THE MOLECULAR CHAPERONE CALNEXIN BINDS GLC(1)MAN(9)GLCNAC(2) OLIGOSACCHARIDE AS AN INITIAL STEP IN RECOGNIZING UNFOLDED GLYCOPROTEINS

Calnexin is a molecular chaperone that resides in the membrane of the endoplasmic reticulum, Most proteins that calnexin binds are N-glycosy lated, and treatment of cells with tunicamycin or inhibitors of initia l glucose trimming steps interferes with calnexin binding. To test if calnexin is a lectin that binds early oligosaccharide processing inter mediates, a recombinant soluble calnexin was created, Incubation of so luble calnexin with a mixture of Glc(0-3)Man(9)GlcNAc(2) oligosacchari des resulted in specific binding of the Glc(1)Man(9)GlcNAc(2) species, Furthermore, Glc(1)Man(5-7)GlcNAc(2) oligosaccharides bound relativel y poorly, suggesting that, in addition to a requirement for the single terminal glucose residue, at least one of the terminal mannose residu es was important for binding, To assess the involvement of oligosaccha ride-protein interactions in complexes of calnexin and newly synthesiz ed glycoproteins, alpha(1)-antitrypsin or the heavy chain of the class I histocompatibility molecule were purified as complexes with calnexi n and digested with endoglycosidase H. All oligosaccharides on either glycoprotein were accessible to this probe and could be removed withou t disrupting the association with calnexin, Furthermore, the addition of 1 M alpha-methyl glucoside or alpha-methyl mannoside had no effect on complex stability, These findings suggest that once complexes betwe en calnexin and glycoproteins are formed, oligosaccharide binding does not contribute significantly to the overall interaction, However, it is likely that the binding of Glc(1)Man(9)GlcNAc(2) oligosaccharides i s a crucial event during the initial recognition of newly synthesized glycoproteins by calnexin.