Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins

Abstract

Here, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC-MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtool^MS software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.

Figure 1

Workflow of the site-specific N-glycopeptide analysis of ZIKV proteins.

Figure 2

Site-specific N-glycosylation of the NS1 protein of ZIKV produced in EB66 cells. The structure of the homodimer NS1 (PDB: 5k6k) is exemplary illustrated. Monomers are visualized using different colors (green and cyan). Potential N-glycosylation sites are highlighted in magenta. Annotated N-glycans represent only possible N-glycan structures based on our results obtained by MS spectra analysis. Symbolic representation of N-glycan structures was generated with GlycoWorkbench Version 1.1 following the guideline of Symbol Nomenclature for Graphical Representation of Glycans.

Figure 3

Exemplary illustrated structure of the M/E protein of ZIKV. The structure of the heterodimer protein M and E complex (PDB: 5h37) is illustrated. This complex is formed from an M protein homodimer (monomer visualized in green) and an E protein homodimer (monomer visualized in cyan). The potential N-glycosylation site N444 is highlighted in magenta.

Figure 4

MS/MS fragment ion spectrum of the de-N-glycosylated and deamidated N-glycopeptide from the ZIKV E protein produced in EB66 cells. The fragment ion spectrum of the tryptic peptide IMLSVHGSQHSGMIVN(deamidated)DTGHETDNR after N-glycan release is shown. Diagnostic y-ions (red) and b-ions (blue) are annotated in the ion spectrum and highlighted in the peptide sequence at the upper right corner. The potential N-glycosylation site N444 is deamidated (red).