Conformational and Functional Differences between Recombinant Human Lens αA- and αB-Crystallin

Abstract

Human and other mammalian lens proteins are composed of three major crystallins: ␣-, ␤-, and ␥-crystallin. ␣-Crystallin plays a prominent role in the supramolecular assembly required to maintain lens transparency. With age, the crystallins, especially ␣-crystallin, undergo posttranslational modifications that may disrupt the supramolecular assembly, and the lens becomes susceptible to other stresses resulting in cataract formation. Because these modifications occur even at a relatively young age, it is difficult to obtain pure, unmodified crystallins for in vitro experiments. ␣-Crystallin is composed of two subunits, ␣A and ␣B. Before the application of recombinant DNA technology, these two ␣-crystallin subunits were separated from calf lens in the denatured state and reconstituted by the removal of the denaturant, but they were not refolded properly. In the present studies, we applied the recombinant DNA technology to prepare native, unmodified ␣A-and ␣B-crystallins for conformational and functional studies. The expressed proteins from Escherichia coli are in the native state and can be studied directly. First, ␣A and ␣B cDNAs were isolated from a human lens epithelial cell cDNA library. The cDNAs were cloned into a pAED4 expression vector and then expressed in E. coli strain BL21(DE3). Pure recombinant ␣A-and ␣B-crystallins were obtained after purification by gel filtration and DEAE liquid chromatography. They were subjected to conformational studies involving various spectroscopic measurements and an assessment of chaperone-like activity. ␣A-and ␣B-crystallins have not only different secondary structure, but also tertiary structure. 1-Anilino-8-naphthalene sulfonate fluorescence indicates that ␣B-crystallin is more hydrophobic than ␣A-crystallin. The chaperone-like activity, as measured by the ability to protect insulin aggregation, is about 4 times greater for ␣B-than for ␣A-crystallin. The resulting data provide a base line for further studies of human lens ␣-crystallin.␣-Crystallin is the major lens structural protein responsible for the supramolecular assembly necessary to maintain lens transparency. It is isolated as a large water-soluble aggregate with a mass of 800 kDa and is composed of two homologous subunits, ␣A and ␣B, each with a molecular mass of 20 kDa. With aging and cataract formation, ␣-crystallins gradually become a high molecular weight aggregate and eventually an insoluble protein (1). This is manifested by the observation that the major crystallin component in the insoluble protein fraction of aged and cataractous lenses is ␣-crystallin (2). The aggregation and insolubilization of ␣-crystallin are believed to arise from posttranslational modifications, which accumulate significantly because of the low turnover rate of lens crystallins. Two-dimensional gel electrophoresis indicated that human lens crystallins were modified extensively even at a relatively young age (3). The significance of the modification is that crystallins become partially unfolded, and this partially unfolding exposes ...

The eye lens chaperone α-crystallin forms defined globular assemblies

“…The secondary and tertiary structures of the preparations were assessed by circular dichroism spectroscopy (Fig. S1 E and F) and found to correspond to published data (36).…”

Human αA- and αB-crystallins bind to Bax and Bcl-XS to sequester their translocation during staurosporine-induced apoptosis

“…[39][40][41][42][43][44][45][46][62][63][64][65][66][67][68][69][70][71][72][73][74] One of the most important functions is the ability to protect cell from induced apoptosis. 39-46 a-Crystallin and Hsp27 are closely related family members and protect cells from apoptosis induced by a large numbers of stress factors.…”

“…[19][20]61 aA/B-crystallins are initially known as major lens structural proteins that play an essential role in maintaining the transparency of the ocular lens. 62 Later, a-crystallins are found to act as molecular chaperones [63][64][65][66][67] and also display autokinase activity. 68,69 As antiapoptotic regulators, acrystallins are initially shown to protect cells from thermal, 70 osmotic 71 and oxidative insult.…”

Differential Temperature-dependent Chaperone-like Activity of αA- and αB-crystallin Homoaggregates

“…␣-Crystallin is a heteroaggregate of two gene products, ␣A-and ␣B-crystallin. Both the subunits can homoaggregate and function as chaperones, albeit to different extents (11,22). The roles of the two proteins and their contribution to the structural and functional properties of ␣-crystallin are not well understood.…”