Cathepsin B localizes to plasma membrane caveolae of differentiating myoblasts and is secreted in an active form at physiological pH.
Our in vitro studies support a functional link between the induction of cathepsin B gene expression and the catabolic restructuring associated with myotube formation during myogenesis in vivo. We have tested two predictions that are basic to this hypothesis: (1) that active cathepsin B is localized to plasma membrane caveolae of fusing myoblasts; and (2) that active cathepsin B is secreted from fusing myoblasts at physiological pH. During differentiation, L6 rat myoblasts demonstrated a fusion-related increase in activity associated with the 25/26-kDa, fully processed, active form of cathepsin B. Immunocytochemical studies demonstrated a redistribution of lysosomal cathepsin B protein toward the membrane of fusing myoblasts, and a colocalization of cathepsin B with caveolin-3, the muscle-specific structural protein of membrane caveolae. Sucrose density fractionation and Western blot analysis demonstrated that an active form of cathepsin B localizes to caveolar fractions along with caveolin-3, annexin-VII, beta-dystroglycan and dystrophin. Finally, 'real-time' activity assays and Western blot analysis demonstrated that active cathepsin B is secreted from fusing myoblasts at physiological pH. Collectively, these studies support an association of active cathepsin B with plasma membrane caveolae and the secretion of active cathepsin B from differentiating myoblasts during myoblast fusion.
Jane
Derek T
DT
Biological Sciences, University of Windsor, Windsor N9B 3P4, ON, Canada.
Morvay
Les
L
Dasilva
Luis
L
Cavallo-Medved
Dora
D
Sloane
Bonnie F
BF
Dufresne
Michael J
MJ
D000818:Animals; D002401:Cathepsin B / Q000737:chemistry / Q000378:metabolism*; D021941:Caveolae / Q000201:enzymology* / Q000648:ultrastructure; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000648:ultrastructure; D002478:Cells, Cultured; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D051381:Rats
D002401:Cathepsin B