Mutations in calpain 3 associated with limb girdle muscular dystrophy: analysis by molecular modeling and by mutation in m-calpain.
Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder characterized by selective atrophy of the proximal limb muscles. Its occurrence is correlated, in a large number of patients, with defects in the human CAPN3 gene, a gene that encodes the skeletal muscle-specific member of the calpain family, calpain 3 (or p94). Because calpain 3 is difficult to study due to its rapid autolysis, we have developed a molecular model of calpain 3 based on the recently reported crystal structures of m-calpain and on the high-sequence homology between p94 and m-calpain (47% sequence identity). On the basis of this model, it was possible to explain many LGMD2A point mutations in terms of calpain 3 inactivation, supporting the idea that loss of calpain 3 activity is responsible for the disease. The majority of the LGMD2A mutations appear to affect domain/domain interaction, which may be critical in the assembly and the activation of the multi-domain calpain 3. In particular, we suggest that the flexibility of protease domain I in calpain 3 may play a critical role in the functionality of calpain 3. In support of the model, some clinically observed calpain 3 mutations were generated and analyzed in recombinant m-calpain. Mutations of residues forming intramolecular domain contacts caused the expected loss of activity, but mutations of some surface residues had no effect on activity, implying that these residues in calpain 3 may interact in vivo with other target molecules. These results contribute to an understanding of structure-function relationships and of pathogenesis in calpain 3.
Jia
Z
Z
Department of Biochemistry, Queen's University and The Protein Engineering Network of Centres of Excellence, Kingston, Ontario K7L 3N6, Canada. jia@post.queensu.ca
Petrounevitch
V
V
Wong
A
A
Moldoveanu
T
T
Davies
P L
PL
Elce
J S
JS
Beckmann
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D002154:Calpain / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009124:Muscle Proteins; D009136:Muscular Dystrophies / Q000201:enzymology* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D051381:Rats; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D009124:Muscle Proteins; C105884:CAPN3 protein, human; D002154:Calpain; C491372:Capn3 protein, rat