Endosomal proteolysis of diphtheria toxin without toxin translocation into the cytosol of rat liver in vivo.
A detailed proteolysis study of internalized diphtheria toxin (DT) within rat liver endosomes was undertaken to determine whether DT-resistant species exhibit defects in toxin endocytosis, toxin activation by cellular enzymes or toxin translocation to its cytosolic target. Following administration of a saturating dose of wild-type DT or nontoxic mutant DT (mDT) to rats, rapid endocytosis of the intact 62-kDa toxin was observed coincident with the endosomal association of DT-A (low association) and DT-B (high association) subunits. Assessment of the subsequent post-endosomal fate of internalized mDT revealed a sustained endo-lysosomal transfer of the mDT-B subunit accompanied by a net decrease in intact mDT and mDT-A subunit throughout the endo-lysosomal apparatus. In vitro proteolysis of DT, using an endosomal lysate, was observed at both neutral and acidic pH, with the subsequent generation of DT-A and DT-B subunits (pH 7) or DT fragments with low ADP-ribosyltransferase activity (pH 4). Biochemical characterization revealed that the neutral endosomal DT-degrading activity was due to a novel luminal 70-kDa furin enzyme, whereas the aspartic acid protease cathepsin D (EC 3.4.23.5) was identified as being responsible for toxin degradation at acidic pH. Moreover, an absence of in vivo association of the DT-A subunit with cytosolic fractions was identified, as well as an absence of in vitro translocation of the DT-A subunit from cell-free endosomes into the external milieu. Based on these findings, we propose that, in rat, resistance to DT may originate from two different mechanisms: the ability of free DT-A subunits to be rapidly proteolyzed by acidic cathepsin D within the endosomal lumen, and/or the absence of DT translocation across the endosomal membrane, which may arise from the absence of a functional cytosolic translocation factor previously reported to participate in the export of DT from human endosomes.
El Hage
Tatiana
T
INSERM U756, Université Paris-Sud, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, Châtenay-Malabry, France.
Decottignies
Paulette
P
Authier
François
F
D000818:Animals; D002384:Catalysis; D002403:Cathepsins / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D009154:Mutation / Q000235:genetics; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley
D004167:Diphtheria Toxin; D002403:Cathepsins