ArticleFrontiers in chemistry2021
A Linkage-specific Sialic Acid Labeling Strategy Reveals Different Site-specific Glycosylation Patterns in SARS-CoV-2 Spike Protein Produced in CHO and HEK Cell Substrates.
Article in Frontiers in chemistry, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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12 citing papers in PubMed, 19 citations in OpenAlex.
- Defining the molecular interaction between influenza hemagglutinin and MHC-II.bioRxiv : the preprint server for biology · 2026Article
- Investigating the effects and underlying mechanisms of glycosylation sites on the immunogenicity of COVID-19 vaccines.Signal transduction and targeted therapy · 2026Article
- Glycoprofile Comparison of the SARS-CoV-2 Spike Proteins Expressed in CHO and HEK Cell Lines.Molecular biotechnology · 2025Article
- Identification of SARS-CoV-2-binding lectins on a commercial lectin array.Scientific reports · 2025Article
- Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance.PLoS biology · 2024Article
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- Formulation development and comparability studies with an aluminum-salt adjuvanted SARS-CoV-2 Spike ferritin nanoparticle vaccine antigen produced from two different cell lines.bioRxiv : the preprint server for biology · 2023Article
- Glycoproteomic Analyses of Influenza A Viruses Using timsTOF Pro MS.Journal of proteome research · 2023Article
- Clinical and Diagnostic Significance of Sialic Acids Determination in Biological Material.Biochemistry (Moscow) Supplement. Series B, Biomedical chemistry · 2022Article
- Site-Specific Glycosylation Patterns of the SARS-CoV-2 Spike Protein Derived From Recombinant Protein and Viral WA1 and D614G Strains.Frontiers in chemistry · 2021Article
- Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2021-2022.Mass spectrometry reviewsReview
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12 authors at 3 institutions in 2 countries.
Funding
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Abstract
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.
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