Identification of mu-, m-calpains and calpastatin and capture of mu-calpain activation in endothelial cells.
The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange chromatography, Western blot analysis, and Northern blot analysis. On DEAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively. For half-maximal activity, the protease required 800 microM Ca2+, comparable to the Ca2+ requirement of m-calpain. By Western blot analysis, the large subunit of mu-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) and calpastatin were detected in the respective active fractions. By Northern blot analysis, mRNAs for large subunits of mu- and m-calpains were detected in single bands, each corresponding to approximately 3.5 Kb. Calpastatin mRNA was observed in two bands corresponding to approximately 3.8 and 2.6 Kb. Furthermore, the activation of mu-calpain in HUVEC by a calcium ionophore was examined, using an antibody specifically recognizing an autolytic intermediate form of mu-calpain large subunit (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a calcium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytoskeleton was preceded by the appearance of the autolytic intermediate form of mu-calpain, while the fully autolyzed postautolysis form of mu-calpain (76 kDa) remained below detectable levels at all time points examined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that mu-calpain may be involved in endothelial cell function mediated by Ca2+ via the limited proteolysis of various proteins.
Fujitani
K
K
Department of Surgery II, Osaka University Medical School, Suita, Japan.
Kambayashi
J
J
Sakon
M
M
Ohmi
S I
SI
Kawashima
S
S
Yukawa
M
M
Yano
Y
Y
Miyoshi
H
H
Ikeda
M
M
Shinoki
N
N
Monden
M
M
D000918:Antibody Specificity; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002154:Calpain / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002478:Cells, Cultured; D002848:Chromatography, DEAE-Cellulose; D003598:Cytoskeletal Proteins / Q000187:drug effects / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation / Q000276:immunology; D004792:Enzyme Precursors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D005110:Extracellular Space / Q000502:physiology; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis; D014471:Umbilical Veins / Q000166:cytology
D002135:Calcium-Binding Proteins; D003598:Cytoskeletal Proteins; D004151:Dipeptides; D004792:Enzyme Precursors; D012333:RNA, Messenger; C071482:calpeptin; D000001:Calcimycin; C033668:calpastatin; D002154:Calpain; C098484:mu-calpain proenzyme; D002118:Calcium