Site-specific steric control of SARS-CoV-2 spike glycosylation

Abstract

A central tenet in the design of recombinant vaccines is the display of native-like antigens in the elicitation of protective immunity. However, the diversity of global vaccine strategies against Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses challenges to benchmark antigens across global vaccine programs. Here, we investigate the glycosylation of a variety of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against the glycan shield of an infectious virus. The site-specific stalling of glycan maturation is a highly sensitive reporter of local protein structure and we find there is remarkable conservation of this feature across all samples. Analysis of molecular dynamics simulations of a fully glycosylated spike supports a model of steric restrictions that shape enzymatic processing of the glycans. Furthermore, we show that there is a conserved glycosylation pattern across the monomeric receptor binding domain (RBD) protein and the complete trimeric spike (S) protein. This is in contrast to RBD glycosylation in Middle East respiratory syndrome coronavirus (MERS-CoV) where quaternary architecture limits glycan processing when in the context of full-length MERS-CoV S protein. These results suggest that spike-based immunogen glycosylation reproducibly recapitulates viral glycosylation.

Probing Affinity, Avidity, Anti-Cooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses

“…To estimate the expected mass contribution of the glycan shield, we calculated the average N-glycan masses derived from the glycoproteomic data of Allen and coworkers. 43 The calculated glycan (92.0 kDa) and resultant total S-trimer (482.4 kDa) masses agree quite well (within 2%) with the masses measured by both MP and CD-MS. The glycan mass contribution measured here is somewhat lower than the recent results of Miller and co-workers 44 who reported large mass discrepancies of ∼40% from similar glycoproteomic experiments.…”

Distinct shifts in site-specific glycosylation pattern of SARS-CoV-2 spike proteins associated with arising mutations in the D614G and Alpha variants

“…Outside the RBD, site N657 carries mostly complex type structures, while both N603 and N616 have a mixture of oligomannose and complex type structures with the former consistently less processed. Notably, the value for N603 (and several other sites) as determined from analyses of different S-protein sources varies considerably 16 and not well represented by the reported average value (Fig. 3), which was skewed by atypically high amount of complex type structures at this site in the Swiss and Harvard samples 16 .…”

“…On the other hand, a few emerging characteristics homing in on the overall degree of glycan processing on specific sites as inferred from the relative amount of oligomannose versus hybrid/complex type structures appear to be very consistently reproduced by each reported analysis to date. Allen et al 16 has applied the same data processing method to analyze the available datasets from 5 different sources of prefusion state stabilized recombinant trimeric spike proteins (Amsterdam Medical Centre, Harvard Medical School, Switzerland, The Wellcome centre for Human genetics, and University of Southampton/University of Texas at Austin), very similar to our wildtype S-fm2P used here. They further calculated the average oligomannose/hybrid versus complex type glycan compositions carried on each site from these 5 samples.…”

Predicted pH-dependent stability of SARS-CoV-2 spike protein trimer from interfacial acidic groups

“…including studies of receptor binding [18], drug binding to the linoleic acid binding pocket [19], the opening of cryptic pockets as potential drug targets [20], temperature effects [21], and the effect of glycosylation [22].…”

“…Molecular dynamics simulations are a common tool for structure/function analyses, particular those involving structural transitions. They have been extensively applied to SARS-CoV-2 S protein [17], including studies of receptor binding [18], drug binding to the linoleic acid binding pocket [19], the opening of cryptic pockets as potential drug targets [20], temperature effects [21], and the effect of glycosylation [22].…”