title: Residue R120 is essential for the quaternary structure and functional integrity of human alphaB-crystallin.
abstract: The missense mutation Arg-120 to Gly (R120G) in the human alphaBeta-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 alphaBeta-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 alphaBeta-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.
authors.affiliation: EA 300, Université Paris 7, Case 7136, 75251 Paris Cedex 05, France. stef.labo@gmail.com
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Simon
authors.forename: Magalie
authors.initials: M
authors.lastname: Michiel
authors.forename: Fériel
authors.initials: F
authors.lastname: Skouri-Panet
authors.forename: Jean Pierre
authors.initials: JP
authors.lastname: Lechaire
authors.forename: Patrick
authors.initials: P
authors.lastname: Vicart
authors.forename: Annette
authors.initials: A
authors.lastname: Tardieu
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D051379:Mice; D046032:Microscopy, Energy-Filtering Transmission Electron; D020125:Mutation, Missense; D041681:NIH 3T3 Cells; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D038203:alpha-Crystallin B Chain / Q000032:analysis / Q000737:chemistry* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D038203:alpha-Crystallin B Chain; D001120:Arginine