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.
Highlights
The major components of the mammalian lens fiber cells are the ␣, , and ␥-crystallins, which constitute an estimated 35% wet weight of the lens
Like other small heat shock proteins (sHSP), ␣-crystallin is a large (0.35 to Ͼ1 MDa) oligomeric complex and has the ability to suppress nonspecific aggregation of various proteins and enzymes denatured by heat [11], UV irradiation [12], and chemical modification [13]
Since crystallins exist for the lifespan of the host due to the virtual absence of protein turnover in mature lens fiber cells, it seems likely that mechanisms must exist to maintain these proteins in their native conformations throughout decades of exposure to metabolic and environmental insult
Summary
The major components of the mammalian lens fiber cells are the ␣-, -, and ␥-crystallins, which constitute an estimated 35% wet weight of the lens. Structure-function studies to probe the mechanism of chaperone-like activity of ␣-crystallin can be assisted with the use of recombinant ␣-crystallin subunits and protein substrates.
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