Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control.
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.
Hammond
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C
Department of Cell Biology, Yale School of Medicine, New Haven, CT 06510.
Braakman
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I
Helenius
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A
D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000737:chemistry / Q000378:metabolism; D008562:Membrane Glycoproteins; D008954:Models, Biological; D009844:Oligosaccharides / Q000378:metabolism*; D009975:Orthomyxoviridae; D011485:Protein Binding; D017510:Protein Folding; D014721:Vesicular stomatitis Indiana virus; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000378:metabolism
D002135:Calcium-Binding Proteins; C028391:G protein, vesicular stomatitis virus; D006023:Glycoproteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D014759:Viral Envelope Proteins; D037281:Calnexin; D005947:Glucose