Distinct shifts in site-specific glycosylation pattern of SARS-CoV-2 spike proteins associated with arising mutations in the D614G and Alpha variants
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Abstract
Extensive glycosylation of the spike protein of severe acute respiratory syndrome coronavirus 2 virus not only shields the major part of it from host immune responses, but glycans at specific sites also act on its conformation dynamics and contribute to efficient host receptor binding, and hence infectivity. As variants of concern arise during the course of the coronavirus disease of 2019 pandemic, it is unclear if mutations accumulated within the spike protein would affect its site-specific glycosylation pattern. The Alpha variant derived from the D614G lineage is distinguished from others by having deletion mutations located right within an immunogenic supersite of the spike N-terminal domain (NTD) that make it refractory to most neutralizing antibodies directed against this domain. Despite maintaining an overall similar structural conformation, our mass spectrometry-based site-specific glycosylation analyses of similarly produced spike proteins with and without the D614G and Alpha variant mutations reveal a significant shift in the processing state of N-glycans on one specific NTD site. Its conversion to a higher proportion of complex type structures is indicative of altered spatial accessibility attributable to mutations specific to the Alpha variant that may impact its transmissibility. This and other more subtle changes in glycosylation features detected at other sites provide crucial missing information otherwise not apparent in the available cryogenic electron microscopy-derived structures of the spike protein variants.
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SciScore for 10.1101/2021.07.21.453140: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
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Experimental Models: Cell Lines Sentences Resources The expression vectors encoding for all S proteins were transiently transfected into HEK293 Freestyle (HEK293F) cells with polyethylenimine (PEI, linear, 25 kDa, Polysciences, USA) at a ratio of DNA: PEI = 1:2. HEK293Fsuggested: RRID:CVCL_6642)Recombinant DNA Sentences Resources The DNA sequence corresponding to residues 1-1208 of the S protein was subcloned from the full-length S sequence, appended with the T4 fibritin foldon sequence at the C-terminus, followed by a c-Myc sequence and a hexahistidine (His6) tag, and inserted into the mammalian expression vector pcDNA3.4-TOPO … SciScore for 10.1101/2021.07.21.453140: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Experimental Models: Cell Lines Sentences Resources The expression vectors encoding for all S proteins were transiently transfected into HEK293 Freestyle (HEK293F) cells with polyethylenimine (PEI, linear, 25 kDa, Polysciences, USA) at a ratio of DNA: PEI = 1:2. HEK293Fsuggested: RRID:CVCL_6642)Recombinant DNA Sentences Resources The DNA sequence corresponding to residues 1-1208 of the S protein was subcloned from the full-length S sequence, appended with the T4 fibritin foldon sequence at the C-terminus, followed by a c-Myc sequence and a hexahistidine (His6) tag, and inserted into the mammalian expression vector pcDNA3.4-TOPO (Invitrogen, USA). pcDNA3.4-TOPOsuggested: NoneSoftware and Algorithms Sentences Resources The buffer viscosity (η) was set to 0.8945 cP at 25 °C based on the theoretical estimate using SEDNTERP. SEDNTERPsuggested: (Sednterp, RRID:SCR_016253)Those parameters were applied in ASTRA 6.0 software (Wyatt Technology, USA) ASTRAsuggested: (ASTRA, RRID:SCR_016255)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
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