Mass Spectrometry Analysis of Newly Emerging Coronavirus HCoV-19 Spike Protein and Human ACE2 Reveals Camouflaging Glycans and Unique Post-Translational Modifications

Abstract

The COVID-19 pandemic has led to worldwide efforts to understand the biological traits of the newly identified HCoV-19 virus. In this mass spectrometry (MS)-based study, we reveal that out of 21 possible glycosites in the HCoV-19 S protein, 20 are completely occupied by N -glycans, predominantly of the oligomannose type. All seven glycosylation sites in human angiotensin I converting enzyme 2 (hACE2) were found to be completely occupied, mainly by complex N -glycans. However, glycosylation did not directly contribute to the binding affinity between HCoV-19 S and hACE2. Additional post-translational modifications (PTM) was identified, including multiple methylated sites in both proteins and multiple sites with hydroxylproline in hACE2. Refined structural models of HCoV-19 S and hACE2 were built by adding N -glycan and PTMs to recently published cryogenic electron microscopy (cryo-EM) structures. The PTM and glycan maps of HCoV-19 S and hACE2 provide additional structural details for studying the mechanisms underlying host attachment and the immune response of HCoV-19, as well as knowledge for developing desperately needed remedies and vaccines.

Covid-19 and blood groups: ABO antibody levels may also matter

“…Several structural analyses of the SARS-CoV-2 S protein glycans have been reported, showing the presence of a large fraction of mature N- and O-glycans, alongside immature forms. However, none of these studies described the expression of blood group antigens on the S protein ( Gao et al, 2020 , Sanda et al, 2020 , Shajahan et al, 2020 , Sun et al, 2020 , Watanabe et al, 2020 ). Yet, all analyses were performed on recombinant S protein produced in HEK-293T cells which do not express blood group antigens, unlike epithelial cells of the larynx and the bronchial mucosa where infectious viral particles are likely produced.…”

The SARS-CoV-2 Spike Glycoprotein Biosynthesis, Structure, Function, and Antigenicity: Implications for the Design of Spike-Based Vaccine Immunogens

“…The remaining eight sites were found to be dominated by oligomannose-type glycans, which are divergent from those founded on host glycoproteins (63). Although glycosylation sites (N165, N234, N343) proximal to the receptor-binding sites on the SARS-CoV-2 S protein can be observed, ACE2 bound to the glycosylated and deglycosylated S ectodomains with nearly identical affinity (1.7 nM vs 1.5 nM) determined by a biolayer interferometry binding assay (64). This observation suggests that the high binding affinity between the SARS-CoV-2 S protein and ACE2 does not depend on the S protein glycosylation.…”

SARS-CoV-2 structural features may explain limited neutralizing-antibody responses

“…S is cleaved by furin and the serine proteases TMPRSS2 and TMPRSS4, enabling fusion of viral and cellular membranes, and consequent entry of viral RNA into the host cell 8 . This cleavage site may also be a target for neutralizing antibodies 9,10 . Overall, the RBD/RBM is the immunological Achilles heel of the virus.…”