title: Interaction of αA-crystallin F71L mutant with wild type αA- and αB-crystallins by mammalian two hybrid assay.
abstract: Incidence of age related cataract (ARC) increases by a variety of factors including metabolic and environmental factors. Nonetheless, genetic mutations are responsible for the altered structural stability of the proteins, especially; the F71L mutation in αA-crystallin has been shown to be responsible for the incidence of cataracts. However, structural characteristics and chaperone function of this mutant and its interaction with wild type (WT) crystallins may aid to decipher its role in cataractogenesis. The aim of the present study is to show the interaction of F71L mutant protein with the WT α-crystallins. The F71L mutant used in this study was created by site-directed mutagenesis, overexpressed and purified. Biophysical characteristics determined by size exclusion HPLC, DLS, CD spectrometry, tryptophan fluorescence and surface hydrophobicity did not show significant structural changes in the mutant protein compared to WT counterpart. Interestingly, the F71L mutant displayed a significant loss in homogenous interaction with WT αA-crystallin and F71L mutant as well as heterogeneous interaction with αB-crystallin as evaluated by mammalian two hybrid system. Our findings suggest that F71L loses the ability to form homo and hetero-oligomers seems to result in the loss in chaperone like activity (CLA) and refractive index resulting in the development of cataracts.
authors.affiliation: Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India.
authors.forename: Srinivasagan
authors.initials: S
authors.lastname: Ramkumar
authors.affiliation: Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India.
authors.forename: Bency
authors.initials: B
authors.lastname: Thankappan
authors.affiliation: Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka 590 0494, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Fujii
authors.affiliation: Department of Microbiology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India.
authors.forename: Kalimuthusamy
authors.initials: K
authors.lastname: Natarajaseenivasan
authors.affiliation: Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India. Electronic address: anbubdu@gmail.com.
authors.forename: Kumarasamy
authors.initials: K
authors.lastname: Anbarasu
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D038202:alpha-Crystallin A Chain / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D038203:alpha-Crystallin B Chain / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain
keywords: Mammalian two hybrid system; Protein–protein interaction; α-Crystallin, F71L mutation