The protease core of the muscle-specific calpain, p94, undergoes Ca2+-dependent intramolecular autolysis.
Limb girdle muscular dystrophy type 2A is linked to a skeletal muscle-specific calpain isoform known as p94. Isolation of the intact 94-kDa enzyme has been difficult to achieve due to its rapid autolysis, and uncertainty has arisen over its Ca2+-dependence for activity. We have expressed a C-terminally truncated form of the enzyme that comprises the protease core (domains I and II) along with its insertion sequence, IS1, and N-terminal leader sequence, NS. This 47-kDa p94I-II mini-calpain was stable during purification. In the presence of Ca2+, p94I-II cleaved itself within the NS and IS1 sequences. Mapping of the autolysis sites showed that NS and IS1 have the potential to be removed without damage to the protease core. Ca2+-dependent autolysis must be an intramolecular event because the inactive p94I-II C129S mutant was not cleaved by incubation with wild-type p94I-II. In addition, the rate of autolysis of p94I-II was independent of the concentration of the enzyme.
Rey
Michelle A
MA
Department of Biochemistry and Protein Engineering Network of Centres of Excellence Queen's University, K7L 3N6, Kingston, ON, Canada.
Davies
Peter L
PL
D001329:Autolysis; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000494:pharmacology*; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D018076:DNA, Complementary; D010455:Peptides; D002154:Calpain; D003546:Cysteine Endopeptidases; C078134:calpain p94; D002118:Calcium