Vulnerabilities in coronavirus glycan shields despite extensive glycosylation

Yasunori Watanabe; Zachary T. Berndsen; Jayna Raghwani; Gemma E. Seabright; Joel D. Allen; Jason S. McLellan; Ian A. Wilson; Thomas A. Bowden; Andrew B. Ward; Max Crispin

Following UPLC and IM-ESI MS analysis of released N-linked glycans, we performed glycopeptide analysis to ascertain the compositions of glycans present at all of the potential Nlinked glycosylation sites (PNGs). MERS, SARS and HKU1 recombinant S proteins were reduced, alkylated and digested with an assortment of proteases to yield glycopeptides, which were subjected to in-line liquid-chromatography mass spectrometry (LC-MS). This analysis revealed that each site presents differential levels of oligomannose, hybrid, and complex-type glycan populations ( Fig. 2A & 2B ). Using structures of the trimeric MERS and SARS S proteins (PDB ID: 5X59 and 5X58, respectively), we generated models of fully glycosylated coronavirus spikes using our experimentally determined glycan compositions ( Fig. 3A and 3B ). This analysis revealed that underprocessed oligomannose-type glycans on MERS S colocalize to a specific cluster on the head of the S protein, consisting of glycans at Asn155, Asn166, and Asn236 (Fig. 3A) . We hypothesized that the fully oligomannose-type glycan . CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.02.20.957472 doi: bioRxiv preprint population in this cluster arises due to the hindered accessibility of glycan processing enzymes to access the substrate glycan 33 . As such, we performed mutagenesis to knock out glycosylation sites with N155A, N166A, and N236A mutations. Site-specific analysis of these glycan-KO mutants revealed enhanced trimming of mannose residues, i.e. increased processing, when clustering of glycans was reduced (SI Fig. 4) . The presence of clustered oligomannose-type glycans is reminiscent of that found on other viral glycoproteins, including HIV-1 Env and LASV GPC 29, 36, 46 .