The Role of the Calpain System in Neuromuscular Disease

Abstract

The first report of a neutral calcium-activated proteinase appeared in 1964 in a paper describing a Ca2+-stimulated proteolytic activity having a pH optimum between 7.7 and 8.0 in 0.25 M sucrose supernatants of rat brain extracts1. The proteolytic activity was partly purified by using ammonium sulfate precipitation at 40% saturation and chromatography on a DEAE-anion exchange column, where the activity eluted between 0.27 and 0.30 mM NaC1. Activity of the partly purified enzyme was dependent on the presence of Ca2+, was completely inhibited by 1 mM EDTA, stimulated by 2-mercaptoethanol, optimal between pH 7.1-7.3 (partial purification seemed to lower the pH optimum), and inhibited by Zn2+, Co2+, Cu2+, Hg2+, and iodoacetate, but affected very little by Mn2+, Mg2+, Ba2+,or diisopropyl fluorophosphate. These properties are identical to those described for m-calpain when it was first purified2,3. and it seems very likely that the proteolytic activity described by Guroff1 was m-calpain4. Interestingly, although Guroff found Ca2+-dependent proteolytic activity in extracts of brains from a number of species, including bovine, porcine, mouse, guinea pig, and rabbit, he did not detect it in kidney or spleen extracts, although these tissues are now known to contain substantial quantities of both μ-calpain and m-calpain5,6. It seems likely that Guroff's failure to detect Ca2+-dependent proteolytic activity in kidney or spleen extracts was due to the presence of calpastatin in these extracts. Although brain also contains calpastatin, the calpastatin levels in rat brain are slightly lower than the calpain levels, whereas calpastatin is present in excess over calpain in the kidney and spleen5,6. If Guroff had assayed the fractions of his DEAE-cellulose column for inhibitory activity, he would have also identified calpastatin, which was not identified until 12 years later7. The calpain system has been implicated in a number of neuromuscular diseases and other tissue pathologies. In general, inappropriate activity of the calpains is suspected whenever an alteration in Ca2+ homeostasis occurs, and this alteration is accompanied by limited degradation of cytoskeletal proteins such the neurofilament proteins, spectrin/fodrin, talin, vinculin, myelin basic protein, MAP2, and tau8,9. Administration of synthetic inhibitors that have some selectivity for inhibiting the calpains has often been used to support the conclusion that the calpains are responsible for the observed degradation. It has been impossible, however, to obtain a molecule that inhibits only the calpains9,10 (other than its natural inhibitor, calpastatin), and it is necessary to use a battery of different inhibitors with differing selectivities for different proteolytic enzymes such as the proteasome and cathepsins B, L, and S before concluding that the calpains are involved. Even then, the conclusion should be tempered by the possibility that other proteases may also be involved. Similarly, the cytoskeletal substrates of the calpains are degraded by a number of proteolytic enzymes, and with the exception of spectrin, where the bond cleaved by the calpains has been