title: Cloning, expression, and chaperone-like activity of human alphaA-crystallin.
abstract: One of the major protein components of the ocular lens, alpha-crystallin, is composed of alphaA and alphaB chain subunits that have structural homology to the family of mammalian small heat shock proteins. Like other small heat shock proteins, alpha-crystallin subunits associate to form large oligomeric aggregates that express chaperone-like activity, as defined by the ability to suppress nonspecific aggregation of proteins destabilized by treatment with a variety of denaturants including heat, UV irradiation, and chemical modification. It has been proposed that age-related loss of sequences at the C terminus of the alphaA chain subunit may be a factor in the pathogenesis of cataract due to diminished capacity of the truncated crystallin to protect against nonspecific aggregation of lens proteins. To evaluate the functional consequences of alpha-crystallin modification, two mutant forms of alphaA subunits were prepared by site-directed mutagenesis. Like wild type (WT), aggregates of approximately 540 kDa were formed from a tryptophan-free alphaA mutant (W9F). When added in stoichiometric amounts, both WT and W9F subunits completely suppressed the heat-induced aggregation of aldose reductase. In contrast, subunits encoded by a truncation mutant in which the C-terminal 17 residues were deleted (R157STOP), despite having spectroscopic properties similar to WT, formed much larger aggregates with a marked reduction in chaperone-like activity. Similar results were observed when the chaperone-like activity was assessed through inhibition of gamma-crystallin aggregation induced by singlet oxygen. These results demonstrate that the structurally conservative substitution of Phe for Trp-9 has a negligible effect on the functional interaction of alphaA subunits, and that deletion of C-terminal sequences from the alphaA subunit results in substantial loss of chaperone-like activity, despite overall preservation of secondary structure.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA. petrash@am.seer.wustl.edu
authors.forename: U P
authors.initials: UP
authors.lastname: Andley
authors.forename: S
authors.initials: S
authors.lastname: Mathur
authors.forename: T A
authors.initials: TA
authors.lastname: Griest
authors.forename: J M
authors.initials: JM
authors.lastname: Petrash
mesh_terms: D000449:Aldehyde Reductase / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D018833:Chaperonins / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003459:Crystallins / Q000737:chemistry / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation
chemical_list: D003459:Crystallins; D000449:Aldehyde Reductase; D018833:Chaperonins