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Cotranslational folding and calnexin binding during glycoprotein synthesis
W Chen1, J Helenius, I Braakman
1Department of Cell Biology, Yale School of Medicine, New Haven, CT 06510-8002, USA.
Summary
Influenza hemagglutinin undergoes cotranslational folding in the endoplasmic reticulum, involving disulfide bond formation and N-linked glycosylation. The chaperone calnexin plays a role in this process, influenced by oligosaccharide modifications.
Area of Science:
- Molecular Biology
- Protein Folding
- Virology
Background:
- Influenza hemagglutinin (HA) is a crucial viral glycoprotein involved in host cell entry.
- Understanding the cotranslational folding of HA is essential for comprehending viral assembly and infectivity.
- The endoplasmic reticulum (ER) is the primary site for glycoprotein folding and maturation.
Purpose of the Study:
- To investigate the cotranslational folding pathway of influenza hemagglutinin within live cells.
- To elucidate the roles of disulfide bond formation, N-linked glycosylation, and chaperone interactions during HA synthesis.
Main Methods:
- Utilized short pulses of radiolabeling followed by immunoprecipitation.
- Employed a two-dimensional SDS-PAGE system (non-reducing then reducing) to separate nascent polypeptides.
- Analyzed polypeptide length, oxidation state, and interactions with calnexin.
Main Results:
- Provided evidence for cotranslational disulfide bond formation and the generation of conformational epitopes.
- Demonstrated N-linked glycosylation occurring during translation.
- Showed oligosaccharide-dependent binding of calnexin to incompletely folded HA via trimmed oligosaccharides.
Conclusions:
- N-linked oligosaccharides and calnexin are integral to the cotranslational folding of hemagglutinin.
- Inhibition of glycosylation or oligosaccharide trimming perturbs the HA folding pathway.
- These findings highlight the interplay between nascent polypeptide modifications and chaperone-assisted folding.