Abstract
Abstract: :
Purpose: To understand protein–derived molecular mechanisms of cataract formation due to a genetic mutation, and age–related truncation of human γD and ßB1–crystallins respectively. Methods: We expressed recombinant proteins (native γD–crystallin, its P23T mutant, native ßB1–crystallin, its N–terminal truncated form (ßB1ΔN41)) in E. coli, and compared in vitro, the solution properties of pure native, mutant and truncated proteins. Results: The cataract–causing mutation P23T shows no significant structural change relative to native γD–crystallin. However, the solubility of P23T is dramatically lowered, in marked contrast to the native. Reduced solubility of P23T results from the condensation of the mutant protein into clusters (observed by electron and light microscopies). The cluster size distribution observed by us in vitro is comparable to that of the "dense globular deposits" seen in the cataractous lens in vivo by others. The monomer–cluster equilibrium is represented by a solubility curve in the phase diagram. The solubility of P23T shows an inverse dependence on temperature, suggesting that the insoluble phase is formed by hydrophobic interactions. The solubility of P23T can be significantly altered by introducing specific mutations at or next to residue 23. For example, among the mutants we expressed and examined (P23S, P23V, P23TinsP24 and P23TN24K), the latter two mutations restore the solubility of P23T. We also compared native ßB1–crystallin with ßB1ΔN41, which models the truncation observed with aging in the lens in vivo. Solutions of ßB1ΔN41 undergo two distinct types of phase transitions. In the first, rod–like assemblies are formed which evolve into crystals. In the second, liquid–liquid phase separation is observed analogous to that of the γ crystallins, but the dense liquid phase undergoes gelation. These phase transitions are absent in the native protein. Conclusions: The condensed phases reported here are the most likely cause of the light scattering and opacity observed in these cataracts. These phases are the latest among an increasing variety of protein–derived condensates (e.g. liquid droplets, disulfide cross–linked aggregates, crystals, rod–like assemblies, gels), that we have identified in recent years, which arise from mutations and modifications in the γ and ß crystallins associated with human cataracts. Our work not only provides plausible mechanisms for cataract formation, but also suggests strategies for the inhibition of these light scattering elements.
Keywords: crystallins • protein structure/function • cataract