Proteolysis of glucagon within hepatic endosomes by membrane-associated cathepsins B and D.
The acidic glucagon-degrading activity of hepatic endosomes has been attributed to membrane-bound forms of cathepsins B and D. Endosomal lysates processed full-length nonradiolabeled glucagon to 32 different peptides that were identified by amino acid analysis and full-length sequencing. These indicated C-terminal carboxypeptidase, endopeptidase as well as N-terminal tripeptidyl-aminopeptidase activities in endosomes. Glucagon proteolysis was inhibited 95% by E-64 and pepstatin A, inhibitors of cathepsins B and D, respectively. This was confirmed by the pH 6-dependent chemical cross-linking of [125I]iodoglucagon to a polypeptide of 30 kDa, which was immunodepleted by polyclonal anti-cathepsin B antibody, and the removal of greater than 80% of glucagon-degrading activity by polyclonal antibodies to cathepsins B and D. By similar criteria, insulin-degrading enzyme was ruled out as a candidate enzyme for endosomal proteolysis of glucagon. Lysosomal contamination was unlikely since all forms of cathepsin B in endosomes, i.e. the major 45-kDa inactive precursor as well as the lesser amounts of the 32- and 28-kDa active forms, were tightly bound to endosomal membranes. Furthermore the mature 29-kDa single-chain and 22-kDa heavy-chain forms of cathepsin L were undetectable in endosomes, although high levels of the 37-kDa proform were observed. Membrane association of the cathepsins B and D was not to the mannose 6-phosphate receptor since association was unaffected by mannose 6-phosphate and/or EDTA, thereby indicating a distinct endosomal receptor. Hence, a pool of active cathepsins B and D as well as a poorly defined tripeptidyl aminopeptidase is maintained in endosomes by selective membrane retention. These hydrolases degrade glucagon internalized into liver parenchyma early in endocytosis.
Authier
F
F
Institut National de la Santé et de la Recherche Médicale U30, Hôpital des Enfants Malades, Paris, France.
Mort
J S
JS
Bell
A W
AW
Posner
B I
BI
Bergeron
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis; D002401:Cathepsin B / Q000502:physiology*; D002402:Cathepsin D / Q000502:physiology*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D011992:Endosomes / Q000378:metabolism*; D005934:Glucagon / Q000378:metabolism*; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley
D002352:Carrier Proteins; D007328:Insulin; D005934:Glucagon; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D002401:Cathepsin B; D002402:Cathepsin D