Tissue Nonspecific Alkaline Phosphatase Is Activated via a Two-step Mechanism by Zinc Transport Complexes in the Early Secretory Pathway
Tissue Nonspecific Alkaline Phosphatase Is Activated via a Two-step Mechanism by Zinc Transport Complexes in the Early Secretory Pathway
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DOI：
10.1074/jbc.m111.227173
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发表时间：
2011-05-06
影响因子：
4.8
通讯作者：
Kambe, Taiho
中科院分区：
文献类型：
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作者：
Fukunaka, Ayako;Kurokawa, Yayoi;Kambe, Taiho
许多酶在通过早期分泌途径的过程中通过与锌结合而发挥功能。锌转运蛋白（ZnTs）在这一过程中起重要作用。我们以前已经表明，两个锌转运复合物，ZnT 5/ZnT 6异二聚体和ZnT 7同源寡聚体，需要激活碱性磷酸酶，通过将它们从脱辅基转换为全形式。在这里，我们研究了这种激活的分子机制。在鸡DT 40细胞中表达的ZnT 1和ZnT 4对组织非特异性碱性磷酸酶（TNAP）的激活没有贡献。TNAP在DT 40细胞中的两个ZnT复合物缺乏的活性降低，没有恢复锌的补充，也没有通过外源性表达的其他ZnTs，增加锌含量的分泌途径。此外，我们表明，用锌转运缺陷型ZnT 5突变体重建的ZnT 5/ZnT 6异二聚体的表达不能恢复TNAP活性，但可以稳定TNAP蛋白作为载脂蛋白形式，而与锌状态无关。这些研究结果表明，TNAP被激活，而不是简单的被动锌结合，但通过一个精心设计的两步机制，通过蛋白质稳定化，然后从载脂蛋白的酶转化为全形式与锌负载的ZnT复合物在早期分泌途径。
A number of enzymes become functional by binding to zinc during their journey through the early secretory pathway. The zinc transporters (ZnTs) located there play important roles in this step. We have previously shown that two zinc transport complexes, ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers, are required for the activation of alkaline phosphatases, by converting them from the apo- to the holo-form. Here, we investigated the molecular mechanisms of this activation. ZnT1 and ZnT4 expressed in chicken DT40 cells did not contribute to the activation of tissue nonspecific alkaline phosphatase ( TNAP). The reduced activity of TNAP in DT40 cells deficient in both ZnT complexes was not restored by zinc supplementation nor by exogenous expression of other ZnTs that increase the zinc content in the secretory pathway. Moreover, we showed that expression of ZnT5/ZnT6 heterodimers reconstituted with zinc transport-incompetent ZnT5 mutant failed to restore TNAP activity but could stabilize the TNAP protein as the apo- form, regardless of zinc status. These findings demonstrate that TNAP is activated not simply by passive zinc binding but by an elaborate two-step mechanism via protein stabilization followed by enzyme conversion from the apo- to the holo-form with zinc loaded by ZnT complexes in the early secretory pathway.