Analysis of glycosylation and disulfide bonding of wild-type SARS-CoV-2 spike glycoprotein

Abstract

The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by coexpression of SARS-CoV-2 S, M, E and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to enrich the Golgi-resident fraction of a wild-type SARS-CoV-2 S glycoprotein trimer, and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on soluble and virion S trimers, is predominantly modified in the Golgi by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions.

SARS-CoV-2 activates ER stress and Unfolded protein response

“…SARS-CoV-2 hijacks several host machineries to control immune reaction 46 , viral protein translation 47 , viral genome packing into nascent viral particles as well as support the release of mature virus particles. Host cellular machineries are redirected to synthesize and remodel viral proteins through post-translational modifications such as proteolytic cleavages, disulfide bridges formation, phosphorylation, ubiquitination and glycosylation [48][49][50][51][52][53] . Thus, in this study we sought out which processes and pathways could be changed by the viral infection.…”

“…Glycosylation of viral proteins is an important process that regulates viral assembly and infectivity. The structural and functional role of glycosylation in the SARS-CoV-2 spike protein has been widely investigated 52,[60][61][62][63][64][65][66] . Of note is the fact that 35% of the SARS-CoV-2 spike glycoprotein contains carbohydrate moieties, which have profound influence on the viral infectivity, susceptibility to antibody neutralization 67,68 .…”

Isolation and Characterization of Mouse Monoclonal Antibodies That Neutralize SARS-CoV-2 and Its Variants of Concern Alpha, Beta, Gamma and Delta by Binding Conformational Epitopes of Glycosylated RBD With High Potency

“…The RBD undergoes hinge-like conformational movements and constantly switches between an open conformational state (RBD standing-up position) for receptor binding and a closed conformational state (RBD lying-down position). A complex topology corresponds to this dynamism including several co-translational modifications by reason of nine cysteine residues (eight of them forming disulfide bonds) ( 36 ), two N-glycosylations at sites N331 and N343 and at least one O-glycosylation at Thr323 and a possible O-glycosylation at Ser325 ( 19 , 35 , 37 ). All these modifications have a relevant role on the spike protein correct folding, dynamics and stability, but glycosylation of RBD seems to be not determinant for its interaction whit ACE2 receptor, since a refolded non-glycosylated rRBD produced in E. coli was shown by surface plasmon resonance to bind the receptor ( 38 ).…”

The opportunity cost of automated glycopeptide analysis: case study profiling the SARS-CoV-2 S glycoprotein

“…The protein expression and purification have been described previously, [29], and the detailed protocol is included here for the reader's convenience. SARS-CoV-2 S glycoprotein expression vector Inducible expression of the wild-type SARS-CoV-2 S glycoprotein (with Asp 614) was achieved using a self-inactivating lentivirus v e c t o r c o m p r i s i n g T R E 3 g -S A R S -C o V -2 -S p i k e -6His.IRS6A.Puro-T2A-GFP (K5650).…”