Characterization of α-Crystallin-Plasma Membrane Binding

Abstract

␣-Crystallin, a large lenticular protein complex made up of two related subunits (␣A-and ␣B-crystallin), is known to associate increasingly with fiber cell plasma membranes with age and/or the onset of cataract. To understand better the binding mechanism, we developed a sensitive membrane binding assay using lens plasma membranes and recombinant human ␣A-and ␣B-crystallins conjugated to a small fluorescent tag (Alexa350 ® ). Both ␣A and ␣B homopolymer complexes, as well as a reconstituted 3:1 heteromeric complex, bind to lens membranes in a specific, saturable, and partially irreversible manner that is sensitive to both time and temperature. The amount of ␣-crystallin that binds to the membrane increases under acidic pH conditions and upon removal of exposed intrinsic membrane protein domains but is not affected at high ionic strength, suggesting that ␣-crystallin binds to the fiber cell plasma membranes mainly through hydrophobic interactions. The binding capacity and affinity for the reconstituted 3:1 heteromeric complex were measured to be 3.45 ؎ 0.11 ng/g of membrane and 4.57 ؎ 0.50 ؋ 10 ؊4 g ؊1 of membrane, respectively. The present membrane binding data support the hypothesis that the physical properties of a mixed ␣-crystallin complex may hold particular relevance for the function of ␣-crystallin within the lens.

αA-Crystallin and αB-Crystallin Reside in Separate Subcellular Compartments in the Developing Ocular Lens

“…It must be recognized that both ␣A as well as ␣B have been considered as soluble proteins of the ocular lens, yet the data presented thus far in this investigation and elsewhere (14,15,31,33,35) provide definitive evidence that both these proteins are membrane-associated. Although the exact function of ␣A remains to be elucidated, its presence in the ER has been related to ER-associated degradation of denatured proteins; it may be involved in quality control of proteins destined for the cell surface as has been recently suggested for epithelial Na (ϩ) channel expression in mouse cultured collecting duct cells (29).…”

“…Historically, a fraction of ␣-crystallin (both ␣A and ␣B) has been reported to be membrane-associated in the normal adult transparent lens. These observations about the association of ␣-crystallins with the membranes were first made about four decades ago (32)(33)(34)(35). We therefore examined ␣A status first in the lens epithelial explants in culture and then in the early developing lens.…”

The Small Heat Shock Protein IbpA of Escherichia coli Cooperates with IbpB in Stabilization of Thermally Aggregated Proteins in a Disaggregation Competent State

“…The studies performed for B. japonicum sHsps reveal that heterooligomers display chaperone activities indistinguishable from homooligomeric complexes (34). In contrast, the properties of homo-and hetero-oligomeric ␣-crystallin complexes differ, because it was reported that these complexes have different lens plasma membrane-binding properties (47). It was also shown that ␣A and ␣B-crystallin homooligomers possess unique chaperone activities that are additionally differently modulated in response to temperature changes (48,49).…”

Different anti-aggregation and pro-degradative functions of the members of the mammalian sHSP family in neurological disorders

“…This ACD plays an important role in the modulation of both structural and functional properties of the HSPBs. Indeed, monomers of the HSPB proteins associate (partially via their ACDs) into dimers that are thought to act as basic units/building blocks, capable of generating oligomers ranging from ca 200 to 600 kDa [49,53]. The various HSPB monomers can form both homo-and hetero-dimers as well as Table 1.…”