Endosomal proteolysis of internalized insulin at the C-terminal region of the B chain by cathepsin D
Endosomal proteolysis of internalized insulin at the C-terminal region of the B chain by cathepsin D
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DOI：
10.1074/jbc.m110188200
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发表时间：
2002-03-15
影响因子：
4.8
通讯作者：
Briand, G
中科院分区：
文献类型：
--
作者：
Authier, F;Métioui, M;Briand, G
肝实质细胞的内体区室含有酸性内肽酶，内体酸性胰岛素酶，其水解内化的胰岛素并产生主要的初级终产物A（1-21）-B1-24胰岛素，其由残基Phe（B24）-Phe（B25）处的主要裂解产生。本研究讨论了大鼠肝脏中相关内肽酶活性的性质，该活性负责体内大多数受体介导的胰岛素降解。基于与纯化CD在以下方面的生化相似性，显示内体活性为天冬氨酸蛋白酶组织蛋白酶D（CD）：1）底物裂解的速率和位点，2）最适pH值，3）对胃酶抑素A的敏感性，以及4）与胃酶抑素A-琼脂糖的结合。通过使用CD的多克隆抗体去除与内体裂解物相关的大于90%的胰岛素降解活性，免疫学确认蛋白酶的身份。此外，凝胶过滤TSK-GEL G3000 SWXL高效液相色谱柱上的内体酸性胰岛素酶活性的洗脱曲线与免疫反应性45 kDa成熟形式的内体CD的洗脱曲线完全一致。使用非变性免疫沉淀和免疫印迹程序，其他内体天冬氨酸蛋白酶，如组织蛋白酶E和β-位点淀粉样前体蛋白裂解酶（BACE）被排除为内化胰岛素内体降解的候选酶。免疫荧光研究显示，在大鼠肝细胞中，部分与CD共定位的内化胰岛素主要呈囊泡染色模式。体内胃酶抑素A治疗对内体的胰岛素受体含量没有任何可观察到的影响，但在胰岛素注射后增加了内体胰岛素受体的磷酸酪氨酸含量。这些结果表明，CD是内体酸性胰岛素酶活性，它催化Phe（B24）-Phe（B25）键体内切割的限速步骤，产生无活性的A（1-21）-B1-24胰岛素中间体。
The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase, which hydrolyzes internalized insulin and generates the major primary end product A(1-21)-B1-24 insulin resulting from a major cleavage at residues Phe(B24)-Phe(B25). This study addresses the nature of the relevant endopeptidase activity in rat liver that is responsible for most receptor-mediated insulin degradation in vivo. The endosomal activity was shown to be aspartic acid protease cathepsin D (CD), based on biochemical similarities to purified CD in 1) the rate and site of substrate cleavage, 2) pH optimum, 3) sensitivity to pepstatin A, and 4) binding to pepstatin A-agarose. The identity of the protease was immunologically confirmed by removal of greater than 90% of the insulin-degrading activity associated with an endosomal lysate using polyclonal antibodies to CD. Moreover, the elution profile of the endosomal acidic insulinase activity on a gel-filtration TSK-GEL G3000 SWXL high performance liquid chromatography column corresponded exactly with the elution profile of the immunoreactive 45-kDa mature form of endosomal CD. Using nondenaturating immunoprecipitation and immunoblotting procedures, other endosomal aspartic acid proteases such as cathepsin E and beta-site amyloid precursor protein-cleaving enzyme (BACE) were ruled out as candidate enzymes for the endosomal degradation of internalized insulin. Immunofluorescence studies showed a largely vesicular staining pattern for internalized insulin in rat hepatocytes that colocalized partially with CD. In vivo pepstatin A treatment was without any observable effect on the insulin receptor content of endosomes but augmented the phosphotyrosine content of the endosomal insulin receptor after insulin injection. These results suggest that CD is the endosomal acidic insulinase activity which catalyzes the rate-limiting step of the in vivo cleavage at the Phe(B24)-Phe(B25) bond, generating the inactive A(1-21)-B1-24 insulin intermediate.