Glycoproteomic and single-protein glycomic analyses reveal zwitterionic N-glycans on natural and recombinant proteins derived from insect cells
Glycoproteomic and single-protein glycomic analyses reveal zwitterionic N-glycans on natural and recombinant proteins derived from insect cells
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 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 modelling 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 haemagglutinins 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 haemagglutinin had only traces of the former type, while the latter were 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.
Animals, Glycomics/methods, Glycoproteins/metabolism, Glycosylation, Humans, Polysaccharides/chemistry, Proteomics/methods, Recombinant Proteins/metabolism, Sf9 Cells, Spodoptera/metabolism, Tandem Mass Spectrometry
100981
Yan, Shi
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Vanbeselaere, Jorick
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Ives, Callum
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Stenitzer, David
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Nuschy, Lena
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Wöls, Florian
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Paschinger, Katharina
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Fadda, Elisa
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Stadlmann, Johannes
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Wilson, Iain B H
ca78eff4-c7f2-4c58-a49d-29071105d2a7
1 June 2025
Yan, Shi
b03df485-f1e7-4af0-abd5-dfa7b6e25dd2
Vanbeselaere, Jorick
31167b3e-00d3-4763-a3e8-f3467759f3a6
Ives, Callum
b8c798a7-ddf0-40ac-8194-c757032b85e2
Stenitzer, David
d38461a3-0242-42e6-83d3-73d3875765b9
Nuschy, Lena
e3274fd5-33be-4d08-a3b4-59719ee60b5c
Wöls, Florian
23034b3c-af7d-4d05-8be7-8c91a43e895d
Paschinger, Katharina
d1c54dd6-11f3-4f10-bd74-ddfa12f5c420
Fadda, Elisa
11ba1755-9585-44aa-a38e-a8bcfd766abb
Stadlmann, Johannes
e35debd6-a9d2-4104-976b-1058dbcbacd5
Wilson, Iain B H
ca78eff4-c7f2-4c58-a49d-29071105d2a7
Yan, Shi, Vanbeselaere, Jorick, Ives, Callum, Stenitzer, David, Nuschy, Lena, Wöls, Florian, Paschinger, Katharina, Fadda, Elisa, Stadlmann, Johannes and Wilson, Iain B H
(2025)
Glycoproteomic and single-protein glycomic analyses reveal zwitterionic N-glycans on natural and recombinant proteins derived from insect cells.
Molecular & Cellular Proteomics, 24 (6), , [100981].
(doi:10.1016/j.mcpro.2025.100981).
Abstract
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 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 modelling 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 haemagglutinins 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 haemagglutinin had only traces of the former type, while the latter were 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.
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PIIS1535947625000799
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e-pub ahead of print date: 5 May 2025
Published date: 1 June 2025
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Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.
Keywords:
Animals, Glycomics/methods, Glycoproteins/metabolism, Glycosylation, Humans, Polysaccharides/chemistry, Proteomics/methods, Recombinant Proteins/metabolism, Sf9 Cells, Spodoptera/metabolism, Tandem Mass Spectrometry
Identifiers
Local EPrints ID: 503156
URI: http://eprints.soton.ac.uk/id/eprint/503156
ISSN: 1535-9476
PURE UUID: c6f56320-3452-4a21-9513-f28663110ef2
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Date deposited: 22 Jul 2025 17:03
Last modified: 13 Sep 2025 02:33
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Author:
Shi Yan
Author:
Jorick Vanbeselaere
Author:
Callum Ives
Author:
David Stenitzer
Author:
Lena Nuschy
Author:
Florian Wöls
Author:
Katharina Paschinger
Author:
Elisa Fadda
Author:
Johannes Stadlmann
Author:
Iain B H Wilson
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