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 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.

Implications of the Immune Polymorphisms of the Host and the Genetic Variability of SARS-CoV-2 in the Development of COVID-19

“…This modification was enriched in the S1 and S2 subunit, responsible for receptor binding and membrane fusion, respectively [78]. It was also demonstrated that S protein is an O-glycoprotein, displaying a variety composition of O-glycosites and O-glycan in a host cell type-dependent manner [79]. Additionally, the size of O-glycan in the glycosylated S protein appears as a factor that modulates the affinity of virus-receptor interaction.…”

SARS-CoV-2: A Master of Immune Evasion

“…Numerous viruses have demonstrated the relevance of the N-associated glycosylation of envelope proteins for immunological escape, including hepatitis C virus [ 16 , 17 ], HIV-1 [ 18 , 19 , 20 ], hepatitis B virus [ 21 ], herpes simplex virus [ 22 ], and coronaviruses [ 23 , 24 , 25 , 26 ]. In addition to playing a crucial role in epitope camouflage, which represents an efficient method of immune evasion [ 24 , 27 , 28 ], the glycan barrier also enhances viral bonding, entrance, and membrane fusion [ 29 ].…”

“…Besides N-associated glycans, low levels of O-glycans in SARS-CoV-2 spike protein have recently been discovered [ 24 , 27 , 28 , 29 ]. While the oligomannose glycan content of SARS-CoV-2 (28%) is greater compared to normal host glycans [ 24 ], it is much lower when compared with the HIV-1 Env, which has 60% oligomannose-type glycans [ 20 , 30 ].…”

Targeting Mucin Hypersecretion in COVID-19 Therapy

“…Zhang et al reported the mucin-type O-glycosylation landscapes of SARS-CoV-2 S proteins [ 11 ]. The heavily glycosylated S proteins exposed on the SARS-CoV-2 surface support the attachment, entry and fusion of the virus with host cell membranes.…”

“…Site-specific O-glycosylation profiling of SARS-CoV-2 S proteins identified 43 non-sialylated O-glycosites in the insect cell-expressed S protein and 30 sialylated O-glycosites in the human S protein. The investigations revealed that the clustered mucin-type O-glycans modified the SARS-CoV-2 S protein, where the O-glycan and O-glycosite composition varied with the host cell type [ 11 ]. He et al reported mucin-induced bronchoalveolar dysfunction in COVID-19 patients [ 12 ].…”