Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control.

TLDR

The study proposes that ER calnexin functions as a chaperone that binds proteins with partially glucose‑trimmed N‑linked oligosaccharides, forming a unique folding and quality‑control machinery. The authors used pulse‑chase immunoprecipitation to show that nascent HA and VSV G transiently bind calnexin during folding, and they propose that ER glucosidases I/II and UGGT act as signal modifiers and folding sensors in a calnexin‑mediated quality‑control pathway. Inhibiting N‑linked glycosylation or glucosidases I/II blocked calnexin binding, while alpha‑mannosidase inhibition had no effect; binding correlated with oligosaccharide composition, with monoglucosylated species showing the strongest interaction.

Abstract

Using a pulse-chase approach combined with immunoprecipitation, we showed that newly synthesized influenza virus hemagglutinin (HA) and vesicular stomatitis virus G protein associate transiently during their folding with calnexin, a membrane-bound endoplasmic reticulum (ER) chaperone. Inhibitors of N-linked glycosylation (tunicamycin) and glucosidases I and II (castanospermine and 1-deoxynojirimycin) prevented the association, whereas inhibitors of ER alpha-mannosidases did not. Our results indicated that binding of these viral glycoproteins to calnexin correlated closely with the composition of their N-linked oligosaccharide side chains. Proteins with monoglucosylated oligosaccharides were the most likely binding species. On the basis of our data and existing information concerning the role of monoglucosylated oligosaccharides on glycoproteins, we propose that the ER contains a unique folding and quality control machinery in which calnexin acts as a chaperone that binds proteins with partially glucose-trimmed carbohydrate side chains. In this model glucosidases I and II serve as signal modifiers and UDP-glucose:glycoprotein glucosyltransferase, as a folding sensor.