Role of cysteine amino acid residues in calnexin
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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.
KeywordsCalnexin Protein folding Endoplasmic reticulum Glycoprotein Cysteine Histidine Mutation ERp57 Structure Function
This work was supported by a grant to M.M. from the Canadian Institutes of Health Research (MOP-53050). H.C. and J.J. are supported by a studentship from the Alberta Innovates-Health Solutions.
Conflict of interest
These authors have no conflicting interest.
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