SARS2 spike- N- glykosylaatioista , myös maininta parista O-glykosylaatiosta

Site-specific glycan analysis of the SARS-CoV-2 spike

1. View ORCID ProfileYasunori Watanabe¹,²,³,*,
2. View ORCID ProfileJoel D. Allen¹,*,
3. View ORCID ProfileDaniel Wrapp⁴,
4. View ORCID ProfileJason S. McLellan⁴,
5. View ORCID ProfileMax Crispin¹,†

See all authors and affiliations

Science  04 May 2020:
eabb9983
DOI: 10.1126/science.abb9983  

Abstract

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.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative pathogen of COVID-19 (1, 2), induces fever, severe respiratory illness and pneumonia. SARS-CoV-2 utilizes an extensively glycosylated spike (S) protein that protrudes from the viral surface to bind to angiotensin-converting enzyme 2 (ACE2) to mediate host-cell entry (3). The S protein is trimeric class I fusion protein, composed of two functional subunits, responsible for receptor binding (S1 subunit) and membrane fusion (S2 subunit) (4, 5). Remarkably, the surface of the envelope spike is dominated by host-derived glycans with each trimer displaying 66 N-linked glycosylation sites. The S protein is a key target in vaccine design efforts (6), and understanding the glycosylation of recombinant viral spikes can reveal fundamental features of viral biology and guide vaccine design strategies (7, 8).

Viral glycosylation has wide-ranging roles in viral pathobiology, including mediating protein folding and stability, and shaping viral tropism (9). Glycosylation sites are under selective pressure as they facilitate immune evasion by shielding specific epitopes from antibody neutralization. However, we note the low mutation rate of SARS-CoV-2, and as yet that there have been no observed mutations to N-linked glycosylation sites (10). 

Surfaces with an unusually high density of glycans can also enable immune recognition (9, 11, 12). The role of glycosylation in camouflaging immunogenic protein epitopes has been studied for other coronaviruses (10, 13, 14). 

Coronaviruses form virions by budding into the lumen of endoplasmic reticulum-Golgi intermediate compartments (ERGIC) (15, 16). 

However, observations of complex-type glycans on virally derived material suggests that the viral glycoproteins are subjected to Golgi-resident processing enzymes (13, 17).

High viral glycan density and local protein architecture can sterically impair the glycan maturation pathway. Impaired glycan maturation resulting in the presence of oligomannose-type glycans can be a sensitive reporter of native-like protein architecture (8), and site-specific glycan analysis can be used to compare different immunogens and monitor manufacturing processes (18). 

Additionally, glycosylation can influence the trafficking of recombinant immunogen to germinal centers (19).

To resolve the site-specific glycosylation of SARS-CoV-2 S protein and visualize the distribution of glycoforms across the protein surface, we expressed and purified three biological replicates of recombinant soluble material in an identical manner to that which was used to obtain the high-resolution cryo-electron microscopy (cryo-EM) structure, albeit without glycan processing blockade using kifunensine (4). 

This variant of the S protein contains all 22 glycans on the SARS-CoV-2 S protein (Fig. 1A). Stabilization of the trimeric prefusion structure was achieved using the “2P” stabilizing mutations (20) at residues 986 and 987, a “GSAS” substitution at the furin cleavage site (residues 682–685), and a C-terminal trimerization motif. This helps to maintain quaternary architecture during glycan processing. Prior to analysis, supernatant containing the recombinant SARS-CoV-2 S was purified by size-exclusion chromatography ensure only native-like trimeric protein was analyzed (Fig. 1B and fig. S1). The trimeric conformation of the purified material was validated using negative-stain electron microscopy (Fig. 1C).

 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).

 To determine the site-specific glycosylation of SARS-CoV-2 S, we employed trypsin, chymotrypsin, and alpha-lytic protease to generate three glycopeptide samples. These proteases were selected to generate glycopeptides that contain a single N-linked glycan sequon. The glycopeptides were analyzed by liquid-chromatography-mass spectrometry (LC-MS), and the glycan compositions were determined for all 22 N-linked glycan sites (Fig. 2). To convey the main processing features at each site, the abundances of each glycan are summed into oligomannose-, hybrid- and categories of complex-type glycosylation based on branching and fucosylation. The detailed, expanded graphs showing the diverse range of glycan compositions is presented in table S1 and fig. S2.

 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.

 https://science.sciencemag.org/content/sci/early/2020/05/01/science.abb9983/F2.large.jpg

There are two sites on SARS-CoV-2 S that are principally oligomannose-type: N234 and N709. The predominant oligomannose-type glycan structure observed across the protein, with the exception of N234, is Man₅GlcNAc₂, which demonstrates that these sites are largely accessible to α1,2-mannosidases but are poor substrates for GlcNAcT-I, which is the gateway enzyme in the formation of hybrid- and complex-type glycans in the Golgi apparatus. The stage at which processing is impeded is a signature related to the density and presentation of glycans on the viral spike. For example, the more densely glycosylated spikes of HIV-1 Env and Lassa virus GPC exhibit numerous sites dominated by Man₉GlcNAc₂ (21–24).

A mixture of oligomannose- and complex-type glycans can be found at sites N61, N122, N603, N717, N801 and N1074 (Fig. 2). Of the 22 sites on the S protein, 8 contain significant populations of oligomannose-type glycans, highlighting how the processing of the SARS-CoV-2 S glycans is divergent from host glycoproteins (25). The remaining 14 sites are dominated by processed, complex-type glycans.

Although unoccupied glycosylation sites were detected on SARS-CoV-2 S, when quantified they were revealed to form a very minor component of the total peptide pool (table S2). In HIV-1 immunogen research, the holes generated by unoccupied glycan sites have been shown to be immunogenic and potentially give rise to distracting epitopes (26). The high occupancy of N-linked glycan sequons of SARS-CoV-2 S indicates that recombinant immunogens will not require further optimization to enhance site occupancy.

Using the cryo-EM structure of the trimeric SARS-CoV-2 S protein (PDB ID 6VSB) (4), we mapped the glycosylation status of the coronavirus spike mimetic onto the experimentally determined 3D structure (Fig. 3). This combined mass spectrometric and cryo-EM analysis reveals how the N-linked glycans occlude distinct regions across the surface of the SARS-CoV-2 spike.https://science.sciencemag.org/content/sci/early/2020/05/01/science.abb9983/F3.large.jpg

Tauko.