Zhou Y, Rovere C, Kitabgi P, Lindberg I
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112, USA.
J Biol Chem. 1995 Oct 20;270(42):24702-6. doi: 10.1074/jbc.270.42.24702.
The proteinase mPC1, a neuroendocrine member of the mammalian family of subtilisin-like enzymes, has previously been shown to be converted to a carboxyl-terminally truncated 66-kDa form during transport through the secretory pathway. The cleavage site and the function of this carboxyl-terminal truncation event are unknown. We have performed site-directed mutagenesis of two paried basic sites in the mPC1 carboxyl-terminal tail and expressed these constructs in PC12 cells, a rat pheochromocytoma known to lack endogenous PC1. We found that the most likely site for the truncation event was at Arg590-Arg591 since mutation of this site to Lys-His prevented processing of 87-kDa PC1. A PC1 mutant carboxyl-terminally truncated at this site and expressed in PC12 cells was efficiently routed to the secretory pathway and stored in secretory granules, indicating that the carboxyl-terminal extension is not required for sorting of this enzyme. The function of the various PC1 constructs was assessed by analyzing proneurotensin cleavage to various forms. The carboxyl-terminally truncated PC1 mutant was found to perform most of the cleavages of this precursor as well as wild-type PC1; however, the blockade mutant processed proneurotensin much less efficiently. Differences between the site preferences of the various enzymes were noted. Our results support the notion that carboxyl-terminal processing of PC1 serves to regulate PC1 activity.
蛋白酶mPC1是哺乳动物类枯草杆菌蛋白酶样酶家族的神经内分泌成员，先前已表明其在通过分泌途径转运过程中会转化为羧基末端截短的66 kDa形式。该羧基末端截短事件的切割位点和功能尚不清楚。我们对mPC1羧基末端尾巴中的两个成对碱性位点进行了定点诱变，并在PC12细胞中表达了这些构建体，PC12细胞是一种已知缺乏内源性PC1的大鼠嗜铬细胞瘤。我们发现截短事件最可能的位点是在Arg590 - Arg591处，因为将该位点突变为Lys - His可阻止87 kDa的PC1进行加工。在该位点羧基末端截短并在PC12细胞中表达的PC1突变体被有效地导向分泌途径并储存在分泌颗粒中，这表明该酶的分选不需要羧基末端延伸。通过分析前神经降压素切割成各种形式来评估各种PC1构建体的功能。发现羧基末端截短的PC1突变体与野生型PC1一样能进行该前体的大部分切割；然而，阻断突变体处理前神经降压素的效率要低得多。注意到了各种酶在切割位点偏好上的差异。我们的结果支持这样一种观点，即PC1的羧基末端加工有助于调节PC1的活性。
