title: Miniglucagon (MG)-generating endopeptidase, which processes glucagon into MG, is composed of N-arginine dibasic convertase and aminopeptidase B.
abstract: Miniglucagon (MG), the C-terminal glucagon fragment, processed from glucagon by the MG-generating endopeptidase (MGE) at the Arg17-Arg18 dibasic site, displays biological effects opposite to that of the mother-hormone. This secondary processing occurs in the glucagon- and MG-producing alpha-cells of the islets of Langerhans and from circulating glucagon. We first characterized the enzymatic activities of MGE in culture media from glucagon and MG-secreting alphaTC1.6 cells as made of a metalloendoprotease and an aminopeptidase. We observed that glucagon is a substrate for N-arginine dibasic convertase (NRDc), a metalloendoprotease, and that aminopeptidase B cleaves in vitro the intermediate cleavage products sequentially, releasing mature MG. Furthermore, immunodepletion of either enzyme resulted in the disappearance of the majority of MGE activity from the culture medium. We found RNAs and proteins corresponding to both enzymes in different cell lines containing a MGE activity (mouse alphaTC1.6 cells, rat hepatic FaO, and rat pituitary GH4C1). Using confocal microscopy, we observed a granular immunostaining of both enzymes in the alphaTC1.6 and native rat alpha-cells from islets of Langerhans. By immunogold electron microscopy, both enzymes were found in the mature secretory granules of alpha-cells, close to their substrate (glucagon) and their product (MG). Finally, we found NRDc only in the fractions from perfused pancreas that contain glucagon and MG after stimulation by hypoglycemia. We conclude that MGE is composed of NRDc and aminopeptidase B acting sequentially, providing a molecular basis for this uncommon regulatory process, which should be now addressed in both physiological and pathophysiological situations.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale Unité 376, Centre Hospitalier Universitaire Arnaud de Villeneuve, 371, Rue du Doyen G. Giraud, 34295 Montpellier, Cedex 5, France.
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Fontés
authors.forename: Anne-Dominique
authors.initials: AD
authors.lastname: Lajoix
authors.forename: François
authors.initials: F
authors.lastname: Bergeron
authors.forename: Sandrine
authors.initials: S
authors.lastname: Cadel
authors.forename: Annik
authors.initials: A
authors.lastname: Prat
authors.forename: Thierry
authors.initials: T
authors.lastname: Foulon
authors.forename: René
authors.initials: R
authors.lastname: Gross
authors.forename: Stéphane
authors.initials: S
authors.lastname: Dalle
authors.forename: Dung
authors.initials: D
authors.lastname: Le-Nguyen
authors.forename: Florence
authors.initials: F
authors.lastname: Tribillac
authors.forename: Dominique
authors.initials: D
authors.lastname: Bataille
mesh_terms: D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D003035:Cobalt / Q000494:pharmacology; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D005934:Glucagon / Q000096:biosynthesis* / Q000378:metabolism*; D007003:Hypoglycemia / Q000378:metabolism; D007515:Islets of Langerhans / Q000201:enzymology*; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010446:Peptide Fragments / Q000096:biosynthesis*; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology
chemical_list: D010446:Peptide Fragments; D010618:Phenanthrolines; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D003035:Cobalt; C039265:glucagon (19-29); D005934:Glucagon; D010450:Endopeptidases; D000626:Aminopeptidases; C019721:aminopeptidase B; D008666:Metalloendopeptidases; C087976:nardilysin; D015032:Zinc; D002118:Calcium; C025205:1,10-phenanthroline