Fontés Ghislaine, Lajoix Anne-Dominique, Bergeron François, Cadel Sandrine, Prat Annik, Foulon Thierry, Gross René, Dalle Stéphane, Le-Nguyen Dung, Tribillac Florence, Bataille Dominique
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.
Endocrinology. 2005 Feb;146(2):702-12. doi: 10.1210/en.2004-0853. Epub 2004 Nov 11.
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.
小胰高血糖素（MG）是胰高血糖素的C末端片段，由产生MG的内肽酶（MGE）在精氨酸17 - 精氨酸18双碱性位点对胰高血糖素进行加工而成，其生物学效应与母激素相反。这种二次加工发生在胰岛中产生胰高血糖素和MG的α细胞以及循环中的胰高血糖素中。我们首先将来自分泌胰高血糖素和MG的αTC1.6细胞的培养基中的MGE酶活性鉴定为由一种金属内蛋白酶和一种氨肽酶组成。我们观察到胰高血糖素是金属内蛋白酶N - 精氨酸双碱性转化酶（NRDc）的底物，并且氨肽酶B在体外依次切割中间切割产物，释放出成熟的MG。此外，对其中任何一种酶进行免疫去除都会导致培养基中大部分MGE活性消失。我们在含有MGE活性的不同细胞系（小鼠αTC1.6细胞、大鼠肝脏FaO细胞和大鼠垂体GH4C1细胞）中发现了与这两种酶相对应的RNA和蛋白质。使用共聚焦显微镜，我们在来自胰岛的αTC1.6细胞和天然大鼠α细胞中观察到这两种酶的颗粒状免疫染色。通过免疫金电子显微镜，在α细胞的成熟分泌颗粒中发现了这两种酶，靠近它们的底物（胰高血糖素）和产物（MG）。最后，我们仅在低血糖刺激后含有胰高血糖素和MG的灌注胰腺馏分中发现了NRDc。我们得出结论，MGE由依次作用的NRDc和氨肽酶B组成，为这种不常见的调节过程提供了分子基础，现在应该在生理和病理生理情况下对其进行研究。
