N-glycosylation profiles of the SARS-CoV-2 spike D614G mutant and its ancestral protein characterized by advanced mass spectrometry

Abstract

N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins.

Glycosylation in SARS-CoV-2 variants: A path to infection and recovery

“…However, the glycosylation pattern of the remaining N-glycosylation sequons altered their glycan distribution. Sequons at N122, N234, N603, N709, and N801 of S-614G showed reduced complex and hybrid structures 35 . Whereas sequons at N165, N282, N616 N1098 and N1134 expressed higher hybrid and lower complex glycans in the S-614G variant.…”

“…Whereas sequons at N165, N282, N616 N1098 and N1134 expressed higher hybrid and lower complex glycans in the S-614G variant. Sequon N717 did not express any complex sugars for the S-614 G or S-614 D variants, respectively 35 . This study also found that the S-614G mutation decreased the amount of complex-type glycans by up to 45% and increased the amount of oligomannose glycans by up to 33%.…”

“…Numerous derivation techniques, including esterification amidation, permethylation, tandem mass tagging, and others, can also be used to improve the glycopeptide's poor ionisation efficiency 33 . The glycan compositions at each of the sites on the SARS-CoV-2 S protein were determined by LC-MS analysis of the glycopeptide pools with high-energy collision-induced dissociation (HCD) or electron transfer dissociation (ETD) fragmentation [34] , [35] along with an advanced data analysis platform.…”

Conserved Neutralizing Epitopes on the N-Terminal Domain of Variant SARS-CoV-2 Spike Proteins

“…C1717 light chain CDRL2 and CDRL3 loops contact the C-terminus of the NTD (residues 286-303) and the NTD loop 210-218, respectively (Figures 5I and 5J). In addition, light chain FWR3 buries against the N282 NTD -glycan, a complex-type N-glycan(Wang et al, 2021a) that wedges against the SD1 domain of the adjacent protomer (Figures 5F-G, I). Modeling of Omicron mutations found in NTD loop 210-218 shows accommodation of the 214-insertion and potential establishment of backbone contacts with the NTD loop and R95 LC in CDRL3 (Figure 5K).…”

“…C1717 light chain CDRL2 and CDRL3 loops contact the C-terminus of the NTD (residues 286-303) and the NTD loop 210-218, respectively (Figures 5I and 5J). In addition, light chain FWR3 buries against the N282NTD-glycan, a complex-type N-glycan (Wang et al, 2021a) that wedges against the SD1 domain of the adjacent protomer (Figures 5F-G, I).…”