N-Glycosylation Network Construction and Analysis to Modify Glycans on the Spike (S) Glycoprotein of SARS-CoV-2

Abstract

Background: The N-glycan structure and composition of the spike (S) protein of SARS-CoV-2 are pertinent to vaccine development and efficacy.Methods: We reconstructed the glycosylation network based on previously published mass spectrometry data using GNAT, a glycosylation network analysis tool. Our compilation of the network tool had 26 glycosyltransferase and glucosidase enzymes and could infer the pathway of glycosylation machinery based on glycans in the virus spike protein. Once the glycan biosynthesis pathway was generated, we simulated the effect of blocking specific enzymes—swainsonine or deoxynojirimycin for blocking mannosidase-II and indolizidine for blocking alpha-1,6-fucosyltransferase—to see how they would affect the biosynthesis network and the glycans that were synthesized.Results: The N-glycan biosynthesis network of SARS-CoV-2 spike protein shows an elaborate enzymatic pathway with several intermediate glycans, along with the ones identified by mass spectrometric studies. Of the 26 enzymes, the following were involved—Man-Ia, MGAT1, MGAT2, MGAT4, MGAT5, B3GalT, B4GalT, Man-II, SiaT, ST3GalI, ST3GalVI, and FucT8. Blocking specific enzymes resulted in a substantially modified glycan profile of SARS-CoV-2.Conclusion: Variations in the final N-glycan profile of the virus, given its site-specific microheterogeneity, are factors in the host response to the infection, vaccines, and antibodies. Heterogeneity in the N-glycan profile of the spike (S) protein and its potential effect on vaccine efficacy or adverse reactions to the vaccines remain unexplored. Here, we provide all the resources we generated—the glycans in the glycoCT xml format and the biosynthesis network for future work.

Site‐specific glycosylation of SARS‐CoV‐2: Big challenges in mass spectrometry analysis

“…Differences in data are not only influenced by experimental variation, but also by data misinterpretation as well. For example, bioinformatics mass data analysis by Krishnan et al., has mixed nonidentified N‐glycosites in some cases as unoccupied [ 103 ]. Furthermore, Segreto et al., misinterpred O‐glycosylation data obtained by EthcD as data obtained by HCD fragmentation with modulated collision energy [ 104 ].…”

Proteomics‐based mass spectrometry profiling of SARS‐CoV‐2 infection from human nasopharyngeal samples

“…Glycosylation of recombinant trimeric S‐protein expressed in different systems have been studied by various groups around the world. While the identified N ‐ and O ‐glycosites are mostly consistent across different studies, the glycan compositions and occupancies on each site are unique depending on the analysis platform and expression system, respectively (Krishnan & Krishnan, 2021 ). As an example, the micro‐heterogeneity of N ‐ and O ‐glycoforms observed for only HEK293‐derived S‐protein from different research groups are depicted in Figure 3B .…”

“Multiomics” Approaches to Understand and Treat COVID-19: Mass Spectrometry and Next-Generation Sequencing

“…Techniques based on mass spectrometry have been critical for understanding the glycosylation of SARS-CoV-2 spike protein [27,[101][102][103][104][105][106]. Glycosylation of the spike protein of coronavirus and other viral proteins are crucial to the infection process because the modification shields immunogenic epitopes, and impact immunological pressure across the protein surface [107,108].…”