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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 |

REPOSITORIES: biostudies-literature

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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.

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

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