van Horssen A M, van den Hurk W H, Bailyes E M, Hutton J C, Martens G J, Lindberg I
Department of Animal Physiology, University of Nijmegen, Toernooiveld, The Netherlands.
J Biol Chem. 1995 Jun 16;270(24):14292-6. doi: 10.1074/jbc.270.24.14292.
The highly conserved polypeptide 7B2 and the subtilisin-related prohormone convertases PC1/PC3 and PC2 are broadly distributed in neurons and endocrine cells and are localized to secretory granules. We recently showed that recombinant 7B2 is in vitro a potent inhibitor of PC2 activity, but not of PC1/PC3, and that newly synthesized 7B2 is transiently associated with proPC2 in vivo. In the present study, in vitro mutagenesis was used to identify the region within the 7B2 sequence responsible for the inhibition of PC2. Mutant proteins were produced in a prokaryotic expression system and their effects on PC1/PC3 and PC2 activities were studied by two different in vitro enzyme assays. None of the 7B2 mutant proteins inhibited PC1/PC3 activity. Truncation studies revealed that a short segment within the COOH-terminal portion of 7B2 is critical for its inhibitory effect on PC2. This segment contains a pair of basic amino acid residues which may represent a recognition motif for PC2. Single amino acid substitutions within this Lys171-Lys172 site strongly diminished and a double mutation abolished the inhibitory potency of 7B2. Our results indicate that, although amino acid residues directly surrounding this dibasic pair also contribute to PC2 inhibition, the Lys171-Lys172 site is particularly important for the ability of 7B2 to inhibit PC2.
高度保守的多肽7B2以及枯草杆菌蛋白酶相关的激素原转化酶PC1/PC3和PC2广泛分布于神经元和内分泌细胞中，并定位于分泌颗粒。我们最近发现，重组7B2在体外是PC2活性的有效抑制剂，但不是PC1/PC3的抑制剂，并且新合成的7B2在体内与前体PC2短暂结合。在本研究中，利用体外诱变来确定7B2序列中负责抑制PC2的区域。在原核表达系统中产生突变蛋白，并通过两种不同的体外酶分析研究它们对PC1/PC3和PC2活性的影响。7B2突变蛋白均未抑制PC1/PC3活性。截短研究表明，7B2羧基末端部分的一个短片段对其对PC2的抑制作用至关重要。该片段包含一对碱性氨基酸残基，这可能代表PC2的识别基序。在这个Lys171-Lys172位点内的单氨基酸取代大大降低了7B2的抑制效力，而双突变则消除了其抑制效力。我们的结果表明，尽管直接围绕这个双碱基对的氨基酸残基也有助于抑制PC2，但Lys171-Lys172位点对于7B2抑制PC2的能力尤为重要。
