A proteomic study of calpain-3 and its involvement in limb girdle muscular dystrophy type 2a.
Limb-girdle muscular dystrophy type 2A is an autosomal recessive disorder generated by inactivating mutations in the gene coding for the muscle specific protease calpain-3. It is mainly expressed in skeletal muscle as a monomeric multidomain protein characterized by three unique insertion sequences (NS, IS1, IS2). It is unstable, and undergoes very rapid autolysis in solution, therefore, its heterologous expression and purification have been difficult. So far, calpain-3 substrates have been only identified in vitro and with indirect approaches. We have therefore decided to perform a comprehensive study of the substrates of the protease by comparing the 2D electrophoretic profile of myotubes from obtained from calpain-3 knockout and wild type mice. Digestion of differentially expressed spots was followed by mass spectrometry analysis. We could identify 16 proteins which differed in knockout and wild type mice. Among them: desmin, nestin, spectrin and PDLIM1 were of particular interest. In vitro experiments have then revealed that only PDLIM1 is cleaved directly by the protease, and that a fragment of about 8 kDa is released from the C-terminal portion of the protein.
Bertipaglia
Ilenia
I
Department of Biochemistry University of Padova, and Venetian Institute of Molecular Medicine, Padova, Italy.
Bourg
Natalie
N
Richard
Isabelle
I
Pahlman
Anna-Karin
AK
Andersson
Liselotte
L
James
Peter
P
Carafoli
Ernesto
E
D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D002478:Cells, Cultured; D003893:Desmin / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D007381:Intermediate Filament Proteins / Q000737:chemistry; D060588:LIM Domain Proteins; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000737:chemistry*; D018485:Muscle Fibers, Skeletal; D009124:Muscle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D049288:Muscular Dystrophies, Limb-Girdle / Q000503:physiopathology; D009419:Nerve Tissue Proteins / Q000737:chemistry; D064231:Nestin; D013049:Spectrin / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003893:Desmin; D007381:Intermediate Filament Proteins; D060588:LIM Domain Proteins; C106649:Ldb2 protein, mouse; D008840:Microfilament Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D014157:Transcription Factors; D013049:Spectrin; D002154:Calpain; C491373:Capn3 protein, mouse