Mutational analysis of calnexin.
Calnexin is a type I endoplasmic reticulum lectin-like chaperone protein. In this study, we have used site-specific mutagenesis to investigate the functional importance of glutamate E351 found at the tip of the P-domain of calnexin, and tryptophan W428 found in the carbohydrate binding region of the globular domain of the protein. The E351 and W428 calnexin mutants lost the ability to inhibit aggregation of IgY (glycosylated substrate). The E351 mutation led to slightly enhanced ERp57 binding to calnexin, whereas W428 greatly enhanced binding of ERp57 to calnexin. These findings indicate that modification of a residue(s) in the carbohydrate binding region may have a profound effect on the structural and functional properties of the P-domain and consequently on association of calnexin with the folding enzyme ERp57.
Groenendyk
Jody
J
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
Dabrowska
Monika
M
Michalak
Marek
M
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry / Q000494:pharmacology; D037281:Calnexin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D007136:Immunoglobulins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013696:Temperature; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000737:chemistry / Q000494:pharmacology
C016512:IgY; D007136:Immunoglobulins; D050505:Mutant Proteins; D037281:Calnexin; D018698:Glutamic Acid; D014364:Tryptophan; D000255:Adenosine Triphosphate; C513195:Pdia3 protein, mouse; D019704:Protein Disulfide-Isomerases; D015032:Zinc; D002118:Calcium