The Cataract-associated R14C Mutant of Human γD-Crystallin Shows a Variety of Intermolecular Disulfide Crosslinks: A Raman Spectroscopic Study

摘要

The Arg14 to Cys (R14C) mutation in the human γD-crystallin (HGD) gene has been associated with a juvenile-onset hereditary cataract. We showed earlier () that rapid oxidation of Cys14 in the mutant leads to the formation of intermolecular, disulfide-crosslinked aggregates at physiological pH. Here we present Raman spectroscopic analysis of R14C and HGD and show that R14C forms such aggregates even at pH 4.5. The lower pH enabled us to monitor the evolution of a variety of disulfide crosslinks with distinct conformations around the CC-SS-CC dihedral angles. At least three cysteine residues are involved, forming protein-protein crosslinks through disulfide-exchange reactions. From the pattern of the S-S and Trp Raman bands, we infer that Cys32 is likely to be involved in the crosslinking. The data suggest that protein precipitation in the mutant may not be the direct result of disulfide crosslinking, although such crosslinking is the initiating event. Thus, our Raman data not only enhance the understanding of the reactivity of Cys14 in the R14C mutant and the mechanism of opacity, but also shed light on the mechanism of oxidative degradation during long-term storage of thiol-containing pharmaceuticals.