Calpain 3 (CAPN3) is a muscle-specific,
calcium-dependent
proteinase that is mutated in Limb Girdle Muscle Dystrophy type 2A. Most pathogenic missense mutations in
LGMD2A affect CAPN3's proteolytic activity; however, two mutations, D705G and R448H, retain activity but nevertheless cause
muscular dystrophy. Previously, we showed that D705G and R448H mutations reduce CAPN3s ability to bind to
titin in vitro. In this investigation, we tested the consequence of loss of
titin binding in vivo and examined whether this loss can be an underlying pathogenic mechanism in
LGMD2A. To address this question, we created transgenic mice that express R448H or D705G in muscles, on wild-type (WT) CAPN3 or knock-out background. Both mutants were readily expressed in insect cells, but when D705G was expressed in skeletal muscle, it was not stable enough to study. Moreover, the D705G mutation had a dominant negative effect on endogenous CAPN3 when expressed on a WT background. The R448H
protein was stably expressed in muscles; however, it was more rapidly degraded in muscle extracts compared with WT CAPN3. Increased degradation of R448H was due to non-
cysteine, cellular
proteases acting on the autolytic sites of CAPN3, rather than
autolysis. Fractionation experiments revealed a significant decrease of R448H from the myofibrillar fraction, likely due to the mutant's inability to bind
titin. Our data suggest that R448H and D705G mutations affect both CAPN3s anchorage to
titin and its stability. These studies reveal a novel mechanism by which mutations that spare enzymatic activity can still lead to
calpainopathy.