The proteolytic maturation of prohormone convertase 2 (PC2) is a pH-driven process.
The proteolytic maturation of prohormone convertase 2 (PC2) is a pH-driven process.
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DOI：
10.1006/abbi.1998.1033
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发表时间：
1999-02
影响因子：
3.9
通讯作者：
N. Lamango;E. Apletalina;June Liu;Iris Lindberg
中科院分区：
文献类型：
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作者：
N. Lamango;E. Apletalina;June Liu;Iris Lindberg
从过表达激素原转化酶（PC）前体proPC 2和神经内分泌蛋白7 B2的21-kDa氨基末端部分的CHO细胞的培养基中纯化的重组proPC 2可以自发地转化为活性物质。在本报告中，我们的特点proPC 2酶原转化过程。成熟的66 kDa的酶的测序揭示了一个单一的切割位点在成对的碱性位点氨基末端的GYRDI序列。与成熟的PC 2活性相反，proPC 2转化既不被真核枯草杆菌蛋白酶抑制剂pCMS抑制，也不被特异性PC 2抑制剂7 B2 CT肽抑制，这表明proPC 2转化反应与成熟的PC 2对合成底物的水解之间存在显著差异。为了支持这一观点，proPC 2转化不依赖于钙，并且不受5 mM EDTA的影响。proPC 2的转化率保持相似，酶原浓度相差10倍，这意味着分子内而不是分子间的激活机制。有趣的是，proPC 2转化率是非常pH依赖性的，最广泛地发生在pH 4.0和4.9之间。两者合计，我们的研究结果表明，细胞proPC 2成熟发生通过自催化，分子内的过程控制，而不是由7 B2抑制，也不是由钙水平，但由pH梯度下降沿着分泌途径。
Recombinant proPC2 purified from the medium of CHO cells overexpressing both the prohormone convertase (PC) precursor proPC2 and the 21-kDa amino terminal portion of the neuroendocrine protein 7B2 can spontaneously convert to an active species. In the present report, we have characterized the proPC2 zymogen conversion process. Sequencing of the mature 66 kDa enzyme revealed a single site of cleavage at the paired basic site amino terminal to the GYRDI sequence. In contrast to mature PC2 activity, proPC2 conversion was inhibited neither by the eukaryotic subtilisin inhibitor pCMS nor by the specific PC2 inhibitor, 7B2 CT peptide, suggesting significant differences between the proPC2 conversion reaction and the hydrolysis of synthetic substrates by mature PC2. In support of this idea, proPC2 conversion was not calcium dependent and was unaffected by 5 mM EDTA. The rate of conversion of proPC2 remained similar with a 10-fold difference in zymogen concentration, implicating an intramolecular rather than intermolecular mechanism of activation. Interestingly, the rate of proPC2 conversion was extremely pH dependent, occurring most extensively between pHs 4.0 and 4.9. Taken together, our results suggest that cellular proPC2 maturation occurs via an autocatalytic, intramolecular process controlled not by 7B2 inhibition nor by calcium levels, but by the decreasing pH gradient along the secretory pathway.