Molecular Biochemical Study of the Cataract-Associated Mutants in Human gammaD-Crystallin.

摘要

Cataract is a vision impairment disease that leads to light scattering and opacity of the eye lens. It is recognized as the most prevalent cause of blindness. Most cataracts occur due to aging, but those that occur in childhood or in young adults are typically of genetic origin. In the young (developing) eye, post-surgical complications such as glaucoma are common after cataract surgery. Hence there is a great need to develop non-surgical intervention strategies for the treatment of cataract. Here, two cataract-associated mutants of human γD-crystallin (HGD): i) Arg76 to Ser (R76S) and ii) a truncation mutation at Trp156 (W156X or W156stop), were compared with the wild-type protein.;For R76S, preliminary data suggest an aggregation pattern that is distinct from that of HGD. Oligomers of R76S are smaller in mass (∼135 kDa compared to ∼175 kDa for HGD oligomers), but are much more resistant to dissociation.;For the W156Xmutation, the mutant protein when expressed in E. coli, is an inclusion body (IB) protein, and here I re-investigated the purification methods for this mutant. The molecular basis of light scattering and opacity due to this mutation is not well understood; hence innovative procedures for exploratory future work are suggested. Preliminary data presented here suggest that under certain solution conditions (e.g. higher concentrations of L-Arginine at pH 10), or by the use of a chemical chaperone (4-PBA), the protein can be partitioned into the soluble and insoluble phases and shows a distribution of monomer and oligomer forms in both.