Site-specific glycan analysis of SARS-CoV-2 peak

Vaccine development for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused on the trimeric spike protein that causes infection. Each protomer in the trimeric spike has 22 glycosylation sites. How these sites are glycosylated may affect which cells the virus infects and may protect certain epitopes from antibody neutralization. Watanabe waited. The recombinant glycosylated spike trimer was expressed and purified, proteolyzed to produce glycopeptides containing single glycans, and the composition of glycan sites was determined by mass spectrometry. This analysis provides a benchmark that can be used to measure antigen quality when developing vaccines and antibody tests.
β-coronavirus, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the 2019 coronavirus disease (COVID-19), posing a huge threat to human health worldwide. Vaccine development has focused on the main target of the humoral immune response, the synaptic (S) glycoprotein, which mediates cell entry and membrane fusion. Each SARS-CoV-2 S gene encodes 22 N-linked glycosyl sequences, which may play a role in protein folding and immune escape. Here, using site-specific mass spectrometry, we revealed the glycan structure on the recombinant SARS-CoV-2 S immunogen. This analysis can map the glycan processing state across the trimeric virus peak. We show the difference between SARS-CoV-2 S glycan and typical host glycan processing, which may have an impact on virus pathology and vaccine design.
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative pathogen (1, 2) of Coronavirus 2019 (COVID-19), which can cause fever, severe respiratory diseases and pneumonia. SARS-CoV-2 uses a widely glycosylated spike (S) protein protruding from the surface of the virus to bind to angiotensin converting enzyme 2 (ACE2) to mediate host cell entry (3). S protein is a trimeric class I fusion protein, composed of two functional subunits, responsible for receptor binding (S1 subunit) and membrane fusion (S2 subunit) (4, 5). The surface of the coated nail is mainly controlled by host-derived glycans, and each trimer displays 66 N-linked glycosylation sites. S protein is a key goal in vaccine design work (6). Understanding the glycosylation of recombinant virus spikes can reveal the basic characteristics of virus biology and guide vaccine design strategies (7, 8).
Viral glycosylation has a wide range of roles in viral pathobiology, including mediating protein folding and stability and shaping viral tropism (9). Glycosylation sites are under selective pressure because they promote immune escape by protecting specific epitopes from antibody neutralization. However, we have noticed that the mutation rate of SARS-CoV-2 is very low, and so far, no N-linked glycosylation site mutation has been observed (10). Surfaces with abnormally high density of glycans can also achieve immune recognition (9, 11, 12). For other coronaviruses, the role of glycosylation in camouflaging immunogenic protein epitopes has been studied (10, 13, 14). Coronaviruses germinate into the endoplasmic reticulum-Golgi cavity to form virions (15, 16). However, when complex types of glycans were observed on virus-derived materials, it was found that the viral glycoprotein was affected by the processing enzymes that resided in Golgi (13, 17).
High viral glycan density and local protein structure spatially impair the glycan maturation pathway. The impaired glycan maturation that leads to the presence of oligomannose-type glycans may be a sensitive reporter of natural protein structure (8), and site-specific glycan analysis can be used to compare different immunogens and monitor production processes (18). In addition, glycosylation can affect the transport of recombinant immunogen to the germinal center (19).
In order to solve the site-specific glycosylation of SARS-CoV-2 S protein and visualize the distribution of glycoproteins on the entire protein surface, we expressed and purified the three biological aspects of recombinant soluble materials in the same manner as used to obtain proteins. replica. High-resolution cryo-electron microscopy (cryo-EM) structure, despite the lack of glycan processing blocking effect of kifennonine (4). This variant of the S protein contains all 22 glycans on the SARS-CoV-2 S protein (Figure 1A). By using 2P stable mutations of residues 986 and 987 (20), GSAS (Gly-Ser-Ala-Ser) substitution (residues 682 to 685) and C at the furin cleavage site, the trimer pre-fusion structure is realized Of stability. -Terminal trimerization motif. This helps maintain the quaternary structure during glycan processing. Before analysis, the supernatant containing recombinant SARS-CoV-2 S was purified by size exclusion chromatography to ensure that only natural-like trimeric proteins were analyzed (Figure 1B and Figure S1). The trimer conformation of the purified material was verified by using negative staining EM (Figure 1C).
(A) Schematic diagram of SARS-CoV-2 S glycoprotein. The position of the N-linked glycosylation sequence (NXS/T, where X≠P) is shown as a branch (N, Asn; X, any residue; S, Ser; T, Thr; P, Pro). Describes protein domains: N-terminal domain (NTD), receptor binding domain (RBD), fusion peptide (FP), heptapeptide repeat 1 (HR1), central helix (CH), connector domain (CD) ) And transmembrane domain (TM)). (B) SDS-polyacrylamide gel electrophoresis analysis of SARS-CoV-2 S protein (indicated by arrow) expressed in human embryonic kidney (HEK) 293F cells. Lane 1: Supernatant filtered from transfected cells; Lane 2: Flow-through from StrepTactin resin; Lane 3: Washing with StrepTactin resin; Lane 4: Elution from StrepTactin resin. (C) Mean value of EM 2D negative of SARS-CoV-2 S protein. The 2D average value of SARS-CoV-2 S protein is shown, confirming that the protein adopts a trimer pre-fusion conformation, which matches the material used to determine the structure (4).
In order to determine the site-specific glycosylation of SARS-CoV-2 S, we used trypsin, chymotrypsin and α-lytic protease to generate three glycopeptide samples. These proteases are selected to produce glycopeptides containing a single N-linked glycan sequence. The glycopeptides were analyzed by liquid chromatography-mass spectrometry, and the glycan composition of all 22 N-linked glycan sites was determined (Figure 2). In order to convey the main processing characteristics of each site, according to the branching and fucosylation, the abundance of each glycan is classified into oligomannose, hybrid and complex glycosylation categories. Table S1 and Figure 6 give detailed expanded views showing various glycan compositions. S2.
The diagram illustrates the color codes for the main types of glycans that can appear along the maturation pathway from oligomannose to hybrid types of glycans. These figures summarize the quantitative mass spectrometry analysis of the glycan population present at a single N-linked glycosylation site, reduced to the glycan category. The oligomannose type glycan series (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) are green, non-glycosylated and fucosylated hybrid glycans (hybrid and F hybrid), the dotted line is pink, according to the number of antennae The complex glycan grouping core fucosylation (A1 to FA4) is pink in the presence of conditions. Vacancies in N-linked glycan sites are shown in gray. The pie chart summarizes the quantification of these glycans. Glycan sites are colored according to the content of oligomannose-type glycans, and the glycan sites are marked as green (80% to 100%), orange (30% to 79%) and pink (0% to 29%). An expanded version of the site-specific analysis showing the heterogeneity within each category can be found in Table S1 and Figure 6. S2. The bar graph represents the average of three biological replicates, and the error bars represent the standard error of the average.
The two sites on SARS-CoV-2 S are mainly oligomannose: N234 and N709. Except for N234, the main oligomannose glycan structure observed in the whole protein is Man5GlcNAc2 (Man, mannose; GlcNAc, N-acetylglucosamine), which indicates that these sites can be largely α-1,2-mannosidase access, but the substrate of GlcNAcT-1 is weak, GlcNAcT-1 is the key enzyme in the formation of heterozygous and complex glycans in the Golgi apparatus. The stage that hinders processing is a characteristic related to the density and presentation of glycans on the viral spikes. For example, the high glycosylation peaks of HIV-1 Env and Lassa virus (LASV) GPC show many sites dominated by Man9GlcNAc2 (21-24).
A mixture of oligomannose and complex glycans can be found at sites N61, N122, N603, N717, N801 and N1074 (Figure 2). Of the 22 sites in the S protein, 8 contain a large number of oligomannose-type glycans, which highlights the difference between the processing of SARS-CoV-2 S glycans and the host glycoprotein (25). The remaining 14 sites are dominated by processed complex types of glycans.
Although unoccupied glycosylation sites were detected on SARS-CoV-2 S, they were found to account for a very small part of the total peptide library during quantification (Table S2). In HIV-1 immunogen studies, the voids created by unoccupied glycan sites have been shown to be immunogenic and may cause distracting epitopes (26). The high occupancy rate of the N-linked glycan sequence of SARS-CoV-2 S indicates that the recombinant immunogen will not require further optimization to enhance site occupancy.
Using the frozen EM structure of the trimeric SARS-CoV-2 S protein [Protein Database (PDB) ID 6VSB] (4), the glycosylation state of the coronavirus spike mimic was mapped to the experimentally determined three-dimensional (3D) Structure (Figure 3). The combination of mass spectrometry and low-temperature EM analysis revealed how N-linked glycans trap different areas on the entire surface of the SARS-CoV-2 spike.
Model representative glycans on the pre-fusion structure of trimeric SARS-CoV-2 S glycoprotein (PDB ID 6VSB) (4), where one RBD is in the “up” conformation, and the other two RBDs are in the “direction”. Down” conformation. The glycans are colored according to the oligomannose content defined by the bond. The ACE2 receptor binding site is highlighted in light blue. The S1 and S2 subunits are represented by semi-transparent surfaces, respectively, drawn in light and dark gray. The flexible loops where the N74 and N149 glycan sites are located are represented by gray dashed lines, and the glycan sites on the loops are mapped in their approximate regions.
It can be observed that the receptor binding site on the SARS-CoV-2 spike is shielded by the proximal glycosylation sites (N165, N234, N343), especially when the receptor binding domain is in the “down” conformation. As observed in SARS-CoV-1 S (10, 13), HIV-1 Env (27), influenza hemagglutinin (28, 29) and LASV GPC, the shielding of receptor binding sites by glycans It is a common feature of viral glycoproteins. (twenty four). Considering the functional limitations of the receptor binding site and the low mutation rate of these residues, there may be selective pressure to use N-linked glycans to camouflage one of the most conserved and potentially vulnerable regions of their respective glycoproteins (30, 31).
We noticed the dispersion of oligomannose-type glycans on the S1 and S2 subunits. This is in contrast to other viral glycoproteins. For example, dense glycan clusters in several HIV-1 Env strains can induce oligomannose-type glycans recognized by antibodies (32, 33). In SARS-CoV-2 S, the oligomannose structure is likely to be protected by protein components, such as N234 glycans (partially sandwiched between the N-terminus and the receptor binding domain) (Figure 3).
We characterized the N-linked glycans on the extended flexible loop structures (N74 and N149) and the proximal C-terminus of the membrane (N1158, N1173, N1194), and these molecules were unresolved in the cryoEM images (4). These were identified as complex glycans, consistent with the spatial accessibility of these residues.
Although the content of oligomannose glycans (28%) (Table S2) is higher than that on typical host glycoproteins, it is lower than other viral glycoproteins. For example, one of the most densely glycosylated viral spike proteins is HIV-1 Env, which exhibits ~60% oligomannose-type glycans (21, 34). This indicates that compared with other viral glycoproteins (including HIV-1 Env and LASV GPC), the glycosylation of SARS-CoV-2 S protein is weaker, and the formation of glycans is less shielded, which may be useful for triggering neutralizing antibodies Useful.
In addition, the processing of complex glycans is an important consideration in immunogen engineering, especially considering that the epitope of the neutralizing antibody against SARS-CoV-2 S may contain fucosylated glycans at N343 (35 ). Among the 22 N-linked glycosylation sites, 52% of the fucosylation and 15% of the glycans contained at least one sialic acid residue (Table S2 and Figure S3). Our analysis showed that N343 is highly fucosylated by 98% of the test glycans with fucose residues. The modification of glycans will be severely affected by the cell expression system used. We have previously demonstrated for HIV-1 Env glycosylation that the processing of complex glycans is driven by production cells, but the level of oligomannose glycans is largely independent of the expression system and is related to protein structure. Closely related to glycan density (36).
As observed on LASV GPC and HIV-1 Env, the high-density glycan shield has so-called mannose clusters (22, 24) on the protein surface (Figure 4). Although small mannose-type clusters were found on the S1 subunit of Middle East Respiratory Syndrome (MERS)-CoV S (10), this phenomenon was not observed with SARS-CoV-1 or SARS-CoV-2 S proteins. The site-specific glycosylation analysis reported here shows that the SARS-CoV-2 S glycan shield is consistent with other coronaviruses, and the entire glycan shield also exhibits many vulnerabilities (10) . Finally, we detected trace amounts of O-linked glycosylation at Thr323/Ser325 (T323/S325), where more than 99% of the sites were unmodified (Figure S4), which indicates that when the structure is native-like.
From left to right are MERS-CoV S (10), SARS-CoV-1 S (10), SARS-CoV-2 S, LASV GPC (24) and HIV-1 Env (8, 21). The site-specific N-linked glycan oligomannose quantification is colored according to keywords. Except LASV GPC, all glycoproteins are expressed as soluble trimers in HEK 293F cells, while LASV GPC is derived from virus-like particles of Madin-Darby canine kidney II cells.
Our glycosylation analysis of SARS-CoV-2 provides a detailed benchmark for the site-specific glycan characteristics characteristic of naturally folded trimer spikes. With the development of more and more candidate vaccines based on glycoproteins, detailed glycan analysis provides a way to compare the integrity of immunogens, and as production scales expand to clinical use, monitoring will also be very important . Therefore, in the manufacture of serological test kits, glycan profile analysis will also be an important indicator of antigen quality. Finally, with the advent of nucleotide-based vaccines, it is important to understand how these delivery mechanisms affect the processing and presentation of immunogens.
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Mass spectrometry analysis revealed the glycan composition of all glycosylation sites on the SARS-CoV-2 spike protein.
Mass spectrometry analysis revealed the glycan composition of all glycosylation sites on the SARS-CoV-2 spike protein.
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Post time: Jan-21-2021

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