Mutations Causing Early Cataract Development In Mice Destabilize Human gammaD-crystallin

Abstract

The human eye lens is composed of layers of elongated fiber cells packed with crystallin proteins at concentrations up to 400 mg/ml. Human γD-crystallin (HγD-Crys), one of the three major γ-crystallins, is a monomeric, two-domain protein found in the lens nucleus, the central region of the lens formed earliest during development. Genetic screens for mutations resulting in cataract in mice identified three mutations affecting mouse γ-crystallins. These amino acid substitutions were introduced into HγD-Crys by site-specific mutation of the cloned gene. The three mutant proteins L5S, V75D, and I90F were expressed and purified from E. coli. Equilibrium unfolding/refolding experiments were performed to measure the thermodynamic stability of the mutant proteins compared to wild type. Wild-type HγD-Crys was previously shown to exhibit a three-state unfolding/refolding pathway. This pathway is sequential with the N-terminal domain unfolding first, followed by the C-terminal domain. L5S and V75D also displayed three-state unfolding/refolding transitions with populated intermediates. In both cases, the first transition midpoint was shifted to lower denaturant concentrations, 0.7 M GdnHCl for L5S and 0.8 M for V75D compared to 2.2 M for the wild type. I90F exhibited a two-state unfolding/refolding transition with a single midpoint at 1.7 M. The mutant proteins all exhibited decreased thermal stability compared with wild type. Kinetic unfolding experiments confirmed that wild type unfolded through a three-state mechanism. The N-terminal domains of L5S and V75D unfolded extremely fast (t1/2@2 s) at lower denaturant concentrations than those required for wild type. I90F was globally destabilized and unfolded through a two-state mechanism faster than wild type. These results support models of cataract formation in which generation of partially unfolded intermediates - whether due to mutation or to covalent damage - are precursors to the aggregated cataractous states responsible for light scattering.