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. Sub...

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Autores principales:	Alexander Pralow, Alexander Nikolay, Arnaud Leon, Yvonne Genzel, Erdmann Rapp, Udo Reichl
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/22d1a00117f2457d84ccc5377000bc89
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spelling	oai:doaj.org-article:22d1a00117f2457d84ccc5377000bc892021-12-02T13:34:32ZSite-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins10.1038/s41598-021-84682-z2045-2322https://doaj.org/article/22d1a00117f2457d84ccc5377000bc892021-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-84682-zhttps://doaj.org/toc/2045-2322Abstract 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.Alexander PralowAlexander NikolayArnaud LeonYvonne GenzelErdmann RappUdo ReichlNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-7 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Alexander Pralow Alexander Nikolay Arnaud Leon Yvonne Genzel Erdmann Rapp Udo Reichl Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins
description	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 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.
format	article
author	Alexander Pralow Alexander Nikolay Arnaud Leon Yvonne Genzel Erdmann Rapp Udo Reichl
author_facet	Alexander Pralow Alexander Nikolay Arnaud Leon Yvonne Genzel Erdmann Rapp Udo Reichl
author_sort	Alexander Pralow
title	Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins
title_short	Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins
title_full	Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins
title_fullStr	Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins
title_full_unstemmed	Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins
title_sort	site-specific n-glycosylation analysis of animal cell culture-derived zika virus proteins
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/22d1a00117f2457d84ccc5377000bc89
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Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins

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