title: Structural and functional consequences of chaperone site deletion in αA-crystallin.
abstract: The chaperone-like activity of αA-crystallin has an important role in maintaining lens transparency. Previously we identified residues 70-88 as a chaperone site in αA-crystallin. In this study, we deleted the chaperone site residues to generate αAΔ70-76 and αAΔ70-88 mutants and investigated if there are additional substrate-binding sites in αA-crystallin. Both mutant proteins when expressed in E. coli formed inclusion bodies, and on solubilizing and refolding, they exhibited similar structural properties, with a 2- to 3-fold increase in molar mass compared to the molar mass of wild-type protein. The deletion mutants were less stable than the wild-type αA-crystallin. Functionally αAΔ70-88 was completely inactive as a chaperone, while αAΔ70-76 demonstrated a 40-50% reduction in anti-aggregation activity against alcohol dehydrogenase (ADH). Deletion of residues 70-88 abolished the ADH binding sites in αA-crystallin at physiological temperature. At 45°C, cryptic ADH binding site(s) became exposed, which contributed subtly to the chaperone-like activity of αAΔ70-88. Both of the deletion mutants were completely inactive in suppressing aggregation of βL-crystallin at 53°C. The mutants completely lost the anti-apoptotic property that αA-crystallin exhibits while they protected ARPE-19 (a human retinal pigment epithelial cell line) and primary human primary lens epithelial (HLE) cells from oxidative stress. Our studies demonstrate that residues 70-88 in αA-crystallin act as a primary substrate binding site and account for the bulk of the total chaperone activity. The β3 and β4 strands in αA-crystallin comprising 70-88 residues play an important role in maintenance of the structure and in preventing aggregation of denaturing proteins.
authors.affiliation: Department of Ophthalmology, University of Missouri School of Medicine, Columbia, MO 65212, United States. Electronic address: Puttursa@health.missouri.edu.
authors.forename: Puttur
authors.initials: P
authors.lastname: Santhoshkumar
authors.affiliation: Department of Ophthalmology, University of Missouri School of Medicine, Columbia, MO 65212, United States.
authors.forename: Srabani
authors.initials: S
authors.lastname: Karmakar
authors.affiliation: Department of Ophthalmology, University of Missouri School of Medicine, Columbia, MO 65212, United States; Department of Biochemistry, University of Missouri School of Medicine, Columbia, MO 65212, United States. Electronic address: sharmak@health.missouri.edu.
authors.forename: Krishna K
authors.initials: KK
authors.lastname: Sharma
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D003459:Crystallins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
chemical_list: C511246:CRYAA protein, human; D003459:Crystallins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase
keywords: Aggregation; Apoptosis; Chaperone; Crystallin; Deletion; Structure-function