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Characterization of site-specific O- and N-glycopeptides from recombinant spike and ACE2 glycoproteins using LC-MS/MS analysis

ABSTRACT: The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of infections and deaths globally. Although vaccination campaigns are mitigating the pandemic, emerging viral variants continue to pose challenges. The spike (S) protein of SARS-CoV-2 plays a critical role in viral entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, making both proteins essential targets for therapeutic and vaccine development. Glycosylation of these proteins influences their structure and function, underscoring the need for detailed site-specific glycoproteomic analysis. In this study, we characterized the N- or O-glycosylation profiles of recombinant receptor-binding domain (RBD) of spike protein and ACE2 proteins expressed from HEK293 cells, as well as S2 subunit of spike protein expressed in plants cells. Using a high-resolution Orbitrap Eclipse Tribrid mass spectrometer integrated with the Ultimate 3000 RSLCnano and IQ-GPA (Identification and Quantification of GlycoProteome Analyzer), 148 N- and 28 O-glycopeptides from RBD, 71 N-glycopeptides from the S2 subunit, and 139 N-glycopeptides from ACE2 were characterized. Additionally, we report novel post-translational modifications (PTMs) in RBD glycopeptides, including mannose-6-phosphate (M6P) and GlcNAc-1-phosphate-6-O-mannose of N-glycan, and O-acetylation of O-glycan, identified for the first time. These findings provide valuable insights into the glycosylation patterns that influence protein function and immunogenicity, offering new perspectives for the development of vaccines and antibody-based therapies against COVID-19.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Sars Coronavirus (ncbitaxon:227859) Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ju Yeon Lee

PROVIDER: MSV000096403 | MassIVE | Wed Nov 13 09:10:00 GMT 2024

SECONDARY ACCESSION(S): PXD057852

REPOSITORIES: MassIVE

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Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes an extensively glycosylated spike protein that protrudes from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the predominant recombinant spike protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, recombinant full-size spike protein was produced transiently in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on spike glycans, 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 glycans and glycopeptides from spike protein. We determined global and site-specific N-linked glycosylation patterns in soluble SARS-CoV-2 spike using MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation. We identified the glycan compositions at 21 and 19 out of the 22 predicted N-glycosylation sites of the SARS-CoV-2 spike proteins produced in CHO and HEK, respectively. The N-glycan site at 1158 position (N1158) and the N-glycan sites at 122 ,282 and 1158 positions (N122, N282 and N1158) were found to be unoccupied on spike secreted from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human spike proteins revealed that CHO-Spike presented more complex and higher sialylation (α2,3-linked) content while HEK-Spike displayed more high-mannose and minor content of α2,3- and α2,6-linked sialic acids. The N74 site represents the most heavily N-glycosylated site on both spike proteins while some high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801 and N1134) on HEK-Spike may differentially shield the virus compared to CHO-spike and provide a different host immune system interaction strategy. Collectively, these data underscore the importance of characterizing site-specific glycosylation on recombinant human spike protein from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in diagnostics and vaccines.

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Characterization of site-specific O- and N-glycopeptides from recombinant spike and ACE2 glycoproteins using LC-MS/MS analysis

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