Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike S protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications

Abstract

The pneumonia-causing COVID-19 pandemia has prompt worldwide efforts to understand its biological and clinical traits of newly identified HCoV-19 virus. In this study, post-translational modification (PTM) of recombinant HCoV-19 S and hACE2 were characterized by LC-MSMS. We revealed that both proteins were highly decorated with specific proportions of N-glycan subtypes. Out of 21 possible glycosites in HCoV-19 S protein, 20 were confirmed completely occupied by N-glycans, with oligomannose glycans being the most abundant type. All 7 possible glycosylation sites in hACE2 were completely occupied mainly by complex type N-glycans. However, we showed that glycosylation did not directly contribute to the binding affinity between SARS-CoV spike protein and hACE2. Additionally, we also identified multiple sites methylated in both proteins, and multiple prolines in hACE2 were converted to hydroxylproline. Refined structural models were built by adding N-glycan and PTMs to recently published cryo-EM structure of the HCoV-19 S and hACE2 generated with glycosylation sites in the vicinity of binding surface. The PTM and glycan maps of both HCoV-19 S and hACE2 provide additional structural details to study mechanisms underlying host attachment, immune response mediated by S protein and hACE2, as well as knowledge to develop remedies and vaccines desperately needed nowadays.

Data, Reagents, Assays and Merits of Proteomics for SARS-CoV-2 Research and Testing

“…Although not covered in this work, it is well established that virus-host interactions are heavily reliant on post-translational modifications (PTMs) on virus and host proteins. Both the virus spike protein as well as the host receptors ACE2 and TMPRSS2 are glycoproteins and several studies are emerging that characterize the glycosylation pattern of individual proteins ( 60 , 61 , 62 , 63 ). Unfortunately, global and protein-specific surface glycosylation profiling is still challenging but could become an important future avenue to understand better how complex glycan patterns both of the virus as well as host cells determine the tropism of SARS-CoV-2.…”

COVID-19: Mechanisms of Vaccination and Immunity

“…Consequently, the antibodies induced by the vaccines may potentially be cross-reactive and non-protective. Interestingly, however, the receptor interaction site (RIS) directly binding to ACE2 is not glycosylated, indicating that this RIS may potentially be an ideal vaccine candidate [ 54 , 55 ] ( Figure 2 B).…”

The flexibility of ACE2 in the context of SARS-CoV-2 infection

“…ACE2 and RBD glycosylation was defined according to glycoanalytic data (45,47) and modeled using the Glycan Reader & Modeler tool (52) integrated into Glycan Reader (53) in CHARMM-GUI (54). In total, 7 complex, bi-antennary N glycans and 1 O glycan were added to ACE2, as well as 1 N glycan to RBD (Supplementary Table 1).…”

“…Seven complex N-glycans and one O-glycan in ACE2 were modeled according to glycoanalytic data (45)(46)(47), as well as glycan N343 in RBD for the RBD-bound simulations (Figure 1b). The B 0 AT1 transporter solved in the cryo-EM structure was not included in our simulations in order to inform on the intrinsic dynamics of ACE2.…”

“…Here, we perform all-atom molecular dynamics (MD) simulations of the full length, membrane-embedded and glycosylated ACE2 homodimer both in the apo state and in complex with RBD to study the molecular origins of the ACE2-S flexibility on the host receptor side. Seven complex N-glycans and one O-glycan in ACE2 were modeled according to glycoanalytic data ( 45 – 47 ), as well as glycan N343 in RBD for the RBD-bound simulations ( Figure 1b ). The B 0 AT1 transporter solved in the cryo-EM structure was not included in our simulations in order to inform on the intrinsic dynamics of ACE2.…”