Site-Specific Glycosylation Patterns of the SARS-CoV-2 Spike Protein Derived From Recombinant Protein and Viral WA1 and D614G Strains

The SARS-CoV-2 spike protein is heavily glycosylated, having 22 predicted N-glycosylation sites per monomer. It is also O-glycosylated, although the number of O-glycosites is less defined. Recent studies show that spike protein glycans play critical roles in viral entry and infection. The spike mono...

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Autores principales:	Yuan Tian, Lisa M. Parsons, Ewa Jankowska, John F. Cipollo
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	SARS-CoV-2 N-glycosylation O-glycosylation glycan shield cell substrate spike protein Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/1ba96580f82d40399da7d8c71e88c94b
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spelling	oai:doaj.org-article:1ba96580f82d40399da7d8c71e88c94b2021-11-19T07:54:05ZSite-Specific Glycosylation Patterns of the SARS-CoV-2 Spike Protein Derived From Recombinant Protein and Viral WA1 and D614G Strains2296-264610.3389/fchem.2021.767448https://doaj.org/article/1ba96580f82d40399da7d8c71e88c94b2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fchem.2021.767448/fullhttps://doaj.org/toc/2296-2646The SARS-CoV-2 spike protein is heavily glycosylated, having 22 predicted N-glycosylation sites per monomer. It is also O-glycosylated, although the number of O-glycosites is less defined. Recent studies show that spike protein glycans play critical roles in viral entry and infection. The spike monomer has two subdomains, S1 and S2, and a receptor-binding domain (RBD) within the S1 domain. In this study, we have characterized the site-specific glycosylation patterns of the HEK293 recombinant spike RBD and S1 domains as well as the intact spike derived from the whole virus produced in Vero cells. The Vero cell-derived spike from the WA1 strain and a D614G variant was analyzed. All spike proteins, S1, and RBDs were analyzed using hydrophilic interaction chromatography (HILIC) and LC-MS/MS on an Orbitrap Eclipse Tribrid mass spectrometer. N-glycans identified in HEK293-derived S1 were structurally diverse. Those found in the HEK293-derived RBD were highly similar to those in HEK293 S1 where N-glycosites were shared. Comparison of the whole cell-derived WA1 and D614G spike proteins revealed that N-glycosites local to the mutation site appeared to be more readily detected, hinting that these sites are more exposed to glycosylation machinery. Moreover, recombinant HEK293-derived S1 was occupied almost completely with complex glycan, while both WA1 and D614G derived from the Vero E6 cell whole virus were predominantly high-mannose glycans. This stands in stark contrast to glycosylation patterns seen in both CHO- and HEK cell-derived recombinant S1, S2, and the whole spike previously reported. Concerning O-glycosylation, our analyses revealed that HEK293 recombinant proteins possessed a range of O-glycosites with compositions consistent with Core type 1 and 2 glycans. The O-glycosites shared between the S1 and RBD constructs, sites T323 and T523, were occupied by a similar range of Core 1 and 2 type O-glycans. Overall, this study reveals that the sample nature and cell substrate used for production of these proteins can have a dramatic impact on the glycosylation profile. SARS-CoV-2 spike glycans are associated with host ACE2 receptor interaction efficiency. Therefore, understanding such differences will serve to better understand these host–pathogen interactions and inform the choice of cell substrates to suite downstream investigations.Yuan TianLisa M. ParsonsEwa JankowskaJohn F. CipolloFrontiers Media S.A.articleSARS-CoV-2N-glycosylationO-glycosylationglycan shieldcell substratespike proteinChemistryQD1-999ENFrontiers in Chemistry, Vol 9 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	SARS-CoV-2 N-glycosylation O-glycosylation glycan shield cell substrate spike protein Chemistry QD1-999
spellingShingle	SARS-CoV-2 N-glycosylation O-glycosylation glycan shield cell substrate spike protein Chemistry QD1-999 Yuan Tian Lisa M. Parsons Ewa Jankowska John F. Cipollo Site-Specific Glycosylation Patterns of the SARS-CoV-2 Spike Protein Derived From Recombinant Protein and Viral WA1 and D614G Strains
description	The SARS-CoV-2 spike protein is heavily glycosylated, having 22 predicted N-glycosylation sites per monomer. It is also O-glycosylated, although the number of O-glycosites is less defined. Recent studies show that spike protein glycans play critical roles in viral entry and infection. The spike monomer has two subdomains, S1 and S2, and a receptor-binding domain (RBD) within the S1 domain. In this study, we have characterized the site-specific glycosylation patterns of the HEK293 recombinant spike RBD and S1 domains as well as the intact spike derived from the whole virus produced in Vero cells. The Vero cell-derived spike from the WA1 strain and a D614G variant was analyzed. All spike proteins, S1, and RBDs were analyzed using hydrophilic interaction chromatography (HILIC) and LC-MS/MS on an Orbitrap Eclipse Tribrid mass spectrometer. N-glycans identified in HEK293-derived S1 were structurally diverse. Those found in the HEK293-derived RBD were highly similar to those in HEK293 S1 where N-glycosites were shared. Comparison of the whole cell-derived WA1 and D614G spike proteins revealed that N-glycosites local to the mutation site appeared to be more readily detected, hinting that these sites are more exposed to glycosylation machinery. Moreover, recombinant HEK293-derived S1 was occupied almost completely with complex glycan, while both WA1 and D614G derived from the Vero E6 cell whole virus were predominantly high-mannose glycans. This stands in stark contrast to glycosylation patterns seen in both CHO- and HEK cell-derived recombinant S1, S2, and the whole spike previously reported. Concerning O-glycosylation, our analyses revealed that HEK293 recombinant proteins possessed a range of O-glycosites with compositions consistent with Core type 1 and 2 glycans. The O-glycosites shared between the S1 and RBD constructs, sites T323 and T523, were occupied by a similar range of Core 1 and 2 type O-glycans. Overall, this study reveals that the sample nature and cell substrate used for production of these proteins can have a dramatic impact on the glycosylation profile. SARS-CoV-2 spike glycans are associated with host ACE2 receptor interaction efficiency. Therefore, understanding such differences will serve to better understand these host–pathogen interactions and inform the choice of cell substrates to suite downstream investigations.
format	article
author	Yuan Tian Lisa M. Parsons Ewa Jankowska John F. Cipollo
author_facet	Yuan Tian Lisa M. Parsons Ewa Jankowska John F. Cipollo
author_sort	Yuan Tian
title	Site-Specific Glycosylation Patterns of the SARS-CoV-2 Spike Protein Derived From Recombinant Protein and Viral WA1 and D614G Strains
title_short	Site-Specific Glycosylation Patterns of the SARS-CoV-2 Spike Protein Derived From Recombinant Protein and Viral WA1 and D614G Strains
title_full	Site-Specific Glycosylation Patterns of the SARS-CoV-2 Spike Protein Derived From Recombinant Protein and Viral WA1 and D614G Strains
title_fullStr	Site-Specific Glycosylation Patterns of the SARS-CoV-2 Spike Protein Derived From Recombinant Protein and Viral WA1 and D614G Strains
title_full_unstemmed	Site-Specific Glycosylation Patterns of the SARS-CoV-2 Spike Protein Derived From Recombinant Protein and Viral WA1 and D614G Strains
title_sort	site-specific glycosylation patterns of the sars-cov-2 spike protein derived from recombinant protein and viral wa1 and d614g strains
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/1ba96580f82d40399da7d8c71e88c94b
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Site-Specific Glycosylation Patterns of the SARS-CoV-2 Spike Protein Derived From Recombinant Protein and Viral WA1 and D614G Strains

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