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

In order to generate a soluble mimic of the viral S proteins, we used the 2P stabilised nativelike SARS and MERS S protein antigens, the design and structures of which have been described previously by Pallesen et al. 41 . SARS, MERS and HKU1 S genes encode a large number of N-linked glycan sequons 23, 23 and 29, respectively (Fig. 1A) . We initially sought to quantitatively assess the composition of the carbohydrate structures displayed on the S glycoproteins. To this end, N-linked glycans were enzymatically released, fluorescently labelled, and subjected to hydrophilic interaction chromatography-ultra-performance liquid chromatography (HILIC-UPLC). Cleavage of the fluorescently labelled glycans by endoglycosidase H (Endo H) revealed a population (SARS 32.2%; MERS 33.8%, HKU1 25.0%) of under-processed oligomannose-type glycans (Fig. 1B) . This observation of both complex and oligomannose-type glycans reveals that the majority of N-linked glycans are able to be processed, although there is limited processing at specific sites across the S proteins. It is also interesting to note that the distribution of oligomannose-type glycans was broad, with