Peter James
Professor
A proteomic study of calpain-3 and its involvement in limb girdle muscular dystrophy type 2A.
Author
Summary, in English
Abstract in Undetermined
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. (C) 2009 Elsevier Ltd. All rights reserved.
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. (C) 2009 Elsevier Ltd. All rights reserved.
Department/s
- Departments at LTH
- Department of Immunotechnology
Publishing year
2009
Language
English
Pages
356-363
Publication/Series
Cell Calcium
Volume
46
Issue
5-6
Document type
Journal article
Publisher
Elsevier
Topic
- Immunology in the medical area
Keywords
- Calpain
- Muscular dystrophy
- Proteomics
- PDLIM 1
Status
Published
ISBN/ISSN/Other
- ISSN: 0143-4160