Mucin-type O-glycosylation Landscapes of SARS-CoV-2 Spike Proteins

ABSTRACT: 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 comprehensive 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 O-glycosites and the 6 most common O-glycans occupying them, via Byonic identification and manual validation. Finally, 43 O-glycosites were identified by higher energy collision-induced dissociation (HCD), and 11 O-glycosites were verified 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, 30 O-glycosites were identified by HCD, and 14 O-glycosites were verified by EThcD in the human cell-expressed S protein S1 subunit. 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 are the first to reveal that the SARS-CoV-2 S protein is a mucin-type glycoprotein; clustered O-glycans often occur in the N- and the C-termini of the S protein, and the O-glycosite and O-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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Yong Zhang  

LAB HEAD: Yong Zhang

PROVIDER: PXD022896 | Pride | 2021-11-03

REPOSITORIES: Pride