Topological N-glycosylation and site-specific N-glycan sulfation of influenza proteins in the highly expressed H1N1 candidate vaccines.

Abstract

The outbreak of a pandemic influenza H1N1 in 2009 required the rapid generation of high-yielding vaccines against the A/California/7/2009 virus, which were achieved by either addition or deletion of a glycosylation site in the influenza proteins hemagglutinin and neuraminidase. In this report, we have systematically evaluated the glycan composition, structural distribution and topology of glycosylation for two high-yield candidate reassortant vaccines (NIBRG-121xp and NYMC-X181A) by combining various enzymatic digestions with high performance liquid chromatography and multiple-stage mass spectrometry. Proteomic data analyses of the full-length protein sequences determined 9 N-glycosylation sites of hemagglutinin, and defined 6 N-glycosylation sites and the glycan structures of low abundance neuraminidase, which were occupied by high-mannose, hybrid and complex-type N-glycans. A total of ~300 glycopeptides were analyzed and manually validated by tandem mass spectrometry. The specific N-glycan structure and topological location of these N-glycans are highly correlated to the spatial protein structure and the residential ligand binding. Interestingly, sulfation, fucosylation and bisecting N-acetylglucosamine of N-glycans were also reliably identified at the specific glycosylation sites of the two influenza proteins that may serve a crucial role in regulating the protein structure and increasing the protein abundance of the influenza virus reassortants.

• Authors: Yi-Min She, Aaron Farnsworth, Xuguang Li, Terry D Cyr
• Journal: Scientific reports (Sci Rep) Vol. 7 Issue 1 Pg. 10232 (08 31 2017) ISSN: 2045-2322 [Electronic] England
• PMID: 28860626 (Publication Type: Journal Article)

Chemical References

• Hemagglutinins
• Influenza Vaccines
• Polysaccharides
• Viral Proteins
• NA protein, influenza A virus
• Neuraminidase

Topics

• Amino Acid Sequence
• Chromatography, High Pressure Liquid
• Glycosylation
• Hemagglutinins (analysis, chemistry, genetics)
• Humans
• Influenza A Virus, H1N1 Subtype (chemistry, metabolism)
• Influenza Vaccines (chemistry, metabolism)
• Models, Molecular
• Neuraminidase (analysis, chemistry)
• Polysaccharides (analysis, chemistry)
• Protein Conformation
• Proteomics (methods)
• Reassortant Viruses (chemistry, metabolism)
• Tandem Mass Spectrometry
• Viral Proteins (analysis, chemistry)