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

Autor: Yvonne Genzel, Alexander Nikolay, Udo Reichl, Alexander Pralow, Arnaud Leon, Erdmann Rapp
Rok vydání: 2020
Předmět:
0301 basic medicine
PNGase F
Antigenicity
Glycosylation
animal structures
Molecular biology
Science
Diseases
Viral Nonstructural Proteins
Virus Replication
Biochemistry
Virus
Article
Zika virus
Cell Line
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
N-linked glycosylation
Viral envelope
Viral Envelope Proteins
Tandem Mass Spectrometry
Chlorocebus aethiops
Animals
Humans
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Asparagine
Vero Cells
Multidisciplinary
biology
Zika Virus Infection
Zika Virus
biology.organism_classification
Virology
carbohydrates (lipids)
030104 developmental biology
chemistry
Medicine
lipids (amino acids
peptides
and proteins)
Structural biology
030217 neurology & neurosurgery
Brazil
Biomarkers
Chromatography
Liquid
Biotechnology
Zdroj: Scientific Reports
Scientific Reports, Vol 11, Iss 1, Pp 1-7 (2021)
ISSN: 2045-2322
Popis: 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 glyXtoolMS 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.
Databáze: OpenAIRE
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