Crystal cataracts: Human genetic cataract caused by protein crystallization

Abstract

Several human genetic cataracts have been linked recently to point mutations in the ␥D crystallin gene. Here we provide a molecular basis for lens opacity in two genetic cataracts and suggest that the opacity occurs because of the spontaneous crystallization of the mutant proteins. Such crystallization of endogenous proteins leading to pathology is an unusual event. Measurements of the solubility curves of crystals of the Arg-58 to His and Arg-36 to Ser mutants of ␥D crystallin show that the mutations dramatically lower the solubility of the protein. Furthermore, the crystal nucleation rate of the mutants is enhanced considerably relative to that of the wild-type protein. It should be noted that, although there is a marked difference in phase behavior, there is no significant difference in protein conformation among the three proteins.H uman ␥D crystallin is a member of a highly homologous family of mammalian lens proteins called the ␥ crystallins (1). Together with the ␣ and ␤ crystallins, these proteins are essential for maintaining lens transparency. However, the ␥ crystallins differ from the ␣ and ␤ crystallins in one important respect: the interactions between the ␥ crystallins are attractive (2). This feature reduces the osmotic pressure in the lens, but it also makes the ␥ crystallins more susceptible to aggregation and phase separation, phenomena that diminish the homogeneity of the lens and lead to cataract (3). Yet, despite these attractive interactions, the ␥ crystallins remain soluble for many years at high concentrations and with little turnover, maintaining the proper refractive index gradient of the lens (4).It is well known that random mutations in proteins dramatically affect their solubility (5). In this article, we show that the Arg-58 to His (R58H) mutant [linked to the aculeiform cataract (Fig. 1a; ref. 6)] and the Arg-36 to Ser (R36S) mutant [linked to another form of genetic (congenital) cataract ( Fig. 1b; ref. 7)] are much less soluble than the wild-type human ␥D crystallin protein (HGD). We also show that these mutants are more prone to crystallize than the wild-type. Indeed, Kmoch et al. (7) recently extracted crystals of the R36S mutant from the eye of a young patient. To determine the mechanism of cataract formation caused by these mutations, we compared the conformation, stability, and phase behavior of the recombinant HGD, R58H, and R36S proteins in solution. Materials and MethodsCloning, Expression, and Isolation of Proteins. Recombinant human ␥D crystallin was prepared by the amplification of the coding sequence from a human fetal lens cDNA library as detailed (8). Overexpression of ␥D crystallin, and isolation and purification of the protein, were all done according to procedures as described (8). Mutant proteins were prepared as follows.(i) The R58H mutant. To introduce a histidine mutation in place of Arg-58, the following oligonucleotide primers were made: 5Ј-CCAGTACTTCCTGCACCGCGGCGACTATGC-3Ј as the forward primer and 5Ј-GCATAGTCGCCGCGGTGCAG-GAAGTACTGG-3Ј as the reverse prime...

β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

“…70 In addition, point mutations associated with congenital cataracts also involve surface polar residues of c-crystallins. 71,72 Taken together, aforementioned experimental observations suggested that the surface charge network, together with the hydrophobic interactions, plays a major role in maintaining the stability of native cD-crys. The greater abundance of acidic and basic residues within the C-td most likely adds to its higher stability over the N-td in the native state.…”

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

“…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)(9)(10)(11)(12)(13). These studies revealed a class of γ-crystallin mutations in which small changes to the protein surface were sufficient to bring about severe solubility changes, without unfolding the protein, and were in direct contrast to other mutations (i.e., other missense mutations, insertions, and splice mutations) that are accompanied by alterations in protein fold and stability (1,14,15).…”

Altered phase diagram due to a single point mutation in human γD-crystallin

“…Mutation at site 23 of HGD is only one of a number of single point mutations, including the R14C, R58H, and R36S mutations, that occur on the CRGD gene and that have been linked with early-onset cataract disease (5)(6)(7)(8)(9). Physicochemical characterization of the mutant proteins shows why these changes result in the formation of either covalently linked aggregates in the case of R14C (5,6) or crystals in the case of the R58H and R36S mutants (7)(8)(9). The P23T mutation results in decreased solubility of the protein, leading to protein aggregation and light scattering, and hence to lens opacity.…”