Protein Oxidative Damage in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis
Protein Oxidative Damage in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis
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DOI：
10.1046/j.1471-4159.1998.71052041.x
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发表时间：
1998-11
影响因子：
4.7
通讯作者：
P. K. Andrus;T. Fleck;M. Gurney;E. Hall
中科院分区：
文献类型：
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作者：
P. K. Andrus;T. Fleck;M. Gurney;E. Hall
摘要：在一些家族性肌萎缩侧索硬化症（FALS）患者中发现的Cu，Zn超氧化物歧化酶（Cu，Zn-SOD）基因（SOD 1）中的Gly 93 →Ala突变已被证明会导致突变酶产生的羟基自由基异常增加，这可能会导致脊髓运动神经元的氧化损伤。在本研究中，我们分析了与非转基因小鼠相比，过表达SOD 1突变[TgN（SOD 1-G93 A）G1 H]的转基因FALS小鼠的腰和颈脊髓蛋白的氧化损伤程度。通过二硝基苯肼（DNPH）测定法分光光度法测量组织蛋白羰基含量，检查总蛋白氧化。调查了四个年龄段：30天（运动神经元前病变和临床疾病）、60天（病变开始后，但处于疾病前）、100天（运动神经元和功能丧失> 50%）和120天（接近完全后肢瘫痪）。30天大的TgN（SOD 1-G93 A）G1 H小鼠的蛋白质羰基含量是年龄匹配的非转基因小鼠的两倍。然而，在60和100日龄时，水平是相同的。然后，在100至120日龄之间，与非转基因小鼠或过表达野生型人Cu，Zn-SOD [TgN（SOD 1）N29]的转基因动物相比，TgN（SOD 1-G93 A）G1 H小鼠中的水平显著增加（557%）。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离和蛋白质印迹免疫测定证实了脊髓蛋白羰基水平在100-120天内的增加，这使得能够使用针对DNPH衍生蛋白的单克隆抗体鉴定重度氧化的单个蛋白质。通过免疫沉淀法鉴定了一条氧化程度更高的蛋白条带（14 kDa），主要是Cu，Zn-SOD。对Cu，Zn-SOD蛋白羰基化程度的蛋白质印迹比较显示，120天龄TgN（SOD 1-G93 A）G1 H小鼠脊髓样本中的水平显著高于年龄匹配的非转基因或TgN（SOD 1）N29小鼠。这些结果表明，与G93 A SOD 1突变和/或脂质过氧化衍生的自由基种类（过氧基或烷氧基）相关的羟基自由基产生增加导致广泛的蛋白质氧化损伤，并且Cu，Zn‐SOD本身是一个关键目标，这可能会损害其抗氧化功能。
Abstract: The Gly93→Ala mutation in the Cu,Zn superoxide dismutase (Cu,Zn‐SOD) gene (SOD1) found in some familial amyotrophic lateral sclerosis (FALS) patients has been shown to result in an aberrant increase in hydroxyl radical production by the mutant enzyme that may cause oxidative injury to spinal motor neurons. In the present study, we analyzed the extent of oxidative injury to lumbar and cervical spinal cord proteins in transgenic FALS mice that overexpress the SOD1 mutation [TgN(SOD1‐G93A)G1H] in comparison with nontransgenic mice. Total protein oxidation was examined by spectrophotometric measurement of tissue protein carbonyl content by the dinitrophenylhydrazine (DNPH) assay. Four ages were investigated: 30 (pre‐motor neuron pathology and clinical disease), 60 (after initiation of pathology, but pre‐disease), 100 (∼50% loss of motor neurons and function), and 120 (near complete hindlimb paralysis) days. Protein carbonyl content in 30‐day‐old TgN(SOD1‐G93A)G1H mice was twice as high as the level found in age‐matched nontransgenic mice. However, at 60 and 100 days of age, the levels were the same. Then, between 100 and 120 days of age, the levels in the TgN(SOD1‐G93A)G1H mice increased dramatically (557%) compared with either the nontransgenic mice or transgenic animals that overexpress the wild‐type human Cu,Zn‐SOD [TgN(SOD1)N29]. The 100–120‐day increase in spinal cord protein carbonyl levels was confirmed by sodium dodecyl sulfate‐polyacrylamide gel electrophoretic separation and western blot immunoassay, which enabled the identification of heavily oxidized individual proteins using a monoclonal antibody against DNPH‐derivatized proteins. One of the more heavily oxidized protein bands (14 kDa) was identified by immunoprecipitation as largely Cu,Zn‐SOD. Western blot comparison of the extent of Cu,Zn‐SOD protein carbonylation revealed that the level in spinal cord samples from 120‐day‐old TgN(SOD1‐G93A)G1H mice was significantly higher than that found in age‐matched nontransgenic or TgN(SOD1)N29 mice. These results suggest that the increased hydroxyl radical production associated with the G93A SOD1 mutation and/or lipid peroxidation‐derived radical species (peroxyl or alkoxyl) causes extensive protein oxidative injury and that the Cu,Zn‐SOD itself is a key target, which may compromise its antioxidant function.