engleski

Measuring calpain 3 proteolytic activity using an in vitro substrate

Limb-girdle muscular dystrophy type 2A (LGMD2A ; OMIM 253600) is an autosomal recessive muscular disorder caused by mutations in the gene coding for calpain 3, a calcium-dependant protease. In order to improve presently available LGMD2A diagnosis, we developed an in vitro assay that can detect the proteolytic activity of calpain 3 in a muscle sample. This assay is based on the use of an inactive calpain 3 that can still be a substrate for active calpain 3 molecules that may be present in the sample to be tested. A total of 80 human biopsies consisting of 43 LGMD2A muscles and 37 controls have been analyzed with this assay. In all controls, normal calpain 3 level and activity were observed. A reduced or absent proteolytic activity was found in 32 (74%) of the LGMD2A biopsies. In the remaining 11 biopsies, normal proteolytic activity was found despite the presence of calpain 3 mutation(s), suggesting that other calpain 3 properties, possibly recognition of substrates or improper localization, should be impaired in those cases to give rise to the LGMD2A phenotype. Although the false positive results are possible, we believe that this assay is a quick and simple approach that improves LGMD2A diagnosis as it is able to test at once several calpain 3 properties.

LGMD 2A; calpain 3 proteolytic activity; diagnosis; dysferlin

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