Structural and Functional Changes in the αA-Crystallin R116C Mutant in Hereditary Cataracts

Abstract

alpha-Crystallin, the major protein component of vertebrate lenses, forms a large complex comprised of two homologous subunits, alphaA- and alphaB-crystallin. It has the ability to suppress stress-induced protein aggregation in vitro, bind saturably to lens plasma membranes, and aid in light refraction through short-range ordering. Recently, a missense mutation in alphaA-crystallin that changes arginine 116 to a cysteine residue (R116C) was genetically linked to one form of autosomal dominant congenital cataracts. This point mutation is reported to cause structural alterations at many levels as well as a 4-fold reduction in chaperone-like activity. To extend these findings, we examined the quaternary stability of the alphaA R116C mutant protein and its effect on chaperone-like activity, subunit exchange, and membrane association. Homocomplexes of mutant subunits become highly polydisperse following incubation at 37 degrees C, reflecting the likely in vivo distribution of the complexes. Chaperone-like activity of the alphaA R116C mutant is approximately 4-fold lower than wild type, whether measured before or after conversion to a polydisperse population with incubation. alphaA R116C complexes also have a 4-fold reduced ability to exchange subunits with wild-type complexes. Finally, membrane binding capacity measurements of mutant subunits showed a 10-fold increase over wild type. Our results, in conjunction with previous reports, suggest that the changes in complex polydispersity, the reduction of subunit exchange, and increased membrane binding capacity are all potential factors in the pathogenesis of alphaA R116C associated congenital cataracts.

Cell death triggered by a novel mutation in the alphaA-crystallin gene underlies autosomal dominant cataract linked to chromosome 21q

“…56 Second, when expressed in HLE B-3 cells the R49C mutant was localized to both nuclear and cytoplasmic compartments (Figures 4 and 5). In contrast, the R116C-CRYAA mutant was mainly restricted to the cytoplasm.…”

“…53 In addition, bacterial expression studies of recombinant CRYAA have shown that the R116C missense substitution resulted in reduced prevention of heatinduced protein aggregation in vitro (Bfour-fold) compared to wild type. 54 -56 However, this loss of chaperone-like function was largely regained when R116C-CRYAA was mixed with wild-type CRYAA and CRYAB subunits in the stoichiometric amounts (1.5:1.5:1, respectively) predicted to occur in the lens of an affected (heterozygous) individual, 56 indicating the absence of dominant negative effects. Moreover, heterozygous loss of Cryaa in mice 52 and, theoretically, CRYAA in humans 16 is not sufficient to cause cataract.…”

“…57 Biochemical studies have shown that the aggregate size of R116C-CRYAA was Bfour-fold higher (42 MDa) that that of wild-type CRYAA homocomplexes 53 and these mutant aggregates also exhibited a significant increase (B10-fold) in binding to cell membranes in vitro compared with wildtype CRYAA. 56 Although our attempts to isolate R49C-CRYAA from HLE B-3 transfectants were unsuccessful, due to the high level of cell death, two pieces of immuno- Figure 5 Cryoimmunoelectron microscopy of HLE B-3 cells expressing either wild-type CRYAA or the R49C mutant. (a) Immunogold labeling showed that wild-type CRYAA was restricted to the cytoplasm.…”

Mechanism of a Hereditary Cataract Phenotype

“…Such loss of chaperone efficiency, however, cannot be responsible for the congenital phenotype since ␣A-crystallin knock-out mice are born with morphologically normal lenses and develop early onset cataract (31). Therefore, Cobb and Petrash (27) argued that the mutations must result in a toxic gain of function. Enhanced binding to membranes was proposed to be an important factor in the pathogenesis associated with the ␣A-R116C mutation.…”

Genetic disorders involving molecular-chaperone genes: A perspective

“…The stability of the quaternary structure of the alpha-Acrystallin R116C mutant was investigated, along with this mutated protein's chaperoning activity, its subunit-exchange capability, and its membrane association abilities. 75 The mutant-protein complexes became highly polydisperse at 37°C. Under these conditions, the chaperoning activity was four times lower than that of the wild-type complexes.…”