Dataset Information

Site-specific O-glycosylation analysis of SARS-CoV-2 spike protein produced in insect and human cells.

ABSTRACT: Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S - insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.

SUBMITTER: Bagdonaite I

PROVIDER: S-EPMC7872350 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Site-specific O-glycosylation analysis of SARS-CoV-2 spike protein produced in insect and human cells.

Bagdonaite Ieva I Thompson Andrew J AJ Wang Xiaoning X Søgaard Max M Fougeroux Cyrielle C Frank Martin M Diedrich Jolene K JK Yates John R JR Salanti Ali A Vakhrushev Sergey Y SY Paulson James C JC Wandall Hans H HH

bioRxiv : the preprint server for biology 20210210

Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence ...[more]

PMID: 33564762

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Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes the extensively glycosylated spike (S) protein protruding from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the main recombinant S protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, a recombinant S protein truncated at the transmembrane domain and engineered to express a C-terminal trimerization motif was transiently produced in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on S protein, a two-step amidation process, employing dimethylamine and ammonium hydroxide reactions in a solid support system, was developed to differentially modify the sialic acid linkages on the glycans and glycopeptides from the S protein. The process also adds a charge to Asp and Glu which aids in ionization. We used MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation to determine global and site-specific N-linked glycosylation patterns. We identified 21 and 19 out of the 22 predicted N-glycosites of the SARS-CoV-2 S proteins produced in CHO and HEK, respectively. It was found that the N-glycosite at 1,158 position (N1158) and at 122, 282 and 1,158 positions (N122, N282 and N1158) were absent on S from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human S proteins reveals that CHO-Spike exhibits more complex and higher sialylation (α2,3-linked) content while HEK-Spike exhibits more high-mannose and a small amount of α2,3- and α2,6-linked sialic acids. The N74 site represents the most abundant glycosite on both spike proteins. The relatively higher amount of high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801, and N1134) on HEK-Spike suggests that glycan-shielding may differ among the two constructs. HEK-Spike can also provide different host immune system interaction profiles based on known immune system active lectins. Collectively, these data underscore the importance of characterizing the site-specific glycosylation of recombinant human spike proteins from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in research, diagnostics and vaccines.

Project description:The SARS-CoV-2 spike protein is heavily glycosylated, having 22 predicted N-glycosylation sites per monomer. It is also O-glycosylated, although the number of O-glycosites is less defined. Recent studies show that spike protein glycans play critical roles in viral entry and infection. The spike monomer has two subdomains, S1 and S2, and a receptor-binding domain (RBD) within the S1 domain. In this study, we have characterized the site-specific glycosylation patterns of the HEK293 recombinant spike RBD and S1 domains as well as the intact spike derived from the whole virus produced in Vero cells. The Vero cell-derived spike from the WA1 strain and a D614G variant was analyzed. All spike proteins, S1, and RBDs were analyzed using hydrophilic interaction chromatography (HILIC) and LC-MS/MS on an Orbitrap Eclipse Tribrid mass spectrometer. N-glycans identified in HEK293-derived S1 were structurally diverse. Those found in the HEK293-derived RBD were highly similar to those in HEK293 S1 where N-glycosites were shared. Comparison of the whole cell-derived WA1 and D614G spike proteins revealed that N-glycosites local to the mutation site appeared to be more readily detected, hinting that these sites are more exposed to glycosylation machinery. Moreover, recombinant HEK293-derived S1 was occupied almost completely with complex glycan, while both WA1 and D614G derived from the Vero E6 cell whole virus were predominantly high-mannose glycans. This stands in stark contrast to glycosylation patterns seen in both CHO- and HEK cell-derived recombinant S1, S2, and the whole spike previously reported. Concerning O-glycosylation, our analyses revealed that HEK293 recombinant proteins possessed a range of O-glycosites with compositions consistent with Core type 1 and 2 glycans. The O-glycosites shared between the S1 and RBD constructs, sites T323 and T523, were occupied by a similar range of Core 1 and 2 type O-glycans. Overall, this study reveals that the sample nature and cell substrate used for production of these proteins can have a dramatic impact on the glycosylation profile. SARS-CoV-2 spike glycans are associated with host ACE2 receptor interaction efficiency. Therefore, understanding such differences will serve to better understand these host-pathogen interactions and inform the choice of cell substrates to suite downstream investigations.

Project description:The glycosylation on the spike (S) protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, modulates the viral infection by altering conformational dynamics, receptor interaction and host immune responses. Several variants of concern (VOCs) of SARS-CoV-2 have evolved during the pandemic, and crucial mutations on the S protein of the virus have led to increased transmissibility and immune escape. In this study, we compare the site-specific glycosylation and overall glycomic profiles of the wild type Wuhan-Hu-1 strain (WT) S protein and five VOCs of SARS-CoV-2: Alpha, Beta, Gamma, Delta and Omicron. Interestingly, both N- and O-glycosylation sites on the S protein are highly conserved among the spike mutant variants, particularly at the sites on the receptor-binding domain (RBD). The conservation of glycosylation sites is noteworthy, as over 2 million SARS-CoV-2 S protein sequences have been reported with various amino acid mutations. Our detailed profiling of the glycosylation at each of the individual sites of the S protein across the variants revealed intriguing possible association of glycosylation pattern on the variants and their previously reported infectivity. While the sites are conserved, we observed changes in the N- and O-glycosylation profile across the variants. The newly emerged variants, which showed higher resistance to neutralizing antibodies and vaccines, displayed a decrease in the overall abundance of complex-type glycans with both fucosylation and sialylation and an increase in the oligomannose-type glycans across the sites. Among the variants, the glycosylation sites with significant changes in glycan profile were observed at both the N-terminal domain and RBD of S protein, with Omicron showing the highest deviation. The increase in oligomannose-type happens sequentially from Alpha through Delta. Interestingly, Omicron does not contain more oligomannose-type glycans compared to Delta but does contain more compared to the WT and other VOCs. O-glycosylation at the RBD showed lower occupancy in the VOCs in comparison to the WT. Our study on the sites and pattern of glycosylation on the SARS-CoV-2 S proteins across the VOCs may help to understand how the virus evolved to trick the host immune system. Our study also highlights how the SARS-CoV-2 virus has conserved both N- and O- glycosylation sites on the S protein of the most successful variants even after undergoing extensive mutations, suggesting a correlation between infectivity/ transmissibility and glycosylation.

Dataset Information

Site-specific O-glycosylation analysis of SARS-CoV-2 spike protein produced in insect and human cells.

Publications

Site-specific O-glycosylation analysis of SARS-CoV-2 spike protein produced in insect and human cells.

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