Eskeland N L, Zhou A, Dinh T Q, Wu H, Parmer R J, Mains R E, O'Connor D T
Department of Medicine and Center for Molecular Genetics, University of California, San Diego, USA.
J Clin Invest. 1996 Jul 1;98(1):148-56. doi: 10.1172/JCI118760.
Chromogranins A and B and secretogranin II are a family of acidic proteins found in neuroendocrine secretory vesicles; these proteins contain multiple potential cleavage sites for proteolytic processing by the mammalian subtilisin-like serine endoproteases PC1 and PC2 (prohormone convertases 1 and 2), and furin. We explored the role of these endoproteases in chromogranin processing in AtT-20 mouse pituitary corticotropes. Expression of inducible antisense PC1 mRNA virtually abolished PC1 immunoreactivity on immunoblots. Chromogranin A immunoblots revealed chromogranin A processing, from both the NH2 and COOH termini, in both wild-type AtT-20 and AtT-20 antisense PC1 cells. After antisense PC1 induction, an approximately 66-kD chromogranin A NH2-terminal fragment as well as the parent chromogranin A molecule accumulated, while an approximately 50 kD NH2-terminal and an approximately 30 kD COOH-terminal fragment declined in abundance. Chromogranin B and secretogranin II immunoblots showed no change after PC1 reduction. [35S]Methionine/cysteine pulse-chase metabolic labeling in AtT-20 antisense PC1 and antisense furin cells revealed reciprocal changes in secreted chromogranin A COOH-terminal fragments (increased approximately 82 kD and decreased approximately 74 kD forms, as compared with wild-type AtT-20 cells) indicating decreased cleavage, while AtT-20 cells overexpressing PC2 showed increased processing to and secretion of approximately 71 and approximately 27 kD NH2-terminal chromogranin A fragments. Antisense PC1 specifically abolished regulated secretion of both chromogranin A and beta-endorphin in response to the usual secretagogue, corticotropin-releasing hormone. Moreover, immunocytochemistry demonstrated a relative decrease of chromogranin A in processes (where regulated secretory vesicles accumulate) of AtT-20 cells overexpressing either PC1 or PC2. These results demonstrate that chromogranin A is a substrate for the endogenous endoproteases PC1 and furin in vivo, and that such processing influences its trafficking into the regulated secretory pathway; furthermore, lack of change in chromogranin B and secretogranin II cleavage after diminution of PCl suggests that the action of PC1 on chromogranin A may be specific within the chromogranin/secretogranin protein family.
嗜铬粒蛋白A、B和分泌粒蛋白II是一类酸性蛋白，存在于神经内分泌分泌小泡中；这些蛋白含有多个潜在的切割位点，可被哺乳动物枯草杆菌蛋白酶样丝氨酸内切蛋白酶PC1和PC2（激素原转化酶1和2）以及弗林蛋白酶进行蛋白水解加工。我们探讨了这些内切蛋白酶在AtT-20小鼠垂体促肾上腺皮质激素细胞中嗜铬粒蛋白加工过程中的作用。诱导型反义PC1 mRNA的表达实际上消除了免疫印迹上的PC1免疫反应性。嗜铬粒蛋白A免疫印迹显示，在野生型AtT-20和AtT-20反义PC1细胞中，嗜铬粒蛋白A从NH2和COOH末端都有加工过程。反义PC1诱导后，一个约66-kD的嗜铬粒蛋白A NH2末端片段以及亲本嗜铬粒蛋白A分子积累，而一个约50 kD的NH2末端片段和一个约30 kD的COOH末端片段丰度下降。嗜铬粒蛋白B和分泌粒蛋白II免疫印迹显示PC1减少后无变化。AtT-20反义PC1和反义弗林蛋白酶细胞中的[35S]甲硫氨酸/半胱氨酸脉冲追踪代谢标记显示，分泌的嗜铬粒蛋白A COOH末端片段发生了相反的变化（与野生型AtT-20细胞相比，约82 kD的形式增加，约74 kD的形式减少），表明切割减少，而过表达PC2的AtT-20细胞显示出对约71 kD和约27 kD NH2末端嗜铬粒蛋白A片段的加工增加和分泌增加。反义PC1特异性消除了嗜铬粒蛋白A和β-内啡肽对通常的促分泌剂促肾上腺皮质激素释放激素的调节分泌。此外，免疫细胞化学显示，在过表达PC1或PC2的AtT-20细胞的突起（调节性分泌小泡积累的部位）中，嗜铬粒蛋白A相对减少。这些结果表明，嗜铬粒蛋白A在体内是内源性内切蛋白酶PC1和弗林蛋白酶的底物，这种加工影响其进入调节性分泌途径的运输；此外，PC1减少后嗜铬粒蛋白B和分泌粒蛋白II切割没有变化，表明PC1对嗜铬粒蛋白A的作用在嗜铬粒蛋白/分泌粒蛋白家族中可能是特异性的。
