Glycoproteomic and Single-Protein Glycomic Analyses Reveal Zwitterionic N-Glycans on Natural and Recombinant Proteins Derived From Insect Cells

Title (eng)
Glycoproteomic and Single-Protein Glycomic Analyses Reveal Zwitterionic N-Glycans on Natural and Recombinant Proteins Derived From Insect Cells
Author
Shi Yan
Author
Jorick Vanbeselaere
Author
Callum Ives
Author
David Stenitzer
Author
Lena Nuschy
Author
Florian Woels
Author
Katharina Paschinger
Author
Elisa Fadda
Author
Johannes Stadlmann
Author
Iain Wilson
Abstract (eng)
Insect cells are a convenient cell factory to produce recombinant glycoproteins. Their glycosylation potential is believed to be simple, needing primarily addition of glycosyltransferases to humanize the recombinant products. In this study, the native glycoproteome of Spodoptera frugiperda Sf9 and Trichoplusia ni High Five cells, examined using an LC-MS/MS approach, revealed not only which proteins are N-glycosylated but also indicated that the N-glycomes contain novel glucuronylated and phosphorylcholine-modified glycans, in addition to typical oligomannosidic and fucosylated structures. These data were corroborated by a parallel MALDI-TOF MS/MS analysis of N-glycosidase-released oligosaccharides. Molecular modeling analysis of one endogenous Sf9 glycoprotein correlated the occurrence of complex and oligomannosidic N-glycans with the accessibility of the occupied N-glycosylation sites. Further, we showed that the N-glycans of influenza hemagglutinins and SARS-CoV-2 spike glycoprotein produced in Spodoptera cells possess a number of glycan structures modified with phosphorylcholine, but core difucosylation was minimal; in contrast, the Trichoplusia-produced hemagglutinin had only traces of the former type, while the latter was dominant. Detection of phosphorylcholine on these glycoproteins correlated with binding to human C-reactive protein. In conclusion, not just oligomannosidic or truncated paucimannosidic N-glycans, but structures with immunogenic features occur on both natural and recombinant glycoproteins derived from insect cell lines.
Keywords (eng)
BaculovirusFucoseGlucuronic AcidInsect CellsN-glycansPhosphorylcholine
Type (eng)
journal article
Language
[eng]
Persistent identifier
https://phaidra.vetmeduni.ac.at/o:4833
DOI
10.1016/j.mcpro.2025.100981
Is in series
Title (eng)
Molecular & Cellular Proteomics
Volume
24
Issue
6
ISSN
1535-9484
Issued
2025
Number of pages
19
Publication
American Society for Biochemistry and Molecular Biology
Version type (eng)
version of record (published version)
Date issued
2025
Access rights (eng)
open access
License
CC BY 4.0 International
Rights statement (eng)
© 2025 The Author(s)