Aggregation of Trp > Glu point mutants of human gamma‐D crystallin provides a model for hereditary or UV‐induced cataract

Abstract

Numerous mutations and covalent modifications of the highly abundant, long-lived crystallins of the eye lens cause their aggregation leading to progressive opacification of the lens, cataract. The nature and biochemical mechanisms of the aggregation process are poorly understood, as neither amyloid nor native-state polymers are commonly found in opaque lenses. The bccrystallin fold contains four highly conserved buried tryptophans, which can be oxidized to more hydrophilic products, such as kynurenine, upon UV-B irradiation. We mimicked this class of oxidative damage using TrpfiGlu point mutants of human cD-crystallin. Such substitutions may represent a model of UV-induced photodamage-introduction of a charged group into the hydrophobic core generating "denaturation from within." The effects of TrpfiGlu substitutions were highly position dependent. While each was destabilizing, only the two located in the bottom of the double Greek key fold-W42E and W130E-yielded robust aggregation of partially unfolded intermediates at 378C and pH 7. The aB-crystallin chaperone suppressed aggregation of W130E, but not W42E, indicating distinct aggregation pathways from damage in the N-terminal vs C-terminal domain. The W130E aggregates had loosely fibrillar morphology, yet were nonamyloid, noncovalent, showed little surface hydrophobicity, and formed at least 208C below the melting temperature of the native bsheets. These features are most consistent with domain-swapped polymerization. Aggregation of partially destabilized crystallins under physiological conditions, as occurs in this class of point mutants, could provide a simple in vitro model system for drug discovery and optimization.

An Internal Disulfide Locks a Misfolded Aggregation-Prone Intermediate in Cataract-Linked Mutants of Human Gamma-D Crystallin

“…Even in the WT protein, the N-terminal domain was less stable than the C-terminal domain, also consistent with past experimental results (18,55,56) and simulations (57). Also consistent with experiments, the W130E mutation largely eliminated the differential domain stability, thus leading to a more cooperative unfolding transition (50). Partially unfolded structures from W42R simulations at intermediate temperature show that separation of the two N-terminal Greek keys and detachment of the N-terminal hairpin are early events in the unfolding process ( Figure 3C).…”

“…Oxidative aggregation was found to be highly temperature-dependent, consistent with a requirement of conformational change (39,50). Thus, W42Q showed no aggregation at 32 °C even under the most oxidizing conditions employed; oxidationdependent aggregation at 37 °C; and strong aggregation even at low OxD values at 42 °C ( Figure 7B,C,D).…”

“…Trp 42, located in the bottom of the N-terminal domain core ( Figure 1A), is particularly sensitive to substitution, as both oxidation-mimicking W42E and W42Q substitutions in vitro and the congenital cataract-linked W42R substitution destabilize the protein and lead to aggregation (39,50,51). The congenital and oxidation-mimicking substitutions differ in their charge.…”

“…We have previously reported that the aggregates formed by the temperature-sensitive Trp mutants of γD-crystallin under near-physiological conditions are neither disulfide-bridged nor amyloid and expose few hydrophobic residues (39,50). These observations raised the question of whether a seeding effect exists in this aggregation pathway.…”

Cataract-associated P23T γD-crystallin retains a native-like fold in amorphous-looking aggregates formed at physiological pH

“…Deposits assembled from intertwined or 3D domain-swapped proteins, as previously proposed for cataract-associated aggregates8910, would also be predicted to have native-like spectra37. On the basis of known domain-swapped multimers as large as tetramers, it has been proposed that ‘runaway' domain swapping in a daisy-chain manner could be a mechanism of protein aggregation.…”