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S. Mruthinti, J.E. Hansen, A.M. Kersh, V. Srinivas, C. Mohan Rao, S. Swamy-Mruthinti; Role of C-Terminus of AQP0 During Thermal-Stress Induced Interaction With Alpha Crystallin . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2366.
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Purpose: In the avascular lens, Aquaporin0 (AQP0) channels are essential for homeostasis. We have recently shown that AQP0 undergoes thermal aggregation, and α crystallin prevents via chaperone-like activity [EER, 72 (suppl. 2), 40, 2002]. This study investigates the role of the C-terminus of AQP0 in this interaction. Methods: Thermal denaturation of calf AQP0, in the presence or absence of α crystallin, was studied either in the lens membranes or after octyl glucoside solubilization. AQP0-α crystallin complex was immunoprecipitated using anti-AQP0 antibody. AQP0 was glycated in vitro by incubating calf lens membranes with 1 M glucose. The C-terminus of AQP0 was cleaved with Glu-C protease. Thermal aggregation was measured by light scattering assay or by SDS-PAGE. Protein structural changes were monitored by circular dichroism (CD). Results: AQP0 undergoes time and temperature dependent aggregation, which was prevented by α crystallin. αA offered higher protection than αB. Immunoprecipitation showed that α crystallin binds to AQP0 when the latter was undergoing thermal denaturation. CD data suggests changes in the secondary structure of AQP0 during thermal aggregation. Modification of C-terminus by in vitro glycation or truncation with Glu-C protease of AQP0 has no effect either on the kinetics of thermal aggregation or on the chaperone function of α crystallin. Conclusions: AQP0 undergoes thermal denaturation both in the membranes (between 80-950C) and in the detergent micellar (between 40-550C) environments, and α crystallin renders protection via its chaperone-like activity. The fact that C-terminus does not play any role suggests that either the transmembrane helices or connecting loops of AQP0 are involved in the AQP0-α crystallin interaction. Selectivity of αA over αB may have physiological significance, as αA and AQP0 are exclusively expressed in the lens fiber cells and an increasing amount of αA has been shown to associate with membranes during aging and in cataracts.
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