Mass spectrometry and molecular modeling evidence for an unusual disulfide crosslink between cysteines 24 and 36 in the gammaS-crystallin linked to cataracts.

Abstract

During normal aging and cataract formation the predominant proteins in the lens of the eye, crystallins, undergo extensive deamidation and disulfide formation. One of the major crystallins, gammaS-crystallin, has multiple potential deamidation sites and 7 cysteines with all but 3 buried. GammaS-crystallin is found in the isolated insoluble proteins from aged lenses. We have previously identified the major deamidation sites occurring at Asn14, 76, and 143. In this present series of experiments, we subjected recombinantly expressed gammaS-crystallin with deamidations mimicked at the in vivo sites to oxidation by incubating anaerobically for 5 days with excess oxidized glutathione. Samples were then analyzed by mass spectrometry to measure either whole masses or tryptic peptides. The major mass in the deamidated gammaS-crystallin suggested deamidation increased disulfide bond formation and that the major disulfide was between Cys24 and Cys36, with lesser amount between Cys24 and Cys82. Given the buried nature of Cys36, molecular modeling was performed. The structural rearrangement to form 24-36 was surprisingly minimal. The second strand in the structure must relax for the bond to form, providing much more flexibility to C22 and to C26. We conclude that while the unique arrangement of Cys 22, 24, and 26 suggests a possible role in maintaining the redox potential of the protein, deamidation disrupts this potential facilitating an unusual bond between Cys24 and 36 associated with insolubilization.