The molecular chaperone calnexin binds Glc1Man9GlcNAc2 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-glycosylated, and treatment of cells with tunicamycin or inhibitors of initial glucose trimming steps interferes with calnexin binding. To test if calnexin is a lectin that binds early oligosaccharide processing intermediates, a recombinant soluble calnexin was created. Incubation of soluble calnexin with a mixture of Glc0-3Man9GlcNAc2 oligosaccharides resulted in specific binding of the Glc1Man9GlcNAc2 species. Furthermore, Glc1Man5-7GlcNAc2 oligosaccharides bound relatively poorly, suggesting that, in addition to a requirement for the single terminal glucose residue, at least one of the terminal mannose residues was important for binding. To assess the involvement of oligosaccharide-protein interactions in complexes of calnexin and newly synthesized glycoproteins, alpha 1-antitrypsin or the heavy chain of the class I histocompatibility molecule were purified as complexes with calnexin and digested with endoglycosidase H. All oligosaccharides on either glycoprotein were accessible to this probe and could be removed without 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 between calnexin and glycoproteins are formed, oligosaccharide binding does not contribute significantly to the overall interaction. However, it is likely that the binding of Glc1Man9GlcNAc2 oligosaccharides is a crucial event during the initial recognition of newly synthesized glycoproteins by calnexin.
Ware
F E
FE
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041.
Vassilakos
A
A
Peterson
P A
PA
Jackson
M R
MR
Lehrman
M A
MA
Williams
D B
DB
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D002240:Carbohydrate Sequence; D004285:Dogs; D004330:Drosophila; D006023:Glycoproteins / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D014407:Tumor Cells, Cultured
D002135:Calcium-Binding Proteins; C092383:Glc(1)Man(9)GlcNAc(2) oligosaccharide; D006023:Glycoproteins; D006027:Glycosides; D008024:Ligands; D009844:Oligosaccharides; D037281:Calnexin