụtala otu protein sperm-egosipụta phenotypically yiri IZUMO1 na JUNO20,30,31,32,33,34,35.Achọpụtara protein 6 (SPACA6) nke jikọtara spam na sperm acrosomal dị ka ihe dị mkpa maka fatịlaịza na nnukwu ọmụmụ mutagenesis murine.Ntinye nke transgene n'ime mkpụrụ ndụ Spaca6 na-emepụta spermatozoa na-enweghị ike ime, ọ bụ ezie na spermatozoa ndị a na-abanye na oghere perivitelline 36.Ọmụmụ knockout na-esote na ụmụ oke kwadoro na Spaca6 chọrọ maka gamete fusion 30,32.A na-egosipụta SPACA6 fọrọ nke nta ka ọ bụrụ nanị na testes ma nwee usoro nhazi nke yiri nke IZUMO1, ya bụ n'ime intima nke spermatozoa tupu mmeghachi omume acrosomal, wee kwaga na mpaghara equatorial mgbe mmeghachi omume acrosomal 30,32 gasịrị.Spaca6 homologues dị n'ụdị dị iche iche nke mammals na ndị ọzọ eukaryotes 30 na mkpa ya maka mmadụ gamete fusion ka egosipụtara site na mgbochi nke fatịlaịza mmadụ na vitro site na iguzogide SPACA6 30.N'adịghị ka IZUMO1 na JUNO, nkọwa nke nhazi, mmekọrịta, na ọrụ nke SPACA6 ka edoghị anya.
Iji ghọta nke ọma usoro dị n'okpuru njikọ nke spam na àkwá mmadụ, nke ga-eme ka anyị mara ihe ga-eme n'ọdịnihu na nhazi ezinụlọ na ọgwụgwọ ọmụmụ, anyị mere SPACA6 structural and biochemical studies.Ọdịdị kristal nke ngalaba extracellular nke SPACA6 na-egosi ngwugwu anọ-helical (4HB) na ngalaba immunoglobulin-dị ka (Ig-dị ka) jikọtara site na mpaghara ndị na-agbanwe agbanwe.Dị ka e buru n'amụma na ọmụmụ ndị gara aga, 7,32,37 ngalaba nhazi nke SPACA6 yiri nke mmadụ IZUMO1, na protein abụọ ahụ na-ekerịta ihe na-adịghị ahụkebe: 4HB na elu triangular helical na otu ụzọ CXXC jikọtara disulfide.Anyị na-atụ aro na IZUMO1 na SPACA6 ugbu a na-akọwapụta nnukwu ezinụlọ protein nwere njikọ chiri anya nke jikọtara ya na ngwakọta gamete.N'iji atụmatụ pụrụ iche nke ezinụlọ karịrị akarị, anyị mere nchọcha ike maka proteome structural human AlphaFold, na-achọpụta ndị ọzọ so na nnukwu ezinụlọ a, gụnyere ọtụtụ ndị otu na-etinye aka na ngwakọta gamete na/ma ọ bụ fatịlaịza.Ugbu a, ọ dị ka enwere mkpokọta nhazi na nnukwu ezinụlọ nke protein jikọtara ya na ngwakọta gamete, na usoro anyị na-enye maapụ molekụla nke akụkụ a dị mkpa nke usoro ngwakọta gamete mmadụ.
SPACA6 bụ protein transmembrane nke nwere otu ngafe nwere otu glycan jikọtara ya na njikọ disulfide isii putative (Figures S1a na S2).Anyị gosipụtara ngalaba extracellular nke mmadụ SPACA6 (nke fọdụrụ 27-246) na mkpụrụ ndụ Drosophila S2 ma mee ka protein dị ọcha site na iji nickel affinity, mgbanwe cation, na chromatography nke mwepu (Fig S1b).SPACA6 ectodomain a sachara nke ọma kwụsiri ike yana otu.Nyocha site na iji chromatography mwepu nha jikọtara ya na mgbasa ọkụ polygonal (SEC-MALS) kpughere otu ọnụ ọgụgụ kacha elu nwere nha molekụla gbakọrọ nke 26.2 ± 0.5 kDa (Fig S1c).Nke a kwekọrọ na nha SPACA6 monomeric ectodomain, na-egosi na oligomerization emeghị n'oge a na-eme ka ọ dị ọcha.Na mgbakwunye, okirikiri dichroism (CD) spectroscopy kpughere usoro α / β agwakọtara na ebe agbaze nke 51.3 Celsius C (Fig S1d, e).Deconvolution nke ụdịdị CD dị iche iche kpughere 38.6% α-helical na 15.8% β-stranded ọcha (Ọnụ ọgụgụ S1d).
SPACA6 ectodomain bụ crystallized site na iji random matrix seeding38 rụpụtara na data setịpụrụ na mkpebi nke 2.2 Å (Table 1 na Figure S3).N'iji nchikota nke nchikota nke ihe ngbanwe molekul na SAD phasing data na bromide ikpughe maka nhazi nhazi (Table 1 na Figure S4), ihe nchacha nke ikpeazụ nwere ihe fọduru 27-246.N'oge ekpebie ihe owuwu a, enweghị nnwale ma ọ bụ ihe nrụpụta AlphaFold dị.Ihe SPACA6 ectodomain na-atụ 20 Å × 20 Å × 85 Å, nwere helices asaa na β-strands itoolu, ma nwee akwa akwa elongated tertiary fold kwadoro site na njikọ disulfide isii (Fig 1a, b).Njupụta eletrọn na-adịghị ike na njedebe nke yinye akụkụ Asn243 na-egosi na ihe fọdụrụ a bụ glycosylation njikọ N.Ihe owuwu ahụ nwere ngalaba abụọ: ngwugwu N-terminal anọ-helix (4HB) na ngalaba C-terminal Ig nke nwere mpaghara mkpuchi etiti n'etiti ha (Fig 1c).
Ihe owuwu nke ngalaba extracellular nke SPACA6.Eserese nke ngalaba extracellular nke SPACA6, agba nke yinye site na N ruo C-terminus site na acha anụnụ anụnụ ruo na-acha ọbara ọbara.Cysteines na-etinye aka na njikọ disulfide na-apụta ìhè na magenta.b Topology nke ngalaba extracellular nke SPACA6.Jiri otu atụmatụ agba dị na eserese 1a.c SPACA6 ngalaba extracellular.Ihe eserese ngalaba 4HB, hinge na Ig-dị ka nke nwere agba oroma, akwụkwọ ndụ akwụkwọ ndụ na acha anụnụ anụnụ, n'otu n'otu.A naghị adọta akwa akwa na nha.
Ngalaba 4HB nke SPACA6 na-agụnye isi ihe anọ (helices 1-4), nke a na-ahazi n'ụdị helix helix (Fig 2a), na-agbanwe n'etiti mmekọrịta antiparallel na parallel (Fig 2b).A na-edobere obere helix otu ntụgharị (helix 1′) kwụ ọtọ na ngwugwu ahụ, na-akpụ triangle nwere helices 1 na 2. Nke a triangle na-agbanwe ntakịrị n'ime ngwugwu gbagọrọ agbagọ nke nnukwu mkpọkọ nke helices 3 na 4. Fig. 2a).
4HB N-terminal warara eserese.b Nlele elu nke ngwugwu nke helices anọ, heliks ọ bụla gosipụtara na-acha anụnụ anụnụ na N-terminus na uhie gbara ọchịchịrị na C-terminus.c Eserese wheel spiral nke dị n'elu maka 4HB, nke egosiri nke ọ bụla dị ka okirikiri nke ejiri koodu amino acid nwere otu mkpụrụedemede;naanị amino acid anọ dị n'elu wheel ka a na-agụ.Ihe fọdụrụ na-abụghị polar bụ agba edo edo, ihe fọdụrụ anaghị akwụ ụgwọ na-acha akwụkwọ ndụ akwụkwọ ndụ, ihe fọdụrụ ebubo nke ọma na-acha anụnụ anụnụ, ihe fọdụrụ ebubo na-adịghị mma na-acha ọbara ọbara.d Ihu triangular nke ngalaba 4HB, nwere 4HBs na oroma na mkpirisi na akwụkwọ ndụ akwụkwọ ndụ.Ntinye abụọ a na-egosi njikọ disulfide nwere ụdị mkpanaka.
4HB gbadoro anya na isi hydrophobic nke dị n'ime nke mejuputara nke aliphatic na aromatic residues (Fig 2c).Isi ihe nwere njikọ disulfide n'etiti Cys41 na Cys55 na-ejikọta helices 1 na 2 ọnụ na triangle dị elu (Fig 2d).E mebere njikọ disulfide abụọ ọzọ n'etiti motif CXXC na Helix 1′ na ihe ọzọ CXXC achọtara n'ọnụ ọnụ β-hairpin na mpaghara hinge (Fig. 2d).Ihe fọdụrụ nke arginine na-agbanwe agbanwe nke nwere ọrụ amaghị ama (Arg37) dị n'ime triangle oghere nke helices 1′, 1, na 2. Aliphatic carbon atom Cβ, Cγ, na Cδ Arg37 na-emekọ ihe na isi hydrophobic, na otu guanidine ya na-agagharị cyclically. n'etiti helices 1 'na 1 site na mmekọrịta n'etiti Thr32 azụ ọkpụkpụ na n'akụkụ yinye (Fig. S5a,b).Tyr34 gbatịrị n'ime oghere na-ahapụ obere oghere abụọ nke Arg37 nwere ike iji ihe mgbaze mee ihe.
Ig-dị ka β-sanwichi ngalaba bụ nnukwu ezinụlọ nke ndị na-edozi ahụ na-ekekọrịta akụkụ nke abụọ ma ọ bụ karịa multi-stranded amphipathic β-sheets na-emekọrịta site na isi hydrophobic 39. Ngalaba C-terminal Ig-dị ka ngalaba nke SPACA6 nwere otu ụkpụrụ ahụ. na mejupụtara abụọ n'ígwé (Fig. S6a).Mpempe akwụkwọ 1 bụ β-ibé akwụkwọ nke eriri anọ (eriri D, F, H, na I) ebe strands F, H, na m na-etolite nhazi ihe mgbochi, na strands I na D na-enwe mmekọrịta yiri ya.Tebụl 2 bụ obere mpempe akwụkwọ beta nwere eriri abụọ na-emegide myirịta (eriri E na G).A hụrụ njikọ disulfide dị n'ime n'etiti C-terminus nke yinye E na etiti nke agbụ H (Cys170-Cys226) (Fig. S6b).Njikọ disulfide a yiri njikọ disulfide dị na ngalaba β-sandwich nke immunoglobulin40,41.
Mpempe akwụkwọ β-akara anọ na-agbagọ n'ogologo ya dum, na-eme akụkụ asymmetrical nke dị iche na ọdịdị na electrostatics.The thinner onu bụ ewepụghị hydrophobic gburugburu ebe obibi n'elu nke pụtara pụtara tụnyere ndị fọdụrụ uneven na electrostatically iche iche na-ebupụta na SPACA6 (Fig. S6b, c).A halo nke ọkpụkpụ azụ carbonyl/amino ekpughere na ụdọ akụkụ polar gbara gburugburu hydrophobic elu (Fig. S6c).A na-ekpuchi oke oke ahụ site na akụkụ helical capped nke na-egbochi akụkụ N-terminal nke isi hydrophobic wee mepụta njikọ hydrogen atọ na otu polar mepere emepe nke ọkpụkpụ azụ azụ F (Fig. S6d).Akụkụ C-terminal nke nsọtụ a na-etolite nnukwu akpa nke nwere isi hydrophobic nke ekpughere nke ọma.A na-eji ụgwọ dị mma gbara akpa ahụ gburugburu n'ihi nhazi atọ nke abụọ arginine residues (Arg162-Arg221, Arg201-Arg205 na Arg212-Arg214) na Central histidine (His220) (Figure S6e).
Mpaghara hinge bụ akụkụ dị mkpirikpi n'etiti ngalaba helical na ngalaba Ig-dị ka ngalaba, nke nwere otu mgbochi atọ stranded β-layer (eriri A, B na C), obere 310 helix, na ọtụtụ ogologo oge helical ngalaba.(Fig. S7).Otu netwọk nke kọntaktị covalent na electrostatic dị na mpaghara hinge na-egosi na ọ na-eme ka nghazi dị n'etiti 4HB na ngalaba Ig-dị ka ya.Enwere ike kewaa netwọk ahụ ụzọ atọ.Akụkụ nke mbụ gụnyere ihe abụọ CXXC motifs (27CXXC30 na 139CXXC142) nke na-etolite otu ụzọ disulfide n'etiti β-hairpin na hinge na 1′ helix na 4HB.Akụkụ nke abụọ na-agụnye mmekọrịta electrostatic n'etiti ngalaba Ig-dị ka na mgbanaka.Glu132 n'ime hinge na-etolite akwa mmiri nwere Arg233 na ngalaba Ig-dị ka Arg135 na mgbanaka.Akụkụ nke atọ gụnyere njikọ dị n'etiti ngalaba Ig-dị ka mpaghara na mpaghara hinge.Njikọ disulfide abụọ (Cys124-Cys147 na Cys128-Cys153) jikọọ hinge loop na njikọ nke na-eme ka mmekọrịta electrostatic dị n'etiti Gln131 na otu ọrụ ọkpụkpụ azụ, na-enye ohere ịnweta ngalaba mbụ Ig-like.yinye.
A na-eji usoro nke SPACA6 ectodomain na ihe owuwu nke 4HB na Ig-dị ka ngalaba iji chọọ ndekọ ahaziri ahazi na nchekwa data protein 42.Anyị chọpụtara egwuregwu nwere akara Dali Z dị elu, obere ndapụta ọkọlọtọ, yana nnukwu akara LALI (nke ikpeazụ bụ ọnụ ọgụgụ nke ihe fọrọ nke nta ka ọ bụrụ ahaziri).Ọ bụ ezie na 10 nke mbụ sitere na nchọta ectodomain zuru ezu (Table S1) nwere Z-score nke> 842 a na-anakwere, ọchụchọ maka 4HB ma ọ bụ Ig-dị ka ngalaba naanị gosiri na ọtụtụ n'ime hits ndị a kwekọrọ na β-sandwichi naanị.ogige ebe niile a na-achọta n'ọtụtụ protein.Nchọ atọ niile dị na Dali weghachiri naanị otu nsonaazụ: IZUMO1.
Ọ dịla anya a tụrụ aro na SPACA6 na IZUMO1 na-ekerịta myirịta nhazi7,32,37.Ọ bụ ezie na ectodomains nke protein abụọ a na-ejikọta gamete fusion na-ekekọrịta naanị njirimara usoro 21% (Ọgụgụ S8a), ihe akaebe dị mgbagwoju anya, gụnyere usoro njikọ disulfide echekwara na ngalaba C-terminal Ig-dị ka nke a na-ebu amụma na SPACA6, kwere ka mgbalị mbụ iji wuo a. ụdị homology nke A òké SPACA6 iji IZUMO1 dị ka template37.Ihe owuwu anyị na-akwado amụma ndị a ma na-egosi oke myirịta n'ezie.N'ezie, ihe owuwu SPACA6 na IZUMO137,43,44 na-ekerịta otu ụlọ ọrụ abụọ (Fig. S8b) na mpaghara 4HB na Ig-like β-sandwich jikọtara site na mpaghara hinge (Fig. S8c).
IZUMO1 na SPACA6 4HB nwere ọdịiche dị iche iche site na ngwugwu gburugburu.Ụdị 4HB, dị ka ndị ahụ dị na SNARE protein complexes na-etinye aka na endosomal fusion 45,46, nwere helices na-eme ka ọkpụkpụ na-adịgide adịgide na-adịgide adịgide gburugburu axis Central 47. N'ụzọ dị iche, ngalaba helical na ma IZUMO1 na SPACA6 gbagọrọ agbagọ, na-agbanwe agbanwe na curvature. mbukota na-ezighi ezi (Foto S8d).Ntugharị ahụ, ikekwe site na triangle kpụrụ site na helices 1′, 1 na 2, ka edobere na IZUMO1 na SPACA6 wee kwụsie ike site na otu CXXC motif na helix 1′.Otú ọ dị, njikọ disulfide ndị ọzọ a chọtara na SPACA6 (Cys41 na Cys55 na-ejikọta helices 1 na 2 n'elu) na-emepụta apex dị nkọ na triangle apex, na-eme ka SPACA6 gbagọrọ agbagọ karịa IZUMO1, na-enwekwu triangles oghere.Na mgbakwunye, IZUMO1 enweghị Arg37 hụrụ n'etiti oghere a na SPACA6.N'ụzọ dị iche, IZUMO1 nwere ihe a na-ahụkarị hydrophobic isi nke aliphatic na aromatic residues.
IZUMO1 nwere ngalaba Ig-dị ka nke nwere eriri abụọ nwere eriri abụọ yana β-sheet43.Uro ọzọ dị na IZUMO1 na-edochi eriri igwe na SPACA6, nke na-emekọrịta ihe na eriri F iji gbochie njikọ nke hydrogen azụ na eriri ahụ.Isi ihe na-atọ ụtọ ntụnyere bụ ụgwọ elu amụma nke ngalaba Ig dị ka protein abụọ ahụ.A na-ebufe elu IZUMO1 na-adịghị mma karịa elu SPACA6.Ụgwọ ọzọ dị n'akụkụ C-terminus chere ihu n'ahụ akpụkpọ ahụ spam.Na SPACA6, otu mpaghara ahụ na-anọpụ iche ma ọ bụ na-akwụ ụgwọ nke ọma (Fig S8e).Dịka ọmụmaatụ, elu hydrophobic (akụkụ dị nro) na olulu ndị dị mma (ọnụ ọnụ) na SPACA6 na-ebubo ebubo na IZUMO1.
Ọ bụ ezie na mmekọrịta na ihe nhazi nke abụọ dị n'etiti IZUMO1 na SPACA6 na-echekwa nke ọma, nhazi nhazi nke ngalaba Ig-dị ka ngalaba abụọ ahụ dị iche na nhazi ha n'ozuzu ha na ibe ha (Fig S9).A na-atụgharị ngwugwu gburugburu nke IZUMO1 gbasara β-sandwichi, na-ekepụta ọdịdị "boomerang" nke akọwara na mbụ na ihe dịka 50 ° site na etiti etiti.N'ụzọ dị iche, ọkụ ọkụ na SPACA6 na-atụgharị ihe dịka 10 ° n'akụkụ nke ọzọ.Ọdịiche dị na nghazi ndị a nwere ike ịbụ n'ihi ọdịiche dị na mpaghara hinge.N'ọkwa usoro nke mbụ, IZUMO1 na SPACA6 na-ekerịta obere usoro myirịta na mgbanaka, ma e wezụga cysteine, glycine, na aspartic acid residues.N'ihi ya, hydrogen bond na electrostatic netwọk dị nnọọ iche.IZUMO1 na SPACA6 na-ekerịta ihe nhazi nke abụọ β-ibé, ọ bụ ezie na agbụ dị na IZUMO1 dị ogologo ogologo na 310 helix (helix 5) bụ ihe pụrụ iche na SPACA6.Esemokwu ndị a na-ebute ụzọ ngalaba dị iche iche maka protein abụọ yiri ya.
Ọchịchọ ihe nkesa Dali anyị kpughere na SPACA6 na IZUMO1 bụ naanị ihe arụpụtara nnwale abụọ echekwara na nchekwa data protein nwere ụdị 4HB a (Table S1).N'oge na-adịbeghị anya, DeepMind (Alphabet/Google) ewepụtala AlphaFold, sistemụ akwara akwara nke nwere ike ibu amụma n'ụzọ ziri ezi na usoro 3D nke protein site na usoro izizi48.N'oge na-adịghị anya ka anyị dozichara usoro SPACA6, ewepụtara nchekwa data AlphaFold, na-enye ụdị usoro amụma na-ekpuchi 98.5% nke protein niile dị na proteome mmadụ48,49.N'iji nhazi SPACA6 anyị edoziziri dị ka ihe nchọta, nchọpụta homology nhazi maka ihe nlere anya na AlphaFold human proteome achọpụtara ndị na-aga ime nwere myiri nhazi na SPACA6 na IZUMO1.N'iburu n'ụzọ ziri ezi nke AlphaFold na-ebu amụma SPACA6 (Fig. S10a) - karịsịa 1.1 Å ctodomain ectodomain tụnyere usoro anyị kpebiri (Fig S10b) - anyị nwere ike inwe obi ike na egwuregwu SPACA6 achọpụtara nwere ike ịbụ eziokwu.
Na mbụ, PSI-BLAST chọrọ ụyọkọ IZUMO1 nwere protein atọ ndị ọzọ metụtara spam: IZUMO2, IZUMO3, na IZUMO450.AlphaFold buru amụma na protein ezinụlọ IZUMO ndị a na-abanye na ngalaba 4HB nwere otu usoro njikọ disulfide dị ka IZUMO1 (Ọgụgụ 3a na S11), n'agbanyeghị na ha enweghị ngalaba Ig.A na-eche na IZUMO2 na IZUMO3 bụ protein akpụkpọ anụ nwere otu akụkụ dị ka IZUMO1, ebe IZUMO4 yiri ka e zoro ezo.Achọpụtabeghị ọrụ nke IZUMO 2, 3, na 4 protein na ngwakọta gamete.A maara IZUMO3 ka ọ na-ekere òkè na acrosome biogenesis n'oge mmepe spam51 na protein IZUMO achọpụtala na ọ na-emepụta mgbagwoju anya50.Nchekwa nke protein IZUMO n'ime mammals, anụ ufe, na amphibians na-atụ aro na ọrụ ha nwere ike kwekọrọ na nke protein ndị ọzọ na-ejikọta gamete-fusion, dị ka DCST1/2, SOF1, na FIMP.
Eserese nke ngalaba ihe owuwu nke ezinụlọ superfamily IST, nwere 4HB, hinge, na ngalaba Ig nke agbapụtara na oroma, akwụkwọ ndụ akwụkwọ ndụ na acha anụnụ anụnụ, otu.IZUMO4 nwere mpaghara C-terminal pụrụ iche nke yiri oji.Egosiputa njikọ disulfide enwetara na ntinye site na ahịrị siri ike na nwere ntụpọ, n'otu n'otu.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) na-egosipụta n'otu agba agba dị ka panel A. Disulfide bond na-egosi na magenta.TMEM95, IZUMO2 na IZUMO3 transmembrane helices adịghị egosi.
N'adịghị ka protein IZUMO, a na-eche na protein SPACA ndị ọzọ (ya bụ, SPACA1, SPACA3, SPACA4, SPACA5, na SPACA9) dị iche na SPACA6 (Fig S12).Naanị SPACA9 nwere 4HB, mana a naghị atụ anya inwe otu ntụzịaka mgbochi mgbochi ma ọ bụ otu njikọ disulfide dị ka SPACA6.Naanị SPACA1 nwere ngalaba Ig yiri ya.AlphaFold buru amụma na SPACA3, SPACA4 na SPACA5 nwere usoro dị iche kpamkpam karịa SPACA6.N'ụzọ na-akpali mmasị, SPACA4 makwaara na ọ na-ekere òkè na fatịlaịza, mana ọ ka ukwuu karịa SPACA6, kama na-eme ka mmekọrịta dị n'etiti spam na oocyte zona pellucida52.
Ọchịchọ AlphaFold anyị chọtara egwuregwu ọzọ maka IZUMO1 na SPACA6 4HB, TMEM95.TMEM95, otu protein transmembrane sperm-kpọmkwem, na-eme ka ụmụ oke nwoke ghara ịmụ nwa mgbe a kwụsịrị 32,33.Spermatozoa enweghị TMEM95 nwere ọdịdị ọdịdị nkịtị, motility, na ike ịbanye na zona pellucida ma jikọta na akpụkpọ akwa akwa, mana enweghị ike ijikọ ya na akpụkpọ ahụ oocyte.Nnyocha ndị gara aga egosila na TMEM95 na-ekerịta myirịta nhazi na IZUMO133.N'ezie, ihe nlereanya AlphaFold kwadoro na TMEM95 bụ 4HB nwere otu ụzọ CXXC motifs dị ka IZUMO1 na SPACA6 na otu ihe mgbakwunye disulfide n'etiti helices 1 na 2 dị na SPACA6 (Fig. 3a na S11).Ọ bụ ezie na TMEM95 enweghị ngalaba Ig, ọ nwere mpaghara nwere usoro njikọ disulfide yiri mpaghara SPACA6 na IZUMO1 hinge (Fig 3b).N'oge e bipụtara ihe odide a, ihe nkesa preprint kọrọ nhazi nke TMEM95, na-akwado nsonaazụ AlphaFold53.TMEM95 yiri SPACA6 na IZUMO1 na evolushọn echekwabara ugbua na amphibians (Fig 4 na S13).
Nchọta PSI-BLAST jiri NCBI SPACA6, IZUMO1-4, TMEM95, DCST1, DCST2, FIMP, na ọdụ data SOF1 iji chọpụta ọnọdụ nke usoro ndị a na osisi nke ndụ.Egosighị anya n'etiti isi alaka ụlọ ọrụ na nha.
Myirịta n'ozuzu ya dị egwu n'etiti SPACA6 na IZUMO1 na-egosi na ha na-etolite ndị otu nnukwu ezinụlọ echekwabara nke gụnyere protein TMEM95 na IZUMO 2, 3 na 4.ndị ama ama: IZUMO1, SPACA6 na TMEM95.N'ihi na ọ bụ naanị mmadụ ole na ole nwere ngalaba Ig-dị ka, akara ngosi nke IST superfamily bụ ngalaba 4HB, nke nwere njirimara pụrụ iche nke jikọrọ protein ndị a niile: 1) Ejikọtara 4HB na helices ahaziri n'usoro ihe mgbochi / nke yiri ya (Fig). .5a), 2) ngwugwu ahụ nwere ihu triangular nke nwere helices abụọ n'ime ngwugwu na heliks kwụ ọtọ nke atọ (Fig. isi mpaghara (Fig 5c). A maara CXXC motif, nke dị na protein thioredoxin dị ka ọ na-arụ ọrụ. dị ka ihe mmetụta redox 54,55,56, ebe motif na ndị òtù ezinụlọ IST nwere ike jikọta ya na protein disulfide isomerases dị ka ERp57 na gamete fusion. A na-ejikọta ọrụ 57,58.
A na-akọwapụta ndị otu IST superfamily site na njirimara njirimara atọ nke ngalaba 4HB: helice anọ na-agbanwe n'etiti myirịta na ntụgharị ihu, ihu ba-triangular helical, yana ca CXXC okpukpu abụọ kpụrụ n'etiti obere ụmụ irighiri ihe.) N-terminal helixes (oroma) na mpaghara hinge β-hairpin (green).
N'inyere myirịta dị n'etiti SPACA6 na IZUMO1, a nwalere ike nke mbụ iji jikọta na IZUMO1 ma ọ bụ JUNO.Biolayer interferometry (BLI) bụ usoro njikọ nke dabere na kinetic nke ejirila na mbụ kọwaa mmekọrịta dị n'etiti IZUMO1 na JUNO.Mgbe incubation nke biotin-labeled sensọ na IZUMO1 dị ka bait na nnukwu mkpokọta nke JUNO analyte, a chọpụtara ihe mgbaàmà siri ike (Fig. S14a), na-egosi mgbanwe na-ejikọta na ọkpụrụkpụ nke biomaterial na-ejikọta na ọnụ ihe mmetụta.Ihe mgbaàmà ndị yiri ya (ntụgharị, JUNO jikọtara ya na ihe mmetụta dị ka ọnyà megide nyocha IZUMO1) (Fig. S14b).Enweghị mgbaàmà ọ bụla achọpụtara mgbe ejiri SPACA6 mee ihe nyocha megide IZUMO1 na-ejikọta ihe mmetụta ma ọ bụ JUNO (Figure S14a,b).Enweghị mgbaàmà a na-egosi na ngalaba extracellular nke SPACA6 anaghị emekọrịta ihe na ngalaba extracellular nke IZUMO1 ma ọ bụ JUNO.
N'ihi na nyocha BLI dabeere na biotinylation nke free lysine residues na protein bait, mgbanwe a nwere ike igbochi njikọ ma ọ bụrụ na ihe fọdụrụ lysine na-etinye aka na mmekọrịta.Na mgbakwunye, nghazi nke njikọ njikọ na ihe mmetụta nwere ike ịmepụta ihe mgbochi siri ike, ya mere, a na-emekwa nyocha mgbada na SPACA6 recombinant, IZUMO1 na JUNO ectodomains.N'agbanyeghị nke a, SPACA6 emeghị ka ọ pụta ìhè na IZUMO1 nke akara ya ma ọ bụ akara ya JUNO (Fig. S14c, d), na-egosi na ọ dịghị mmekọrịta kwekọrọ na nke a hụrụ na nyocha BLI.Dị ka njikwa dị mma, anyị kwadoro mmekọrịta nke JUNO na akara ya IZUMO1 (Figures S14e na S15).
N'agbanyeghị myirịta dị n'etiti SPACA6 na IZUMO1, enweghị ike nke SPACA6 iji jikọta JUNO abụghị ihe ijuanya.Elu nke mmadụ IZUMO1 nwere ihe karịrị 20 residues na-emekọ ihe na JUNO, gụnyere ihe ndị fọdụrụ na nke ọ bụla n'ime mpaghara atọ (ọ bụ ezie na ọtụtụ n'ime ha dị na mpaghara hinge) (Fig. S14f).N'ime ihe ndị a fọdụrụnụ, naanị otu ka echekwara na SPACA6 (Glu70).Ọ bụ ezie na ọtụtụ ngbanwe fọdụrụnụ na-ejigide ihe ndị dị na biochemical mbụ ha, ihe fọdụrụnụ Arg160 dị na IZUMO1 bụ nke Asp148 na-ezighị ezi na SPACA6 nọchiri ya;Ọmụmụ ihe gara aga egosila na ngbanwe Arg160Glu na IZUMO1 fọrọ nke nta ka ọ kwụsị kpamkpam na-ejikọta na JUNO43.Tụkwasị na nke ahụ, ọdịiche dị na nhazi ngalaba dị n'etiti IZUMO1 na SPACA6 mụbara elu elu nke saịtị JUNO nke mpaghara kwekọrọ na SPACA6 (Fig. S14g).
N'agbanyeghị mkpa a maara maka SPACA6 maka gamete fusion na myirịta ya na IZUMO1, SPACA6 apụtaghị na ọ nwere ọrụ njikọ JUNO kwekọrọ.Ya mere, anyị achọla ijikọ data nhazi anyị na ihe akaebe nke mkpa ọ dị site na usoro ndu evolushọn.Usoro nhazi nke SPACA6 homologues na-egosi ichekwa ihe owuwu a na-ahụkarị karịa anụ mamma.Dịka ọmụmaatụ, ihe fọdụrụ na cysteine ​​dị ugbu a ọbụna na ndị amphibians nwere njikọ dị anya (Fig 6a).N'iji ihe nkesa ConSurf, ọtụtụ data njigide nhazi usoro nke usoro 66 ka edobere n'elu SPACA6.Ụdị nyocha a nwere ike igosi ihe fọdụrụnụ echekwara n'oge evolushọn protein ma nwee ike igosi mpaghara ndị dị n'elu na-arụ ọrụ.
Usoro nhazi nke SPACA6 ectodomains sitere na ụdị iri na abụọ dị iche iche a kwadoro site na iji CLUSTAL OMEGA.Dị ka nyocha nke ConSurf si kwuo, a na-akanye ọkwá kachasị mma na-acha anụnụ anụnụ.A na-akọwapụta ihe fọdụrụ na Cysteine ​​​​na ọbara ọbara.A na-egosi oke ngalaba na ihe nhazi nke abụọ n'elu nhazi, ebe akụ na-egosi β-strands na ebili mmiri na-egosi helices.Ihe nchọpụta nnweta NCBI nwere usoro ndị a bụ: mmadụ (Homo sapiens, NP_001303901), mandrill (Mandrilus leucophaeus, XP_011821277), enwe capuchin (Cebus mimic, XP_017359366), ịnyịnya (Equus01352359366) 23 XP_032_034).), atụrụ (Ovis aries, XP_014955560), elephant (Loxodonta africana, XP_010585293), nkịta (Canis lupus familyis, XP_025277208), òké (Mus musculus, NP_001156381), Tasmanian devils (Sarc1ophilus) , 8) , 61_89 na Bullfrog (Bufo bufo, XP_040282113).Ọnụọgụ ahụ dabere n'usoro mmadụ.b Ngosipụta elu nke usoro SPACA6 nwere 4HB n'elu yana ngalaba Ig dị na ala, agba dabere na atụmatụ nchekwa sitere na sava ConSurf.Akụkụ kachasị mma echekwara bụ na-acha anụnụ anụnụ, akụkụ ndị a na-echekwa nke ọma na-acha ọcha, na nke kachasị mma na-acha odo odo.cysteine ​​odo odo.A na-egosi patches elu atọ nke na-egosipụta oke nchebe dị elu na ntinye akara akara 1, 2 na 3. A na-egosi eserese 4HB na ntinye n'elu aka nri (otu atụmatụ agba).
Ọdịdị SPACA6 nwere mpaghara elu atọ echekwara nke ukwuu (Fig 6b).Patch 1 na-agbatị 4HB na mpaghara hinge ma nwee àkwà mmiri CXXC disulfide abụọ echekwara, Arg233-Glu132-Arg135-Ser144 hinge network (Fig. S7), na ihe mpụta aromatic atọ echekwara (Phe31, Tyr73, Phe137).obosara obosara nke ngalaba Ig-dị ka (Fig. S6e), nke na-anọchi anya ọtụtụ ihe fọdụrụ n'ụzọ ziri ezi n'elu spam.N'ụzọ na-akpali mmasị, patch a nwere epitope mgbochi mmadụ nke egosiri na mbụ iji gbochie ọrụ SPACA6 30.Mpaghara 3 na-agbatị mgbanaka na otu akụkụ nke ngalaba Ig-dị ka;mpaghara a nwere prolines echekwara (Pro126, Pro127, Pro150, Pro154) na ihe fọdụrụ na-eche pola/ chajị.N'ụzọ dị ịtụnanya, ọtụtụ n'ime ihe ndị dị n'elu 4HB na-agbanwe nke ukwuu (Fig 6b), ọ bụ ezie na a na-echekwa n'ogige atụrụ ahụ dum SPACA6 homologue (dị ka e gosiri site na conservatism nke hydrophobic bundle core) na karịa IST superfamily.
Ọ bụ ezie na nke a bụ mpaghara kacha nta na SPACA6 nwere obere ihe nhazi nke abụọ a na-achọpụta, ọtụtụ mpaghara remnants (gụnyere mpaghara 3) na-echekwa nke ọma n'etiti SPACA6 homologues, nke nwere ike igosi na nhazi nke ngwugwu helical na β-sandwich na-ekere òkè.dị ka onye mgbanwe.Otú ọ dị, n'agbanyeghị nnukwu njikọ hydrogen na netwọk electrostatic dị na mpaghara hinge nke SPACA6 na IZUMO1, a pụrụ ịhụ ihe àmà nke mgbanwe dị n'ime ya na nhazi nke ọtụtụ ihe owuwu a kwadoro nke IZUMO137,43,44.Ndozi nke ngalaba nke onye ọ bụla gbakọtara nke ọma, mana nhazi nke ngalaba ndị metụtara ibe ha dịgasị iche site na 50 ° ruo 70 ° site na etiti etiti (Fig S16).Iji ghọta mgbanwe mgbanwe nke SPACA6 na ngwọta, e mere nnwale SAXS (Fig. S17a,b).Ab initio nwughari nke SPACA6 ectodomain kwekọrọ na mkpara crystal Ọdịdị (Fig. S18), ọ bụ ezie na Kratky ibé gosiri ụfọdụ ogo mgbanwe (Fig. S17b).Nkwekọrịta a dị iche na IZUMO1, nke protein na-adịghị ejikọta ya na-ewere ọdịdị boomerang ma na lattice na ngwọta43.
Iji chọpụta kpọmkwem mpaghara na-agbanwe agbanwe, hydrogen-deuterium Exchange mass spectroscopy (H-DXMS) mere na SPACA6 ma jiri ya tụnyere data enwetara na IZUMO143 (Fig. 7a,b).SPACA6 doro anya karịa mgbanwe karịa IZUMO1, dị ka egosipụtara site na mgbanwe deuterium dị elu n'ime usoro ahụ mgbe 100,000 s nke mgbanwe gasịrị.N'ime ụlọ abụọ ahụ, akụkụ C-terminal nke mpaghara hinge na-egosi mgbanwe mgbanwe dị elu, nke nwere ike na-enye ohere ntụgharị oke nke ngalaba 4HB na Ig-dị ka ngalaba n'akụkụ ibe ha.N'ụzọ na-akpali mmasị, akụkụ C-terminal nke SPACA6 hinge, nke nwere ihe fọdụrụnụ 147CDLPLDCP154, bụ mpaghara 3 nke echekwara nke ọma (Fig 6b), ikekwe na-egosi na mgbanwe interdomain bụ ihe evolushọn echekwara nke SPACA6.Dị ka nyocha nke mgbanwe mgbanwe, data CD thermal melt data gosiri na SPACA6 (Tm = 51.2 ° C) adịghị ike karịa IZUMO1 (Tm = 62.9 ° C) (Fig. S1e na S19).
ihe onyonyo H-DXMS nke SPACA6 na b IZUMO1.Achọpụtara mgbanwe pasentị deuterium n'oge egosipụtara.A na-egosi ọkwa mgbanwe hydrogen-deuterium site na agba n'ọ̀tụ̀tụ̀ gradient site na acha anụnụ anụnụ (10%) ruo uhie (90%).Igbe ojii na-anọchite anya mpaghara mgbanwe dị elu.Oke nke 4HB, hinge na ngalaba Ig-dị ka a hụrụ na nhazi kristal ka egosiri n'elu usoro izizi.Atụpụtara ọkwa mgbanwe Deuterium na 10 s, 1000 s na 100,000 s na eserese mpempe akwụkwọ nke etinyere n'elu mbara igwe nke SPACA6 na IZUMO1.Akụkụ nke ihe owuwu nwere ọkwa mgbanwe deuterium n'okpuru 50% nwere agba ọcha.Mpaghara dị n'elu 50% H-DXMS mgbanwe na-acha n'ọ̀tụ̀tụ̀ gradient.
Iji CRISPR/Cas9 na mouse gene knockout atumatu mkpụrụ ndụ ihe nketa emela ka amata ọtụtụ ihe dị mkpa maka njikọta spam na akwa na njikọta.Ewezuga mmekọrịta agwara nke ọma nke usoro IZUMO1-JUNO na CD9, ọtụtụ n'ime protein ndị jikọtara ya na ngwakọta gamete na-anọgide na-arụ ọrụ na-arụ ọrụ enigmatic.Nhazi nke biophysical na nhazi nke SPACA6 bụ akụkụ ọzọ nke ihe mgbagwoju anya molecular adhesion/fusion n'oge njikọta spam nwoke na nwanyị.
SPACA6 na ndị ọzọ so na IST superfamily yiri ka echekwabara nke ukwuu n'ime anụ mamma nakwa dị ka nnụnụ n'otu n'otu, anụ ufe, na amphibians;N'ezie, a na-eche na SPACA6 dị mkpa ọbụna maka fatịlaịza na zebrafish 59. Nkesa a yiri protein ndị ọzọ na-ejikọta gamete fusion dị ka DCST134, DCST234, FIMP31, na SOF132, na-atụ aro na ihe ndị a bụ HAP2-erughị eru (nakwa). mara dị ka GCS1) protein ndị na-ahụ maka ọrụ catalytic nke ọtụtụ ndị protists., osisi, na arthropods.Protein fusion fusion 60, 61. N'agbanyeghị myirịta siri ike dị n'etiti SPACA6 na IZUMO1, knockout nke mkpụrụ ndụ ihe nketa na-edobe nke ọ bụla n'ime protein ndị a butere infertility na ụmụ oke nwoke, na-egosi na ọrụ ha na ngwakọta gamete adịghị emepụtaghachi..N'ịgbasawanye nke ọma, ọ dịghị nke ọ bụla n'ime protein sperm mara amara nke achọrọ maka oge adhesion nke ngwakọta bụ nke a na-ejikarị.
Ọ ka bụ ajụjụ mepere emepe ma SPACA6 (na ndị otu IST superfamily) na-ekere òkè na njikọ mmekọrịta, na-etolite netwọkụ intragametic iji weta protein dị mkpa na isi ihe, ma ọ bụ ikekwe na-eme ihe dị ka fusogens na-enweghị atụ.Ọmụmụ ihe mgbochi mgbochi na sel HEK293T gosipụtara mmekọrịta n'etiti ogologo ogologo IZUMO1 na SPACA632.Otú ọ dị, ectodomains recombinant adịghị emekọrịta ihe na vitro, na-atụ aro na mmekọrịta ndị a hụrụ na Noda et al.ehichapụrụ ha abụọ n'ime ihe owuwu ahụ (rịba ama ọdụ cytoplasmic nke IZUMO1, nke egosiri na ọ dịghị mkpa maka fatịlaịza62).N'aka nke ọzọ, IZUMO1 na/ma ọ bụ SPACA6 nwere ike ịchọ ebe a na-ekekọta kpọmkwem nke anyị na-adịghị amụba na vitro, dị ka nhazi physiologically kpọmkwem ma ọ bụ ogige molecular nwere protein ndị ọzọ (amaara ma ọ bụ achọpụtabeghị).Ọ bụ ezie na IZUMO1 ectodomain kwenyere na ọ na-edozi ntinye nke spermatozoa na akwa n'ime oghere perivitelline, ebumnuche nke SPACA6 ectodomain amabeghị.
Nhazi nke SPACA6 na-ekpughe ọtụtụ ebe echekwara nke nwere ike itinye aka na mmekọrịta protein-protein.Akụkụ echekwara nke mpaghara hinge ozugbo dị n'akụkụ CXXC motif (ahapụtara Patch 1 n'elu) nwere ọtụtụ ihe fọdụrụ na-eche ihu na-esi ísì ụtọ na-ejikọta ya na hydrophobic na π-stacking mmekọrịta n'etiti biomolecules.Akụkụ sara mbara nke ngalaba Ig-dị ka (mpaghara 2) na-etolite oghere dị mma nke nwere nnukwu nchekwa Arg na ihe fọdụrụ ya, na ọgwụ mgbochi megide mpaghara a ejirila na mbụ gbochie fusion gamete 30.Ihe mgbochi ahụ na-amata epitope linear 212RIRPAQLTHRGTFS225, nke nwere atọ n'ime ihe fọdụrụ arginine isii yana His220 echekwara nke ukwuu.O doghị anya ma ọ bụ n'ihi nkwụsị nke ihe ndị a kpọmkwem ma ọ bụ mpaghara dum.Ebe oghere a dị nso na C-terminus nke β-sanwichi na-egosi mmekọrịta cis na protein sperm agbata obi, ma ọ bụghị na protein oocyte.Ọzọkwa, njigide nke tangle proline-rich tangle (saịtị 3) na-agbanwe nke ukwuu n'ime eriri nwere ike ịbụ saịtị nke mmekọrịta protein-protein ma ọ bụ, o yikarịrị, na-egosi njide nke mgbanwe n'etiti ngalaba abụọ ahụ.okike dị mkpa maka ọrụ SPACA6 amaghị ama.ngwakọta nke gametes.
SPACA6 nwere njirimara nke protein adhesion intercellular, gụnyere Ig-dị ka β-sanwichi.Ọtụtụ protein nrapado (dịka ọmụmaatụ, cadherins, integrins, adhesins, na IZUMO1) nwere otu ngalaba β-sanwichi ma ọ bụ karịa nke na-agbatị protein site na akpụkpọ ahụ cell na ebumnuche gburugburu ha63,64,65.Ngalaba Ig dị ka SPACA6 nwekwara ebumnuche a na-ahụkarị na β-sandwiches nke adhesion na njikọta: okpukpu abụọ nke eriri ndị yiri ya na nsọtụ β-sandwichi, nke a maara dị ka igwe clamps66.A kwenyere na ebumnuche a na-abawanye iguzogide ndị agha shear, nke bara uru maka protein ndị na-etinye aka na mmekọrịta nke intercellular.Otú ọ dị, n'agbanyeghị myirịta a na adhesins, ọ dịghị ugbu a na-egosi na SPACA6 na-emekọ ihe na akwa ọcha.SPACA6 ectodomain enweghị ike ijikọ na JUNO, na SPACA6 na-egosipụta mkpụrụ ndụ HEK293T, dị ka egosiri ebe a, ọ na-esiri ike imekọrịta ihe na oocytes enweghị zona 32.Ọ bụrụ na SPACA6 guzobe njikọ dị n'etiti, mmekọrịta ndị a nwere ike ịchọ mgbanwe ntụgharị asụsụ ma ọ bụ na-edozi ya site na protein spam ndị ọzọ.N'ịkwado echiche nke ikpeazụ, IZUMO1-deficient spermatozoa na-ejikọta na oocytes, na-egosi na ụmụ irighiri ihe ndị ọzọ karịa IZUMO1 na-etinye aka na nkwụsị nke gamete nzọụkwụ 27.
Ọtụtụ protein viral, cellular, na mmepe mmepe nwere akụrụngwa na-ebu amụma ọrụ ha dị ka fusogens.Dịka ọmụmaatụ, glycoproteins fusion viral (klas I, II na III) nwere peptide fusion hydrophobic ma ọ bụ loop na njedebe nke protein a na-etinye n'ime akpụkpọ anụ ụlọ.Map nke hydrophilicity nke IZUMO143 na nhazi (kpebisiri ike na amụma) nke ezinụlọ IST enweghị ihe ọ bụla pụtara ìhè peptide hydrophobic fusion.Ya mere, ọ bụrụ na protein ọ bụla dị na ezinụlọ superfamily IST na-arụ ọrụ dị ka fusogens, ha na-eme ya n'ụzọ dị iche na ihe atụ ndị ọzọ a ma ama.
Na mmechi, ọrụ nke ndị otu IST superfamily nke protein ndị metụtara njikọ gamete ka bụ ihe omimi na-akpali akpali.Ihe e ji mara SPACA6 recombinant molecule na nhazi ya nke edoziri ga-enye nghọta na mmekọrịta dị n'etiti ihe ndị a na-ekekọrịta na ọrụ ha na mgbakwunye gamete na ngwakọta.
Usoro DNA nke dabara na SPACA6 ectodomain mmadụ buru amụma (NCBI accession number NP_001303901.1; residues 27-246) bụ codon-kachasị mma maka nkwupụta na mkpụrụ ndụ Drosophila melanogaster S2 ma mejupụta ya dị ka mkpụrụ ndụ mkpụrụ ndụ nke nwere usoro ntinye koodu Kozak (Eurofin Gezak)., mgbama nzuzo nke BiP na 5' na 3' kwekọrọ na njedebe maka ligation-independent cloning nke mkpụrụ ndụ ihe nketa a n'ime pMT okwu vector dabere na ihe nkwalite metallothionein gbanwere maka nhọrọ na puromycin (pMT-puro).Vector pMT-puro na-edobe ebe mgbawa thrombin na-esochi mkpado 10x-Ya C-terminal (Njirimara S2).
Ntugharị kwụsiri ike nke SPACA6 pMT-puro vector n'ime sel D. melanogaster S2 (Gibco) ka emere n'otu aka ahụ na protocol ejiri maka IZUMO1 na JUNO43.A na-agbaze mkpụrụ ndụ S2 ma tolite na Schneider's medium (Gibco) gbakwunyere na njedebe ikpeazụ nke 10% (v / v) serum nwa ebu n'afọ na-ekpo ọkụ (Gibco) na 1X antimycotic ọgwụ nje (Gibco).Edebere mkpụrụ ndụ ngafe mbụ (sel 3.0 x 106) n'ime olulu mmiri nke ọ bụla nke efere olulu mmiri 6 (Corning).Mgbe awa 24 nke incubation gasịrị na 27 Celsius C, a na-ebufe mkpụrụ ndụ site na ngwakọta nke 2 mg nke SPACA6 pMT-puro vector na Effectene transfection reagent (Qiagen) dị ka usoro onye nrụpụta si dị.A na-etinye mkpụrụ ndụ ndị a gbanwere maka awa 72 wee were 6 mg / ml puromycin wee weta ya.E kewapụrụ mkpụrụ ndụ site na usoro Schneider zuru oke ma tinye ya na serum free Insect-XPRESS medium (Lonza) maka mmepụta protein buru ibu.A 1 L batch nke omenala S2 cell toro ka ọ bụrụ sel 8-10 × 106 ml-1 n'ime 2 L ventilated flat-bottomed polypropylene Erlenmeyer flask wee mee ya na ntinye ikpeazụ nke 500 µM CuSO4 (Millipore Sigma) na sterile filter.kpaliri.A na-etinye omenala ndị a kpalitere na 27 ° C. na 120 rpm maka ụbọchị anọ.
Ihe mkpuchi nwere SPACA6 kewapụrụ ya site na centrifugation na 5660×g na 4°C sochiri Centramate tangential flow filtration system (Pall Corp) nwere akpụkpọ anụ 10 kDa MWCO.Tinye usoro etinye uche nwere SPACA6 na kọlụm Ni-NTA agarose resin (Qiagen) 2 ml.A na-asa resin Ni-NTA na mpịakọta kọlụm 10 (CV) nke ihe nchekwa A wee tinye 1 CV nke ihe nchekwa A iji nye mkpokọta imidazole ikpeazụ nke 50 mM.Ejiri 10 ml nke ihe nchekwa kpuchie SPACA6 na imidazole na ntinye ikpeazụ nke 500 mM.Agbakwunyere thrombin klas mmachi (Millipore Sigma) ozugbo na tubing dialysis (MWCO 12-14 kDa) na 1 unit kwa mg SPACA6 vs. 1 L 10 mM Tris-HCl, pH 7.5 na 150 mM NaCl (ihe nchekwa B) maka dialysis.) na 4 Celsius C maka awa 48.A na-agbajikwa SPACA6 nke thrombin nke gbawara agbawa ugboro atọ iji belata ntinye nnu wee tinye ya na kọlụm mgbanwe mgbanwe nke 1 ml MonoS 5/50 GL (Cytiva/GE) na 10 mM Tris-HCl, pH 7.5.Ejiri 3 CV nke 10 mM Tris-HCl, pH 7.5, wee sachaa onye na-agbanwe cation na 0 ruo 500 mM NaCl na linear gradient na 10 mM Tris-HCl, pH 7.5 maka 25 CV.Mgbe mgbanwe ion chromatography gasịrị, SPACA6 gbadoro anya na 1 ml wee pụta n'ụzọ dịpụrụ adịpụ site na kọlụm ENrich SEC650 10 x 300 (BioRad) jikọtara ya na ihe nchekwa B. Dị ka chromatogram, ọdọ mmiri na ntinye uche nwere SPACA6.Eletrọphoresis nwere ntụpọ Coomassie na-achịkwa ịdị ọcha na gel 16% SDS-polyacrylamide.A na-agbakọ mkpokọta protein site na nnabata na 280 nm site na iji iwu Beer-Lambert na ọnụọgụ mkpochapụ molar.
A na-enyocha SPACA6 dị ọcha n'otu abalị megide 10 mM sodium phosphate, pH 7.4 na 150 mM NaF wee gbanye ya na 0.16 mg/mL tupu nyocha nke CD spectroscopy.A na-anakọta nyocha Spectral nke CD nke nwere ogologo 185 ruo 260 nm na Jasco J-1500 spectropolarimeter na-eji quartz cuvettes nwere ogologo ụzọ anya anya 1 mm (Helma) na 25 ° C na ọnụego 50 nm / min.A na-agbaziri ihe nkiri CD dị n'usoro ntọala, gbakọọ ihe karịrị ihe 10 nwetara, wee gbanwee ka ọ bụrụ elliptical residual (θMRE) na ogo cm2/dmol:
ebe MW bụ molekụla arọ nke ihe nlele ọ bụla na Da;N bụ ọnụ ọgụgụ nke amino asịd;θ bụ elliptical na millidegrees;d kwekọrọ n'ogologo ụzọ anya na cm;itinye uche protein na nkeji.