Proteolytic activation of internalized cholera toxin within hepatic endosomes by cathepsin D.
We have defined the in vivo and in vitro metabolic fate of internalized cholera toxin (CT) in the endosomal apparatus of rat liver. In vivo, CT was internalized and accumulated in endosomes where it underwent degradation in a pH-dependent manner. In vitro proteolysis of CT using an endosomal lysate required an acidic pH and was sensitive to pepstatin A, an inhibitor of aspartic acid proteases. By nondenaturating immunoprecipitation, the acidic CT-degrading activity was attributed to the luminal form of endosomal cathepsin D. The rate of toxin hydrolysis using an endosomal lysate or pure cathepsin D was found to be high for native CT and free CT-B subunit, and low for free CT-A subunit. On the basis of IC(50) values, competition studies revealed that CT-A and CT-B subunits share a common binding site on the cathepsin D enzyme, with native CT and free CT-B subunit displaying the highest affinity for the protease. By immunofluorescence, partial colocalization of internalized CT with cathepsin D was confirmed at early times of endocytosis in both hepatoma HepG2 and intestinal Caco-2 cells. Hydrolysates of CT generated at low pH by bovine cathepsin D displayed ADP-ribosyltransferase activity towards exogenous Gsalpha protein suggesting that CT cytotoxicity, at least in part, may be related to proteolytic events within endocytic vesicles. Together, these data identify the endocytic apparatus as a critical subcellular site for the accumulation and proteolytic degradation of endocytosed CT, and define endosomal cathepsin D an enzyme potentially responsible for CT cytotoxic activation.
Merlen
Clémence
C
Institut National de la Santé et de la Recherche Médicale U510, Faculté de Pharmacie Paris XI, Châtenay-Malabry, France.
Fayol-Messaoudi
Domitille
D
Fabrega
Sylvie
S
El Hage
Tatiana
T
Servin
Alain
A
Authier
François
F
D000818:Animals; D001673:Biodegradation, Environmental; D001711:Biotransformation; D018938:Caco-2 Cells; D002402:Cathepsin D / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002772:Cholera Toxin / Q000737:chemistry / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D021122:Protein Subunits; D051381:Rats; D017207:Rats, Sprague-Dawley
D021122:Protein Subunits; D002772:Cholera Toxin; D002402:Cathepsin D