Residue R120 is essential for the quaternary structure and functional integrity of human αB-Crystallin

The missense mutation Arg-120 to Gly (R120G) in the human alpha Beta-crystallin sequence has been reported to be associated with autosomal dominant myopathy, cardiomyopathy, and cataract. Previous studies of the mutant showed a significant ability to aggregate in cultured cells and an increased oligomeric size coupled to an important loss of the chaperone-like activity in vitro. The aim of this study was to further analyze the role of the R120 residue in the structural and functional properties of alpha Beta-crystallin. The following mutants were generated, Arg-120 to Gly (R120G), Cys (R120C), Lys (R120K), and Asp (R120D). In cellulo, after expression in two cultured cell lines, NIH-3T3 and Cos-7, the capacity of the wild-type and mutant crystallins to aggregate was evaluated and the protein location was determined by immunofluorescence. In vitro, the wild-type and mutant crystallins were expressed in Escherichia coli cells, purified by size exclusion chromatography, and characterized using dynamic light scattering, electron microscopy, and chaperone-like activity assays. Aggregate sizes in cellulo and in vitro were analyzed. The whole of the data showed that the preservation of an Arg residue at position 120 of alpha Beta-crystallin is critical for the structural and functional integrity of the protein and that each mutation results in specific changes in both structural and functional characteristics.