Influence of Mutations and N-Glycosylation Sites in the Receptor-Binding Domain (RBD) and the Membrane Protein of SARS-CoV-2 Variants of Concern on Antibody Binding in ELISA

Simple Summary Many proteins are glycosylated, for example, on asparagine residues, which can affect protein function in a variety of ways, but can also shield immunogenic sites in viral proteins from the immune system. This is also the case for the highly glycosylated SARS-CoV-2 proteins. In this study, different receptor-binding domains (RBDs) of several SARS-CoV-2 variants of concern (VOCs), a subunit of the highly glycosylated spike protein, and the membrane protein, which has not been extensively studied, were produced in human cells and bacteria for the relevant VOCs. The resulting RBDs were analyzed for their N-glycosylation sites by mass spectrometry and for their ability to bind to IgG antibodies produced in response to SARS-CoV-2 infection by ELISA. Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect human cells by first attaching to the ACE-2 receptor via its receptor-binding domain (RBD) in the spike protein. Here, we report the influence of N-glycosylation sites of the RBD and the membrane (M) protein on IgG antibody binding in serum samples from patients infected with the original SARS-CoV-2 strain in Germany. The RBDs of the wildtype, alpha, beta, gamma, and kappa variants expressed in HEK293S GnTI− cells were all N-glycosylated at Asn331, Asn334, Asn343, and Asn360 or Asn370, whereas the M-protein was glycosylated at Asn5. An ELISA using a coated RBD and probed with anti-RBD IgG antibodies gave a sensitivity of 96.3% and a specificity of 100% for the wildtype RBD, while the sensitivity decreased by 5% to 10% for the variants of concern, essentially in the order of appearance. Deglycosylation of the wildtype RBD strongly reduced antibody recognition by ~20%, considering the mean of the absorbances recorded for the ELISA. This effect was even stronger for the unglycosylated RBD expressed in Escherichia coli , suggesting structural changes affecting epitope recognition. Interestingly, the N-glycosylated M-protein expressed in HEK293S GnTI− cells gave good sensitivity (95%), which also decreased to 65% after deglycosylation, and selectivity (100%). In conclusion, N-glycosylation of the M-protein, the RBD, and most likely the spike protein are important for proper antibody binding and immunological assays, whereas the type of N-glycosylation is less relevant.