Ukwakhiwa komgomo we-acute acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) kugxile kumaprotheni e-trimeric spike abangela ukutheleleka. I-protomer ngayinye ku-trimeric spike inezindawo ze-glycosylation ezingama-22. Ukuthi lawa masayithi afakwa kanjani i-glycosylated kungase kuthinte ukuthi yimaphi amaseli igciwane elithelelayo futhi angase avikele ama-epitopes athile ekwakhiweni kwe-antibody. Watanabe walinda. I-recombinant glycosylated spike trimer yavezwa futhi yahlanjululwa, i-proteolyzed ukukhiqiza ama-glycopeptides aqukethe i-glycans eyodwa, futhi ukwakheka kwezindawo ze-glycan kwanqunywa i-mass spectrometry. Lokhu kuhlaziya kunikeza ibhentshimark engasetshenziswa ukukala ikhwalithi ye-antigen lapho kwenziwa imithi yokugoma nokuhlolwa kwe-antibody.
I-β-coronavirus, ukuvela kwe-acute acute respiratory syndrome 2 (SARS-CoV-2) iyimbangela yesifo se-coronavirus sika-2019 (COVID-19), esibeka engcupheni enkulu impilo yabantu emhlabeni jikelele. Ukuthuthukiswa komuthi wokugoma kugxile ekuhlosweni okuyinhloko kokusabela kwe-humoral immune, i-synaptic (S) glycoprotein, elamula ukungena kwamaseli kanye nokuhlangana kwe-membrane. Ufuzo ngalunye lwe-SARS-CoV-2 S lufaka ama-22 N-axhumene ne-glycosyl ukulandelana, okungase kube neqhaza ekugoqeni amaprotheni kanye nokuphunyuka kokuzivikela komzimba. Lapha, sisebenzisa i-mass spectrometry eqondene nendawo ethile, sembule ukwakheka kwe-glycan ku-immunogen ephindayo ye-SARS-CoV-2 S. Lokhu kuhlaziya kungenza kumephu isimo sokucutshungulwa kwe-glycan kulo lonke inani eliphakeme legciwane le-trimeric. Sibonisa umehluko phakathi kwe-SARS-CoV-2 S glycan kanye nokucubungula okujwayelekile kwe-glycan, okungase kube nomthelela ku-pathology yegciwane kanye nokwakhiwa komgomo.
ISevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) iyimbangela (1, 2) yeCoronavirus 2019 (COVID-19), engabangela umkhuhlane, izifo ezinzima zokuphefumula kanye nenyumoniya. I-SARS-CoV-2 isebenzisa iphrotheni ebanzi ye-glycosylated spike (S) ephuma ebusweni begciwane ukubopha i-angiotensin eguqula i-enzyme 2 (ACE2) ukuze ilamule ukungena kweseli lomsingathi (3). Iprotheni i-S iyiphrotheni ehlanganisayo yekilasi le-trimeric I, elakhiwe amayunithi amabili asebenzayo, anesibopho sokubopha isamukeli (i-S1 subunit) kanye ne-membrane fusion (i-S2 subunit) (4, 5). Ingaphezulu le-nail eboshwe ikakhulukazi lilawulwa ama-glycans atholakala ku-host, futhi i-trimer ngayinye ibonisa izingosi ze-glycosylation ezingu-66 N-linked. I-S protein ingumgomo oyinhloko emsebenzini wokuklama umgomo (6). Ukuqonda i-glycosylation ye-recombinant virus spikes kungaveza izici eziyisisekelo zebhayoloji yegciwane kanye namasu okuklama umgomo wokugoma (7, 8).
I-Viral glycosylation inenqwaba yezindima ku-viral pathobiology, okuhlanganisa ukulamula ukugoqa kwamaprotheni nokuzinza kanye nokubumba i-viral tropism (9). Izingosi ze-Glycosylation zingaphansi kwengcindezi ekhethiwe ngoba zikhuthaza ukuphunyuka kwamasosha omzimba ngokuvikela ama-epitopes athile ekwakhiweni kwe-antibody. Kodwa-ke, siqaphele ukuthi izinga lokuguqulwa kwe-SARS-CoV-2 liphansi kakhulu, futhi kuze kube manje, akukho ukuguqulwa kwesayithi le-glycosylation elixhunywe ku-N okuye kwabonwa (10). Indawo enokuminyana okuphezulu ngokungavamile kwama-glycans nayo ingafinyelela ukuqashelwa kokuzivikela komzimba (9, 11, 12). Kwamanye ama-coronavirus, indima ye-glycosylation ekufihleni i-immunogenic protein epitopes isihloliwe (10, 13, 14). Ama-Coronaviruses ahlumela ku-endoplasmic reticulum-Golgi cavity ukuze akhe ama-virions (15, 16). Kodwa-ke, lapho izinhlobo eziyinkimbinkimbi zama-glycans zibonwa ezintweni ezithathwe yigciwane, kwatholakala ukuthi i-viral glycoprotein ithintwa ama-enzyme okucubungula ahlala eGolgi (13, 17).
Ukuminyana kwe-viral glycan ephezulu kanye nesakhiwo samaprotheni endawo kuphazamisa indawo indlela yokuvuthwa kwe-glycan. Ukuvuthwa kwe-glycan okukhubazekile okuholela ekubeni khona kwama-glycans ohlobo lwe-oligomannose kungase kube intatheli ebucayi yokwakheka kwamaprotheni emvelo (8), futhi ukuhlaziywa kwe-glycan okuqondene nesayithi kungasetshenziswa ukuqhathanisa ama-immunogens ahlukene nokuqapha izinqubo zokukhiqiza (18). Ngaphezu kwalokho, i-glycosylation ingathinta ukuthuthwa kwe-recombinant immunogen ukuya esikhungweni se-germinal (19).
Ukuze uxazulule i-glycosylation eqondene nendawo ethile yephrotheni ye-SARS-CoV-2 S futhi ubone ngeso lengqondo ukusatshalaliswa kwama-glycoprotein kuyo yonke indawo yamaprotheni, sizwakalise futhi sahlanza izici ezintathu zebhayoloji zezinto ezincibilika kabusha ngendlela efanayo naleyo esetshenziselwa ukuthola amaprotheni. . ikhophi. Isakhiwo sokulungiswa okuphezulu kwe-cryo-electron microscopy (cryo-EM), naphezu kokuntuleka komphumela wokuvinjwa kwe-glycan we-kifennonine (4). Lokhu okuhlukile kwephrotheni ye-S kuqukethe wonke ama-glycans angama-22 kuphrotheni ye-SARS-CoV-2 S (Umfanekiso 1A). Ngokusebenzisa ukuguqulwa okuzinzile kwe-2P kwezinsalela 986 kanye no-987 (20), esikhundleni se-GSAS (Gly-Ser-Ala-Ser) (izinsalela 682 kuya ku-685) kanye no-C endaweni yokuhlukanisa i-furin, ukwakheka kwe-trimer pre-fusion kuyabonakala Ngokuzinza. -I-Terminal trimerization motif. Lokhu kusiza ukugcina isakhiwo se-quaternary ngesikhathi sokucubungula i-glycan. Ngaphambi kokuhlaziywa, i-supernatant equkethe kabusha i-SARS-CoV-2 S yahlanzwa nge-chromatography yokukhishwa kosayizi ukuze kuqinisekiswe ukuthi kwahlaziywa kuphela amaprotheni emvelo afana ne-trimeric (Umfanekiso 1B kanye noMfanekiso S1). Ukucutshungulwa kwe-trimer kwento ehlanziwe kwaqinisekiswa kusetshenziswa ibala elibi i-EM (Umfanekiso 1C).
(A) Umdwebo ohleliwe we-SARS-CoV-2 S glycoprotein. Indawo yokulandelana kwe-glycosylation exhunywe ku-N (NXS/T, lapho i-X≠P) iboniswa njengegatsha (N, Asn; X, noma iyiphi insalela; S, Ser; T, Thr; P, Pro). Ichaza izizinda zamaprotheni: isizinda se-N-terminal (NTD), isizinda esibopha i-receptor (RBD), i-fusion peptide (FP), i-heptapeptide repeat 1 (HR1), i-central helix (CH), isizinda sesixhumi (CD) ) Nesizinda se-transmembrane (TM) ). (B) Ukuhlaziywa kwejeli ye-SDS-polyacrylamide ye-electrophoresis yephrotheni ye-SARS-CoV-2 S (ekhonjiswa ngomcibisholo) evezwa kumaseli ezinso zombungu womuntu (HEK) 293F. Umzila 1: I-Supernatant ihlungwa kumaseli adlulisiwe; Umzila 2: Ukugeleza kusuka ku-StrepTactin resin; Umzila 3: Ukugeza nge-StrepTactin resin; Umzila 4: I-Elution evela ku-StrepTactin resin. (C) Inani elimaphakathi le-EM 2D negative lephrotheni ye-SARS-CoV-2 S. Inani elimaphakathi le-2D lephrotheni ye-SARS-CoV-2 S liyaboniswa, liqinisekisa ukuthi iphrotheni ithatha i-trimer pre-fusion conformation, efana nokokusebenza okusetshenziselwa ukunquma ukwakheka (4).
Ukuze sinqume i-glycosylation yesayithi ethile ye-SARS-CoV-2 S, sasebenzisa i-trypsin, i-chymotrypsin ne-α-lytic protease ukuze sikhiqize amasampula amathathu e-glycopeptide. Lawa ma-protease akhethwa ukukhiqiza ama-glycopeptides aqukethe ukulandelana kwe-glycan okukodwa kwe-N. Ama-glycopeptides ahlaziywa nge-liquid chromatography-mass spectrometry, futhi ukwakheka kwe-glycan kwazo zonke izingosi ze-glycan ezingama-22 ezixhunywe ku-N kwanqunywa (Umfanekiso 2). Ukuze kudluliselwe izici eziyinhloko zokucubungula zesayithi ngalinye, ngokusho kwe-branching ne-fucosylation, ukuchichima kwe-glycan ngayinye kuhlukaniswa ngezigaba ze-oligomannose, i-hybrid kanye ne-complex glycosylation. Ithebula S1 kanye nomfanekiso wesi-6 zinikeza ukubukwa okunemininingwane okunwetshiwe okubonisa ukuqanjwa okuhlukahlukene kwe-glycan. S2.
Umdwebo ubonisa amakhodi ombala wezinhlobo eziyinhloko zama-glycans ezingavela endleleni yokuvuthwa ukusuka ku-oligomannose kuya ezinhlotsheni ezixubile zama-glycans. Lezi zibalo zifingqa ukuhlaziywa kwe-quantitative mass spectrometry yabantu be-glycan abakhona endaweni eyodwa ye-glycosylation exhunywe ku-N, eyehliselwe esigabeni se-glycan. Uchungechunge lwe-oligomannose yohlobo lwe-glycan (M9 ukuya ku-M5; i-Man9GlcNAc2 ukuya ku-Man5GlcNAc2) iluhlaza, i-non-glycosylated futhi i-fucosylated hybrid glycans (ingxubevange kanye ne-F hybrid), umugqa onamachashazi unombala opinki, ngokwenani lezimpondo I-complex glycan grouping core fucosylation ( I-A1 kuye ku-FA4) inombala opinki uma kunezimo. Izikhala kumasayithi e-glycan axhunywe ku-N zikhonjiswe ngokumpunga. Ishadi likaphayi lifingqa ukulinganisa kwalawa ma-glycans. Izingosi ze-Glycan zinemibala ngokusho kokuqukethwe kwama-glycans ohlobo lwe-oligomannose, kanti izindawo ze-glycan zimakwe njengeziluhlaza (80% kuya ku-100%), osawolintshi (30% kuya ku-79%) kanye nopinki (0% kuya ku-29%). Inguqulo enwetshiwe yokuhlaziywa okuqondene nesayithi ebonisa ukuhlukahluka phakathi kwesigaba ngasinye ingatholakala kuThebula S1 kanye nomfanekiso 6. S2. Igrafu yebha imelela isilinganiso sezimpinda ezintathu zebhayoloji, futhi amabha amaphutha amelela iphutha elijwayelekile lesilinganiso.
Iziza ezimbili eziku-SARS-CoV-2 S ikakhulukazi ziyi-oligomannose: i-N234 ne-N709. Ngaphandle kwe-N234, ukwakheka okuyinhloko kwe-oligomannose glycan okubonwa kulo lonke iphrotheni yi-Man5GlcNAc2 (Indoda, i-mannose; i-GlcNAc, i-N-acetylglucosamine), okubonisa ukuthi lezi zindawo zingafinyelela kakhulu ku-α-1,2-mannosidase, kodwa i-substrate ye-GlcNAcT -1 ibuthakathaka, i-GlcNAcT-1 iyi-enzyme eyinhloko ekwakhekeni kwe-heterozygous kanye ne-glycans eyinkimbinkimbi ku-apparatus ye-Golgi. Isiteji esivimbela ukucubungula isici esihlobene nokuminyana kanye nokwethulwa kwama-glycans kuma-viral spikes. Isibonelo, iziqongo eziphezulu ze-glycosylation ze-HIV-1 Env ne-Lassa virus (LASV) GPC zibonisa amasayithi amaningi abuswa yi-Man9GlcNAc2 (21-24).
Ingxube ye-oligomannose kanye ne-glycans eyinkimbinkimbi ingatholakala kumasayithi N61, N122, N603, N717, N801 kanye ne-N1074 (Umfanekiso 2). Ezingosini ezingama-22 kuphrotheni ye-S, eziyisi-8 ziqukethe inani elikhulu lama-glycans ohlobo lwe-oligomannose, agqamisa umehluko phakathi kokucutshungulwa kwe-SARS-CoV-2 S glycans kanye ne-glycoprotein ephethe (25). Iziza ezisele eziyi-14 zibuswa yizinhlobo eziyinkimbinkimbi ezicutshunguliwe zama-glycans.
Yize kutholwe iziza ze-glycosylation ezingahlali muntu kwa-SARS-CoV-2 S, kutholakale ukuthi zinengxenye encane kakhulu yomtapo wezincwadi we-peptide ophelele ngesikhathi sokulinganisa (Ithebula S2). Ocwaningweni lwe-HIV-1 immunogen, i-voids edalwe yizindawo ze-glycan ezingahlali muntu ikhonjiswe njenge-immunogenic futhi ingabangela ama-epitopi aphazamisayo (26). Izinga eliphezulu lokuhlala kwabantu ngokulandelana kwe-N-linked glycan ye-SARS-CoV-2 S libonisa ukuthi i-recombinant immunogen ngeke idinge ukuthuthukiswa okwengeziwe ukuze kuthuthukiswe ukuhlala kwesiza.
Kusetshenziswa ukwakheka kwe-EM eqandisiwe ye-trimeric SARS-CoV-2 S protein [Protein Database (PDB) ID 6VSB] (4), isimo se-glycosylation se-coronavirus spike silingisa saklanywa ngeSakhiwo se-3-dimensional (3D) esinqunywa ukuhlolwa ( Umfanekiso 3). Inhlanganisela ye-mass spectrometry kanye nokuhlaziywa kwe-EM yezinga lokushisa eliphansi kwembula ukuthi ama-glycans axhumene ne-N abamba kanjani izindawo ezihlukene kuyo yonke indawo ye-SARS-CoV-2 spike.
Ama-glycans amele amamodeli esakhiweni sangaphambi kokuhlanganiswa kwe-trimeric SARS-CoV-2 S glycoprotein (PDB ID 6VSB) (4), lapho i-RBD eyodwa isesimweni “phezulu,” kanti amanye ama-RBD amabili “asendleleni”. Phansi” ukuhambisana. Ama-glycans anemibala ngokusho kokuqukethwe kwe-oligomannose okuchazwe yisibopho. Isayithi elibophezela i-ACE2 receptor ligqanyiswe ngokuluhlaza okwesibhakabhaka. Ama-subunits e-S1 kanye ne-S2 amelwe izindawo ezibonisa ngale, ngokulandelana, zidwetshwe ngokumpunga okukhanyayo nokumnyama. Izihibe eziguquguqukayo lapho izingosi ze-N74 kanye ne-N149 ze-glycan zitholakala khona zimelelwa imigqa empunga edayishiwe, futhi amasayithi e-glycan kumaluphu afakwe imephu ezindaweni ezicishe zifane.
Kungaqashelwa ukuthi isayithi elibophezela ama-receptor ku-SARS-CoV-2 spike livikelwe iziza ze-proximal glycosylation (N165, N234, N343), ikakhulukazi uma isizinda sokubopha i-receptor sisesimweni “phansi”. Njengoba kuphawulwe ku-SARS-CoV-1 S (10, 13), HIV-1 Env (27), influenza hemagglutinin (28, 29) kanye ne-LASV GPC, ukuvikela amasayithi okubopha ama-receptor ngama-glycans Kuyisici esivamile sama-viral glycoprotein. . (amashumi amabili nane). Uma kucatshangelwa ukulinganiselwa kokusebenza kwesizinda sokubopha i-receptor kanye nezinga eliphansi lokuguqulwa kwalezi zinsalela, kungase kube nengcindezi ekhethiwe yokusebenzisa ama-glycans axhunywe i-N ukuze kufihlwe esinye sezifunda ezilondolozwe kakhulu nezingaba sengozini yama-glycoprotein azo (30, 31).
Siqaphele ukusakazeka kwama-glycans ohlobo lwe-oligomannose kuma-subunits e-S1 kanye ne-S2. Lokhu kuphambene namanye ama-viral glycoprotein. Isibonelo, amaqoqo e-glycan aminyene ezinhlotsheni ezimbalwa ze-HIV-1 Env angenza ama-glycans ohlobo lwe-oligomannose abonwa amasosha omzimba (32, 33). Ku-SARS-CoV-2 S, ukwakheka kwe-oligomannose kungenzeka ukuthi kuvikelwe izingxenye zamaprotheni, njenge-N234 glycans (ihlukaniswe ngokwengxenye phakathi kwe-N-terminus nesizinda sokubopha isamukeli) (Umfanekiso 3).
Siphawule ama-glycans axhumene ne-N ezakhiweni ezinwetshiwe ze-loop (N74 kanye ne-N149) kanye ne-C-terminus eseduze ye-membrane (N1158, N1173, N1194), futhi la ma-molecule awazange axazululwe ezithombeni ze-cryoEM (4). Lawa ahlonzwe njengama-glycans ayinkimbinkimbi, ahambisana nokufinyeleleka kwendawo kwalezi zinsalela.
Nakuba okuqukethwe kwe-oligomannose glycans (28%) (Ithebula S2) kungaphezulu kwalokho kuma-glycoprotein abambayo ajwayelekile, kuphansi kunamanye ama-glycoprotein amagciwane. Isibonelo, enye yamaprotheni e-viral spike aminyene kakhulu i-HIV-1 Env, ebonisa ama-glycans angama-60% ohlobo lwe-oligomannose (21, 34). Lokhu kukhombisa ukuthi uma kuqhathaniswa namanye ama-viral glycoprotein (okubandakanya i-HIV-1 Env kanye ne-LASV GPC), i-glycosylation ye-SARS-CoV-2 S protein ibuthaka, futhi ukwakheka kwama-glycans avikelwe kancane, okungaba wusizo ekucupheni amasosha omzimba Awusizo. .
Ngaphezu kwalokho, ukucutshungulwa kwama-glycans ayinkimbinkimbi kuwukucatshangelwa okubalulekile kubunjiniyela be-immunogen, ikakhulukazi uma kucatshangelwa ukuthi i-epitope ye-antibody engathathi hlangothi ngokumelene ne-SARS-CoV-2 S ingase iqukathe ama-glycans e-fucosylated ku-N343 (35). Phakathi kwezindawo ze-glycosylation ezingu-22 ezixhunywe ku-N, i-52% ye-fucosylation kanye ne-15% yama-glycans aqukethe okungenani izinsalela ze-asilic acid eyodwa (Ithebula S2 kanye noMfanekiso S3). Ukuhlaziywa kwethu kubonise ukuthi i-N343 i-fucosylated kakhulu ngama-98% okuhlola ama-glycans anezinsalela ze-fucose. Ukuguqulwa kwama-glycans kuzothikamezwa kakhulu yisistimu ye-cell expression esetshenziswayo. Sike sabonisa ngaphambilini nge-HIV-1 Env glycosylation ukuthi ukucutshungulwa kwama-glycans ayinkimbinkimbi kuqhutshwa amaseli okukhiqiza, kodwa izinga le-oligomannose glycans lizimele kakhulu ohlelweni lokukhuluma futhi lihlobene nesakhiwo samaprotheni. Ihlobene eduze nokuminyana kwe-glycan (36).
Njengoba kuphawulwe ku-LASV GPC kanye ne-HIV-1 Env, isihlangu se-glycan esiminyana kakhulu sinalokho okubizwa ngamaqoqo e-mannose (22, 24) endaweni yamaprotheni (Umfanekiso 4). Yize amaqoqo amancane ohlobo lwe-mannose atholakala ku-S1 subunit ye-Middle East Respiratory Syndrome (MERS)-CoV S (10), lesi simo asizange sibonwe ngamaprotheni akwa-SARS-CoV-1 noma akwa-SARS-CoV-2 S. Ukuhlaziywa kwe-glycosylation okuqondene nesayithi okubikwe lapha kukhombisa ukuthi isihlangu se-glycan sakwa-SARS-CoV-2 S siyahambisana namanye ama-coronavirus, futhi sonke isihlangu se-glycan sibonisa ubungozi obuningi (10) . Ekugcineni, sithole amanani omkhondo we-O-linked glycosylation ku-Thr323/Ser325 (T323/S325), lapho ngaphezu kuka-99% wamasayithi ayengalungiswanga (Figure S4), okubonisa ukuthi lapho isakhiwo sinjengomdabu.
Ukusuka kwesobunxele kuye kwesokudla kukhona i-MERS-CoV S (10), i-SARS-CoV-1 S (10), i-SARS-CoV-2 S, i-LASV GPC (24) ne-HIV-1 Env (8, 21). Umthamo othize wesayithi othize we-N-linked glycan oligomannose unombala ngokuya ngamagama angukhiye. Ngaphandle kwe-LASV GPC, wonke ama-glycoprotein avezwa njengezinqamuli ezincibilikayo kumaseli e-HEK 293F, kuyilapho i-LASV GPC isuselwa ezinhlayiyeni ezifana negciwane zamaseli ezinso ze-Madin-Darby canine II.
Ukuhlaziywa kwethu kwe-glycosylation kwe-SARS-CoV-2 kunikeza ibhentshimakhi enemininingwane yezimpawu zesayithi ezithize ze-glycan zezikhonkwane ezigoqiwe ngokwemvelo. Ngokuthuthukiswa kwemithi yokugoma yamakhandidethi eminingi esekelwe kuma-glycoprotein, ukuhlaziya okuningiliziwe kwe-glycan kunikeza indlela yokuqhathanisa ubuqotho bama-immunogens, futhi njengoba izikali zokukhiqiza zikhula ziye ekusetshenzisweni komtholampilo, ukuqapha nakho kuyoba okubaluleke kakhulu. Ngakho-ke, lapho kukhiqizwa amakhithi okuhlola ama-serological, ukuhlaziywa kwephrofayili ye-glycan nakho kuzoba inkomba ebalulekile yekhwalithi ye-antigen. Okokugcina, ngokufika kwemigomo esekelwe ku-nucleotide, kubalulekile ukuqonda ukuthi lezi zindlela zokulethwa zithinta kanjani ukucubungula nokwethulwa kwama-immunogens.
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Qaphela: Sikucela kuphela ukuthi unikeze ikheli lakho le-imeyili ukuze umuntu omncomayo ekhasini azi ukuthi ufuna abone i-imeyili futhi akuwona ugaxekile. Ngeke sithwebule noma yimaphi amakheli e-imeyili.
Lo mbuzo usetshenziselwa ukuhlola ukuthi ingabe uyisivakashi futhi uvimbele ukuthunyelwa kogaxekile okuzenzakalelayo.
Ukuhlaziywa kwe-mass spectrometry kwembule ukwakheka kwe-glycan yazo zonke izingosi ze-glycosylation kuphrotheni ye-spike ye-SARS-CoV-2.
Ukuhlaziywa kwe-mass spectrometry kwembule ukwakheka kwe-glycan yazo zonke izingosi ze-glycosylation kuphrotheni ye-spike ye-SARS-CoV-2.
©2021 Inhlangano YaseMelika Yokuthuthukiswa Kwesayensi. wonke Amalungelo Agodliwe. I-AAAS inguzakwethu we-HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef kanye ne-COUNTER.Science ISSN 1095-9203.
Isikhathi sokuthumela: Jan-21-2021
