Glycan processing of trimeric SARS and MERS spike proteins
To generate a soluble mimic of the viral S proteins, we used the 2P-stabilised native-like 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 many 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. N-linked glycans were enzymatically released, fluorescently labelled, and subjected to hydrophilic interaction chromatography-ultra-performance liquid chromatography (HILIC-UPLC). Treatment with endoglycosidase H (Endo H) revealed a population (SARS 32.2%; MERS 33.8%, HKU1 25.0%) of underprocessed oligomannose-type glycans (Fig. 1b). This observation of both complex and oligomannose-type glycans reveals that the majority of N-linked glycans can 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 Man5GlcNAc2 to Man9GlcNAc2 glycans all present, without one particular dominant peak, as is the case for some viral glycoproteins, such as HIV-1 Env36. The proportion of oligomannose-type glycans on recombinant coronavirus S proteins is consistent with previous studies performed on virally derived MERS and SARS coronavirus S proteins17,42. Coronaviruses have been previously been reported to form virions by budding into the lumen of endoplasmic reticulum-Golgi intermediate compartments (ERGIC)43,44. Observations of hybrid- and complex-type glycans on virally derived material17,42 would, however, suggest that it is likely that coronavirus virions travel through the Golgi apparatus after virion formation in the ERGIC en route to the cell surface, thus supporting recombinant immunogens as models of viral glycoproteins.
Fig. 1 Compositional analysis of SARS, MERS and HKU1 glycans.
a Schematic representation of SARS, MERS and HKU1 coronavirus S glycoproteins, showing the positions of N-linked glycosylation amino-acid sequons (NXS/T, where X ≠ P) shown as branches. The domains of the S glycoproteins are illustrated: N-terminal domain (NTD), receptor-binding domain (RBD), sub-domain 1/2 (SD1/2), fusion peptide (FP), heptad repeat 1/2 (HR1/2), central helix (CH), and transmembrane domain (TM). b HILIC-UPLC chromatograms of fluorescently labelled N-linked glycans from SARS, MERS and HKU1 S. Oligomannose-type glycans (M5 to M9; Man5GlcNAc2–Man9GlcNAc2) (green) and complex-type glycans (magenta) were identified by Endo H digestion, with quantification of major glycan types summarised as a pie chart. Oligomannose-type glycans are schematically annotated with mannose residues as green circles and GlcNAc residues as blue squares.
To ascertain the precise structures of N-linked glycans, glycan pools of each coronavirus S protein were analysed by negative-ion ion-mobility-electrospray ionisation mass spectrometry (IM-ESI MS) (Supplementary Fig. 1). Consistent with the UPLC data, IM-ESI MS confirmed an array of complex-type glycans ranging from mono- to tetra-antennary, but also oligomannose- and hybrid-type glycans. The glycan compositions characterised in the spectra were largely invariant among the coronaviruses with no major structural differences observed.