Cataract, the leading cause of blindness worldwide, results from age-related misfolding and aggregation of long-lived crystallin proteins in the eye lens. The cytoplasm of fiber cells in the lens core becomes increasingly oxidizing with age, allowing non-native disulfides to drive light-scattering aggregation of γ-crystallins. Despite this vulnerability to non-native disulfides, and despite lacking any native-state disulfides, γ-crystallins are unexpectedly Cys-rich. To understand this paradox, we investigated how replacing all four Cys residues in the aggregation-prone N-terminal domain of γD-crystallin affects its stability and aggregation. Cys removal precludes the disulfide-driven aggregation pathway we reported previously. Here, we characterize two full-length human γD-crystallin variants: C18S/C32S/C41S/C78S ("NCS") and C18T/C32A/C41A/C78A ("NCA/T"). Thermodynamic and kinetic stability measurements indicate the N-terminal domain was greatly destabilized in both variants relative to WT, with NCS more destabilized than NCA/T. Upon mild heating or partial denaturation, both variants formed light-scattering aggregates, which were amorphous by transmission electron microscopy. Surprisingly, the aggregation proceeded exclusively from a dimer of natively folded molecules held together by a C-terminal disulfide bridge. These dimers form readily even in the WT protein, and evidence of them has been found in the lens. Aggregation was strongly suppressed by the lens's native chemical chaperone, myo-inositol. The aggregation rate depended linearly on protein concentration, indicating that the rate limiting step was a transformation of the natively-folded to misfolded molecules within the dimer. We propose that many age-related chemical modifications could destabilize the native fold of human γD-crystallin, favor misfolding within disulfide-bridged dimers, and thereby cause aggregation.
Pu et al. (Mon,) studied this question.