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Endosomal Proteolysis of Glucagon at Neutral pH Generates the Bioactive Degradation Product Miniglucagon-(19–29)

Institut National de la Santé et de la Recherche Médicale Unité 510 (F.A., C.M.), Faculté de Pharmacie Paris XI, 92296 Châtenay-Malabry, France; Department of Anatomy and Cell Biology (P.H.C.), McGill University, Montréal, Québec, Canada H3A 2B2; and Laboratoire de Spectrométrie de Masse (M.K., G.B.), Faculté de Médecine, 59000 Lille, France

Address all correspondence and requests for reprints to: François Authier, Institut National de la Santé et de la Recherche Médicale Unité 510, Faculté de Pharmacie Paris XI, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry, France. E-mail: francois.authier{at}cep.u-psud.fr.

We have investigated the proteolytic mechanisms of glucagon degradation within hepatic endosomes at neutral pH before lumen acidification. Hepatic endosomes incubated at neutral pH rapidly degraded native glucagon into 13 intermediate products, one of which corresponded to the bioactive fragment glucagon-(19–29) (miniglucagon). The serine protease inhibitor phenylmethylsulfonyl fluoride as well as the nonspecific protease inhibitor bacitracin inhibited the endosomal degradation of glucagon at pH 7. In purified endosomal fractions, miniglucagon endopeptidase was undetectable as evaluated by immunoblotting, and immunoprecipitation with antibodies to insulin-degrading enzyme, cathepsins B and D, or furin failed to remove the endosomal neutral glucagonase activity. Incubation of endosomal fractions and [¹²⁵I]iodoglucagon with the zero-length bifunctional cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in specific labeling of a 170-kDa polypeptide. The labeling was completely inhibited by unlabeled glucagon (IC₅₀ value, 5 x 10^-7 M) and bacitracin (IC₅₀ value, 1 µg/ml), suggesting that it may correspond to a bacitracin-sensitive glucagon-degrading enzyme. Treatment of the ¹²⁵I-labeled 170-kDa cross-linked polypeptide with N-glycanase demonstrated that the cross-linked complex contained approximately 30 kDa of N-linked oligosaccharides. Specific cross-linking of the 170-kDa polypeptide was also observed using [¹²⁵I]Tyr¹²-miniglucagon as the radioligand. Together, these data suggest that the 170-kDa glycoprotein represents a novel glucagon-degrading activity that could mediate glucagon proteolysis within endosomes before the acidification step and generate the bioactive (19–29) miniglucagon peptide.

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