A heterozygous mutation of β-actin associated with neutrophil dysfunction and recurrent infection
A heterozygous mutation of β-actin associated with neutrophil dysfunction and recurrent infection
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DOI：
10.1073/pnas.96.15.8693
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发表时间：
1999-07-20
影响因子：
11.1
通讯作者：
Kanegasaki, S
中科院分区：
文献类型：
--
作者：
Nunoi, H;Yamazaki, T;Kanegasaki, S
非肌肉肌动蛋白突变引起的人类疾病尚未报道。我们在此报告一位女性患者的非肌肉肌动蛋白变异，她有反复感染、光敏性和智力迟钝。她还出现中性粒细胞趋化性、超氧化物生成和膜电位反应异常。来自该患者的嗜中性粒细胞和其他细胞类型的蛋白质的二维PAGE分析表明，在42 kDa处迁移的独特蛋白质点，相对于正常β-和γ-肌动蛋白，pI略微向中性移动。酶切肽图谱和蛋白质印迹分析表明该斑点为异常肌动蛋白。通过使用来自患者细胞的mRNA构建全长cDNA文库，并通过体外翻译方法鉴定编码突变型β-肌动蛋白分子的cDNA。克隆的测序表明G-1174被A取代，预测β-肌动蛋白中谷氨酸-364被赖氨酸取代，并消除了在正常β-肌动蛋白序列中发现的HinfI DNA酶限制位点。通过HinfI酶切和测序，证实了患者基因组DNA中一个等位基因的突变。虽然没有检测到肌动蛋白的无细胞聚合缺陷，但这种缺陷存在于与profilin和其他肌动蛋白调节分子结合的重要结构域中。事实上，突变的肌动蛋白与profilin结合的效率低于正常的肌动蛋白。该患者中性粒细胞和其他细胞中突变型β-肌动蛋白的杂合表达可能以显性负性方式对依赖于非肌肉肌动蛋白功能的细胞活性产生不利影响。
A human disorder caused by mutation in nonmuscle actin has not been reported. We report here a variant of nonmuscle actin in a female patient with recurrent infections, photosensitivity, and mental retardation. She also had abnormalities in neutrophil chemotaxis, superoxide production, and membrane potential response. Two-dimensional PAGE analysis of proteins from neutrophils and other cell types from this patient demonstrated a unique protein spot migrating at 42 kDa with pI shifted slightly to neutral relative to normal beta- and gamma-actin. Digestion peptide mapping and Western blotting showed this spot to be an abnormal actin. A full-length cDNA library was constructed by using mRNA from patient's cells and cDNA encoding the mutant beta-actin molecule was identified by an in vitro translation method. Sequencing of the clones demonstrated a G-1174 to A substitution, predicting a glutamic acid-364 to lysine substitution in beta-actin and eliminating a HinfI DNase restriction site found in normal beta-actin sequence. By HinfI digestion and by sequencing, the mutation in one allele of patient's genomic DNA was confirmed. Though no defect in cell-free polymerization of actin was detected, this defect lies in a domain important for binding to profilin and other actin-regulatory molecules. In fact, the mutant actin bound to profilin less efficiently than normal actin did. Heterozygous expression of mutant beta-actin in neutrophils and other cells of this patient may act in a dominant-negative fashion to adversely affect cellular activities dependent on the function of nonmuscle actin.