O-Glycosylation Landscapes of SARS-CoV-2 Spike Proteins

The densely glycosylated spike (S) proteins that are highly exposed on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate viral attachment, entry, and membrane fusion. We have previously reported all the 22  N -glycosites and site-specific N -glycans in the S protein protomer. Herein, we report the O- glycosylation landscapes of SARS-CoV-2 S proteins, which were characterized through high-resolution mass spectrometry. Following digestion with trypsin and trypsin/Glu-C, and de- N- glycosylation using PNGase F, we determined the GalNAc-type O- glycosylation pattern of S proteins, including O -glycosites and the six most common O -glycans occupying them, via Byonic identification and manual validation. Finally, 255 intact O -glycopeptides composed of 50 peptides sequences and 43 O -glycosites were discovered by higher energy collision-induced dissociation (HCD), and three O -glycosites were confidently identified by electron transfer/higher energy collision-induced dissociation (EThcD) in the insect cell-expressed S protein. Most glycosites were modified by non-sialylated O -glycans such as HexNAc(1) and HexNAc(1)Hex (1). In contrast, in the human cell-expressed S protein S1 subunit, 407 intact O -glycopeptides composed of 34 peptides sequences and 30 O -glycosites were discovered by HCD, and 11 O -glycosites were unambiguously assigned by EThcD. However, the measurement of O-glycosylation occupancy hasn’t been made. Most glycosites were modified by sialylated O -glycans such as HexNAc(1)Hex (1)NeuAc (1) and HexNAc(1)Hex (1)NeuAc (2). Our results reveal that the SARS-CoV-2 S protein is an O -glycoprotein; the O -glycosites and O -glycan compositions vary with the host cell type. These comprehensive O -glycosylation landscapes of the S protein are expected to provide novel insights into the viral binding mechanism and present a strategy for the development of vaccines and targeted drugs.