Miyauchi Yuki, Daiho Takashi, Yamasaki Kazuo, Takahashi Hidetoshi, Ishida-Yamamoto Akemi, Danko Stefania, Suzuki Hiroshi, Iizuka Hajime
Departments of Biochemistry and Dermatology, Asahikawa Medical College, Asahikawa 078-8510, Japan.
J Biol Chem. 2006 Aug 11;281(32):22882-95. doi: 10.1074/jbc.M601966200. Epub 2006 Jun 9.
We examined possible defects of sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b) associated with its 51 mutations found in Darier disease (DD) pedigrees, i.e. most of the substitution and deletion mutations of residues reported so far. COS-1 cells were transfected with each of the mutant cDNAs, and the expression and function of the SERCA2b protein was analyzed with microsomes prepared from the cells and compared with those of the wild type. Fifteen mutants showed markedly reduced expression. Among the other 36, 29 mutants exhibited completely abolished or strongly inhibited Ca2+-ATPase activity, whereas the other seven possessed fairly high or normal ATPase activity. In four of the aforementioned seven mutants, Ca2+ transport activity was significantly reduced or almost completely lost, therefore uncoupled from ATP hydrolysis. The other three were exceptional cases as they were seemingly normal in protein expression and Ca2+ transport function, but were found to have abnormalities in the kinetic properties altered by the three mutations, which happened to be in the three DD pedigrees found by us previously (Sato, K., Yamasaki, K., Daiho, T., Miyauchi, Y., Takahashi, H., Ishida-Yamamoto, A., Nakamura, S., Iizuka, H., and Suzuki, H. (2004) J. Biol. Chem. 279, 35595-35603). Collectively, our results indicated that in most cases (48 of 51) DD mutations cause severe disruption of Ca2+ homeostasis by the defects in protein expression and/or transport function and hence DD, but even a slight disturbance of the homeostasis will result in the disease. Our results also provided further insight into the structure-function relationship of SERCAs and revealed critical regions and residues of the enzyme.
我们研究了肌浆（内质）网Ca2+ -ATP酶2b（SERCA2b）的可能缺陷，这些缺陷与其在 Darier病（DD）家系中发现的51种突变相关，即迄今为止报道的大多数残基的替换和缺失突变。用每种突变cDNA转染COS-1细胞，并用从细胞制备的微粒体分析SERCA2b蛋白的表达和功能，并与野生型进行比较。15种突变体表现出明显降低的表达。在其他36种突变体中，29种突变体的Ca2+ -ATP酶活性完全丧失或受到强烈抑制，而其他7种具有相当高或正常的ATP酶活性。在上述7种突变体中的4种中，Ca2+ 转运活性显著降低或几乎完全丧失，因此与ATP水解解偶联。另外3种是例外情况，因为它们在蛋白质表达和Ca2+ 转运功能方面看似正常，但发现其动力学特性因三种突变而改变，这三种突变恰好出现在我们之前发现的三个DD家系中（佐藤，K.，山崎，K.，代保，T.，宫内，Y.，高桥，H.，石田 - 山本，A.，中村，S.，饭冢，H.，和铃木，H.（2004年）《生物化学杂志》279，35595 - 35603）。总体而言，我们的结果表明，在大多数情况下（51种中的48种），DD突变通过蛋白质表达和/或转运功能缺陷导致Ca2+ 稳态的严重破坏，从而引发DD，但即使是稳态的轻微干扰也会导致疾病。我们的结果还进一步深入了解了SERCA的结构 - 功能关系，并揭示了该酶的关键区域和残基。
