Cataract-associated mutant E107A of human γD-crystallin shows increased attraction to α-crystallin and enhanced light scattering

Abstract

Several point mutations in human γD-crystallin (HGD) are now known to be associated with cataract. So far, the in vitro studies of individual mutants of HGD alone have been sufficient in providing plausible molecular mechanisms for the associated cataract in vivo. Nearly all the mutant proteins in solution showed compromised solubility and enhanced light scattering due to altered homologous γ-γ crystallin interactions. In sharp contrast, here we present an intriguing case of a human nuclear cataract-associated mutant of HGD-namely Glu107 to Ala (E107A), which is nearly identical to the wild type in structure, stability, and solubility properties, with one exception: Its pI is higher by nearly one pH unit. This increase dramatically alters its interaction with α-crystallin. There is a striking difference in the liquid-liquid phase separation behavior of E107A-α-crystallin mixtures compared to HGD-α-crystallin mixtures, and the light-scattering intensities are significantly higher for the former. The data show that the two coexisting phases in the E107A-α mixtures differ much more in protein density than those that occur in HGD-α mixtures, as the proportion of α-crystallin approaches that in the lens nucleus. Thus in HGD-α mixtures, the demixing of phases occurs primarily by protein type while in E107A-α mixtures it is increasingly governed by protein density. Analysis of these results suggests that the cataract due to the E107A mutation could result from the instability caused by the altered attractive interactions between dissimilar proteins -i.e., heterologous γ-α crystallin interactions-primarily due to the change in surface electrostatic potential in the mutant protein.T he vertebrate lens contains three families of constituent proteins, the α-, β-, and γ-crystallins, closely packed such that the total protein concentration in the central region of the lens, i.e., nucleus, exceeds 400 mg∕mL (1). Normally, the close protein packing ensures that short-range order is maintained within the fiber cell, such that fluctuations in the refractive index that cause light scattering are minimized, and the lens is transparent (2, 3). Disruption of short-range order associated with aging, altered environment, or mutations in crystallins can cause increased light scattering and cataract.Cataract-associated mutations in the crystallin genes are known to occur in all three crystallin families (1, 4) and show a variety of phenotypes. Over the last decade, we have focused on the point mutations occurring in human γD-crystallin (HGD), a member of the γ-crystallin family expressed at high levels along with γC-and γS-crystallins in the human lens (5). By comparing the properties of several mutants of HGD in solution with those of the wild type, we have identified specific changes in the homologous (i.e., γ-γ interactions) that led to the formation of distinct condensed phases (6, 7) and accounted for the increased light scattering due to these mutations (8-13). These studies revealed a class of γ-crystallin mutations in which smal...

Arrested demixing opens route to bigels

“…Interspecies interactions were shown to play a fundamental role in binary protein mixtures. Eye-lens protein systems, for example, can pass from condensation to demixing by a single point mutation, which is believed to alter the interspecies attraction (12,46). Although this selective interaction is a result of the complex structure of the proteins, research on functionalized colloids unveils unprecedented possibilities of synthesizing particles with tunable interactions.…”

How fluorescent labelling alters the solution behaviour of proteins

“…38,39 In this study, we experimentally assess the phase diagram for human gD-crystallin (HGD), which is a structural protein in the eye lens that undergoes LLPS due to short-ranged attractive interactions which dominate its behaviour at physiological pH (at the pI of the protein). 9,37,39,40 The native protein is remarkably stable in vivo. 41 Several point mutations in HGD have been linked to congenital cataract formation due to significantly decreased solubility of the protein at body temperature leading to increased light scattering and, eventually, blindness.…”

“…41 Several point mutations in HGD have been linked to congenital cataract formation due to significantly decreased solubility of the protein at body temperature leading to increased light scattering and, eventually, blindness. 37,[40][41][42][43][44] Therefore, LLPS of HGD is a readily quantifiable phase transition through which the effect that the addition of a fluorescent dye, by covalent attachment of an organic fluorophore, can be probed. Additionally, the surface chemistry of HGD ensures that the both the position and number of small molecules conjugated to the protein can be controlled.…”

Blind attraction: The mechanism of an inherited congenital cataract

“…The unique aspect of the results described by Banerjee et al (5) for the E107A mutation is that their observations do not follow the scheme outlined above. No condensed phase is produced in vitro.…”