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. 2020 May 4;eabb9983.
doi: 10.1126/science.abb9983. Online ahead of print.

Site-specific Glycan Analysis of the SARS-CoV-2 Spike

Free PMC article

Site-specific Glycan Analysis of the SARS-CoV-2 Spike

Yasunori Watanabe et al. Science. .
Free PMC article


The emergence of the betacoronavirus, SARS-CoV-2, the causative agent of COVID-19, represents a significant threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, which mediates cell entry and membrane fusion. SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.


Fig. 1
Fig. 1. Expression and validation of SARS-CoV-2 S glycoprotein.
(A) Schematic representation of SARS-CoV-2 S glycoprotein. The positions of N-linked glycosylation sequons (N-X-S/T, where X≠P) are shown as branches. Protein domains are illustrated: N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), and transmembrane domain (TM). (B) SDS-PAGE analysis of SARS-CoV-2 S protein expressed in human embryonic kidney 293F cells. Lane 1: filtered supernatant from transfected cells; lane 2: flow-through from StrepTactin resin; lane 3: wash from StrepTactin resin; lane 4: elution from StrepTactin resin. (C) Negative-stain EM 2D class averages of the SARS-CoV-2 S protein. 2D class averages of the SARS-CoV-2 S protein are shown, confirming that the protein adopts the trimeric prefusion conformation matching the material used to determine the structure (4).
Fig. 2
Fig. 2. Site-specific N-linked glycosylation of SARS-CoV-2 S glycoprotein.
The schematic illustrates the color code for the principal glycan types that can arise along the maturation pathway from oligomannose-, hybrid- to complex-type glycans. The graphs summarize quantitative mass spectrometric analysis of the glycan population present at individual N-linked glycosylation sites simplified into categories of glycans. The oligomannose-type glycan series (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) is colored green, afucosylated and fucosylated hybrid-type glycans (Hybrid & F Hybrid) dashed pink, and complex glycans grouped according to the number of antennae and presence of core fucosylation (A1 to FA4) and are colored pink. Unoccupancy of an N-linked glycan site is represented in grey. The pie charts summarize the quantification of these glycans. Glycan sites are colored according to oligomannose-type glycan content with the glycan sites labeled in green (80−100%), orange (30−79%) and pink (0−29%). An extended version of the site-specific analysis showing the heterogeneity within each category can be found in table S1 and fig. S2. The bar graphs represent the mean quantities of three biological replicates with error bars representing the standard error of the mean.
Fig. 3
Fig. 3. Structure-based mapping of SARS-CoV-2 S N-linked glycans.
Representative glycans are modeled onto the prefusion structure of trimeric SARS-CoV-2 S glycoprotein (PDB ID 6VSB) (4), with one RBD in the “up” conformation and the other two RBDs in the “down” conformation. The glycans are colored according to oligomannose content as defined by the key. ACE2 receptor binding sites are highlighted in light blue. The S1 and S2 subunits are rendered with translucent surface representation, colored light and dark grey, respectively. Note that the flexible loops on which N74 and N149 glycan sites reside are represented as dashed lines with glycan sites on the loops mapped at their approximate regions.
Fig. 4
Fig. 4. Under-processing of viral glycan shields.
From left to right, MERS-CoV S (10), SARS-CoV-1 S (10), SARS-CoV-2 S, LASV GPC (24), and HIV-1 Env (8, 21). Site-specific N-linked glycan oligomannose quantifications are colored according to the key. All glycoproteins were expressed as soluble trimers in HEK 293F cells apart from LASV GPC, which was derived from virus-like particles from Madin-Darby canine kidney II cells.

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