Mucin-type 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 22N-glycosites and site-specificN-glycans in the S protein protomer. Herein, we report the comprehensive and precise site-specific O-glycosylation landscapes of SARS-CoV-2 S proteins, which were characterized using high-resolution mass spectrometry. Following digestion using trypsin and trypsin/Glu-C, and de-N-glycosylation using PNGase F, we determined the mucin-type (GalNAc-type) O-glycosylation pattern of S proteins, including unambiguousO-glycosites and the 6 most commonO-glycans occupying them, via Byonic identification and manual validation. Finally, 43O-glycosites were identified in the insect cell-expressed S protein. Most glycosites were modified by non-sialylatedO-glycans such as HexNAc(1) and HexNAc(1)Hex(1). In contrast, 30O-glycosites were identified in the human cell-expressed S protein S1 subunit. Most glycosites were modified by sialylatedO-glycans such as HexNAc(1)Hex(1)NeuAc(1) and HexNAc(1)Hex(1)NeuAc(2). Our results are the first to reveal that the SARS-CoV-2 S protein is a mucin-type glycoprotein; clusteredO-glycans often occur in the N- and the C-termini of the S protein, and theO-glycosite andO-glycan compositions vary with the host cell type. These site-specific O-glycosylation landscapes of the SARS-CoV-2 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.