Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins

Abstract

The glycoprotein spike (S) on the surface of SARS-CoV-2 is a determinant for viral invasion and host immune response. Herein, we characterized the site-specific N-glycosylation of S protein at the level of intact glycopeptides. All 22 potential N-glycosites were identified in the S-protein protomer and were found to be preserved among the 753 SARS-CoV-2 genome sequences. The glycosites exhibited glycoform heterogeneity as expected for a human cell-expressed protein subunit. We identified masses that correspond to 157 N-glycans, primarily of the complex type. In contrast, the insect cell-expressed S protein contained 38 N-glycans, completely of the high-mannose type. Our results revealed that the glycan types were highly determined by the differential processing of N-glycans among human and insect cells, regardless of the glycosites’ location. Moreover, the N-glycan compositions were conserved among different sizes of subunits. Our study indicate that the S protein N-glycosylation occurs regularly at each site, albeit the occupied N-glycans were diverse and heterogenous. This N-glycosylation landscape and the differential N-glycan patterns among distinct host cells are expected to shed light on the infection mechanism and present a positive view for the development of vaccines and targeted drugs.

The SARS-CoV-2 Spike Glycoprotein Biosynthesis, Structure, Function, and Antigenicity: Implications for the Design of Spike-Based Vaccine Immunogens

“…When the site-specific N-linked glycans are mapped onto the prefusion structure of the SARS-CoV-2 S ectodomain ( 63 ), the resulting model exhibited substantially higher levels of glycan-free surface than that revealed by structures of fully glycosylated, trimeric HIV-1 Env ectodomains ( 65 , 66 ). This suggests that the SARS-CoV-2 S protein is covered by a less dense and less effective glycan shield compared to viral glycoproteins from HIV-1 ( 36 , 66 ) and Lassa virus ( 67 ), which may be beneficial for the induction of humoral immunity and could be good news for a SARS-CoV-2 vaccine ( 68 ).…”

N- and O-Glycosylation of the SARS-CoV-2 Spike Protein

“…The studies also detected one O-glycopeptide occupied at sites, distinct from the predicted furin cleavage site at the S1/S2 boundary. 6 , 8 , 9 …”

Proteomics in the COVID‐19 Battlefield: First Semester Check‐Up

“…The exact structures of at least 22 N-linked glycans were successfully established by mass spectrometry [17] and independently confirmed. [18] In addition, the nucleocapsid protein is decorated with O-glycans and N-glycans, at 7 and 2 confirmed sites, respectively. [19] The glycans of the spike protein act as a shield to thwart the host immune response, but a recent study has shown that N-glycans at two sites (N165 & N234) modulate the conformational dynamics of the receptor-binding domain (RBD).…”