Analysis of the SARS-CoV-2 spike protein glycan shield: implications for immune recognition.

Here we have generated 3D structures of glycoforms of the spike (S) glycoprotein from SARS-CoV-2, based on reported 3D structures and glycomics data for the protein produced in HEK293 cells. We also analyze structures for glycoforms representing those present in the nascent glycoproteins (prior to enzymatic modifications in the Golgi), as well as those that are commonly observed on antigens present in other viruses. These models were subjected to molecular dynamics (MD) simulation to determine the extent to which glycan microheterogeneity impacts the antigenicity of the S glycoprotein. Lastly, we have identified peptides in the S glycoprotein that are likely to be presented in human leukocyte antigen (HLA) complexes, and discuss the role of S protein glycosylation in potentially modulating the adaptive immune response to the SARS-CoV-2 virus or to a related vaccine. The 3D structures show that the protein surface is extensively shielded from antibody recognition by glycans, with the exception of the ACE2 receptor binding domain, and also that the degree of shielding is largely insensitive to the specific glycoform. Despite the relatively modest contribution of the glycans to the total molecular weight (17% for the HEK293 glycoform) the level of surface shielding is disproportionately high at 42%.

Analysis of the SARS-CoV-2 spike protein glycan shield reveals implications for immune recognition.

Here we have generated 3D structures of glycoforms of the spike (S) glycoprotein from SARS-CoV-2, based on reported 3D structures and glycomics data for the protein produced in HEK293 cells. We also analyze structures for glycoforms representing those present in the nascent glycoproteins (prior to enzymatic modifications in the Golgi), as well as those that are commonly observed on antigens present in other viruses. These models were subjected to molecular dynamics (MD) simulation to determine the extent to which glycan microheterogeneity impacts the antigenicity of the S glycoprotein. Lastly, we have identified peptides in the S glycoprotein that are likely to be presented in human leukocyte antigen (HLA) complexes, and discuss the role of S protein glycosylation in potentially modulating the innate and adaptive immune response to the SARS-CoV-2 virus or to a related vaccine. The 3D structures show that the protein surface is extensively shielded from antibody recognition by glycans, with the notable exception of the ACE2 receptor binding domain, and also that the degree of shielding is largely insensitive to the specific glycoform. Despite the relatively modest contribution of the glycans to the total molecular weight of the S trimer (17% for the HEK293 glycoform) they shield approximately 40% of the protein surface.

Characterization of heparin and severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein binding interactions.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and continues to spread around the globe at an unprecedented rate. To date, no effective therapeutic is available to fight its associated disease, COVID-19. Our discovery of a novel insertion of glycosaminoglycan (GAG)-binding motif at S1/S2 proteolytic cleavage site (681-686 (PRRARS)) and two other GAG-binding-like motifs within SARS-CoV-2 spike glycoprotein (SGP) led us to hypothesize that host cell surface GAGs may interact SARS-CoV-2 SGPs to facilitate host cell entry. Using a surface plasmon resonance direct binding assay, we found that both monomeric and trimeric SARS-CoV-2 SGP bind more tightly to immobilized heparin (KD = 40 pM and 73 pM, respectively) than the SARS-CoV and MERS-CoV SGPs (500 nM and 1 nM, respectively). In competitive binding studies, the IC50 of heparin, tri-sulfated non-anticoagulant heparan sulfate, and non-anticoagulant low molecular weight heparin against SARS-CoV-2 SGP binding to immobilized heparin were 0.056 µM, 0.12 µM, and 26.4 µM, respectively. Finally, unbiased computational ligand docking indicates that heparan sulfate interacts with the GAG-binding motif at the S1/S2 site on each monomer interface in the trimeric SARS-CoV-2 SGP, and at another site (453-459 (YRLFRKS)) when the receptor-binding domain is in an open conformation. The current study serves a foundation to further investigate biological roles of GAGs in SARS-CoV-2 pathogenesis. Furthermore, our findings may provide additional basis for further heparin-based interventions for COVID-19 patients exhibiting thrombotic complications.