Oxidative damage may be responsible for cataract formation in the S10R mutant of mouse gammaB-crystallin

Abstract

Purpose : The Ser10 to Arg (S10R) mutation in mouse gammaB-crystallin (MGB) was associated with nuclear cataract¹, which could apparently be reversed by over-expression of α3 connexin (Cx46)². However, the molecular mechanism of cataract formation and the role of Cx46 in ameliorating the cataract remain elusive. Here we explore plausible mechanisms for both these observations.

Methods : Recombinant wild-type (WT)-MGB and the S10R mutant were expressed in E.Coli. Electrospray-ionization mass spectrometry and isoelectric focusing were used to characterize the two proteins. Spectroscopic measurements (Circular Dichroism and Tryptophan Fluorescence emission) were carried out to determine the secondary and tertiary structures of the two proteins. Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid)) was used to quantify the number of thiol groups in each protein. SDS-PAGE and light scattering measurements were carried out to determine self-aggregation rates or protein aggregation under different oxidative conditions with H₂O₂, Cu (II), dehydroascorbic acid, and glutathione.

Results : The two proteins are nearly identical in their secondary and tertiary structures. However, the self-aggregation rates of the two proteins at 37 °C are distinct. Under oxidizing conditions, the mutant aggregates more rapidly relative to the WT, and also forms intermolecular disulfide-crosslinked dimers more readily. Such crosslinks may be the precursors to further aggregation. One or more Cys residues in the S10R mutant appear to become more reactive. We are in the process of identifying the reactive Cys residue(s) using trypsin digestion and LC-MS.

Conclusions : The data show that the S10R mutation in MGB leads to enhanced oxidative damage. The mutation indirectly makes the Cys residue(s) of the protein susceptible to oxidation. Cx46, a transporter of extracellular glutathione, may be effective because it nullifies the effects of oxidative damage.


1. Li, L., Chang, B., Cheng, C., Chang, D., Hawes, N. L., Xia, C. H., and Gong, X. (2008) Dense nuclear cataract caused by the gammaB-crystallin S11R point mutation, Investigative ophthalmology & visual science 49(1), 304-309.
2. Li, L., Cheng, C., Xia, C. H., White, T. W., Fletcher, D. A., and Gong, X. (2010) Connexin mediated cataract prevention in mice, PLoS ONE 5(9), e12624.

This is a 2020 ARVO Annual Meeting abstract.