title: Role of cysteine amino acid residues in calnexin.
abstract: Calnexin is an endoplasmic reticulum protein that has a role in folding newly synthesized glycoproteins. In this study, we used site-specific mutagenesis to disrupt cysteine and histidine amino acid residues in the N- and P-domains of calnexin and determined whether these mutations impact the structure and function of calnexin. We identified that disruption of the N-domain cysteines resulted in significant loss of the chaperone activity of calnexin toward the glycosylated substrate, IgY, while disruption of the P-domain cysteines only had a small impact toward IgY. We observed that wild-type calnexin as well as the P-domain double cysteine mutant contained an intramolecular disulfide bond which is lost when the N-domain cysteines are mutated. Mutation to the N-domain histidine and N-domain cysteines resulted in increased binding of ERp57. Mutations to the P-domain cysteines further enhanced ERp57 binding to calnexin. Taken together, these observations indicated that the cysteine residues within calnexin were important for the structure and function of calnexin.
authors.affiliation: Departments of Pediatrics, School of Molecular and Systems Medicine, University of Alberta, Edmonton, AB, T6G 2H7, Canada.
authors.forename: Helen
authors.initials: H
authors.lastname: Coe
authors.forename: Jeannine D
authors.initials: JD
authors.lastname: Schneider
authors.forename: Monika
authors.initials: M
authors.lastname: Dabrowska
authors.forename: Jody
authors.initials: J
authors.lastname: Groenendyk
authors.forename: Joanna
authors.initials: J
authors.lastname: Jung
authors.forename: Marek
authors.initials: M
authors.lastname: Michalak
mesh_terms: D037281:Calnexin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000502:physiology*; D004220:Disulfides; D006639:Histidine; D006801:Humans; D007136:Immunoglobulins; D018832:Molecular Chaperones; D016297:Mutagenesis, Site-Directed; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D021381:Protein Transport
chemical_list: D004220:Disulfides; C016512:IgY; D007136:Immunoglobulins; D018832:Molecular Chaperones; D037281:Calnexin; D006639:Histidine; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human; D003545:Cysteine