Authier F, Mort J S, Bell A W, Posner B I, Bergeron J J
Institut National de la Santé et de la Recherche Médicale U30, Hôpital des Enfants Malades, Paris, France.
J Biol Chem. 1995 Jun 30;270(26):15798-807. doi: 10.1074/jbc.270.26.15798.
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.
肝内体对胰高血糖素的酸性降解活性归因于组织蛋白酶B和D的膜结合形式。内体裂解物将全长未标记的胰高血糖素加工成32种不同的肽段，通过氨基酸分析和全长测序进行鉴定。这些结果表明内体中存在C末端羧肽酶、内肽酶以及N末端三肽基氨基肽酶活性。组织蛋白酶B和D的抑制剂E-64和胃蛋白酶抑制剂A分别使胰高血糖素的蛋白水解作用受到95%的抑制。这通过pH 6依赖性的[125I]碘胰高血糖素与30 kDa多肽的化学交联得到证实，该多肽可被多克隆抗组织蛋白酶B抗体免疫去除，并且多克隆抗组织蛋白酶B和D抗体可去除超过80%的胰高血糖素降解活性。根据类似标准，胰岛素降解酶被排除为胰高血糖素内体蛋白水解的候选酶。内体不太可能受到溶酶体污染，因为内体中所有形式的组织蛋白酶B，即主要的45 kDa无活性前体以及少量的32 kDa和28 kDa活性形式，都紧密结合在内体膜上。此外，在内体中未检测到组织蛋白酶L的成熟29 kDa单链和22 kDa重链形式，尽管观察到高水平的37 kDa前体形式。组织蛋白酶B和D与膜的结合并非通过甘露糖6-磷酸受体，因为这种结合不受甘露糖6-磷酸和/或EDTA的影响，从而表明存在一种独特的内体受体。因此，通过选择性膜保留，内体中维持了一组活性组织蛋白酶B和D以及一种定义不明确的三肽基氨基肽酶。这些水解酶降解在胞吞作用早期内化到肝实质中的胰高血糖素。
