Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificity
Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificity
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DOI：
10.1111/j.1742-4658.2005.04692.x
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发表时间：
2005-06-01
期刊：
影响因子：
5.4
通讯作者：
Ferro, ES
中科院分区：
文献类型：
--
作者：
Oliveira, V;Garrido, PAG;Ferro, ES
金属内肽酶 24.15 (EP24.15) 作为分泌蛋白普遍存在于细胞外环境中。在细胞外，这种酶会降解多种含有 5 至 17 个氨基酸的神经肽（例如促性腺激素释放激素、缓激肽、阿片类药物和神经降压素）。神经胶质瘤 C6 细胞的 EP24.15 组成型分泌被证明可通过降低细胞外钙浓度而线性刺激。在本报告中，我们证明细胞外钙浓度对细胞外（细胞相关+培养基）酶的总量没有影响。事实上，通过共聚焦和电子显微镜进行的免疫细胞化学分析表明，钙的缺乏有利于酶从质膜脱落到培养基中。通过定点诱变改变了 EP24.15 上的两个假定的钙结合位点（D93 和 D159），以研究它们对酶在细胞表面结合的可能贡献。这些突变的重组蛋白在酶活性、二级结构、钙敏感性和免疫反应性方面与野生型酶相似。然而，通过共聚焦显微镜进行的免疫细胞化学分析一致显示，与野生型酶相比，D93A 突变体与神经胶质瘤 C6 细胞质膜结合的能力降低。这些数据和通过X射线衍射确定的酶结构模型表明D93位于酶表面并且与EP24.15的膜缔合一致。此外，还观察到钙会诱导独特荧光底物上 EP24.15 切割位点的重大变化。这些数据表明钙可能是 ep24.15 细胞功能的重要调节剂。
The metalloendopeptidase 24.15 (EP24.15) is ubiquitously present in the extracellular environment as a secreted protein. Outside the cell, this enzyme degrades several neuropeptides containing from 5 to 17 amino acids (e.g. gonadotropin releasing hormone, bradykinin, opioids and neurotensin). The constitutive secretion of EP24.15 from glioma C6 cells was demonstrated to be stimulated linearly by reduced concentrations of extracellular calcium. In the present report we demonstrate that extracellular calcium concentration has no effect on the total amount of the extracellular (cell associated + medium) enzyme. Indeed, immuno-cytochemical analyses by confocal and electron microscopy suggested that the absence of calcium favors the enzyme shedding from the plasma membrane into the medium.. Two putative calcium-binding sites on EP24.15 (D93 and D159) were altered by site-directed mutagenesis to investigate their possible contribution to binding of the enzyme at the cell surface. These mutated recombinant proteins behave similarly to the wild-type enzyme regarding enzymatic activity, secondary structure, calcium sensitivity and immunoreactivity. However, immunocytochemical analyses by confocal microscopy consistently show a reduced ability of the D93A mutant to associate with the plasma membrane of glioma C6 cells when compared with the wild-type enzyme. These data and the model of the enzyme's structure as determined by X-ray diffraction suggest that D93 is located at the enzyme surface and is consistent with membrane association of EP24.15. Moreover, calcium was also observed to induce a major change in the EP24.15 cleavage site on distinctive fluorogenic substrates. These data suggest that calcium may be an important modulator of ep24.15 cell function.