Comparison of N- and O-Glycosylation on Spike Glycoprotein 1 of SARS-CoV-1 and MERS-CoV.

Abstract

SARS-CoV-1 and MERS-CoV were the infective agents of the 2002 and 2012 coronavirus outbreaks, respectively. Here, we report a comparative liquid chromatography/mass spectrometry (LC/MS) Orbitrap N- and O-glycosylation glycoproteomics study of the recombinant S1 spike derived from these two viruses. The former was produced in HEK293 cells and the latter in both HEK293 and insect cells. Both proteins were highly glycosylated, with SARS-CoV-1 S1 having 13 and MERS-CoV S1 having 12 N-glycosites. Nearly all were occupied at 85% or more. Between 2 and 113 unique N-glycan compositions were detected at each N-glycosite across the three proteins. Complex N-glycans dominated in HEK293 cell-derived spike S1 proteins. While glycosylation differs between HEK293 and insect cells, the extent of glycan processing at glycosites was similar for the two MERS-CoV S1 forms. The HEK293-derived SARS-CoV-1 S1 N-glycans were more highly sialylated and fucosylated compared to MERS S1, while the latter had more high-mannose glycosides, particularly in the N-terminus and near the RBD. Seven and 8 O-glycosites were identified in SARS-CoV-1 S1 and MERS-CoV S1, respectively. Mapping of predicted antigenic and glycosylation sites reveals colocalization consistent with a role for glycosylation in immune system avoidance. Glycosylation patterns of these S1 proteins differ from those of other SARS-CoV-1 and MERS-CoV spike reported forms such as recombinant trimeric and virus-propagated forms, which has implications for virus research, including vaccine development, as glycosylation plays a role in spike function and epitope structure.