The 21-base pair deletion mutant Calpain 3 does not inhibit wild-type Calpain 3 activity

Abstract

IntroductionCalpain 3 is one of the calpain protease family members, which is a calcium-dependent proteolytic enzyme predominantly expressed in skeletal muscle. Loss-of-function mutations in the Calpain 3 gene have been related to autosomal recessive Limb-Girdle Muscular Dystrophy 1 (LGMDR1), a common form of muscular dystrophy. Recently, the heterozygous 21-bp deletion mutation of the Calpain 3 gene has been reported to cause autosomal dominant Limb-Girdle Muscular Dystrophy 4 (LGMDD4). According to its dominant inheritance pattern, it has been suggested that the deletion mutant proteins act in a dominant-negative manner. Therefore, we examined whether the mutant protein can suppress the activity of wild-type Calpain 3 and has any dominant toxicity in cell culture and in vivo Drosophila models. MethodsA human cell culture (HeLa cells) model with the transient transfection of human wild-type and mutant Calpain 3 and in vivo Drosophila models overexpressing wild-type and mutant Drosophila Calpain A and B were utilized in this study to assess dominant effects of Calpain 3 21-bp deletion mutant. Western blot analysis was used to determine protein stability and catalytic activity in cell culture. External eye morphology and muscle integrity were examined to observe dominant toxicity in Drosophila models. ResultsThe 21-bp deletion mutation of Calpain 3 resulted in catalytic inactivation, which did not inhibit wild-type Calpain 3 autolytic and catalytic activity against Calpastatin in HeLa cells. In addition, the mutant protein was normally processed by wild-type Calpain 3. Overexpression of wild-type and deletion mutant Calpain 3 in the Drosophila eye and muscles did not exhibit significant developmental and age-related dominant toxicity. DiscussionWe provide evidence that mutant Calpain 3 does not suppress wild-type Calpain 3 activity. Rather, it is a mutant lacking autocatalytic processing activity like many other loss-of-function Calpain 3 mutants causing LGMDR1. Our results implicate that the stability of the heteromeric mutant and wild-type Calpain 3 complexes may be affected without inhibiting the wild-type activity per se. However, a more thorough investigation is necessary to understand the molecular mechanism and dominant inheritance of the heterozygous 21-bp deletion mutation in LGMDD4.