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A. B. De Maria, Y. Shi, N. Kumar, S. Bassnett; Calpain Expression and Activity During Lens Fiber Differentiation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3478.
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© ARVO (1962-2015); The Authors (2016-present)
Calcium-activated proteases (calpains) have been implicated in the etiology of cataract but their role in normal lens physiology and differentiation is not fully understood. In this study we analyzed calpain expression and substrate hydrolysis during fiber cell differentiation in the mouse lens.
DNA microarray and Western Blot were used to analyze calpain distribution in lens fiber cells. Calpain activity was determined using an internally quenched fluorogenic peptide as substrate. To identify potential calpain targets, membrane-cytoskeleton fractions were treated with calpain 2 and cleaved products analyzed by 2 dimensional difference electrophoresis/mass spectrometry.
Calpains 1, 2, 7 and lens-specific calpain 3 (lp82/85) were detected in the mouse lens. Calpains 1, 2 and 7 were most abundant in superficial fibers. In contrast, calpain 3 was most abundant in deep cortical fibers. By microinjecting a fluorogenic calpain substrate into organ-cultured mouse lenses we detected endogenous calpain activity in cells located in the deep cortex. Several cytoskeletal proteins were identified as potential calpain targets, among them II-spectrin. Calpain cleavage of II-spectrin generates a fragment with a unique C-terminus (QQEVY1776 ). An antibody recognizing the calpain-cleaved fragment specifically labeled fiber cells in the deep cortex. Co-labeling with organelle-specific antibodies demonstrated that spectrin was cleaved in cells undergoing organelle breakdown. Based on its distribution, we reasoned that calpain 3 was most likely responsible for spectrin cleavage in the lens. To test this possibility, we analyzed calpain 3-null mice. The QQEVY1776 epitope was absent from calpain 3-deficient lenses implicating calpain 3 in II-spectrin cleavage at QQEVY1776-1777GMMPR.
We have shown that calpains are active during normal lens fiber cell differentiation. Calpain 3 appears to have a unique role in the cleavage of spectrin. Calpains may contribute to organelle breakdown or the remodeling of the fiber cell plasma membrane that occurs during terminal differentiation.
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