304 insimbi engagqwali tubing isakhi samakhemikhali ,Isakhiwo se-SPCA6 ectodomain siqukethe umndeni omkhulu ogciniwe wamaphrotheni ahlotshaniswa ne-gamete fusion.

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Ukucaciswa Okujwayelekile Kwe-ASTM A240 Uhlobo lwe-304 Tube

I-ASTM A240 304 abahlinzeki be-tubing yensimbi engenasici

Imininingwane ASTM A240 / ASME SA240
Ubukhulu 0.5mm-100mm
Ububanzi bangaphandle 10mm, 25.4mm, 38.1mm, 50.8mm, 100mm, 250mm, 300mm, 350mm, njll.
Ubude 2000mm, 2440mm, 3000mm, 5800mm, 6000mm, njll.
Ubuso 2B, 2D, BA, NO.1, NO.4, NO.8, 8K, mirror, checkered, embossed, hair line, sand blast, Brush, etching, njll.
Qeda I-Hot rolled (HR), I-Cold rolled Tube (CR), 2B, 2D, BA NO(8), i-SATIN (Ihlangene ne-Plastic Coated)
Ifomu Ishubhu eliyisikwele Ishubhu elingunxande njll.

304 Ukwakhiwa Kweshubhu ye-Ruond kanye Nezimpawu Zemishini

Ibanga C Mn Si P S Cr Mo Ni N
304 Okuncane.
Ubukhulu.
/
0.08
/
2.0
/
0.75
/
0.045
/
0.030
18.00
20.00
/ 8.00
10.50
/
0.10
304L Okuncane.
Ubukhulu.
/
0.03
/
2.0
/
1.0
/
0.045
/
0.030
18.00
20.00
/ 9.00
11.00
/
304H Okuncane.
Ubukhulu.
0.04
0.10
/
2.0
/
0.75
0.045
/
/
0.030
18.00
20.00
/ 8.00
10.50
/
Ibanga Amandla Okuqina
(MPa)
Isivuno Amandla
0.2% Ubufakazi (MPa)
Ukwelula
(% ku-50mm)
Ukuqina
Rockwell B
(HR B)
Brinell
(HB)
304 515 205 40 92 201
304L 515 205 40 90 187
304H 515 205 40 92 201

Ubukhulu obujwayelekile, ishadi lesisindo kanye namashejuli kasayizi weTube yensimbi engagqwali engama-304

SS 304 Tube Usayizi (mm) SS304 Tube Isisindo per Unit Area (kg/m)
6*1 0.125
6*1.5 0.168
8*1 0.174
8*1.5 0.243
10*1 0.224
10*1.5 0.318
12*1 0.274
12*1.5 0.392
12*2 0.498
14*1 0.324
14*2 0.598
14*3 0.822
16*2 0.697
16*3 0.971
17*3 1.046
18*1 0.423
18*1.5 0.617
18*2 0.797
18*3 1.121
20*1 0.473
20*2 0.897
20*3 1.27
21*3 1.345
22*2 0.996
22*2.5 1.214

I-SPCA6 iyiphrotheni engaphezulu evezwe isidoda ebaluleke kakhulu ekuhlanganisweni kwe-gamete ngesikhathi sokuzalaniswa kocansi kwezilwane ezincelisayo.Ngaphandle kwale ndima eyisisekelo, umsebenzi othize we-SPCA6 awuqondwa kahle.Sicacisa ukwakheka kwekristalu yesizinda se-extracellular se-SPCA6 ngesinqumo se-2.2 Å, sembula iphrotheni enezizinda ezimbili ezakhiwe inqwaba enemigqa emine kanye namasemishi afana ne-Ig ahlanganiswe ama-quasi-flexible linkers.Lesi sakhiwo sifana ne-IZUMO1, enye iphrotheni ehlobene ne-gamete fusion, eyenza i-SPCA6 ne-IZUMO1 amalungu asungula umndeni omkhulu wamaprotheni ahlobene nokuvundiswa okubhekiselwa kuwo lapha njengomndeni omkhulu we-IST.Umndeni omkhulu we-IST uchazwa ngokwesakhiwo ngenqwaba yawo ye-four-helix esontekile kanye nepheya le-disulfide-linked CXXC motifs.Ukusesha kwe-AlphaFold okusekelwe esakhiweni kweproteome yomuntu kuhlonze amalungu engeziwe amaprotheni alo mndeni omkhulu;ngokuphawulekayo, amaningi ala maprotheni ahilelekile ekuhlanganisweni kwe-gamete.Isakhiwo se-SPCA6 kanye nobudlelwano baso namanye amalungu omndeni omkhulu we-IST kunikeza isixhumanisi esingekho olwazini lwethu lokuhlangana kwegeyimu yezilwane ezincelisayo.
Yonke impilo yomuntu iqala ngama-gamete amabili ahlukene e-haploid: isidoda sikababa neqanda likamama.Lesi sidoda singumnqobi wenqubo eqinile yokukhetha lapho izigidi zamangqamuzana esidoda zidlula endaweni yangasese yowesifazane, zinqobe izithiyo ezihlukahlukene1 futhi zibe namandla, okuthuthukisa ukuhamba kwazo kanye nenqubo yezingxenye ezingaphezulu2,3,4.Noma ngabe isidoda kanye ne-oocyte zitholana, inqubo ayikapheli.I-oocyte izungezwe ungqimba lwamaseli e-cumulus kanye nomgoqo we-glycoprotein obizwa ngokuthi i-zona pellucida, lapho isidoda kufanele sidlule khona ukuze singene ku-oocyte.I-Spermatozoa isebenzisa inhlanganisela yama-adhesion molecules angaphezulu kanye nama-enzyme ahlotshaniswa ne-membrane futhi afihliwe ukuze anqobe lezi zithiyo zokugcina5.Lawa ma-molecule nama-enzyme agcinwa ikakhulukazi kulwelwesi lwangaphakathi kanye ne-acrosomal matrix futhi atholwa lapho ulwelwesi lwangaphandle lwesidoda lukhishwa ngesikhathi sokusabela kwe-acrosomal6.Isinyathelo sokugcina kulolu hambo olunamandla umcimbi wokuhlanganisa iqanda nesidoda, lapho amangqamuzana amabili ehlanganisa ulwelwesi lwawo ukuze abe i-diploid organism7.Nakuba le nqubo iyisizinda sokukhiqiza kwabantu, ukuxhumana okudingekayo kwamangqamuzana akuqondakali kahle.
Ngaphezu kokuvundiswa kwama-gametes, i-chemistry yokuhlanganiswa kwama-lipid bilayers amabili iye yacwaningwa kabanzi.Ngokuvamile, ukuhlanganiswa kwe-membrane kuyinqubo engathandeki ngamandla edinga i-protein catalyst ukuze ibhekane noshintsho lwesakhiwo oluletha ulwelwesi oluseduze, luphule ukuqhubeka futhi lubangele ukuhlangana8,9.Lawa ma-protein catalysts aziwa ngokuthi ama-fusogens futhi atholakale ezinhlelweni eziningi zokuhlanganisa.Ayadingeka ukuze kungene igciwane kumaseli aphethe (isb, i-gp160 ku-HIV-1, i-spike kuma-coronavirus, i-hemagglutinin kumagciwane omkhuhlane) 10,11,12 placental (syncytin) 13,14,15 kanye nama-gamete-forming fusions kuma-eukaryote aphansi ( I-HAP2/GCS1 ezitshalweni, ama-protists nama-arthropods) 16,17,18,19.Ama-Fusogens ama-gametes abantu awakakatholakali, nakuba amaprotheni amaningana akhonjiswe ukuthi abalulekile ekunamathiselweni kwe-gamete nokuhlangana.I-CD9 evezwe i-oocyte, iphrotheni ye-transmembrane edingekayo ukuze kuhlanganiswe amagundane nama-gametes abantu, kwaba ngeyokuqala ukutholwa angu-21,22,23.Nakuba umsebenzi wayo oqondile uhlala ungacacile, indima ekunamatheleni, ukwakheka kwe-adhesion foci ku-egg microvilli, kanye / noma indawo efanele yamaprotheni e-oocyte ebusweni kubonakala sengathi i-24,25,26.Amaprotheni avamile amabili abaluleke kakhulu ekuhlanganisweni kwe-gamete iphrotheni yesidoda i-IZUMO127 kanye nephrotheni ye-oocyte JUNO28, futhi ukuhlotshaniswa kwawo kuyisinyathelo esibalulekile ekuqapheliseni kwe-gamete nokunamathela ngaphambi kokuhlanganiswa.Amagundane e-Izumo1 abesilisa kanye namagundane e-Juno knockout abesifazane abayinyumba ngokuphelele, kulawa mamodeli isidoda singena endaweni ye-perivitelline kodwa ama-gamete awahlangani.Ngokufanayo, ukuhlangana kwehliswa lapho ama-gametes ephathwa nge-anti-IZUMO1 noma i-JUNO27,29 yamasosha omzimba ekuhlolweni kokukhulelwa kwe-in vitro.
Muva nje, kutholakale iqembu elisanda kutholwa lamaprotheni aveza isidoda ngendlela efana ne-IZUMO1 kanye ne-JUNO20,30,31,32,33,34,35.I-sperm acrosomal membrane-associated protein 6 (SPACA6) ibonakale njengebalulekile ekuvundiseni esifundweni esikhulu se-murine mutagenesis.Ukufakwa kwe-transgene esakhiweni se-Spaca6 kukhiqiza i-spermatozoa engabonakali, nakuba lezi zidoda zingena endaweni ye-perivitelline 36.Izifundo zokungqongqoza ezalandela kumagundane ziqinisekisile ukuthi i-Spaca6 iyadingeka ekuhlanganiseni kwe-gamete 30,32.I-SPCAA6 ivezwa cishe ngokukhethekile kumasende futhi inephethini yokwenza indawo efana neye-IZUMO1, okungukuthi ngaphakathi kwe-intima yesidoda ngaphambi kokusabela kwe-acrosomal, bese ithuthela esifundeni senkabazwe ngemva kokusabela kwe-acrosomal 30,32.Ama-homologue e-Spaca6 akhona ezinhlobonhlobo zezilwane ezincelisayo nezinye i-eukaryote 30 futhi ukubaluleka kwayo ekuhlanganisweni kwe-gamete yomuntu kuboniswe ngokuvinjelwa kokuvundiswa komuntu ku-vitro ngokumelana ne-SPCAA6 30.Ngokungafani ne-IZUMO1 ne-JUNO, imininingwane yesakhiwo, ukusebenzisana, kanye nomsebenzi we-SPCAA6 ahlala engacacile.
Ukuze siqonde kangcono inqubo eyisisekelo ehlanganisa ukuhlanganiswa kwesidoda namaqanda omuntu, okuzosenza sikwazi ukwazisa intuthuko yesikhathi esizayo ekuhleleni umndeni nokwelashwa kokuzala, senze izifundo ze-SPCA6 zesakhiwo kanye namakhemikhali ezinto eziphilayo.Isakhiwo sekristalu sesizinda se-extracellular se-SPCAA6 sibonisa inqwaba ye-helical emine (4HB) kanye nesizinda se-immunoglobulin-like (Ig-like) esixhunywe izifunda eziguquguqukayo.Njengoba kubikezelwe ezifundweni zangaphambilini, i-7,32,37 isakhiwo sesizinda se-SPCA6 sifana ne-IZUMO1 yomuntu, futhi amaprotheni amabili ahlanganyela nge-motif engavamile: i-4HB ene-helical surface engunxantathu kanye ne-pair of CXXC motifs exhunywe yi-disulfide.Siphakamisa ukuthi i-IZUMO1 kanye ne-SPCA6 manje ichaze umndeni omkhulu, ohlobene nesakhiwo wamaprotheni ahlobene nokuhlangana kwe-gamete.Sisebenzisa izici ezihlukile kumndeni omkhulu, senze ukusesha okuphelele kwe-AlphaFold structural human proteome, sihlonza amalungu engeziwe alo mndeni omkhulu, okuhlanganisa namalungu amaningana ahilelekile ekuhlanganisweni kwe-gamete kanye/noma ukuvundiswa.Manje kubonakala sengathi kukhona ukugoqa okujwayelekile kwesakhiwo kanye nomndeni omkhulu wamaprotheni ahlotshaniswa ne-gamete fusion, futhi isakhiwo sethu sinikeza imephu yamangqamuzana yalesi sici esibalulekile somshini womuntu wokuhlanganisa ama-gamete.
I-SPCA6 iyiphrotheni ye-transmembrane ene-single-pass ene-glycan eyodwa exhunywe ku-N kanye namabhondi ayisithupha e- putative disulfide (Amanani S1a kanye ne-S2).Siveze isizinda se-extracellular se-SPCA6 yomuntu (izinsalela ezingu-27–246) kumaseli e-Drosophila S2 futhi sahlanza amaprotheni sisebenzisa i-nickel affinity, ukushintshaniswa kwe-cation, kanye ne-chromatography yokukhishwa kosayizi (Fig. S1b).I-SPCA6 ectodomain ehlanzekile izinzile kakhulu futhi iyafana.Ukuhlaziywa kusetshenziswa i-chromatography engabandakanyi usayizi ehlanganiswe ne-polygonal light scattering (SEC-MALS) kwembule inani eliphakeme elinesisindo semolekyuli esibaliwe esingu-26.2 ± 0.5 kDa (Fig. S1c).Lokhu kuhambisana nosayizi we-SPCA6 monomeric ectodomain, okubonisa ukuthi i-oligomerization ayizange yenzeke ngesikhathi sokuhlanza.Ukwengeza, i-spectroscopy ye-circular dichroism (CD) yembula isakhiwo esixubile se-α / β esinephuzu lokuncibilika lika-51.3 ° C (Fig. S1d, e).Ukususwa kwe-spectra ye-CD kwembule u-38.6% we-α-helical kanye ne-15.8% yezakhi ze-β-stranded (Figure S1d).
I-SPCA6 ectodomain yenziwe yacwebezela kusetshenziswa i-matrix seeding38 okungahleliwe okuholele kusethi yedatha enesixazululo esingu-2.2 Å (Ithebula 1 kanye noMdwebo S3).Kusetshenziswa inhlanganisela yokushintshwa kwamangqamuzana okususelwe ezingxenyeni kanye nedatha yesigaba ye-SAD enokuchayeka kwe-bromide yokunqunywa kwesakhiwo (Ithebula 1 nomfanekiso we-S4), imodeli yokugcina ecwengisisiwe iqukethe izinsalela ze-27-246.Ngesikhathi kunqunywa isakhiwo, zazingekho izakhiwo zokuhlola noma ze-AlphaFold ezitholakalayo.I-SPCA6 ectodomain ikala u-20 Å × 20 Å × 85 Å, iqukethe amaheliksi ayisikhombisa kanye nemicu engu-β eyisishiyagalolunye, futhi inomkhakha ophakeme ophakeme oqiniswe ngamabhondi ayisithupha e-disulfide (Fig. 1a, b).Ukuminyana kwama-electron abuthaka ekupheleni kochungechunge oluseceleni lwe-Asn243 kubonisa ukuthi le nsalela iyi-glycosylation exhunywe ku-N.Isakhiwo siqukethe izizinda ezimbili: i-N-terminal four-helix bundle (4HB) kanye nesizinda se-C-terminal Ig-like esinesifunda sehinge esiphakathi phakathi kwazo (Fig. 1c).
Isakhiwo sesizinda se-extracellular se-SPCA6.Umdwebo we-strip wesizinda se-extracellular se-SPCA6, umbala weketango ukusuka ku-N ukuya ku-C-terminus ukusuka kokuluhlaza okwesibhakabhaka okumnyama kuya kokubomvu okumnyama.Ama-cysteine ​​​​abandakanyeka kumabhondi e-disulfide aqokonyiswa ku-magenta.b I-Topology yesizinda se-extracellular se-SPCA6.Sebenzisa uhlelo lombala olufanayo njengakuMfanekiso 1a.c SPCA6 isizinda esingaphandle kwamaselula.Amashadi wesizinda sesizinda se-4HB, hinge, kanye ne-Ig anombala osawolintshi, oluhlaza okotshani, noluhlaza okwesibhakabhaka, ngokulandelana.Izendlalelo azidonselwa esikalini.
Isizinda se-4HB se-SPCA6 sihlanganisa ama-helice amane ayinhloko (ama-helices 1-4), ahlelwe ngendlela ye-helical helix (Fig. 2a), ukushintshanisa phakathi kwe-antiparallel kanye nokusebenzisana okufanayo (Fig. 2b).I-helix encane eyengeziwe ye-single-turn (i-helix 1′) ibekwe i-perpendicular to the bundle, yenza unxantathu onamahelice 1 no-2. Lonxantathu ukhubazekile kancane ekupakishweni okusontekile kwe-helical yokupakishwa okuminyene ngokuqhathaniswa kwama-helices 3 no-4 ( Umfanekiso 2a).
Ishadi lomugqa we-4HB N-terminal.b Ukubuka okuphezulu kwenqwaba yamaheliheli amane, i-helix ngayinye igqanyiswe ngokuluhlaza okwesibhakabhaka okumnyama ku-N-terminus nokubomvu okumnyama ku-C-terminus.c Umdwebo wesondo elisontekile eliya phezulu phansi we-4HB, insalela ngayinye iboniswa njengombuthano obhalwe ikhodi ye-amino acid enohlamvu olulodwa;ama-amino acid amane kuphela phezulu kwesondo abaliwe.Izinsalela ezingezona i-polar zinombala ophuzi, izinsalela ze-polar ezingashajiwe zinombala oluhlaza okotshani, izinsalela ezinecala elihle zinombala oluhlaza okwesibhakabhaka, futhi izinsalela ezinecala elibi zinemibala ebomvu.d Ubuso obunxantathu besizinda se-4HB, obunama-4HB anombala osawolintshi namahinge ngokuluhlaza.Womabili ama-inset abonisa amabhondi e-disulfide amise okwenduku.
I-4HB igxile kumongo we-hydrophobic wangaphakathi owakhiwe ikakhulukazi izinsalela ze-aliphatic kanye nephunga elimnandi (Fig. 2c).Umnyombo uqukethe isibopho se-disulfide phakathi kwe-Cys41 ne-Cys55 exhumanisa ama-helices 1 no-2 ndawonye kunxantathu ophakanyisiwe ongaphezulu (Fig. 2d).Izibopho ezimbili ezengeziwe ze-disulfide zakhiwe phakathi kwe-CXXC motif ku-Helix 1' nenye i-CXXC motif etholakala ekugcineni kwe-β-hairpin esifundeni se-hinge (Fig. 2d).Izinsalela ze-arginine ezilondolozayo ezinomsebenzi ongaziwa (i-Arg37) itholakala ngaphakathi kukanxantathu ongenalutho owenziwe ama-helices angu-1′, 1, no-2. Ama-athomu e-aliphatic carbon Cβ, Cγ, kanye ne-Cδ Arg37 asebenzisana ne-hydrophobic core, futhi amaqembu ayo e-guanidine ahamba ngokujikeleza. phakathi kwama-helices 1′ kanye no-1 ngokusebenzisana phakathi komgogodla we-Thr32 kanye neketango laseceleni (Fig. S5a,b).I-Tyr34 inwebela emgodini ishiya izimbotshana ezimbili ezincane lapho i-Arg37 ingakwazi ukuxhumana ne-solvent.
Izizinda ezifana ne-β-sandwich ziwumndeni omkhulu wamaprotheni ahlanganyela isici esivamile samashidi amabili noma ngaphezulu anemigqa eminingi ye-amphipathic asebenzisana nge-hydrophobic core 39. Isizinda esifana ne-C-terminal Ig se-SPCAA6 sinephethini efanayo. futhi siqukethe izingqimba ezimbili (Fig. S6a).Ishidi 1 liyishidi elingu-β elinemicu emine (imicu D, F, H, kanye no-I) lapho imicu F, H, kanye nami sakha ilungiselelo eliphikisana nokufana, futhi imicu I no-D ithatha ukusebenzisana okufanayo.Ithebula lesi-2 ishidi le-beta elincane elinemicu ekabili ephikisana nokuhambisana (imicu E no-G).Isibopho sangaphakathi se-disulfide sibonwe phakathi kwe-C-terminus ye-chain ye-E kanye nendawo ye-H chain (Cys170-Cys226) (Fig. S6b).Lesi sibopho se-disulfide sifana nesibopho se-disulfide esizindeni se-β-sandwich se-immunoglobulin40,41.
Ishidi elingu-β elinemicu emine lisonteka ngobude balo bonke, lenze imiphetho engalinganiyo ehlukile ngokuma kanye nama-electrostatics.Unqenqema oluncane indawo ezungezile ye-hydrophobic eyisicaba egqamayo uma iqhathaniswa nezindawo ezisele ezingalingani futhi ezihlukene ngogesi ku-SPCA6 (Fig. S6b,c).I-halo yamaqembu omgogodla we-carbonyl/amino namaketango aseceleni azungeze i-hydrophobic surface (Fig. S6c).Imajini ebanzi imbozwe ingxenye ye-helical ene-capped evimba ingxenye ye-N-terminal ye-hydrophobic core futhi yakha amabhondi e-hydrogen amathathu neqembu elivulekile le-polar le-F chain backbone (Fig. S6d).Ingxenye ye-C-terminal yalo mkhawulo yenza iphakethe elikhulu elinomgogodla we-hydrophobic ovezwe kancane.Iphakethe lizungezwe ngamashaji avumayo ngenxa yamasethi amathathu ezinsalela ze-arginine (Arg162-Arg221, Arg201-Arg205 kanye ne-Arg212-Arg214) kanye ne-histidine emaphakathi (His220) (Umfanekiso S6e).
Isifunda sehinge siyisigaba esifushane phakathi kwesizinda se-helical kanye nesizinda esifana ne-Ig, esihlanganisa ungqimba olulodwa lwe-antiparallel olunemicu emithathu (imicu A, B kanye no-C), i-helix engu-310 encane, kanye nezingxenye ze-helical eziningana ezinde ezingahleliwe.(Fig. S7).Inethiwekhi yabathintwayo be-covalent kanye ne-electrostatic esifundeni sehinge ibonakala izinza umumo phakathi kuka-4HB nesizinda esifana ne-Ig.Inethiwekhi ingahlukaniswa izingxenye ezintathu.Ingxenye yokuqala ihlanganisa ama-CXXC motifs amabili (27CXXC30 kanye ne-139CXXC142) enza ibhondi ye-disulfide phakathi kwe-β-hairpin kuhinge kanye ne-1′ helix ku-4HB.Ingxenye yesibili ihlanganisa ukusebenzisana kwe-electrostatic phakathi kwesizinda esifana ne-Ig kanye nehinge.I-Glu132 kuhinge yenza ibhuloho likasawoti eline-Arg233 esizindeni esifana ne-Ig kanye ne-Arg135 kuhinge.Ingxenye yesithathu ihlanganisa isibopho sokubambisana phakathi kwesizinda esifana ne-Ig kanye nesifunda sehinge.Amabhondi amabili e-disulfide (Cys124-Cys147 kanye ne-Cys128-Cys153) axhuma i-hinge loop kusixhumi esiqiniswa ukusebenzisana kwe-electrostatic phakathi kwe-Gln131 neqembu elisebenzayo lomgogodla, okuvumela ukufinyelela kusizinda sokuqala esifana ne-Ig.iketango.
Isakhiwo se-SPCA6 ectodomain kanye nezakhiwo ngazinye ze-4HB kanye nezizinda ezifana ne-Ig zasetshenziselwa ukucinga amarekhodi afana nesakhiwo kumininingwane yamaprotheni 42.Sihlonze amameshi anamaphuzu aphezulu e-Dali Z, ukuchezuka okuncane okujwayelekile, kanye nezikolo ezinkulu ze-LALI (okugcina kuyinani lezinsalela ezilingana nesakhiwo).Nakuba amahithi okuqala angu-10 avela kusesho oluphelele lwe-ectodomain (Ithebula S1) libe ne-Z-score eyamukelekayo>842, ukusesha kwesizinda esifana ne-4HB noma se-Ig kukodwa kubonise ukuthi okuningi kwalokhu kushaywa kuhambisana namasemishi we-β kuphela.ukugoqa okutholakala yonke indawo kumaprotheni amaningi.Konke ukusesha okuthathu ku-Dali kubuyise umphumela owodwa kuphela: IZUMO1.
Sekuyisikhathi eside kuphakanyiswa ukuthi i-SPCA6 kanye ne-IZUMO1 babelane ngokufana kwesakhiwo7,32,37.Nakuba ama-ectodomains alawa maprotheni amabili ahlobene ne-gamete fusion abelana kuphela ngobunikazi bokulandelana okungama-21% (Umfanekiso S8a), ubufakazi obuyinkimbinkimbi, kuhlanganise nephethini yebhondi elondoloziwe ye-disulfide kanye nesizinda esibikezelwe njenge-C-terminal Ig-like ku-SPCAA6, kuvumele imizamo yokuqala yokwakha imodeli ye-homology ye-A igundane le-SPCA6 lisebenzisa i-IZUMO1 njengesifanekiso37.Isakhiwo sethu siqinisekisa lezi zibikezelo futhi sibonisa izinga langempela lokufana.Eqinisweni, izakhiwo ze-SPCA6 kanye ne-IZUMO137,43,44 zabelana ngezakhiwo zesizinda ezimbili (I-Fig. S8b) nezizinda ezifanayo ze-4HB ne-Ig-like β-sandwich ezixhunywe isifunda sehinge (Fig. S8c).
I-IZUMO1 kanye ne-SPCA6 4HB inomehluko ojwayelekile kuma-spiral bundles ajwayelekile.Ama-4HB ajwayelekile, afana nalawo atholakala ku-SNARE protein complexes ehilelekile ekuhlanganiseni kwe-endosomal 45,46, anezikhala ezinezikhala ezilinganayo ezigcina ukugoba okungapheli ekseni emaphakathi 47. Ngokuphambene, izizinda ze-helical kuzo zombili i-IZUMO1 kanye ne-SPACA6 bezihlanekezelwe, nokugoba okuguquguqukayo kanye ukupakisha okungalingani (Figure S8d).I-twist, cishe ebangelwa unxantathu owenziwe ngama-helices 1′, 1 no-2, igcinwa ku-IZUMO1 ne-SPCAA6 futhi iqiniswa yi-motif efanayo ye-CXXC ku-helix 1′.Kodwa-ke, ibhondi eyengeziwe ye-disulfide etholakala ku-SPCA6 (Cys41 kanye ne-Cys55 exhumanisa ngokuhlanganyela amahelisethi 1 no-2 ngenhla) idala i-apex ecijile kunxantathu ophakeme, okwenza i-SPACA6 isongwe kakhulu kune-IZUMO1, enonxantathu abayizimbotshana abagqame kakhudlwana.Ngaphezu kwalokho, i-IZUMO1 ayinayo i-Arg37 eqashelwe phakathi nendawo yalesi sikhala ku-SPCA6.Ngokuphambene, i-IZUMO1 inomongo ojwayelekile we-hydrophobic wezinsalela ze-aliphatic kanye nephunga elimnandi.
I-IZUMO1 inesizinda esifana ne-Ig esihlanganisa i-β-sheet43 enemicu ekabili kanye nemigqa emihlanu.I-strand eyengeziwe ku-IZUMO1 ingena esikhundleni sekhoyili eku-SPCA6, esebenzisana ne-F strand ukukhawulela amabhondi e-hydrogen angemuva emucu.Iphuzu elithakazelisayo lokuqhathanisa ukukhokhiswa kwendawo okubikezelwe yezizinda ezifana ne-Ig zamaprotheni amabili.Indawo ye-IZUMO1 ishajwe kabi kakhulu kunobuso be-SPCA6.Ishaja eyengeziwe itholakala eduze kwe-C-terminus ebheke kulwelwesi lwesidoda.Ku-SPCA6, izifunda ezifanayo bezingathathi hlangothi kakhulu noma zikhokhiswe kahle (Fig. S8e).Isibonelo, indawo ye-hydrophobic (imiphetho emincane) kanye nemigodi efakwe kahle (imiphetho ebanzi) ku-SPCAA6 ishajwa kabi ku-IZUMO1.
Nakuba ubudlelwano nezakhi zesakhiwo sesibili phakathi kwe-IZUMO1 ne-SPACA6 zigcinwe kahle, ukuqondanisa kwesakhiwo kwezizinda ezifana ne-Ig kubonise ukuthi lezi zizinda ezimbili ziyahlukahluka ekuqondeni kwazo okujwayelekile ngokuhlobene nomunye nomunye (Fig. S9).I-spiral bundle ye-IZUMO1 igobile mayelana ne-β-sandwich, idala umumo “we-boomerang” ochazwe ngaphambilini cishe ongu-50° ukusuka eksisi emaphakathi.Ngokuphambene, i-helical beam ku-SPCA6 yayitshekiswe cishe ngo-10° ngokuphambene.Umehluko walezi zimo kungenzeka ngenxa yomehluko endaweni yehinge.Ezingeni eliyisisekelo lokulandelana, i-IZUMO1 ne-SPCA6 yabelana ngokufana okuncane kokulandelana kuhinge, ngaphandle kwe-cysteine, i-glycine, nezinsalela ze-aspartic acid.Ngenxa yalokho, amabhondi e-hydrogen namanethiwekhi e-electrostatic ahluke ngokuphelele.Izakhi zesakhiwo sesibili se-β-sheet zabelwa i-IZUMO1 kanye ne-SPCA6, nakuba amaketango ku-IZUMO1 emade kakhulu futhi i-310 helix (helix 5) ihlukile ku-SPCAA6.Lo mehluko uphumela emazingeni ahlukene wesizinda kumaphrotheni amabili afanayo.
Ukusesha kwethu kweseva ye-Dali kwembula ukuthi i-SPCA6 ne-IZUMO1 yizona kuphela izakhiwo ezinqunywe ngokuhlolwa ezigcinwe kusizindalwazi samaprotheni ezinalokhu kugoqa okungu-4HB (Ithebula S1).Muva nje, i-DeepMind (i-Alphabet/Google) ithuthukise i-AlphaFold, isistimu esekelwe kunethiwekhi ye-neural engabikezela ngokunembile izakhiwo ze-3D zamaprotheni kusukela ekulandeleni okuyisisekelo48.Ngokushesha ngemva kokuba sixazulule isakhiwo se-SPCA6, isizindalwazi se-AlphaFold sakhululwa, sinikeza amamodeli esakhiwo esibikezelayo ahlanganisa u-98.5% wawo wonke amaprotheni ku-proteome48,49 yomuntu.Sisebenzisa isakhiwo sethu se-SPCA6 esixazululiwe njengemodeli yokusesha, ukusesha kwe-homology kwesakhiwo kwemodeli ku-AlphaFold human proteome kuhlonze amakhandidethi anokufana kwesakhiwo okungenzeka ku-SPCA6 kanye ne-IZUMO1.Njengoba kunikezwe ukunemba okumangalisayo kwe-AlphaFold ekubikezeleni i-SPCAA6 (Fig. S10a)—ikakhulukazi i-1.1 Å rms ectodomain uma iqhathaniswa nesakhiwo sethu esixazululiwe (Fig. S10b)—singaqiniseka ukuthi okufanayo kwe-SPCAA6 okungenzeka kunembe.
Ngaphambilini, i-PSI-BLAST yayisesha iqoqo le-IZUMO1 namanye amaprotheni amathathu ahambisana nesidoda: IZUMO2, IZUMO3, kanye ne-IZUMO450.I-AlphaFold ibikezele ukuthi lawa maprotheni omndeni we-IZUMO agoqeka esizindeni se-4HB ngephethini yebhondi ye-disulfide efanayo ne-IZUMO1 (Izibalo 3a kanye ne-S11), nakuba bengenawo isizinda esifana ne-Ig.Kucatshangelwa ukuthi i-IZUMO2 ne-IZUMO3 angamaprotheni olwelwesi oluhlangothi olulodwa afana ne-IZUMO1, kuyilapho i-IZUMO4 ibonakala iyimfihlo.Imisebenzi ye-IZUMO 2, 3, kanye ne-4 yamaprotheni ekuhlanganiseni kwe-gamete ayikanqunywa.I-IZUMO3 yaziwa ngokubamba iqhaza ku-acrosome biogenesis ngesikhathi sokukhula kwesidoda51 kanti iphrotheni ye-IZUMO kutholakale ukuthi yenza i-complex50.Ukongiwa kwamaprotheni e-IZUMO ezilwaneni ezincelisayo, ezihuquzelayo, nezilwane eziphila emanzini nasemanzini kuphakamisa ukuthi umsebenzi wazo onamandla uhambisana nowamanye amaprotheni aziwayo ahlobene ne-gamete-fusion, njenge-DCST1/2, SOF1, ne-FIMP.
Umdwebo wesizinda sesakhiwo somndeni omkhulu we-IST, onesizinda esingu-4HB, ihinge, ne-Ig esigqanyiswe ngokusawolintshi, okuluhlaza okotshani, nokuluhlaza okwesibhakabhaka, ngokulandelana.I-IZUMO4 inesifunda esiyingqayizivele se-C esibukeka simnyama.Amabhondi e-disulfide aqinisekisiwe futhi afakayo aboniswa ngemigqa eqinile nenamachashazi, ngokulandelanayo.b IZUMO1 (PDB: 5F4E), SPCA6, IZUMO2 (AlphaFold DB: AF-Q6UXV1-F1), IZUMO3 (AlphaFold DB: AF-Q5VZ72-F1), IZUMO4 (AlphaFold DB: AF-Q1ZYL8-F95), kanye ne-Alpha DB: AF-Q1ZYL8-F1) : AF-Q1ZYL8-F1) : AF-Q3KNT9-F1) aboniswa ebangeni lombala elifanayo njengephaneli A. Amabhondi e-disulfide aboniswa nge-magenta.I-TMEM95, IZUMO2 kanye ne-IZUMO3 i-transmembrane helices ayiboniswa.
Ngokungafani nephrotheni ye-IZUMO, amanye amaprotheni e-SPCA (okungukuthi, i-SPACA1, i-SPCA3, i-SPCA4, i-SPACA5, ne-SPCA9) kucatshangwa ukuthi ahluke ngokwesakhiwo kune-SPCA6 (Fig. S12).I-SPCAA9 kuphela ene-4HB, kodwa akulindelekile ukuthi ibe nomumo ofanayo wokungqubuzana noma ibhondi efanayo ye-disulfide njenge-SPCAA6.I-SPCA1 kuphela enesizinda esifana ne-Ig.I-AlphaFold ibikezela ukuthi i-SPCA3, i-SPACA4 kanye ne-SPCA5 inesakhiwo esihluke ngokuphelele kune-SPCA6.Kuyathakazelisa ukuthi i-SPCA4 yaziwa nangokuthi idlala indima ekuvundiseni, kodwa ngezinga elikhulu kune-SPACA6, esikhundleni salokho yenza kube lula ukusebenzisana phakathi kwesidoda ne-oocyte zona pellucida52.
Ukusesha kwethu kwe-AlphaFold kuthole okunye okufanayo kwe-IZUMO1 kanye ne-SPCA6 4HB, TMEM95.I-TMEM95, iphrotheni eyodwa ye-sperm-specific transmembrane, yenza amagundane wesilisa angabi nanzalo lapho ekhishwa i-32,33.I-Spermatozoa entula i-TMEM95 yayine-morphology evamile, i-motility, kanye nekhono lokungena ku-zona pellucida futhi ibophe kulwelwesi lweqanda, kodwa ayikwazanga ukuhlangana nolwelwesi lwe-oocyte.Ucwaningo lwangaphambilini lubonise ukuthi i-TMEM95 yabelana ngokufana kwesakhiwo ne-IZUMO133.Ngempela, imodeli ye-AlphaFold yaqinisekisa ukuthi i-TMEM95 iyi-4HB enokupheya okufanayo kwe-CXXC motifs njenge-IZUMO1 ne-SPCA6 kanye nebhondi efanayo eyengeziwe ye-disulfide phakathi kwama-helices 1 kanye ne-2 atholakala ku-SPCA6 (Fig. 3a kanye ne-S11).Nakuba i-TMEM95 ingenaso isizinda esifana ne-Ig, inesifunda esinephethini yebhondi ye-disulfide efana ne-SPCA6 ne-IZUMO1 izifunda zamahinge (Fig. 3b).Ngesikhathi sokushicilela lo mbhalo wesandla, iseva yokuphrinta yabika ngesakhiwo se-TMEM95, iqinisekisa umphumela we-AlphaFold53.I-TMEM95 ifana kakhulu ne-SPCA6 kanye ne-IZUMO1 futhi igcinwa ngokuziphendukela kwemvelo kakade ezilwaneni ezihlala emanzini (Fig. 4 and S13).
Usesho lwe-PSI-BLAST lusebenzise izizindalwazi ze-NCBI SPCA6, IZUMO1-4, TMEM95, DCST1, DCST2, FIMP, kanye ne-SOF1 ukuze kutholwe indawo yalokhu kulandelana esihlahleni sokuphila.Amabanga phakathi kwamaphoyinti egatsha awaboniswa ukukala.
Ukufana okumangalisayo kukonke kwesakhiwo phakathi kwe-SPCA6 ne-IZUMO1 kuphakamisa ukuthi bangamalungu asungula omndeni omkhulu wesakhiwo olondoloziwe ohlanganisa i-TMEM95 kanye ne-IZUMO 2, 3, kanye namaprotheni angu-4.amalungu aziwayo: IZUMO1, SPCA6 kanye ne-TMEM95.Ngenxa yokuthi amalungu ambalwa kuphela anezizinda ezifana ne-Ig, uphawu oluphawulekayo lomndeni omkhulu we-IST yisizinda se-4HB, esinezici ezihlukile ezivamile kuwo wonke lawa maprotheni: 1) I-4HB ehlanganisiwe enezindiza ezinophephela emhlane ezihlelwe ngendlela ephikisanayo/efanayo (Fig . 5a), 2) inqwaba inobuso obunxantathu obuhlanganisa ama-helice amabili ngaphakathi kwenqwaba kanye ne-helix yesithathu eqondile (Fig. indawo yokhiye (Fig. 5c). I-CXXC motif, etholakala kumaprotheni afana ne-thioredoxin, yaziwa ngokusebenza. njengenzwa ye-redox 54,55,56 , kuyilapho i-motif kumalungu omndeni we-IST ingahlotshaniswa nama-isomerase amaprotheni disulfide afana ne-ERp57 ekuhlanganisweni kwe-gamete. Izindima zihlotshaniswa 57,58.
Amalungu omndeni omkhulu we-IST achazwa izici ezintathu eziyizici zesizinda se-4HB: ama-helice amane ashintshana phakathi kwe-parallel ne-antiparallel orientation, ubuso be-ba-triangular helical bundle, kanye ne-ca CXXC motif ephindwe kabili eyakhiwe phakathi kwama-molecule amancane.) Amaheliksi e-N-terminal (owolintshi) kanye nesifunda sehinge β-hairpin (eluhlaza).
Uma kubhekwa ukufana phakathi kwe-SPCA6 ne-IZUMO1, ikhono leyangaphambili yokuhlanganisa i-IZUMO1 noma i-JUNO yahlolwa.I-Biolayer interferometry (BLI) iyindlela yokubopha esekwe kinetic eye yasetshenziswa ngaphambilini ukulinganisa ukusebenzisana phakathi kwe-IZUMO1 ne-JUNO.Ngemva kokufakwa kwenzwa ebhalwe nge-biotin ene-IZUMO1 njengesiheho esine-concentration ephezulu ye-JUNO analyte, kutholwe isignali eqinile (Fig. S14a), ebonisa ushintsho olubophezelayo ebugqileni be-biomaterial enamathiselwe ithiphu lenzwa.Izimpawu ezifanayo (okungukuthi, i-JUNO ehlanganiswe nenzwa njengesiyengo ngokumelene ne-IZUMO1 analyte) (Fig. S14b).Ayikho isignali etholakele ngenkathi i-SPCAA6 isetshenziswa njenge-analyte ngokumelene ne-IZUMO1 eboshwe izinzwa noma i-JUNO eboshwe ngenzwa (Figure S14a,b).Ukungabikho kwalesi siginali kubonisa ukuthi isizinda se-extracellular se-SPACA6 asihlanganyeli nesizinda esingaphandle kwamaselula se-IZUMO1 noma i-JUNO.
Ngenxa yokuthi ukuhlolwa kwe-BLI kusekelwe ku-biotinylation yezinsalela ze-lysine zamahhala kuphrotheni ye-bait, lokhu kuguqulwa kungavimbela ukubopha uma izinsalela ze-lysine zihilelekile ekusebenzisaneni.Ngaphezu kwalokho, ukuma kwesihlobo esibophezelayo kunzwa kungakha izithiyo eziqinile, ngakho-ke ukuhlolwa okujwayelekile kokudonsela phansi kwenziwe futhi kuma-recombinant SPCA6, IZUMO1 kanye ne-JUNO ectodomain.Naphezu kwalokhu, i-SPCA6 ayizange ihle nge-IZUMO1 emakiwe Yakhe noma i-JUNO emakiwe Yakhe (Fig. S14c,d), ebonisa ukuthi akukho ukusebenzisana okuhambisana nalokho okubonwe ekuhlolweni kwe-BLI.Njengokulawula okuhle, siqinisekise ukusebenzisana kwe-JUNO okubhalwe ukuthi I-IZUMO1 Yakhe (Izibalo S14e kanye ne-S15).
Ngaphandle kokufana kwesakhiwo phakathi kwe-SPCA6 ne-IZUMO1, ukungakwazi kwe-SPCAA6 ukubopha i-JUNO akumangazi.Ingaphezulu le-IZUMO1 yomuntu linezinsalela ezingaphezu kuka-20 ezisebenzisana ne-JUNO, okuhlanganisa nezinsalela ezivela esifundeni ngasinye kwezithathu (nakuba eziningi zazo zitholakala endaweni eyihinge) (Fig. S14f).Kulezi zinsalela, iyodwa kuphela elondolozwe ku-SPCA6 (Glu70).Nakuba izinsalela eziningi ezishintshwayo zigcine izakhiwo zazo zangempela zamakhemikhali ezinto eziphilayo, izinsalela ze-Arg160 ezibalulekile ku-IZUMO1 zathathelwa indawo yi-Asp148 efakwe kabi ku-SPCA6;ucwaningo lwangaphambilini lubonise ukuthi ukuguqulwa kwe-Arg160Glu ku-IZUMO1 kucishe kukuqede nya ukubophezela ku-JUNO43.Ngaphezu kwalokho, umehluko wokuma kwesizinda phakathi kwe-IZUMO1 ne-SPCA6 ukhulise kakhulu indawo engaphezulu yesayithi ebophezela i-JUNO yesifunda esifanayo ku-SPCA6 (Fig. S14g).
Naphezu kwesidingo esaziwayo se-SPCA6 sokuhlanganisa i-gamete nokufana kwayo ne-IZUMO1, i-SPCAA6 ayibonakali inomsebenzi wokubophezela olinganayo we-JUNO.Ngakho-ke, siye sazama ukuhlanganisa idatha yethu yesakhiwo nobufakazi bokubaluleka obunikezwa yisayensi yokuziphendukela kwemvelo.Ukuhlelwa kokulandelana kwama-homologue e-SPCAA kukhombisa ukulondolozwa kwesakhiwo esivamile ngale kwezilwane ezincelisayo.Isibonelo, izinsalela ze-cysteine ​​​​zikhona nakuma-amphibians ahlobene kude (Fig. 6a).Kusetshenziswa iseva ye-ConSurf, idatha yokugcinwa kokuqondanisa okulandelanayo kokulandelana okungama-66 ifakwe imephu endaweni ye-SPCA6.Lolu hlobo lokuhlaziya lungabonisa ukuthi yiziphi izinsalela ezilondolozwe ngesikhathi sokuvela kwamaprotheni futhi lungabonisa ukuthi yiziphi izindawo ezingaphezulu ezidlala indima emsebenzini.
ukuhlelwa kokulandelana kwe-SPCA6 ectodomains evela ezinhlotsheni ezihlukene eziyi-12 ezilungiselelwe kusetshenziswa i-CLUSTAL OMEGA.Ngokusho kokuhlaziywa kwe-ConSurf, izikhundla ezilondoloza kakhulu zimakwe ngokuluhlaza okwesibhakabhaka.Izinsalela ze-cysteine ​​​​zigqanyiswe ngokubomvu.Imingcele yesizinda nezinto zesakhiwo sesibili ziboniswa phezulu kokuqondanisa, lapho imicibisholo ibonisa ama-β-strands namagagasi abonisa ama-helices.Izihlonzi zokufinyelela ze-NCBI eziqukethe ukulandelana yilezi: umuntu (Homo sapiens, NP_001303901), i-mandrill (Mandrilus leucophaeus, XP_011821277), i-capuchin monkey (Cebus mimic, XP_017359366), ihhashi (Equus 1002 Orphale3) 23 XP_032_034) .), izimvu (Ovis aries, XP_014955560), indlovu (Loxodonta africana, XP_010585293), inja (Canis lupus familyis, XP_025277208), igundane (Mus musculus, NP_001156381), Tasmanian africana, XPus3, XPPlasi, XPPlasi, XP_Plarisi1 , 8) , 61_89 kanye neBullfrog (Bufo bufo, XP_040282113).Izinombolo zisekelwe ngokulandelana kwabantu.b Ukumelwa okungaphezulu kwesakhiwo se-SPACA6 esine-4HB phezulu kanye nesizinda esifana ne-Ig ngezansi, imibala esekelwe ezilinganisweni zokulondoloza ezivela kuseva ye-ConSurf.Izingxenye ezilondolozwe kahle kakhulu ziluhlaza okwesibhakabhaka, izingxenye ezilondolozwe ngokusesilinganisweni zimhlophe, kanti ezigcinwe kancane zinombala ophuzi.i-cysteine ​​ensomi.Amapheshana amathathu angaphezulu abonisa izinga eliphezulu lokuvikela akhonjiswe ku-inset ebhalwe iziqephu 1, 2 kanye no-3. Ikhathuni engu-4HB iboniswa kusihlokwana phezulu kwesokudla (uhlelo lombala olufanayo).
Isakhiwo se-SPCA6 sinezindawo ezingaphezulu ezilondolozwe kakhulu ezintathu (Fig. 6b).Isiqephu 1 sihlanganisa i-4HB kanye nesifunda sehinge futhi siqukethe amabhuloho amabili alondoloziwe e-CXXC e-disulfide, inethiwekhi yehinge ye-Arg233-Glu132-Arg135-Ser144 (Fig. S7), nezinsalela ezintathu zangaphandle ezilondolozwe ezinephunga elimnandi (Phe31, Tyr73, Phe137).unqenqema olubanzi lwesizinda esifana ne-Ig (Fig. S6e), esimele izinsalela ezimbalwa ezinecala elihle endaweni yesidoda.Kuyathakazelisa ukuthi lesi siqeshana siqukethe i-epitope ye-antibody eye yaboniswa ngaphambilini iphazamisa umsebenzi we-SPCAA6 30.Isifunda sesi-3 sihlanganisa ihinge nohlangothi olulodwa lwesizinda esifana ne-Ig;lesi sifunda siqukethe ama-prolines alondoloziwe (i-Pro126, i-Pro127, i-Pro150, i-Pro154) kanye nezinsalela ezibheke ngaphandle ezibheke ngaphandle/ezishajiwe.Ngokumangalisayo, izinsalela eziningi ezingaphezulu kwe-4HB ziyahlukahluka kakhulu (Fig. 6b), nakuba ukugoqa kugcinwa kulo lonke i-homologue ye-SPCAA6 (njengoba kuboniswa yi-conservatism ye-hydrophobic bundle core) nangale kwe-IST superfamily.
Nakuba lesi kuyisifunda esincane kunazo zonke ku-SPCA6 esinezici ezimbalwa ezibonakala zesakhiwo sesibili, izinsalela eziningi zesifunda sehinge (kuhlanganise nesifunda 3) zilondolozwe kakhulu phakathi kwama-homologue e-SPCAA6, okungase kubonise ukuthi ukuma kwe-helical bundle kanye ne-β-sandwich kudlala indima.njengomuntu ohloniphayo.Nokho, naphezu kwebhondi ye-hydrogen ebanzi namanethiwekhi kagesi endaweni yehinge ye-SPCA6 ne-IZUMO1, ubufakazi bokuguquguquka kwangaphakathi bungabonakala ekuqondaneni kwezakhiwo eziningi ezivunyelwe ze-IZUMO137,43,44.Ukuqondanisa kwezizinda ngazinye kudlulele kahle, kodwa ukuma kwezizinda ngokuhlobene kwahluka kusuka ku-50 ° kuya ku-70 ° ukusuka eksisi emaphakathi (Fig. S16).Ukuze uqonde i-conformational dynamics ye-SPCA6 kusixazululo, ukuhlolwa kwe-SAXS kwenziwa (Fig. S17a,b).Ukwakhiwa kabusha kwe-Ab initio ye-SPCA6 ectodomain ihambisane nesakhiwo sekristalu yenduku (Fig. S18), nakuba isiqephu se-Kratky sibonise izinga elithile lokuguquguquka (Fig. S17b).Lokhu kuvumelana kuphambene ne-IZUMO1, lapho iphrotheni engaboshiwe ithatha isimo se-boomerang kokubili ku-lattice kanye nesisombululo43.
Ukuhlonza ngokuqondile isifunda esiguquguqukayo, i-hydrogen-deuterium exchange mass spectroscopy (H-DXMS) yenziwa ku-SPCAA6 futhi uma kuqhathaniswa nedatha etholwe ngaphambilini ku-IZUMO143 (Fig. 7a,b).I-SPCA6 ngokusobala ivumelana nezimo kakhulu kune-IZUMO1, njengoba kufakazelwa ukushintshaniswa okuphezulu kwe-deuterium kuso sonke isakhiwo ngemva kwama-100,000 s wokushintshana.Kuzo zombili izakhiwo, ingxenye ye-C-terminal yesifunda sehinge ikhombisa izinga eliphezulu lokushintshisana, okungenzeka ukuthi livumela ukuzungezisa okulinganiselwe kwesizinda se-4HB ne-Ig-like ngokuhlobene.Kuyathakazelisa ukuthi ingxenye ye-C-terminal yehinge ye-SPCAA6, ehlanganisa izinsalela ze-147CDLPLDCP154, isifunda esilondolozwe kakhulu 3 (Umfanekiso we-6b), okungenzeka okubonisa ukuthi ukuguquguquka kwesizinda kuyisici esilondoloziwe ngokuziphendukela kwemvelo se-SPCAA6.Ngokusho kokuhlaziywa kokuguquguquka, idatha ye-CD thermal melt ibonise ukuthi i-SPACA6 (Tm = 51.2 ° C) izinzile kancane kune-IZUMO1 (Tm = 62.9 ° C) (Fig. S1e ne-S19).
a izithombe ze-H-DXMS ze-SPCA6 kanye ne-b IZUMO1.Iphesenti lokushintshisana kwe-deuterium linqunywe ngezikhathi ezibonisiwe.Amazinga okushintshaniswa kwe-hydrogen-deuterium akhonjiswa ngombala esikalini se-gradient ukusuka kokuluhlaza okwesibhakabhaka (10%) kuya kokubomvu (90%).Amabhokisi amnyama amelela izindawo zokuhwebelana okuphezulu.Imingcele ye-4HB, i-hinge nesizinda esifana ne-Ig esibonwe esakhiweni sekristalu ikhonjiswe ngaphezu kokulandelana okuyisisekelo.Amazinga okushintshana e-Deuterium ku-10 s, 1000 s, kanye ne-100,000 s adwetshwe eshadini lomdweshu elibekwe phezulu endaweni yamangqamuzana ebonisa ngale ye-SPCA6 ne-IZUMO1.Izingxenye zezakhiwo ezinezinga lokushintshanisa i-deuterium elingaphansi kuka-50% zinombala omhlophe.Izindawo ezingaphezulu kuka-50% we-H-DXMS zinemibala esikalini se-gradient.
Ukusetshenziswa kwe-CRISPR/Cas9 kanye namasu ofuzo okugoqa igundane kuye kwaholela ekuhlonzweni kwezici ezimbalwa ezibalulekile zesidoda kanye nokubopha kwamaqanda nokuhlangana.Ngaphandle kokusebenzelana okuphawuleka kahle kwe-IZUMO1-JUNO kanye nesakhiwo se-CD9, amaprotheni amaningi ahlotshaniswa ne-gamete fusion ahlala ehlelekile futhi esebenza ngokungaqondakali.Ukuchazwa kwe-biophysical kanye nesakhiwo se-SPCA6 kungolunye ucezu lwephazili ye-adhesion/fusion yamangqamuzana ngesikhathi sokuvundiswa.
I-SPCA6 namanye amalungu omndeni omkhulu we-IST abonakala elondolozwa kakhulu ezilwaneni ezincelisayo kanye nezinyoni ngazinye, izilwane ezihuquzelayo, nezilwane ezihlala emanzini;empeleni, kucatshangwa ukuthi i-SPCAA6 iyadingeka ngisho nasekuvundiseni ku-zebrafish 59. Lokhu kusatshalaliswa kufana namanye amaprotheni aziwayo ahlobene ne-gamete fusion njenge-DCST134, DCST234, FIMP31, kanye ne-SOF132, okuphakamisa ukuthi lezi zici azinayo i-HAP2 (futhi eyaziwa ngokuthi yi-GCS1) amaprotheni anesibopho somsebenzi wokuvuselela ama-protists amaningi., izitshalo, nama-arthropods.Amaprotheni ahlanganisiwe ahlanganisiwe 60, 61. Naphezu kokufana okuqinile kwesakhiwo phakathi kwe-SPCA6 ne-IZUMO1, ukuphuma kofuzo ekubhaleni enye yala maprotheni kubangele ukungazali kumagundane abesilisa, okubonisa ukuthi imisebenzi yawo ekuhlanganisweni kwe-gamete ayiphindaphindeki..Ngobubanzi, awekho amaprotheni esidoda awaziwayo adingekayo esigabeni sokunamathela sokuhlanganisa awasadingeki.
Kusengumbuzo ovulekile ukuthi ingabe i-SPCA6 (kanye namanye amalungu omndeni omkhulu we-IST) babamba iqhaza ekuhlanganeni kwe-intergametic, benze amanethiwekhi e-intragametic ukuze kubuthelwe amaprotheni abalulekile ezindaweni zokuhlanganisa, noma mhlawumbe benze njengama-fusogen angaqondakali.Ucwaningo lwe-Co-immunoprecipitation kumaseli e-HEK293T lwembule ukusebenzisana phakathi kobude obugcwele be-IZUMO1 ne-SPCA632.Kodwa-ke, i-ectodomain yethu ehlanganisiwe ayizange ihlanganyele ku-vitro, iphakamisa ukuthi ukusebenzisana okubonwe ku-Noda et al.womabili asuliwe ekwakhiweni (qaphela umsila we-cytoplasmic we-IZUMO1, okuboniswe ukuthi awudingeki ekuvundiseni62).Kungenjalo, i-IZUMO1 kanye/noma i-SPACA6 ingase idinge izindawo ezithile ezibophezelayo esingazenzi kabusha ku-vitro, njengokuhlanganisa okuqondile ngokomzimba noma ama-molecular complexes aqukethe amanye amaprotheni (aziwayo noma angakatholakali).Nakuba i-IZUMO1 ectodomain kukholakala ukuthi ilamula ukunamathela kwe-spermatozoa eqandeni elisendaweni ye-perivitelline, inhloso ye-SPCA6 ectodomain ayicacile.
Ukwakheka kwe-SPCA6 kuveza izindawo ezimbalwa ezilondoloziwe ezingase zibandakanyeke ekusebenzisaneni kwamaprotheni namaprotheni.Ingxenye elondoloziwe yesifunda sehinge eseduze ne-CXXC motif (eqokwe Isiqephu 1 ngenhla) inezinsalela ezinephunga elimnandi ezibheke ngaphandle ezivame ukuhlotshaniswa nokusebenzisana kwe-hydrophobic kanye no-π-stacking phakathi kwama-biomolecules.Izinhlangothi ezibanzi zesizinda esifana ne-Ig (isifunda sesi-2) zakha i-groove ene-positive ene-Arg egcinwe kakhulu nezinsalela Zakhe, futhi amasosha omzimba amelene nalesi sifunda aye asetshenziswa ngaphambilini ukuvimba ukuhlangana kwe-gamete 30.I-antibody ibona i-epitope engumugqa 212RIRPAQLTHRGTFS225, enezinsalela ezintathu kweziyisithupha ze-arginine futhi egcinwe kakhulu i-His220.Akucaci ukuthi ukungasebenzi kungenxa yokuvinjwa kwalezi zinsalela ezithile noma isifunda sonke.Indawo yaleli gebe eduze kwe-C-terminus ye-β-sandwich ikhombisa ukusebenzisana kwe-cis namaphrotheni esidoda angomakhelwane, kodwa hhayi nama-oocyte protein.Ngaphezu kwalokho, ukugcinwa kwe-tangle ene-proline-rich eguquguquka kakhulu (isayithi 3) ngaphakathi kwehinge kungase kube indawo yokusebenzisana kwamaprotheni-protein noma, cishe, kubonisa ukugcinwa kokuguquguquka phakathi kwezizinda ezimbili.Ubulili bubalulekile endimeni engaziwa ye-SPCA6.ukuhlanganiswa kwe-gametes.
I-SPCA6 inezakhiwo zama-protein adhesion intercellular, okuhlanganisa ama-sandwich afana ne-Ig.Amaprotheni amaningi anamathelayo (isb., ama-cadherins, ama-interins, ama-adhesin, kanye ne-IZUMO1) anesizinda esisodwa noma ngaphezulu se-β-sandwich esinweba amaprotheni asuka kulwelwesi lwamaseli kuya kokuhlosiwe kwawo kwemvelo63,64,65.Isizinda esifana ne-Ig se-SPCA6 futhi siqukethe i-motif evame ukutholakala kumasemishi angu-β okunamathela nokuhlangana: okuphindwe kabili kwemicu ehambisanayo ekugcineni kwamasemishi angu-β, aziwa ngokuthi ama-clamp mechanical66.Kukholelwa ukuthi le motif yandisa ukumelana namandla okugunda, okubalulekile kumaprotheni ahilelekile ekusebenzisaneni kwe-intercellular.Kodwa-ke, naphezu kwalokhu kufana nama-adhesin, okwamanje abukho ubufakazi bokuthi i-SPCAA6 isebenzisana nabamhlophe beqanda.I-ectodomain ye-SPCA6 ayikwazi ukubophezela ku-JUNO, namaseli e-HEK293T aveza i-SPCA6, njengoba kuboniswe lapha, awahlangani neze nama-oocyte angenayo i-zona 32.Uma i-SPCA6 isungula amabhondi e-intergametic, lokhu kuxhumana kungase kudinge ukuguqulwa kwangemuva kokuhumusha noma kuqiniswe amanye amaprotheni esidoda.Ekusekeleni i-hypothesis yakamuva, i-spermatozoa entula i-IZUMO1 ibophezela kuma-oocyte, okubonisa ukuthi ama-molecule ngaphandle kwe-IZUMO1 ahileleke ku-gamete adhesion step 27.
Amaprotheni amaningi ahlanganisa amagciwane, amaselula, kanye nentuthuko anezinto ezibikezela umsebenzi wawo njengama-fusogens.Isibonelo, i-viral fusion glycoprotein (amakilasi I, II kanye no-III) ane-hydrophobic fusion peptide noma iluphu ekugcineni kwephrotheni efakwa kulwelwesi lomsingathi.Imephu ye-hydrophilicity ye-IZUMO143 kanye nesakhiwo (esinqunywe futhi sabikezelwa) somndeni omkhulu we-IST abonisanga ukuthi ayikho i-hydrophobic fusion peptide ebonakalayo.Ngakho-ke, uma noma imaphi amaprotheni ku-IST superfamily esebenza njengama-fusogens, akwenza ngendlela ehlukile kwezinye izibonelo ezaziwayo.
Sengiphetha, imisebenzi yamalungu omndeni omkhulu we-IST wamaprotheni ahlotshaniswa ne-gamete fusion iseyimpicabadala emangazayo.I-molecule yethu ehlanganisiwe ye-SPCA6 kanye nesakhiwo sayo esixazululiwe izonikeza ukuqonda ebudlelwaneni phakathi kwalezi zakhiwo ezabiwe kanye nendima yazo ekunamathiselweni nasekuhlanganisweni kwe-gamete.
Ukulandelana kwe-DNA okuhambisana ne-ectodomain yomuntu ebikezelwe ye-SPCAA6 (inombolo yokungena ye-NCBI NP_001303901.1; izinsalela 27–246) yenziwe i-codon-optimized ukuze iboniswe kumaseli e-Drosophila melanogaster S2 futhi ahlanganiswa njengocezu lofuzo ngombhalo wekhodi wokulandelana we-Geknofins Koza)., isignali yokuphuma kwe-BiP kanye neziphetho ezihambisanayo ezingu-5′ no-3′ ze-cloning ezimele yalesi sakhi sofuzo ibe ivektha ye-pMT esekelwe kumgqugquzeli we-metallothionein olungiselwe ukukhethwa nge-puromycin (pMT-puro).Ivekhtha ye-pMT-puro ifaka ikhodi yesayithi ye-thrombin cleavage elandelwa umaka we-C-terminal we-10x (Umfanekiso S2).
Ukudluliselwa okuzinzile kwevekhtha ye-SPCA6 pMT-puro kumaseli e-D. melanogaster S2 (Gibco) kwenziwe ngendlela efanayo nephrothokholi esetshenziselwa i-IZUMO1 ne-JUNO43.Amaseli e-S2 ancibilikiswa futhi akhuliswa endaweni ye-Schneider's medium (Gibco) yengezwe ngokugxilisana kokugcina kuka-10% (v/v) neserum yethole le-fetus elingasebenzi (Gibco) kanye ne-1X antimycotic antibiotic (Gibco).Amaseli okudlula akuqala (amaseli angu-3.0 x 106) afakwe emithonjeni ngayinye yamapuleti anemithombo engu-6 (Corning).Ngemva kwamahora angu-24 ekufukamela ku-27°C, amaseli adluliselwa ngengxube engu-2 mg ye-SPCA6 pMT-puro vector kanye ne-Effectene transfection reagent (Qiagen) ngokuya ngephrothokholi yomkhiqizi.Amaseli aguquliwe afukanyelwa amahora angu-72 futhi avunwa nge-puromycin engu-6 mg/ml.Amaseli abe esehlukaniswa ku-Schneider's medium ephelele futhi afakwa ku-serum free Insect-XPRESS medium (Lonza) ukuze kukhiqizwe amaprotheni ngezinga elikhulu.Iqoqo elingu-1 L lesiko leseli le-S2 lakhuliswa laba amaseli angu-8–10 × 106 ml-1 ku-2 L efakwe umoya we-polypropylene Erlenmeyer eyi-2 L futhi yafakwa inzalo ngenhlanganisela yokugcina engu-500 µM CuSO4 (Millipore Sigma) futhi yahlungwa oyinyumba.eyenzelwe.Amasiko ayenziwe afukanyelwa ku-27° C. ku-120 rpm izinsuku ezine.
I-conditioned medium equkethe i-SPCAA6 yahlukaniswa nge-centrifugation ku-5660×g ku-4°C kwalandelwa uhlelo lwe-Centramate tangential flow filtration (Pall Corp) olunolwelwesi lwe-10 kDa MWCO.Sebenzisa okugxilisiwe okuqukethe i-SPCA6 kukholamu engu-2 ml ye-Ni-NTA agarose resin (Qiagen).Iresin ye-Ni-NTA yagezwa ngamavolumu angu-10 wekholomu (CV) ye-buffer A kwase kwengezwa i-CV engu-1 ye-buffer A ukunikeza ukugxila kokugcina kwemidazole okungu-50 mM.I-SPCAA6 ikhishwe ngo-10 ml we-buffer A ehlanganiswe ne-imidazole ekuhlanganiseni kokugcina okungu-500 mM.I-thrombin yesigaba sokukhawulela (i-Millipore Sigma) yengezwe ngokuqondile kushubhu ye-dialysis (MWCO 12-14 kDa) kuyunithi engu-1 nge-mg SPCA6 vs. 1 L 10 mM Tris-HCl, pH 7.5 kanye ne-150 mM NaCl (buffer B) ye-dialysis.) ku-4°C amahora angu-48.I-SPCA6 e-thrombin-cleaved yabe isihlanjululwa kathathu ukuze kwehliswe ukugcwala kukasawoti futhi ilayishwe kukholomu yokushintshisana ye-cation engu-1 ml MonoS 5/50 GL (Cytiva/GE) ehlanganiswe no-10 mM Tris-HCl, pH 7.5.Isishintshi socingo sawashwa nge-3 CV ka-10 mM Tris-HCl, pH 7.5, kwase kuthi i-SPACA6 yakhishwa ngegradient yomugqa engu-0 kuya ku-500 mM NaCl ku-10 mM Tris-HCl, pH 7.5 ku-25 CV.Ngemuva kwe-ion exchange chromatography, i-SPACA6 yagxiliswa ku-1 ml futhi yakhishwa ngokwehlukana kusukela kukholomu ye-ENrich SEC650 10 x 300 (BioRad) ehlanganiswe nebhafa B. Ngokuvumelana ne-chromatogram, i-pool kanye nezingxenye zokugxilisa ingqondo eziqukethe i-SPCAA6.Ukuhlanzeka kwakulawulwa yi-Coomassie-stained electrophoresis kujeli ye-SDS-polyacrylamide engu-16%.Ukugxiliswa kwamaprotheni kulinganiswa ngokumunca ku-280 nm kusetshenziswa umthetho we-Beer-Lambert kanye ne-coefficient ye-molar extinction ecatshangelwayo.
I-SPACA6 ehlanzekile yahlanjululwa ngobusuku ngokumelene ne-10 mM sodium phosphate, pH 7.4 kanye ne-150 mM NaF futhi yahlanjululwa yaba ngu-0.16 mg/mL ngaphambi kokuhlaziywa nge-CD spectroscopy.I-Spectral scanning yama-CD anobude obuyi-185 kuya ku-260 nm yaqoqwa ku-spectropolarimeter ye-Jasco J-1500 kusetshenziswa i-quartz cuvettes enobude bendlela yokubona engu-1 mm (Helma) ku-25°C ngesilinganiso esingu-50 nm/min.Ukubuka kwe-CD kwalungiswa okuyisisekelo, kwaba nesilinganiso sokutholwa okungaphezu kuka-10, futhi kwaguqulwa ukuze kusho i-elliptical residual (θMRE) ngamadigri cm2/dmol:
lapho i-MW iyisisindo se-molecular yesampula ngayinye ku-Da;N inani lama-amino acid;θ i-elliptical in millidegrees;d ihambisana nobude bendlela ye-optical ngo-cm;ukugxila kwamaprotheni ngamayunithi.

 


Isikhathi sokuthumela: Mar-01-2023