Departments of Pediatrics, School of Molecular and Systems Medicine, University of Alberta, Edmonton, AB, T6G 2H7, Canada.
Mol Cell Biochem. 2012 Jan;359(1-2):271-81. doi: 10.1007/s11010-011-1021-0. Epub 2011 Aug 13.
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.
钙连蛋白是内质网中的一种蛋白质，在新合成糖蛋白的折叠中发挥作用。在这项研究中，我们使用定点突变技术破坏钙连蛋白 N 域和 P 域中天冬氨酸和组氨酸残基，以确定这些突变是否影响钙连蛋白的结构和功能。我们发现，破坏 N 域半胱氨酸会导致钙连蛋白对糖基化底物 IgY 的伴侣活性显著丧失，而破坏 P 域半胱氨酸对 IgY 的影响较小。我们观察到，野生型钙连蛋白以及 P 域双半胱氨酸突变体均含有一个分子内二硫键，当 N 域半胱氨酸发生突变时，该键会丢失。突变 N 域组氨酸和 N 域半胱氨酸会导致 ERp57 结合增加。突变 P 域半胱氨酸进一步增强了 ERp57 与钙连蛋白的结合。总之，这些观察结果表明钙连蛋白中的半胱氨酸残基对钙连蛋白的结构和功能很重要。
