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C Fukiage, M Azuma, T Nakajima, K Mizutani, TR Shearer; Function of Retina-Specific Calpain Rt88 (Splice Variant of p94) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2422.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Calpains consist of the ubiquitous m- and µ-calpains, and tissue-specific calpains p94, nCL2, nCL2', nCL4 and Lp82. In rat, we recently discovered a retina-specific calpain termed Rt88, a splice variant of p94. The cDNA for Rt88 was nearly identical to muscle-specific calpain p94, except for a different exon 1 and deletion of exons 15 and 16 in the unique IS2 insert region. Detection of Rt88 protein and it's enzymatic activity in rat retina is difficult because Rt88 contains the IS1 region of p94. The IS1 region is probably involved in promoting the rapid breakdown of Rt88 after translation. Thus, the purpose of the first experiment was to analyze Rt88 after over-expression in bacurovirus expression system. The emerging field of tissue-specific calpains may provide more direct insight into the function of calpains because of their relation to the unique properties of host tissues. For example, mutations in muscle p94 were found to cause limb girdle muscular dystrophy type 2A in man, probably due to loss in proteolytic activity of p94. Thus, the purpose of the second experiment was to determine the involvement of Rt88 in retinal disease in animal models. Methods: Immunoblotting and casein zymography were performed. Changes in the expression mRNA for Rt88 were measured by RT-PCR in retinas from the ischemia-reperfusion model in rat and from WBN/Kob rats. Results: We found that partially purified rRt88 was activated by calcium and inhibited by calpain inhibitor E64. Intact rRt88 was lost after separation by DEAE, and only rRt88 fragments were remained. The rRt88 fragments were still active on casein zymograms. The expression of Rt88 mRNA was down-regulated in retina after ischemia-reperfusion. In WBN/Kob rats, the expression of Rt88 was lost. Conclusion: Rt88 mRNA was stable, but Rt88 protein was unstable after translation. We concluded that Rt88 may be required for maintaining normal retina and that Rt88 may play a specific function in retina related to the unique physiology of this tissue.
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