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317 Stainless hlau coil tubing lwm tus neeg

Chemical composition table ntawm stainless hlau khoom

SPACA6 yog cov phev-expressed nto protein uas yog ib qho tseem ceeb rau gamete fusion thaum mammalian kev sib deev kev sib deev.Txawm hais tias lub luag haujlwm tseem ceeb no, cov haujlwm tshwj xeeb ntawm SPACA6 tsis nkag siab.Peb elucidate cov qauv siv lead ua ntawm lub extracellular domain ntawm SPACA6 ntawm qhov kev daws teeb meem ntawm 2.2 Å, nthuav tawm ob-domain protein uas muaj li ntawm plaub-stranded bundle thiab Ig-zoo li β-sandwiches koom nrog quasi-flexible linkers.Cov qauv no zoo ib yam li IZUMO1, lwm gamete fusion-associated protein, ua SPACA6 thiab IZUMO1 nrhiav cov tswv cuab ntawm superfamily ntawm fertilization-sociated proteins raug xa mus rau hauv no ua IST superfamily.Lub IST superfamily yog cov txheej txheem txhais los ntawm nws cov twisted plaub-helix pob thiab ib khub ntawm disulfide-txuas CXXC motifs.Ib tug qauv-raws li AlphaFold kev tshawb fawb ntawm tib neeg proteome tau txheeb xyuas cov neeg muaj protein ntau ntxiv ntawm no superfamily;tshwj xeeb, ntau ntawm cov proteins no koom nrog gamete fusion.Tus qauv SPACA6 thiab nws txoj kev sib raug zoo rau lwm tus tswv cuab ntawm IST superfamily muab qhov txuas uas ploj lawm hauv peb txoj kev paub txog tsiaj txhu gamete fusion.
Txhua tus tib neeg lub neej pib nrog ob qhov sib cais haploid gametes: leej txiv cov phev thiab leej niam lub qe.Cov phev no yog tus yeej ntawm cov txheej txheem xaiv khaus thaum lub sijhawm ntau lab tus phev hlwb dhau los ntawm poj niam qhov chaw mos, kov yeej ntau yam teeb meem1 thiab dhau mus ua kom muaj peev xwm, uas txhim kho lawv txoj kev txav mus los thiab cov txheej txheem ntawm cov khoom nto 2,3,4.Txawm hais tias cov phev thiab oocyte pom ib leeg, cov txheej txheem tseem tsis tau tiav.Lub oocyte yog ib puag ncig los ntawm ib txheej ntawm cumulus hlwb thiab glycoprotein barrier hu ua zona pellucida, uas cov phev yuav tsum dhau mus rau hauv lub oocyte.Spermatozoa siv kev sib xyaw ua ke ntawm qhov chaw adhesion molecules thiab membrane-sociated thiab secreted enzymes kom kov yeej cov teeb meem zaum kawg 5.Cov molecules thiab enzymes no tsuas yog khaws cia rau hauv daim nyias nyias thiab acrosomal matrix thiab raug kuaj pom thaum lub txheej membrane ntawm cov phev yog lysed thaum lub sij hawm acrosomal cov tshuaj tiv thaiv 6.Cov kauj ruam kawg hauv qhov kev taug kev hnyav no yog qhov tshwm sim ntawm cov phev-qe fusion, uas ob lub hlwb fuse lawv daim nyias nyias los ua ib qho kab mob diploid7.Txawm hais tias tus txheej txheem no yog qhov tsim nyog hauv tib neeg kev yug me nyuam, qhov tsim nyog molecular kev sib cuam tshuam tsis to taub.
Ntxiv nrog rau fertilization ntawm gametes, chemistry ntawm fusion ntawm ob lipid bilayers tau kawm ntau heev.Feem ntau, daim nyias nyias fusion yog cov txheej txheem tsis muaj zog uas yuav tsum muaj cov khoom siv protein ua kom muaj kev hloov pauv ntawm cov qauv uas coj ob daim nyias nyias los ze zog ua ke, rhuav tshem lawv qhov txuas ntxiv thiab ua rau fusion8,9.Cov protein catalysts no hu ua fusogens thiab tau pom nyob rau hauv suav tsis txheeb fusion systems.Lawv yuav tsum tau rau cov kab mob nkag mus rau hauv cov tswv tsev (piv txwv li, gp160 hauv HIV-1, spike in coronaviruses, hemagglutinin hauv cov kab mob khaub thuas) 10,11,12 placental (syncytin) 13,14,15 thiab gamete-forming fusions hauv qis eukaryotes ( HAP2/GCS1 nyob rau hauv cov nroj tsuag, protists thiab arthropods) 16,17,18,19.Fusogens rau tib neeg gametes tseem tsis tau pom, txawm hais tias ntau cov proteins tau pom tias yog qhov tseem ceeb rau gamete txuas thiab fusion.Lub oocyte-expressed CD9, ib tug transmembrane protein xav tau rau fusion ntawm nas thiab tib neeg gametes, yog thawj zaug nrhiav tau 21,22,23.Txawm hais tias nws qhov kev ua haujlwm meej tseem tsis meej, lub luag haujlwm hauv adhesion, cov qauv ntawm adhesion foci ntawm qe microvilli, thiab / lossis qhov tseeb hauv zos ntawm oocyte nto proteins zoo li 24,25,26.Qhov ob feem ntau cov proteins uas tseem ceeb rau gamete fusion yog cov phev protein IZUMO127 thiab oocyte protein JUNO28, thiab lawv cov kev sib koom ua ke yog ib qho tseem ceeb hauv gamete paub thiab adhesion ua ntej fusion.Txiv neej Izumo1 knockout nas thiab poj niam Juno knockout nas yog ua kom tsis muaj menyuam, hauv cov qauv no phev nkag mus rau hauv qhov chaw perivitelline tab sis gametes tsis fuse.Ib yam li ntawd, confluence tau txo qis thaum gametes tau kho nrog anti-IZUMO1 lossis JUNO27,29 cov tshuaj tiv thaiv hauv tib neeg hauv vitro fertilization thwmsim.
Tsis ntev los no, ib pab pawg neeg nrhiav pom tshiab ntawm cov phev-expressed proteins phenotypically zoo ib yam li IZUMO1 thiab JUNO20,30,31,32,33,34,35 tau pom.Sperm acrosomal membrane-associated protein 6 (SPACA6) tau raug txheeb xyuas tias yog qhov tseem ceeb rau kev yug me nyuam hauv kev tshawb fawb murine mutagenesis loj.Ntxig cov transgene rau hauv Spaca6 noob tsim cov spermatozoa nonfusible, tab sis cov spermatozoa infiltrate perivitelline chaw 36.Cov kev tshawb fawb tom qab knockout hauv nas tau lees paub tias Spaca6 yuav tsum muaj rau gamete fusion 30,32 .SPACA6 tau qhia yuav luag tshwj xeeb hauv cov qe ntshav thiab muaj cov qauv hauv zos zoo ib yam li ntawm IZUMO1, uas yog nyob rau hauv lub intima ntawm spermatozoa ua ntej cov tshuaj tiv thaiv acrosomal, thiab tom qab ntawd tsiv mus rau thaj tsam equatorial tom qab cov tshuaj tiv thaiv acrosomal 30,32.Spaca6 homologues muaj nyob rau hauv ntau hom tsiaj thiab lwm yam eukaryotes 30 thiab nws qhov tseem ceeb rau tib neeg gamete fusion tau pom los ntawm inhibition ntawm tib neeg fertilization hauv vitro los ntawm kev tiv thaiv SPACA6 30 .Tsis zoo li IZUMO1 thiab JUNO, cov ntsiab lus ntawm cov qauv, kev sib cuam tshuam, thiab kev ua haujlwm ntawm SPACA6 tseem tsis meej.
Txhawm rau kom nkag siab zoo dua cov txheej txheem hauv qab ntawm kev sib xyaw ntawm tib neeg cov phev thiab qe, uas yuav ua rau peb paub txog kev txhim kho yav tom ntej hauv kev npaj tsev neeg thiab kev kho mob fertility, peb tau ua SPACA6 cov qauv thiab kev tshawb fawb biochemical.Cov qauv siv lead ua ntawm lub extracellular domain ntawm SPACA6 qhia tau hais tias plaub-helical pob (4HB) thiab ib qho immunoglobulin-zoo li (Ig-zoo li) domain txuas los ntawm quasi-flexible regions.Raws li kev kwv yees hauv cov kev tshawb fawb yav dhau los, 7,32,37 tus qauv sau npe ntawm SPACA6 zoo ib yam li tib neeg IZUMO1, thiab ob lub proteins sib koom ib qho txawv txawv motif: 4HB nrog daim duab peb sab helical nto thiab ib khub ntawm disulfide-txuas CXXC motifs.Peb tawm tswv yim tias IZUMO1 thiab SPACA6 tam sim no txhais qhov loj dua, muaj feem cuam tshuam nrog superfamily ntawm cov proteins cuam tshuam nrog gamete fusion.Siv cov yam ntxwv tshwj xeeb rau lub tsev neeg superfamily, peb tau tshawb nrhiav tag nrho rau AlphaFold tus qauv tib neeg proteome, txheeb xyuas cov tswv cuab ntxiv ntawm cov neeg hauv tsev neeg no, suav nrog ntau tus tswv cuab koom nrog gamete fusion thiab / lossis fertilization.Tam sim no nws zoo nkaus li tias muaj ib qho kev sib koom ua ke thiab superfamily ntawm cov proteins txuam nrog gamete fusion, thiab peb cov qauv muab ib daim ntawv qhia molecular ntawm qhov tseem ceeb ntawm tib neeg gamete fusion mechanism.
SPACA6 yog ib qho kev sib kis transmembrane protein nrog ib N-txuas glycan thiab rau putative disulfide bonds (Figures S1a thiab S2).Peb tau nthuav tawm lub extracellular domain ntawm tib neeg SPACA6 (residues 27–246) nyob rau hauv Drosophila S2 hlwb thiab purified cov protein siv npib tsib xee affinity, cation txauv, thiab loj cais chromatography (Fig. S1b).Lub purified SPACA6 ectodomain yog ruaj khov thiab homogeneous.Kev tshuaj xyuas siv qhov loj me me tsis suav nrog chromatography ua ke nrog polygonal lub teeb tawg (SEC-MALS) tau nthuav tawm ib lub ncov nrog cov ntsuas molecular hnyav ntawm 26.2 ± 0.5 kDa (Fig. S1c).Qhov no yog raws li qhov loj ntawm SPACA6 monomeric ectodomain, qhia tias oligomerization tsis tshwm sim thaum lub sij hawm purification.Tsis tas li ntawd, lub voj voog dichroism (CD) spectroscopy tau nthuav tawm cov qauv sib xyaw α / β nrog lub melting point ntawm 51.3 ° C (Fig. S1d, e).Deconvolution ntawm CD spectra qhia 38.6% α-helical thiab 15.8% β-stranded ntsiab (Daim duab S1d).
SPACA6 ectodomain tau crystallized siv random matrix seeding38 ua rau cov ntaub ntawv teeb tsa nrog kev daws teeb meem ntawm 2.2 Å (Table 1 thiab daim duab S3).Siv kev sib xyaw ua ke ntawm kev hloov pauv molecular thiab SAD phasing cov ntaub ntawv nrog bromide raug rau kev txiav txim siab qauv (Table 1 thiab daim duab S4), cov qauv ua kom zoo kawg nkaus muaj cov seem 27-246.Thaum lub sij hawm tus qauv txiav txim, tsis muaj kev sim lossis AlphaFold cov qauv muaj.Lub SPACA6 ectodomain ntsuas 20 Å × 20 Å × 85 Å, muaj xya helices thiab cuaj β-strands, thiab muaj elongated tertiary quav stabilized los ntawm rau disulfide bonds (Fig. 1a, b).Qhov tsis muaj hluav taws xob tsis muaj zog ntawm qhov kawg ntawm Asn243 sab saw qhia tias qhov seem no yog N-txuas glycosylation.Cov qauv muaj ob lub npe: N-terminal plaub-helix pob (4HB) thiab C-terminal Ig-zoo li sau nrog thaj tsam nruab nrab pob khawm ntawm lawv (Daim duab 1c).
ib tug qauv ntawm extracellular domain ntawm SPACA6.Sawb daim duab ntawm lub extracellular domain ntawm SPACA6, xim ntawm cov saw ntawm N rau C-terminus los ntawm tsaus xiav mus rau tsaus liab.Cysteines koom nrog hauv disulfide bonds yog qhov tseem ceeb hauv magenta.b Topology ntawm extracellular domain ntawm SPACA6.Siv tib lub tswv yim xim raws li hauv daim duab 1a.c SPACA6 extracellular domain.Lub 4HB, pob khawm, thiab Ig-zoo li cov kab kos npe kab kos yog xim txiv kab ntxwv, ntsuab, thiab xiav, feem.Cov khaubncaws sab nraud povtseg tsis tau kos rau scale.
4HB domain ntawm SPACA6 suav nrog plaub lub ntsiab helices (helices 1–4), uas yog teem rau hauv daim ntawv ntawm ib tug helical helix (Fig. 2a), alternating ntawm antiparallel thiab parallel kev sib tshuam (Fig. 2b).Ib qho me me ntxiv ib zaug tig helix (helix 1′) yog nteg perpendicular mus rau lub nras, tsim ib daim duab peb sab nrog helices 1 thiab 2. Daim duab peb sab no yog me ntsis deformed nyob rau hauv lub helical-twisted packing ntawm cov tuab tuab ntawm helices 3 thiab 4 ( Fig. 2a).
4HB N-terminal strip chart.b Sab saum toj saib ntawm ib pob ntawm plaub lub helices, txhua lub helix tseem ceeb hauv xiav tsaus ntawm N-terminus thiab tsaus liab ntawm C-terminus.c Sab saum toj-down kauv daim duab kos rau 4HB, nrog rau txhua qhov seem uas pom tias yog lub voj voog uas muaj ib tsab ntawv amino acid code;Tsuas yog plaub lub amino acids nyob rau sab saum toj ntawm lub log yog suav.Cov tsis-polar residues yog xim daj, polar uncharged residues yog xim ntsuab, zoo them residues yog xim xiav, thiab tsis zoo them nqi residue yog xim liab.d Daim duab peb sab ntawm 4HB sau, nrog 4HBs hauv txiv kab ntxwv thiab pob khawm ntsuab.Ob lub insets qhia qws-zoo li tus disulfide bonds.
4HB yog concentrated nyob rau hauv ib tug puab hydrophobic core muaj xws li tsuas yog aliphatic thiab aromatic residues (Fig. 2c).Cov tub ntxhais kawm ntawv muaj ib daim ntawv cog lus disulfide ntawm Cys41 thiab Cys55 uas txuas cov helices 1 thiab 2 ua ke nyob rau hauv daim duab peb sab saum toj (Daim duab 2d).Ob daim ntawv cog lus ntxiv disulfide tau tsim los ntawm CXXC motif hauv Helix 1′ thiab lwm CXXC motif pom nyob rau ntawm qhov kawg ntawm β-hairpin hauv thaj tsam pob khawm (Fig. 2d).Ib qho kev txuag arginine residue nrog qhov tsis paub ua haujlwm (Arg37) yog nyob rau hauv daim duab peb sab hollow tsim los ntawm helices 1′, 1, thiab 2. Aliphatic carbon atoms Cβ, Cγ, thiab Cδ Arg37 cuam tshuam nrog cov tub ntxhais hydrophobic, thiab nws cov pab pawg guanidine cyclically txav. nruab nrab ntawm helices 1′ thiab 1 ntawm kev sib cuam tshuam ntawm Thr32 qaum thiab sab saw (Fig. S5a,b).Tyr34 txuas mus rau hauv cov kab noj hniav tawm hauv ob qhov kab noj hniav me me los ntawm qhov uas Arg37 tuaj yeem cuam tshuam nrog cov kuab tshuaj.
Ig-zoo li β-sandwich domains yog ib tug loj superfamily ntawm cov proteins uas qhia cov yam ntxwv ntawm ob los yog ntau tshaj multi-stranded amphipathic β-phau ntawv interacting ntawm ib tug hydrophobic core 39. C-terminal Ig-zoo li domain ntawm SPACA6 muaj tib tus qauv. thiab muaj ob txheej (Fig. S6a).Daim Ntawv 1 yog β-daim ntawv ntawm plaub strands (strands D, F, H, thiab I) qhov twg strands F, H, thiab kuv tsim ib qho kev tiv thaiv kev sib piv, thiab strands kuv thiab D coj mus rau ib qho kev sib piv.Table 2 yog ib daim ntawv me me los tiv thaiv kev sib txuas ob-stranded beta (strands E thiab G).Ib daim ntawv cog lus nrog disulfide tau pom nyob nruab nrab ntawm C-terminus ntawm E saw thiab qhov chaw ntawm H saw (Cys170-Cys226) (Fig. S6b).Cov nyiaj disulfide no zoo ib yam rau cov nyiaj disulfide hauv β-sandwich domain ntawm immunoglobulin40,41.
Plaub-strand β-daim ntawv twists raws nws tag nrho ntev, tsim asymmetrical npoo uas txawv ntawm cov duab thiab electrostatics.Lub ntug nyias nyias yog ib qho chaw tiaj tus hydrophobic ib puag ncig uas sawv tawm piv rau qhov seem tsis sib xws thiab electrostatically sib txawv ntawm qhov chaw hauv SPACA6 (Fig. S6b,c).Ib qho halo ntawm cov pob txha raug mob carbonyl/amino pawg thiab cov kab nruab nrab sab qaum teb nyob ib puag ncig ntawm qhov chaw hydrophobic (Fig. S6c).Cov npoo dav dav yog npog los ntawm ib qho capped helical ntu uas thaiv N-terminal feem ntawm cov tub ntxhais hydrophobic thiab tsim peb hydrogen bonds nrog qhib polar pawg ntawm F chain backbone (Fig. S6d).C-terminal feem ntawm ntug no ua ib lub hnab ntim loj nrog ib feem ntawm cov tub ntxhais hydrophobic.Lub hnab tshos nyob ib puag ncig los ntawm cov nqi zoo vim yog peb pawg ntawm ob chav arginine residues (Arg162-Arg221, Arg201-Arg205 thiab Arg212-Arg214) thiab lub hauv paus histidine (His220) (Daim duab S6e).
Cov cheeb tsam pob khawm yog ib ntu luv ntawm lub helical domain thiab Ig-zoo li domain, muaj xws li ib tug antiparallel peb-stranded β-txheej (strands A, B thiab C), ib tug me me 310 helix, thiab ob peb ntev random helical ntu.(Fig. S7).Ib lub network ntawm covalent thiab electrostatic tiv tauj hauv cheeb tsam pob khawm zoo li txhim kho kev taw qhia ntawm 4HB thiab Ig-zoo li domain.Lub network tuaj yeem muab faib ua peb ntu.Thawj ntu suav nrog ob CXXC motifs (27CXXC30 thiab 139CXXC142) uas tsim ib khub ntawm disulfide bonds ntawm β-hairpin hauv pob khawm thiab 1′ helix hauv 4HB.Qhov thib ob suav nrog electrostatic kev sib cuam tshuam ntawm Ig-zoo li sau thiab pob khawm.Glu132 nyob rau hauv pob khawm tsim ib tug ntsev choj nrog Arg233 nyob rau hauv lub Ig-zoo li sau thiab Arg135 nyob rau hauv lub pob khawm.Qhov thib peb suav nrog kev sib cog lus ntawm Ig-zoo li sau thiab thaj tsam pob khawm.Ob daim ntawv cog lus disulfide (Cys124-Cys147 thiab Cys128-Cys153) txuas lub pob khawm mus rau qhov sib txuas uas ruaj khov los ntawm kev sib cuam tshuam ntawm electrostatic ntawm Gln131 thiab cov pob txha ua haujlwm, tso cai rau kev nkag mus rau thawj Ig-zoo li tus sau.saw hlau.
Cov qauv ntawm SPACA6 ectodomain thiab tus kheej cov qauv ntawm 4HB thiab Ig-zoo li domains tau siv los tshawb nrhiav cov ntaub ntawv zoo sib xws hauv cov ntaub ntawv protein 42 .Peb tau txheeb xyuas qhov sib tw nrog cov qhab nia Dali Z siab, qhov sib txawv me me, thiab cov qhab nia LALI loj (tom kawg yog tus naj npawb ntawm cov khoom seem sib npaug).Thaum thawj 10 hits los ntawm tag nrho cov kev tshawb fawb ectodomain (Table S1) muaj qhov tau txais Z-cov qhab nia ntawm> 842, kev tshawb fawb rau 4HB lossis Ig-zoo li sau ib leeg pom tau tias feem ntau ntawm cov hits sib xws rau β-sandwiches nkaus xwb.ib qho ubiquitous quav pom nyob rau hauv ntau cov proteins.Tag nrho peb qhov kev tshawb fawb hauv Dali rov qab tsuas yog ib qho txiaj ntsig: IZUMO1.
Nws tau ntev tau pom zoo tias SPACA6 thiab IZUMO1 qhia cov qauv zoo sib xws 7,32,37.Txawm hais tias cov ectodomains ntawm ob gamete fusion-associated proteins sib koom tsuas yog 21% ib ntus tus kheej (Daim duab S8a), cov pov thawj nyuaj, suav nrog cov qauv kev txuag nyiaj disulfide thiab kev kwv yees C-terminal Ig-zoo li sau hauv SPACA6, tso cai rau kev sim ntxov los tsim kom muaj ib qho homology qauv ntawm A ib SPACA6 nas siv IZUMO1 ua tus qauv37.Peb cov qauv lees paub cov kev kwv yees no thiab qhia qhov tseeb ntawm qhov zoo sib xws.Qhov tseeb, SPACA6 thiab IZUMO137,43,44 cov qauv sib koom ua ke ob-ntu architecture (Fig. S8b) nrog 4HB thiab Ig-zoo li β-sandwich domains txuas nrog thaj tsam pob khawm (Fig. S8c).
IZUMO1 thiab SPACA6 4HB muaj qhov sib txawv ntawm cov khoom siv sib xyaw ua ke.Hom 4HBs, zoo li cov pom nyob rau hauv SNARE protein complexes koom nrog hauv endosomal fusion 45,46, muaj qhov sib npaug ntawm cov helices tswj qhov curvature tsis tu ncua nyob ib ncig ntawm lub hauv paus axis 47. Nyob rau hauv sib piv, cov helical domains nyob rau hauv ob qho tib si IZUMO1 thiab SPACA6 tau distorted, nrog sib txawv curvature thiab ntim tsis sib xws (Daim duab S8d).Qhov twist, tej zaum tshwm sim los ntawm daim duab peb sab tsim los ntawm helices 1′, 1 thiab 2, yog khaws cia nyob rau hauv IZUMO1 thiab SPACA6 thiab stabilized los ntawm tib CXXC motif ntawm helix 1′.Txawm li cas los xij, ntxiv cov nyiaj disulfide pom nyob rau hauv SPACA6 (Cys41 thiab Cys55 covalently txuas helices 1 thiab 2 saum toj no) tsim ib tug sharper apex ntawm daim duab peb sab apex, ua SPACA6 ntau twisted tshaj IZUMO1, nrog ntau pronounced kab noj hniav daim duab peb sab.Tsis tas li ntawd, IZUMO1 tsis muaj Arg37 pom nyob hauv nruab nrab ntawm cov kab noj hniav hauv SPACA6.Nyob rau hauv sib piv, IZUMO1 muaj ntau dua hydrophobic core ntawm aliphatic thiab aromatic residues.
IZUMO1 muaj ib qho Ig-zoo li sau muaj xws li ob-stranded thiab tsib-stranded β-sheet43.Qhov ntxiv strand hauv IZUMO1 hloov lub kauj hauv SPACA6, uas cuam tshuam nrog F strand kom txwv cov pob txha hydrogen bonds hauv strand.Ib qho kev nthuav qhia ntawm kev sib piv yog qhov kwv yees qhov chaw them nqi ntawm Ig-zoo li thawj ntawm ob cov proteins.Lub IZUMO1 nto yog qhov tsis zoo them ntau dua li SPACA6 nto.Ib qho nqi ntxiv yog nyob ze ntawm C-terminus ntsib cov phev membrane.Hauv SPACA6, tib cheeb tsam tau nruab nrab lossis raug them nyiaj ntau dua (Fig. S8e).Piv txwv li, hydrophobic nto (thinner edges) thiab zoo them pits (dav ntug) nyob rau hauv SPACA6 yog tsis zoo nqi nyob rau hauv IZUMO1.
Txawm hais tias qhov kev sib raug zoo thiab cov qauv theem nrab ntawm IZUMO1 thiab SPACA6 tau khaws cia zoo, cov qauv kev sib raug zoo ntawm Ig-zoo li thawj tau pom tias ob lub ntsiab lus sib txawv hauv lawv qhov kev qhia dav dav txheeb ze rau ib leeg (Daim duab S9).Lub kauv pob ntawm IZUMO1 yog nkhaus txog β-sandwich, tsim cov yav tas los piav "boomerang" zoo li ntawm 50 ° los ntawm lub hauv paus axis.Nyob rau hauv sib piv, lub helical beam nyob rau hauv SPACA6 yog tilted txog 10 ° nyob rau hauv lub opposite direction.Qhov sib txawv ntawm cov kev taw qhia no yuav yog vim qhov sib txawv hauv cheeb tsam pob khawm.Nyob rau theem thawj theem, IZUMO1 thiab SPACA6 qhia me ntsis kev sib piv ntawm pob khawm, tshwj tsis yog cov cysteine, glycine, thiab aspartic acid residues.Yog li ntawd, hydrogen bonds thiab electrostatic tes hauj lwm sib txawv kiag li.β-daim ntawv theem nrab cov ntsiab lus tau sib koom los ntawm IZUMO1 thiab SPACA6, txawm hais tias cov chains hauv IZUMO1 ntev dua thiab 310 helix (helix 5) yog qhov tshwj xeeb rau SPACA6.Cov kev sib txawv no ua rau muaj kev sib txawv ntawm qhov sib txawv rau ob qho kev sib txawv ntawm cov proteins.
Peb Dali neeg rau zaub mov tshawb nrhiav tau qhia tias SPACA6 thiab IZUMO1 tsuas yog ob qho kev sim txiav txim siab cov qauv khaws cia hauv cov ntaub ntawv protein uas muaj qhov tshwj xeeb 4HB quav (Table S1).Tsis ntev los no, DeepMind (niam ntawv / Google) tau tsim AlphaFold, lub neural network raws li cov txheej txheem uas tuaj yeem kwv yees tau 3D cov qauv ntawm cov protein los ntawm thawj ntu 48.Tsis ntev tom qab peb tau daws cov qauv SPACA6, AlphaFold cov ntaub ntawv tau raug tso tawm, muab cov qauv kev kwv yees uas npog 98.5% ntawm tag nrho cov proteins hauv tib neeg proteome48,49.Siv peb txoj kev daws teeb meem SPACA6 raws li tus qauv tshawb nrhiav, kev tshawb nrhiav homology rau tus qauv hauv AlphaFold human proteome tau txheeb xyuas cov neeg sib tw nrog cov qauv zoo sib xws rau SPACA6 thiab IZUMO1.Muab qhov tseeb tsis txaus ntseeg ntawm AlphaFold hauv kev kwv yees SPACA6 (Daim duab S10a)-tshwj xeeb tshaj yog 1.1 Å rms ectodomain piv rau peb cov qauv daws teeb meem (Daim duab S10b)-peb tuaj yeem ntseeg tau tias qhov kev txheeb xyuas SPACA6 qhov sib tw yog qhov yuav raug.
Yav dhau los, PSI-BLAST tau tshawb nrhiav cov pab pawg IZUMO1 nrog peb lwm cov phev-cov protein uas muaj feem xyuam nrog: IZUMO2, IZUMO3, thiab IZUMO450.AlphaFold tau kwv yees tias cov tsev neeg IZUMO cov proteins quav rau hauv 4HB domain nrog tib lub disulfide daim ntawv cog lus raws li IZUMO1 (Figures 3a thiab S11), txawm hais tias lawv tsis muaj Ig-zoo li sau.Nws yog hypothesized tias IZUMO2 thiab IZUMO3 yog ib leeg-sab membrane proteins zoo ib yam li IZUMO1, thaum IZUMO4 zoo li yuav zais.Kev ua haujlwm ntawm IZUMO 2, 3, thiab 4 proteins hauv gamete fusion tsis tau txiav txim siab.IZUMO3 paub tias ua lub luag haujlwm hauv acrosome biogenesis thaum lub sij hawm spermatozoa txoj kev loj hlob51, thiab cov protein IZUMO tsim cov complex50.Kev txuag ntawm IZUMO cov proteins hauv cov tsiaj tsiaj, tsiaj reptiles, thiab amphibians qhia tias lawv lub peev xwm muaj peev xwm ua tau raws li lwm cov gamete-fusion-sociated proteins, xws li DCST1/2, SOF1, thiab FIMP.
Daim duab kos duab ntawm lub tsev tsim qauv ntawm IST superfamily, nrog 4HB, pob khawm, thiab Ig-zoo li qhov tseem ceeb hauv txiv kab ntxwv, ntsuab, thiab xiav, raws li.IZUMO4 muaj qhov tshwj xeeb C-terminal cheeb tsam uas zoo li dub.Confirmed thiab putative disulfide bonds yog qhia los ntawm cov khoom thiab dotted kab, feem.b IZUMO1 (PDB: 5F4E), SPACA6, IZUMO2 (AlphaFold DB: AF-Q6UXV1-F1), IZUMO3 (AlphaFold DB: AF-Q5VZ72-F1), IZUMO4 (AlphaFold DB: AF-Q1ZYLTM8-F5Al), thiab DB: AF-Q1ZYL8-F1): AF-Q1ZYL8-F1): AF-Q3KNT9-F1) yog tso tawm nyob rau hauv tib xim ntau yam li vaj huam sib luag A. Disulfide bonds muaj nyob rau hauv magenta.TMEM95, IZUMO2 thiab IZUMO3 transmembrane helices tsis qhia.
Tsis zoo li cov protein IZUMO, lwm cov SPACA proteins (piv txwv li, SPACA1, SPACA3, SPACA4, SPACA5, thiab SPACA9) yog xav tias yuav txawv ntawm SPACA6 (Fig. S12).Tsuas yog SPACA9 muaj 4HB, tab sis nws tsis xav tias yuav muaj qhov sib npaug-tiv thaiv kev taw qhia lossis tib daim ntawv cog lus disulfide li SPACA6.Tsuas yog SPACA1 muaj qhov zoo sib xws Ig-zoo li sau npe.AlphaFold kwv yees tias SPACA3, SPACA4 thiab SPACA5 muaj cov qauv sib txawv ntawm SPACA6.Interestingly, SPACA4 tseem paub tias ua lub luag haujlwm hauv fertilization, tab sis ntau dua li SPACA6, es tsis txhob ua kom yooj yim rau kev sib cuam tshuam ntawm phev thiab oocyte zona pellucida52.
Peb qhov kev tshawb nrhiav AlphaFold pom lwm qhov sib tw rau IZUMO1 thiab SPACA6 4HB, TMEM95.TMEM95, ib leeg phev-specific transmembrane protein, ua rau txiv neej nas infertile thaum ablated 32,33.Spermatozoa tsis muaj TMEM95 muaj ib txwm morphology, motility, thiab muaj peev xwm nkag mus rau lub zona pellucida thiab khi rau lub qe membrane, tab sis tsis tuaj yeem fuse nrog oocyte membrane.Cov kev tshawb fawb yav dhau los tau pom tias TMEM95 qhia cov qauv zoo sib xws nrog IZUMO133.Tseeb, tus qauv AlphaFold tau lees paub tias TMEM95 yog 4HB nrog tib khub ntawm CXXC motifs raws li IZUMO1 thiab SPACA6 thiab tib yam ntxiv disulfide daim ntawv cog lus ntawm helices 1 thiab 2 pom hauv SPACA6 (Fig. 3a thiab S11).Txawm hais tias TMEM95 tsis muaj Ig-zoo li tus sau, nws muaj ib cheeb tsam nrog cov qauv disulfide daim ntawv cog lus zoo ib yam li SPACA6 thiab IZUMO1 pob khawm thaj tsam (Daim duab 3b).Thaum lub sijhawm tshaj tawm cov ntawv sau no, cov neeg ua haujlwm sau ua ntej tau tshaj tawm cov qauv ntawm TMEM95, lees paub qhov tshwm sim AlphaFold53.TMEM95 zoo ib yam li SPACA6 thiab IZUMO1 thiab yog evolutionarily khaws cia nyob rau hauv amphibians (Fig. 4 thiab S13).
Kev tshawb nrhiav PSI-BLAST siv NCBI SPACA6, IZUMO1-4, TMEM95, DCST1, DCST2, FIMP, thiab SOF1 cov ntaub ntawv los txiav txim txoj haujlwm ntawm cov kab ke no hauv tsob ntoo ntawm lub neej.Qhov kev ncua deb ntawm ceg taw tes tsis qhia rau qhov ntsuas.
Qhov kev sib tw tag nrho cov qauv zoo sib xws ntawm SPACA6 thiab IZUMO1 qhia tias lawv tab tom nrhiav cov tswv cuab ntawm kev txuag cov txheej txheem superfamily uas suav nrog TMEM95 thiab IZUMO 2, 3, thiab 4 proteins.paub cov tswv cuab: IZUMO1, SPACA6 thiab TMEM95.Vim tias tsuas yog ob peb tus tswvcuab muaj Ig-zoo li tus thawj coj, lub ntsiab lus ntawm IST superfamily yog 4HB domain, uas muaj cov yam ntxwv tshwj xeeb rau tag nrho cov proteins no: 1) Coiled 4HB nrog helices teem nyob rau hauv ib qho kev tiv thaiv kev sib txuas / kev sib txuas (Fig . 5a), 2) lub pob muaj ib lub ntsej muag daim duab peb sab uas muaj ob lub helices nyob rau hauv lub nras thiab ib tug thib peb ntsug helix (Daim duab. 5c). Raws li redox sensor 54,55,56, thaum lub motif hauv IST cov neeg hauv tsev neeg tuaj yeem cuam tshuam nrog cov protein disulfide isomerases xws li ERp57 hauv gamete fusion. Lub luag haujlwm yog txuam nrog 57,58.
Cov tswv cuab ntawm IST superfamily tau txhais los ntawm peb tus yam ntxwv ntawm 4HB domain: plaub lub helices alternating ntawm parallel thiab antiparallel orientation, ba-triangular helical bundle ntsej muag, thiab ib tug ca CXXC ob motif tsim ntawm me me molecules.) N-terminal helixes (txiv kab ntxwv) thiab pob khawm thaj tsam β-hairpin (ntsuab).
Muab qhov zoo sib xws ntawm SPACA6 thiab IZUMO1, lub peev xwm ntawm yav dhau los khi rau IZUMO1 lossis JUNO tau sim.Biolayer interferometry (BLI) yog kinetic-raws li binding txoj kev uas yav tas los tau siv los ntsuas qhov kev cuam tshuam ntawm IZUMO1 thiab JUNO.Tom qab incubation ntawm biotin-labeled sensor nrog IZUMO1 raws li ib tug bait nrog ib tug siab concentration ntawm JUNO analyte, ib tug muaj zog teeb liab tau kuaj pom (Fig. S14a), qhia ib tug binding-induced hloov nyob rau hauv lub thickness ntawm biomaterial txuas mus rau lub sensor tip.Cov teeb liab zoo sib xws (piv txwv li, JUNO txuas rau lub sensor ua ib qho khoom siv tiv thaiv IZUMO1 analyte) (Fig. S14b).Tsis muaj lub teeb liab raug kuaj pom thaum SPACA6 tau siv los ua kev tshuaj ntsuam xyuas qhov ntsuas-txheej txheem IZUMO1 lossis sensor-bound JUNO (Daim duab S14a,b).Qhov tsis muaj cov teeb liab no qhia tau hais tias lub extracellular domain ntawm SPACA6 tsis cuam tshuam nrog lub extracellular domain ntawm IZUMO1 los yog JUNO.
Vim tias BLI kev soj ntsuam yog raws li biotinylation ntawm dawb lysine residues ntawm cov khoom noj protein, qhov kev hloov kho no tuaj yeem tiv thaiv kev khi yog tias lysine residues koom nrog kev sib cuam tshuam.Tsis tas li ntawd, qhov kev taw qhia ntawm kev khi tus txheeb ze rau lub sensor tuaj yeem tsim kev cuam tshuam tsis zoo, yog li cov qauv rub-down kev soj ntsuam kuj tau ua nyob rau hauv recombinant SPACA6, IZUMO1 thiab JUNO ectodomains.Txawm li cas los xij, SPACA6 tsis tau los nag nrog Nws-tagged IZUMO1 lossis Nws-tagged JUNO (Fig. S14c,d), qhia tias tsis muaj kev cuam tshuam nrog qhov pom hauv BLI kev sim.Raws li kev tswj hwm zoo, peb tau lees paub qhov kev sib cuam tshuam ntawm JUNO nrog sau tias Nws IZUMO1 (Figures S14e thiab S15).
Txawm hais tias tus qauv zoo sib xws ntawm SPACA6 thiab IZUMO1, qhov tsis muaj peev xwm ntawm SPACA6 los khi JUNO tsis yog qhov xav tsis thoob.Qhov saum npoo ntawm tib neeg IZUMO1 muaj ntau dua 20 qhov seem uas cuam tshuam nrog JUNO, suav nrog cov seem ntawm txhua qhov ntawm peb cheeb tsam (txawm tias lawv feem ntau nyob hauv thaj tsam pob khawm) (Fig. S14f).Ntawm cov residues no, tsuas yog ib qho yog khaws cia hauv SPACA6 (Glu70).Txawm hais tias muaj ntau qhov kev hloov pauv residue khaws lawv cov khoom qub biochemical, qhov tseem ceeb Arg160 residue hauv IZUMO1 tau hloov los ntawm qhov tsis zoo them Asp148 hauv SPACA6;Cov kev tshawb fawb yav dhau los tau pom tias Arg160Glu kev hloov pauv hauv IZUMO1 yuav luag tag nrho tshem tawm kev khi rau JUNO43.Tsis tas li ntawd, qhov sib txawv ntawm qhov kev taw qhia ntawm IZUMO1 thiab SPACA6 tau nce qhov chaw ntawm JUNO-binding site ntawm thaj tsam sib npaug ntawm SPACA6 (Fig. S14g).
Txawm hais tias qhov kev xav tau ntawm SPACA6 rau gamete fusion thiab nws qhov zoo sib xws rau IZUMO1, SPACA6 tsis tshwm sim kom muaj qhov sib npaug JUNO binding muaj nuj nqi.Yog li ntawd, peb tau nrhiav kev sib txuas peb cov ntaub ntawv qauv nrog cov pov thawj ntawm qhov tseem ceeb los ntawm evolutionary biology.Kev sib xyaw ua ke ntawm SPACA6 homologues qhia txog kev txuag ntawm cov qauv zoo tshaj li cov tsiaj txhu.Piv txwv li, cysteine ​​​​residues yog tam sim no txawm nyob deb ntawm amphibians (Fig. 6a).Siv ConSurf neeg rau zaub mov, ntau qhov sib txuas ua ke khaws cov ntaub ntawv ntawm 66 cov kab ke tau teeb tsa rau SPACA6 nto.Hom kev tshuaj ntsuam no tuaj yeem qhia tau tias cov khoom seem twg tau khaws cia thaum lub sijhawm hloov pauv ntawm cov protein thiab tuaj yeem qhia tau tias thaj chaw ntawm thaj chaw twg ua lub luag haujlwm hauv kev ua haujlwm.
Kev sib txuas ua ke ntawm SPACA6 ectodomains los ntawm 12 hom sib txawv npaj siv CLUSTAL OMEGA.Raws li ConSurf tsom xam, feem ntau kev saib xyuas txoj haujlwm yog cim xiav.Cysteine ​​​​residues tseem ceeb hauv liab.Domain ciam teb thiab cov qauv theem nrab tau pom nyob rau sab saum toj ntawm qhov kev sib dhos, qhov twg cov xub qhia β-strands thiab nthwv dej qhia helices.NCBI Access Identifiers uas muaj cov sequences yog: tib neeg (Homo sapiens, NP_001303901), mandrill (Mandrilus leucophaeus, XP_011821277), capuchin liab (Cebus mimic, XP_017359366), nees (Equus caball5, Equus caball3, Equus 2000, Equus 2000, Equus caball3) _23 XP_032_034).), yaj (Ovis aries, XP_014955560), ntxhw (Loxodonta africana, XP_010585293), aub (Canis lupus familyis, XP_025277208), nas (Mus musculus, NP_001156381), Tasmanian devil (Sarcophii, XP_001001, 2011) s, 8) ib. , 61_89 thiab Bullfrog (Bufo bufo, XP_040282113).Tus naj npawb yog raws li tib neeg kev txiav txim.b Qhov chaw sawv cev ntawm tus qauv SPACA6 nrog 4HB nyob rau sab saum toj thiab Ig-zoo li sau rau hauv qab, xim raws li kev txuag kwv yees los ntawm ConSurf server.Cov khoom khaws cia zoo tshaj plaws yog xiav, qhov chaw khaws cia nruab nrab yog xim dawb, thiab qhov tsawg tshaj plaws khaws cia yog daj.ntshav cysteine.Peb qhov chaw thaj ua rau pom kev tiv thaiv qib siab tau pom nyob rau hauv daim ntawv sau npe thaj ua rau thaj 1, 2 thiab 3. A 4HB tas luav yog qhia nyob rau hauv inset nyob rau sab xis (tib yam xim scheme).
Tus qauv SPACA6 muaj peb thaj chaw ruaj khov heev (Fig. 6b).Patch 1 spans 4HB thiab thaj tsam pob khawm thiab muaj ob qhov chaw khaws cia CXXC disulfide txuas hniav, ib qho Arg233-Glu132-Arg135-Ser144 pob khawm network (Fig. S7), thiab peb khaws cia sab nraud cov ntxhiab tsw qab (Phe31, Tyr73, Phe137).ib txoj kab dav dav ntawm Ig-zoo li tus sau (Fig. S6e), uas sawv cev rau ob peb qhov zoo ntawm cov khoom seem ntawm cov phev nto.Interestingly, lub thaj no muaj cov tshuaj tiv thaiv kab mob epitope uas tau pom yav dhau los cuam tshuam nrog SPACA6 30 muaj nuj nqi.Thaj tsam 3 spans lub pob khawm thiab ib sab ntawm Ig-zoo li sau;thaj av no muaj cov prolines khaws cia (Pro126, Pro127, Pro150, Pro154) thiab sab nraud-ntsej muag polar / charged residues.Kuj ceeb tias, feem ntau ntawm cov residues nyob rau saum npoo ntawm 4HB yog qhov sib txawv heev (Fig. 6b), txawm hais tias lub quav yog khaws cia thoob plaws hauv SPACA6 homologue (raws li qhia los ntawm kev txuag ntawm hydrophobic bundle core) thiab dhau ntawm IST superfamily.
Txawm hais tias qhov no yog thaj tsam me tshaj plaws hauv SPACA6 nrog qhov tsawg tshaj plaws ntawm cov qauv kuaj pom thib ob, ntau thaj tsam pob khawm seem (nrog rau thaj tsam 3) tau txais txiaj ntsig zoo ntawm SPACA6 homologues, uas yuav qhia tau tias kev taw qhia ntawm cov pob helical thiab β-sandwich plays lub luag haujlwm.raws li ib tug conservative.Txawm li cas los xij, txawm hais tias muaj kev sib txuas ntawm hydrogen thiab electrostatic tes hauj lwm nyob rau hauv thaj tsam pob khawm ntawm SPACA6 thiab IZUMO1, cov pov thawj ntawm kev yooj yim ntawm lub cev tuaj yeem pom nyob rau hauv kev sib haum xeeb ntawm ntau cov qauv tso cai ntawm IZUMO137,43,44.Qhov kev sib tw ntawm tus kheej cov thawj coj sib tshooj zoo, tab sis kev taw qhia ntawm cov npe txheeb ze sib txawv ntawm 50 ° txog 70 ° los ntawm lub hauv paus axis (Fig. S16).Txhawm rau kom nkag siab txog qhov sib txawv ntawm SPACA6 hauv kev daws teeb meem, SAXS kev sim tau ua (Fig. S17a,b).Ab initio reconstruction ntawm SPACA6 ectodomain conformed rau ib tug pas nrig siv lead ua qauv (Fig. S18), txawm hais tias lub Kratky zajlus qhia ib co degree ntawm saj zawg zog (Fig. S17b).Qhov kev sib haum xeeb no sib txawv nrog IZUMO1, uas cov protein tsis muaj kev cuam tshuam suav tias yog boomerang zoo li ob qho tib si hauv cov lattice thiab hauv cov tshuaj 43.
Txhawm rau txheeb xyuas thaj tsam hloov tau yooj yim, hydrogen-deuterium pauv huab hwm coj spectroscopy (H-DXMS) tau ua ntawm SPACA6 thiab piv nrog cov ntaub ntawv yav dhau los tau los ntawm IZUMO143 (Fig. 7a,b).SPACA6 yog kom meej meej hloov tau tshaj IZUMO1, raws li qhia los ntawm ib tug ntau dua deuterium pauv thoob plaws hauv lub qauv tom qab 100,000 s pauv.Nyob rau hauv ob qho tib si qauv, C-terminal ib feem ntawm lub pob khawm qhia tau hais tias muaj kev sib pauv theem siab, uas tej zaum tso cai rau kev sib hloov ntawm 4HB thiab Ig-zoo li domains txheeb ze rau ib leeg.Interestingly, C-terminal ib feem ntawm SPACA6 pob khawm, uas muaj 147CDLPLDCP154 residue, yog ib cheeb tsam kev txuag 3 (Fig. 6b), tejzaum nws qhia tau hais tias interdomain yooj yog ib qho evolutionarily conserved feature ntawm SPACA6.Raws li kev txheeb xyuas qhov yooj yim, CD thermal yaj cov ntaub ntawv qhia tias SPACA6 (Tm = 51.2 ° C) tsis ruaj khov dua IZUMO1 (Tm = 62.9 ° C) (Fig. S1e thiab S19).
ib H-DXMS cov duab ntawm SPACA6 thiab b IZUMO1.Qhov feem pua ​​​​ntawm deuterium pauv tau txiav txim siab ntawm lub sijhawm teev cov ntsiab lus.Qib ntawm hydrogen-deuterium pauv yog qhia los ntawm cov xim ntawm gradient scale ntawm xiav (10%) mus rau liab (90%).Cov thawv dub sawv cev rau thaj chaw ntawm kev sib pauv siab.Cov ciam teb ntawm 4HB, pob khawm thiab Ig-zoo li sau tau pom nyob rau hauv cov qauv siv lead ua tau qhia saum toj ntawm thawj theem.Deuterium pauv theem ntawm 10 s, 1000 s, thiab 100,000 s tau npaj rau ntawm daim ntawv qhia strip superimposed ntawm pob tshab molecular ntawm SPACA6 thiab IZUMO1.Qhov chaw ntawm cov qauv nrog deuterium pauv qib qis dua 50% yog xim dawb.Cov cheeb tsam saum toj no 50% H-DXMS pauv yog xim nyob rau hauv gradient scale.
Kev siv CRISPR/Cas9 thiab nas gene knockout cov tswv yim caj ces tau coj mus rau kev txheeb xyuas ntau yam tseem ceeb rau cov phev thiab qe khi thiab fusion.Sib nrug los ntawm kev sib cuam tshuam zoo ntawm IZUMO1-JUNO thiab CD9 qauv, feem ntau ntawm cov proteins uas cuam tshuam nrog gamete fusion nyob twj ywm structurally thiab functionally enigmatic.Lub biophysical thiab structural characterization ntawm SPACA6 yog lwm daim ntawm adhesion / fusion molecular puzzle thaum lub sij hawm fertilization.
SPACA6 thiab lwm tus tswv cuab ntawm IST superfamily zoo li tau txais kev txuag hauv cov tsiaj nyeg nrog rau cov noog, cov tsiaj reptiles, thiab amphibians;Qhov tseeb, nws tau xav tias SPACA6 tseem xav tau rau kev yug me nyuam hauv zebrafish 59. Qhov kev faib tawm no zoo ib yam li lwm cov gamete fusion-associated proteins xws li DCST134, DCST234, FIMP31, thiab SOF132, qhia tias cov yam tseem ceeb no yog HAP2-tsis muaj (tseem. hu ua GCS1) cov proteins uas yog lub luag haujlwm rau kev ua haujlwm catalytic ntawm ntau tus protists., nroj tsuag, thiab arthropods.Fertilized fusion proteins 60, 61. Txawm hais tias muaj kev sib luag zoo sib xws ntawm SPACA6 thiab IZUMO1, knockout ntawm cov noob encoding ib qho ntawm cov proteins no ua rau muaj menyuam tsis muaj menyuam hauv cov txiv neej nas, qhia tias lawv cov haujlwm hauv gamete fusion tsis duplicated..Ntau qhov dav, tsis muaj leej twg paub cov phev proteins uas xav tau rau lub sijhawm adhesion ntawm fusion yog redundant.
Nws tseem yog ib qho lus nug qhib seb SPACA6 (thiab lwm tus tswv cuab ntawm IST superfamily) koom nrog hauv kev sib tshuam sib txuas, tsim cov tes hauj lwm intragametic los nrhiav cov proteins tseem ceeb rau cov ntsiab lus fusion, lossis tej zaum txawm tias ua raws li cov fusogens elusive.Kev tshawb fawb Co-immunoprecipitation hauv HEK293T hlwb qhia kev sib cuam tshuam ntawm tag nrho ntev IZUMO1 thiab SPACA632.Txawm li cas los xij, peb cov recombinant ectodomains tsis cuam tshuam hauv vitro, qhia tias kev sib cuam tshuam pom hauv Noda li al.ob qho tib si raug tshem tawm hauv kev tsim kho (nco lub cytoplasmic tus Tsov tus tw ntawm IZUMO1, uas tau pom tias tsis tsim nyog rau fertilization62).Xwb, IZUMO1 thiab/lossis SPACA6 yuav xav tau ib puag ncig kev khi tshwj xeeb uas peb tsis rov tsim dua tshiab hauv vitro, xws li kev sib haum xeeb tshwj xeeb lossis cov molecular complexes uas muaj lwm cov proteins (paub lossis tseem tsis tau tshawb pom).Txawm hais tias IZUMO1 ectodomain ntseeg tau los kho qhov kev sib txuas ntawm spermatozoa rau lub qe hauv qhov chaw perivitelline, lub hom phiaj ntawm SPACA6 ectodomain tsis meej.
Cov qauv ntawm SPACA6 nthuav tawm ntau qhov chaw khaws cia uas tuaj yeem koom nrog kev sib cuam tshuam ntawm cov protein-protein.Qhov kev txuag ntawm thaj tsam pob khawm tam sim ntawd nyob ib sab ntawm CXXC motif (tsim Patch 1 saum toj no) muaj ob peb sab nraud-ntsej muag residues uas feem ntau cuam tshuam nrog hydrophobic thiab π-stacking kev sib cuam tshuam ntawm biomolecules.Cov sab dav ntawm Ig-zoo li tus sau (thib cheeb tsam 2) tsim ib qho kev them nqi zoo nrog kev txuag Arg thiab Nws cov residues, thiab cov tshuaj tiv thaiv tiv thaiv cheeb tsam no yav dhau los tau siv los thaiv gamete fusion 30 .Cov tshuaj tiv thaiv kab mob lees paub qhov linear epitope 212RIRPAQLTHRGTFS225, uas muaj peb ntawm rau arginine residues thiab txuag His220.Nws tsis paub meej tias qhov kev ua haujlwm tsis zoo yog vim qhov thaiv ntawm cov khoom seem tshwj xeeb lossis tag nrho cheeb tsam.Qhov chaw ntawm qhov sib txawv no ze ntawm C-qhov kawg ntawm β-sandwich qhia tias cis-kev cuam tshuam nrog cov phev protein nyob sib ze, tab sis tsis nrog cov protein oocyte.Tsis tas li ntawd, kev tuav pov hwm ntawm qhov hloov pauv tau yooj yim heev ntawm cov khoom sib txuam (qhov chaw 3) nyob rau hauv lub pob khawm yuav yog qhov chaw ntawm kev sib cuam tshuam ntawm cov protein-protein los yog, feem ntau, qhia tau hais tias kev tuav tswj ntawm kev hloov pauv ntawm ob lub npe.Poj niam los txiv neej yog ib qho tseem ceeb rau lub luag hauj lwm tsis paub ntawm SPACA6.fusion ntawm gametes.
SPACA6 muaj cov khoom ntawm intercellular adhesion proteins, suav nrog Ig-zoo li β-sandwiches.Ntau cov nplaum nplaum (piv txwv li, cadherins, integrins, adhesins, thiab IZUMO1) muaj ib lossis ntau dua β-sandwich domains uas txuas cov proteins ntawm cov cell membrane rau lawv cov hom phiaj ib puag ncig63,64,65.Lub Ig-zoo li tus sau ntawm SPACA6 kuj muaj cov motif feem ntau pom nyob rau hauv β-sandwiches ntawm adhesion thiab cohesion: doublets ntawm parallel strands ntawm qhov kawg ntawm β-sandwiches, hu ua mechanical clamps66.Nws ntseeg tau tias qhov motif no ua rau muaj kev tiv thaiv rau shear rog, uas yog qhov tseem ceeb rau cov proteins uas koom nrog kev sib cuam tshuam ntawm cov cellular.Txawm li cas los xij, txawm tias qhov zoo sib xws rau cov adhesins, tam sim no tsis muaj pov thawj tias SPACA6 cuam tshuam nrog qe dawb.SPACA6 ectodomain tsis tuaj yeem khi rau JUNO, thiab SPACA6-expressing HEK293T hlwb, raws li qhia ntawm no, tsis tshua muaj kev cuam tshuam nrog oocytes tsis muaj zona 32.Yog tias SPACA6 tsim cov ntawv cog lus sib txuas, cov kev sib cuam tshuam no yuav xav tau kev hloov pauv hloov tom qab lossis kev ruaj khov los ntawm lwm cov phev proteins.Hauv kev txhawb nqa ntawm qhov kev xav yav dhau los, IZUMO1-tsis muaj spermatozoa khi rau oocytes, qhia tias cov lwg me me uas tsis yog IZUMO1 koom nrog gamete adhesion kauj ruam 27.
Ntau tus kab mob, cellular, thiab kev loj hlob fusion proteins muaj cov khoom uas kwv yees lawv txoj haujlwm ua fuogens.Piv txwv li, kab mob fusion glycoproteins (chav kawm I, II thiab III) muaj hydrophobic fusion peptide los yog voj ntawm qhov kawg ntawm cov protein uas tau muab tso rau hauv lub party membrane.Daim ntawv qhia hydrophilicity ntawm IZUMO143 thiab cov qauv (kev txiav txim siab thiab kwv yees) ntawm IST superfamily tsis pom tseeb hydrophobic fusion peptide.Yog li, yog tias muaj cov proteins hauv IST superfamily ua haujlwm li fuogens, lawv ua li ntawd txawv ntawm lwm cov piv txwv paub.
Hauv kev xaus, cov haujlwm ntawm cov tswv cuab ntawm IST superfamily ntawm cov proteins cuam tshuam nrog gamete fusion tseem yog qhov tsis paub tsis meej.Peb tus cwj pwm SPACA6 recombinant molecule thiab nws cov qauv daws teeb meem yuav muab kev pom rau kev sib raug zoo ntawm cov qauv sib koom thiab lawv lub luag haujlwm hauv gamete txuas thiab fusion.
Cov DNA ib ntus sib raug rau tib neeg kwv yees SPACA6 ectodomain (NCBI accession naj npawb NP_001303901.1; residues 27–246) yog codon-optimized rau kev qhia nyob rau hauv Drosophila melanogaster S2 hlwb thiab synthesized raws li ib tug gene fragment nrog rau cov kab ke ntawm kev sib txuas lus (Encoding)., BiP secretion teeb liab thiab qhov sib thooj 5′ thiab 3′ xaus rau ligation-yooj yim cloning ntawm cov noob no rau hauv pMT qhia vector raws li tus neeg txhawb nqa metallothionein hloov kho rau kev xaiv nrog puromycin (pMT-puro).Lub pMT-puro vector encodes qhov chaw thrombin cleavage tom qab 10x-Nws C-terminal tag (Daim duab S2).
Stable transfection ntawm SPACA6 pMT-puro vector rau hauv D. melanogaster S2 (Gibco) hlwb tau ua zoo ib yam li cov txheej txheem siv rau IZUMO1 thiab JUNO43.S2 hlwb tau thawed thiab loj hlob nyob rau hauv Schneider qhov nruab nrab (Gibco) supplemented nrog ib tug kawg concentration ntawm 10% (v/v) kub-inactivated fetal calf serum (Gibco) thiab 1X antimycotic tshuaj tua kab mob (Gibco).Cov hlwb thaum ntxov (3.0 x 106 hlwb) tau muab tso rau hauv ib lub qhov dej ntawm 6-zoo daim hlau (Corning).Tom qab 24 teev ntawm incubation ntawm 27 ° C, hlwb tau kis nrog ib tug sib tov ntawm 2 mg ntawm SPACA6 pMT-puro vector thiab Effectene transfection reagent (Qiagen) raws li cov chaw tsim tshuaj paus raws tu qauv.Hloov cov hlwb raug incubated rau 72 teev thiab tom qab ntawd sau nrog 6 mg / ml puromycin.Cells tau raug cais tawm los ntawm kev ua tiav Schneider qhov nruab nrab thiab muab tso rau hauv cov kab mob dawb-XPRESS nruab nrab (Lonza) rau kev tsim cov protein ntau.A 1 L batch ntawm S2 cell kab lis kev cai tau loj hlob mus rau 8-10 × 106 ml-1 hlwb nyob rau hauv 2 L ventilated flat-bottomed polypropylene Erlenmeyer hwj thiab ces sterilized nrog ib tug kawg concentration ntawm 500 µM CuSO4 (Millipore Sigma) thiab sterile lim.induced.Cov kab lis kev cai induced tau incubated ntawm 27 ° C. ntawm 120 rpm rau plaub hnub.
Conditioned nruab nrab uas muaj SPACA6 raug cais los ntawm centrifugation ntawm 5660 × g ntawm 4 ° C ua raws li Centramate tangential flow filtration system (Pall Corp) nrog 10 kDa MWCO membrane.Siv cov concentrated nruab nrab uas muaj SPACA6 rau 2 ml Ni-NTA agarose resin (Qiagen) kab.Ni-NTA resin raug ntxuav nrog 10 kem ntim (CV) ntawm tsis A thiab tom qab ntawd 1 CV ntawm tsis muaj A raug ntxiv kom muab qhov kawg ntawm imidazole concentration ntawm 50 mM.SPACA6 tau eluted nrog 10 ml ntawm buffer A ntxiv nrog imidazole mus rau qhov kawg concentration ntawm 500 mM.Kev txwv cov chav kawm thrombin (Millipore Sigma) tau ntxiv ncaj qha rau hauv lub raj lim ntshav (MWCO 12-14 kDa) ntawm 1 units ib mg SPACA6 vs. 1 L 10 mM Tris-HCl, pH 7.5 thiab 150 mM NaCl (buffer B) rau kev lim ntshav.) ntawm 4 ° C rau 48 teev.Lub thrombin-cleaved SPACA6 ces diluted peb-fold kom txo ntsev concentration thiab loaded mus rau 1 ml MonoS 5/50 GL cation exchange kem (Cytiva/GE) equilibrated nrog 10 mM Tris-HCl, pH 7.5.Cov cation exchanger tau ntxuav nrog 3 OK 10 mM Tris-HCl, pH 7.5, ces SPACA6 yog eluted nrog ib tug linear gradient ntawm 0 mus rau 500 mm NaCl nyob rau hauv 10 mm Tris-HCl, pH 7.5 rau 25 OK.Tom qab ion txauv chromatography, SPACA6 tau concentrated rau 1 ml thiab eluted isocratically los ntawm ENrich SEC650 10 x 300 kem (BioRad) equilibrated nrog tsis B. Raws li cov chromatogram, pas dej ua ke thiab concentrate fractions uas muaj SPACA6.Purity yog tswj los ntawm Coomassie-stained electrophoresis ntawm 16% SDS-polyacrylamide gel.Protein concentration tau ntsuas los ntawm absorbance ntawm 280 nm siv txoj cai Beer-Lambert thiab theoretical molar extinction coefficient.
Purified SPACA6 tau dialyzed ib hmos tawm tsam 10 mM sodium phosphate, pH 7.4 thiab 150 mM NaF thiab diluted rau 0.16 mg / mL ua ntej kev tshuaj xyuas los ntawm CD spectroscopy.Spectral scanning ntawm CDs nrog lub wavelength ntawm 185 mus rau 260 nm tau sau rau ntawm Jasco J-1500 spectropolarimeter siv quartz cuvettes nrog 1 mm optical path length (Helma) ntawm 25 ° C ntawm tus nqi ntawm 50 nm / min.Cov CD spectra tau raug kho, qhov nruab nrab tshaj 10 qhov kev yuav khoom, thiab hloov mus rau qhov seem ellipticity (θMRE) hauv degrees cm2 / dmol:
qhov twg MW yog qhov hnyav molecular ntawm txhua tus qauv hauv Da;N yog tus naj npawb ntawm cov amino acids;θ yog ellipticity nyob rau hauv millidegrees;d sib raug rau qhov ntev ntawm txoj kev kho qhov muag hauv cm;protein concentration nyob rau hauv units.