N-linked oligosaccharides are necessary and sufficient for association of glycosylated forms of bovine RNase with calnexin and calreticulin.
Calnexin and calreticulin are lectin-like molecular chaperones that promote folding and assembly of newly synthesized glycoproteins in the endoplasmic reticulum. While it is well established that they interact with substrate monoglucosylated N-linked oligosaccharides, it has been proposed that they also interact with polypeptide moieties. To test this notion, glycosylated forms of bovine pancreatic ribonuclease (RNase) were translated in the presence of microsomes and their folding and association with calnexin and calreticulin were monitored. When expressed with two N-linked glycans in the presence of micromolar concentrations of deoxynojirimycin, this small soluble protein was found to bind firmly to both calnexin and calreticulin. The oligosaccharides were necessary for association, but it made no difference whether the RNase was folded or not. This indicated that unlike other chaperones, calnexin and calreticulin do not select their substrates on the basis of folding status. Moreover, enzymatic removal of the oligosaccharide chains using peptide N-glycosidase F or removal of the glucoses by ER glucosidase II resulted in dissociation of the complexes. This indicated that the lectin-like interaction, and not a protein-protein interaction, played the central role in stabilizing RNase-calnexin/calreticulin complexes.
Rodan
A R
AR
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520-8002, USA.
Simons
J F
JF
Trombetta
E S
ES
Helenius
A
A
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D037282:Calreticulin; D002417:Cattle; D006031:Glycosylation; D008861:Microsomes / Q000201:enzymology; D009844:Oligosaccharides / Q000737:chemistry*; D010179:Pancreas / Q000201:enzymology; D012260:Ribonucleases / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D013379:Substrate Specificity
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D009844:Oligosaccharides; D012261:Ribonucleoproteins; D037281:Calnexin; D012260:Ribonucleases