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

IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Shi Yan, Jorick Vanbeselaere, Callum Ives, David Stenitzer, Lena Nuschy, Florian Wöls, Katharina Paschinger, Elisa Fadda, Johannes Stadlmann, Iain B H Wilson
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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 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.

糖蛋白组学和单蛋白糖糖分析揭示了天然蛋白和重组蛋白上的两性离子n -聚糖。
昆虫细胞是生产重组糖蛋白的便捷的细胞工厂。它们的糖基化潜力被认为是简单的,主要需要添加糖基转移酶来使重组产物人源化。在这项研究中,使用LC-MS/MS方法检测了Spodoptera frugiperda Sf9和Trichoplusia ni High Five细胞的天然糖蛋白组,不仅揭示了哪些蛋白质是n -糖基化的,而且表明n -糖基化除了典型的寡糖和聚焦结构外,还含有新的葡萄糖醛化和磷酰胆碱修饰的聚糖。这些数据被平行的MALDI-TOF MS/MS分析证实。一种内源性Sf9糖蛋白的分子模型分析将n -糖基化位点的可及性与n -糖基化位点的复合体和寡糖苷n -聚糖的发生联系起来。此外,我们发现在夜蛾细胞中产生的流感血凝素和SARS-CoV-2刺突糖蛋白的n -聚糖具有磷酸化胆碱修饰的许多聚糖结构,但核心二聚焦化很小;相比之下,毛虫产生的血凝素只有前一种类型的痕迹,而后者占主导地位。检测这些糖蛋白上的磷酸胆碱与人c反应蛋白结合相关。综上所述,在昆虫细胞系中提取的天然糖蛋白和重组糖蛋白中,不仅存在寡甘露糖苷或截断的少甘露糖苷n -聚糖,而且存在具有免疫原性的结构。
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来源期刊
Molecular & Cellular Proteomics
Molecular & Cellular Proteomics 生物-生化研究方法
CiteScore
11.50
自引率
4.30%
发文量
131
审稿时长
84 days
期刊介绍: The mission of MCP is to foster the development and applications of proteomics in both basic and translational research. MCP will publish manuscripts that report significant new biological or clinical discoveries underpinned by proteomic observations across all kingdoms of life. Manuscripts must define the biological roles played by the proteins investigated or their mechanisms of action. The journal also emphasizes articles that describe innovative new computational methods and technological advancements that will enable future discoveries. Manuscripts describing such approaches do not have to include a solution to a biological problem, but must demonstrate that the technology works as described, is reproducible and is appropriate to uncover yet unknown protein/proteome function or properties using relevant model systems or publicly available data. Scope: -Fundamental studies in biology, including integrative "omics" studies, that provide mechanistic insights -Novel experimental and computational technologies -Proteogenomic data integration and analysis that enable greater understanding of physiology and disease processes -Pathway and network analyses of signaling that focus on the roles of post-translational modifications -Studies of proteome dynamics and quality controls, and their roles in disease -Studies of evolutionary processes effecting proteome dynamics, quality and regulation -Chemical proteomics, including mechanisms of drug action -Proteomics of the immune system and antigen presentation/recognition -Microbiome proteomics, host-microbe and host-pathogen interactions, and their roles in health and disease -Clinical and translational studies of human diseases -Metabolomics to understand functional connections between genes, proteins and phenotypes
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