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W. Huang, I. Rawe, J.B. Fileta, C.L. Grosskreutz; Calcineurin Cleavage Is Mediated by Calpain in Experimental Glaucoma: A Proteomic Analysis . Invest. Ophthalmol. Vis. Sci. 2006;47(13):196.
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Calcineurin (CaN) is a Ca2+ calmodulin–dependent protein phosphatase that is highly expressed in the central nervous system and retina. We previously demonstrated that elevated intraocular pressure (IOP) leads to CaN cleavage in experimental glaucoma. Caspase 3 and calpain have been shown to cleave CaN in other model systems. Here, proteomic analysis was performed to determine the cleavage site of CaN and to suggest a mechanism leading to activation of CaN.
IOP elevation was induced in rats by hypertonic saline injections into episcleral veins using the Morrison model. Following 10 days of elevated IOP, retinal protein was collected. CaN was isolated by immunoprecipitation and separated on a 10% SDS–PAGE gel. The gel was then stained with colloidal blue followed by in–gel trypsin digestion. For identification of the cleavage site of CaN, peptide masses were analyzed by matrix–assisted laser desorption ionization time–of–flight mass spectrometry (MALDI–TOF/MS).
Comparison of the peptide mass fingerprinting results of the cleaved CaN with that of full–length CaN was performed. The 467–475 amino acid residues were not detected on the cleaved CaN. This observation showed that the cleavage sites on CaN was between residues 460–475. Within this range there were no caspase 3 specific cleavage sites. Taking into consideration the roles of calpain cleavage, we identified the cleavage site of CaN sequence at amino acid 460, which produces a 52–kDa truncated form. The 52 kDa form of CaN loses its Ca(2+)/calmodulin dependence rendering the phosphatase activity constitutively activated upon truncation.
Increased IOP causes full length CaN cleavage resulting in loss of its autoinhibitory domain in the retina. The indentification of the CaN cleavage site suggests that the activation of calpain may mediate this cleavage.
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