Identification and characterization of a temperature-sensitive R268H mutation in the human succinyl-CoA:3-ketoacid CoA transferase (SCOT) gene
Identification and characterization of a temperature-sensitive R268H mutation in the human succinyl-CoA:3-ketoacid CoA transferase (SCOT) gene
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DOI：
10.1016/j.ymgme.2007.07.005
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发表时间：
2007-11-01
影响因子：
3.8
通讯作者：
Kondo, Naomi
中科院分区：
文献类型：
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作者：
Fukao, Toshiyuki;Kursula, Petri;Kondo, Naomi
琥珀酰辅酶A：3-酮酸辅酶A转移酶（SCOT）缺乏会导致发作性酮酸中毒。我们在南非遇到了一个兄弟姐妹的病例，在他们的基因组DNA中发现了一种新的纯合突变（R268 H）。使用免疫印迹分析在其成纤维细胞中非常微弱地检测到突变SCOT蛋白。在37 ℃下对R268 H突变体cDNA的瞬时表达分析显示，清楚地检测到R268 H突变体蛋白，多达50%的野生型，以及40%的残余SCOT活性，因此R268 H首次被认为不是致病突变。由于未发现其他突变，因此通过进一步的瞬时表达分析重新评估R268 H突变。R268 H突变蛋白的积累显示出强烈的温度依赖性;计算出在30 ℃、37 ℃和40 ℃下，在GS 01（S283 X的纯合子）的SV 40转化的成纤维细胞中表达的残余SCOT活性分别为59.7%、34%和4%。R268 H蛋白的SCOT活性比野生型更容易受到50 ℃热处理的影响。这些结果表明，R268 H突变体蛋白在温度敏感性方面明显比野生型更不稳定。此外，对SCOT的三维结构的分析表明，R268 H突变预期会破坏R268和D52之间的保守盐桥，这将预期会导致蛋白质稳定性降低。因此，我们最终得出结论，R268 H突变是一种致病突变。瞬时表达分析中突变蛋白的稳定性并不总是反映患者成纤维细胞中的情况。(C)2007年爱思唯尔公司All rights reserved.
Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency causes episodic ketoacidosis. We encountered a case of siblings in South Africa in whom a novel homozygous mutation (R268H) was found in genomic DNA. Mutant SCOT protein was very faintly detected in their fibroblasts using immunoblot analysis. Transient expression analysis of R268H mutant cDNA at 37 C revealed that the R268H mutant protein was clearly detected, as much as 50% wild-type, together with 40% residual SCOT activities, hence R268H was first regarded as not being a disease-causing mutation. Since no other mutation was identified, R268H mutation was re-evaluated by further transient expression analysis. Accumulation of the R268H mutant protein was revealed to be strongly temperature dependent; residual, SCOT activities were calculated to be 59.7%, 34%, and 4%, respectively, in expression at 30 degrees C, 37 degrees C, and 40 degrees C in SV40-transformed fibroblasts of GS01(a homozygote of S283X). SCOT activity of the R268H protein was more vulnerable than the wild-type to heat treatment at 50 C. These results indicated that the R268H mutant protein was clearly more unstable than the wild-type in a temperature-sensitive manner. Furthermore, an analysis of the three-dimensional structure of SCOT showed that the R268H mutation was expected to break a conserved salt bridge between R268 and D52, which would be expected to lead to decreased stability of the protein. Hence we finally concluded that the R268H mutation is a disease-causing one. The stability of mutant protein in transient expression analysis does not always reflect the condition in patients' fibroblasts. (C) 2007 Elsevier Inc. All rights reserved.