Cloning, Expression, and Chaperone-like Activity of Human αA-Crystallin

Abstract

One of the major protein components of the ocular lens, ␣-crystallin, is composed of ␣A and ␣B chain subunits that have structural homology to the family of mammalian small heat shock proteins. Like other small heat shock proteins, ␣-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 ␣A 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 ␣-crystallin modification, two mutant forms of ␣A subunits were prepared by site-directed mutagenesis. Like wild type (WT), aggregates of ϳ540 kDa were formed from a tryptophan-free ␣A 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 Cterminal 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 ␥-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 ␣A subunits, and that deletion of C-terminal sequences from the ␣A subunit results in substantial loss of chaperone-like activity, despite overall preservation of secondary structure.The major components of the mammalian lens fiber cells are the ␣-, ␤-, and ␥-crystallins, which constitute an estimated 35% wet weight of the lens. The crystallins contribute to the transparency and refractive power of the lens by short range interactions among themselves and cytoskeletal elements in a highly concentrated matrix (1-3). ␣-Crystallin is one of the most abundant of the crystallins in mature lens fiber cells. It is a M r ϳ0.6 -1.0 ϫ 10 6 complex composed of two structurally related subunits, designated ␣A and ␣B, which are encoded by genes localized to chromosomes 21 and 11, respectively (4, 5).

Altered phase diagram due to a single point mutation in human γD-crystallin

“…1). For HGD and the mutant proteins used in this study, an extinction coefficient of 41.4 mM Ϫ1 ⅐cm Ϫ1 at 280 nm was used (40). The volume fraction of the protein is expressed as ϭ c⅐ sp , where c is the concentration in milligrams per milliliter and sp is the partial specific volume, 7.1 ϫ 10 Ϫ4 ml/mg (18).…”

Molecular basis of a progressive juvenile-onset hereditary cataract

“…The final product was analyzed by using electrospray ionization mass spectroscopy. The concentration of human ␥D was determined by using an extinction coefficient of 41.4 mM Ϫ1 ⅐cm Ϫ1 at 280 nm (7). The same extinction coefficient was used for R14C.…”

Cell death triggered by a novel mutation in the alphaA-crystallin gene underlies autosomal dominant cataract linked to chromosome 21q

“…Consensus exon/intron boundaries in CRYAA were verified by aligning the appropriate cDNA sequence 43 with the corresponding genomic sequence 44 assigned to chromosome 21 (BAC clone 445C9). Gene-specific primers (Table 1) were designed to anneal to intronic sequence flanking exon boundaries.…”