Effect of Hailey-Hailey disease mutations on the function of a new variant of human secretory pathway Ca2+/Mn2+-ATPase (hSPCA1)
Effect of Hailey-Hailey disease mutations on the function of a new variant of human secretory pathway Ca2+/Mn2+-ATPase (hSPCA1)
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DOI：
10.1074/jbc.m300509200
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发表时间：
2003-07-04
影响因子：
4.8
通讯作者：
Hovnanian, A
中科院分区：
文献类型：
--
作者：
Fairclough, RJ;Dode, L;Hovnanian, A
ATP 2C 1编码人分泌途径Ca ~（2+）/Mn ~（2+）ATP酶（hSPCA 1），是Hailey-Hailey病（HHD）的缺陷基因。为了探讨HHD的潜在病因，我们分析了HHD患者中发现的突变引起的hSPCA 1表达水平和功能的变化。将突变引入hSPCA 1d，一种在角质形成细胞中表达的新型剪接变体，这里首次描述。编码的全长可选外显子27和28，hSPCA 1d比以前确定的剪接变异体。该蛋白竞争性地将Ca ~（2+）和Mn ~（2+）以同样高的亲和力转运到COS-1细胞的高尔基体中。Ca 2+和Mn 2+依赖性磷酸酶中间体的形成，在正向（ATP-燃料）和反向（P-i-燃料）的方向也被证明。HHD突变蛋白L341 P、C344 Y、C411 R、T570 I和G789 R显示低水平表达，尽管mRNA水平正常且正确靶向高尔基体，这表明突变的hSPCA 1多肽的不稳定性或异常折叠。P201 L对酶循环的影响很小，而I580 V对E（1）的影响类似于P →> E-2-P构象转换。D 742 Y和G309 C缺乏由ATP形成的Ca 2+和Mn 2+依赖性磷酸酶。在这些突变体中保留了从Pi磷酸化的能力，但在D 742 Y中丧失了对Ca 2+和Mn 2+的敏感性，并且在G309 C中优先丧失了对Mn 2+的敏感性。这些结果突出了Asp-742在hSPCA 1离子结合位点的结构中所起的关键作用，并揭示了Gly-309在Mn 2+转运选择性中的作用。
ATP2C1, encoding the human secretory pathway Ca2+/Mn2+ ATPase (hSPCA1), was recently identified as the defective gene in Hailey-Hailey Disease (HHD), an autosomal dominant skin disorder characterized by persistent blisters and erosions. To investigate the underlying cause of HHD, we have analyzed the changes in expression level and function of hSPCA1 caused by mutations found in HHD patients. Mutations were introduced into hSPCA1d, a novel splice variant expressed in keratinocytes, described here for the first time. Encoded by the full-length of optional exons 27 and 28, hSPCA1d was longer than previously identified splice variants. The protein competitively transported Ca2+ and Mn2+ with equally high affinity into the Golgi of COS-1 cells. Ca2+- and Mn2+-dependent phosphoenzyme intermediate formation in forward (ATP-fuelled) and reverse (P-i-fuelled) directions was also demonstrated. HHD mutant proteins L341P, C344Y, C411R, T570I, and G789R showed low levels of expression, despite normal levels of mRNA and correct targeting to the Golgi, suggesting instability or abnormal folding of the mutated hSPCA1 polypeptides. P201L had little effect on the enzymatic cycle, whereas I580V caused a block in the E(1)similar to P --> E-2-P conformational transition. D742Y and G309C were devoid of Ca2+- and Mn2+-dependent phosphoenzyme formation from ATP. The capacity to phosphorylate from Pi was retained in these mutants but with a loss of sensitivity to both Ca2+ and Mn2+ in D742Y and a preferential loss of sensitivity to Mn2+ in G309C. These results highlight the crucial role played by Asp-742 in the architecture of the hSPCA1 ion-binding site and reveal a role for Gly-309 in Mn2+ transport selectivity.