Purification and characterization of insoluble SOD in the animal models of amyotrophic lateral sclerosis

肌萎缩性脊髓侧索硬化症动物模型中不溶性 SOD 的纯化和表征

• 批准号: 15500237
• 负责人: MIYAMOTO Yasuhide
• 金额: $ 1.92万
• 依托单位: Osaka Medical Center for Cancer and Cardiovasclar Diseases
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15500237/
• 关键词: amyotrophic lateral sclerosis; insoluble; SOD; 脊髄; 脳幹; 立体構造; 筋萎縮性側索硬化症; 不溶性画分

The brain and spinal cord of mutant SOD1(G93A, H46R) transgenic animals, which are animal models of amyotrophic lateral sclerosis(ALS), were fractionated into three fractions such as Tris buffered saline(TBS) soluble, NP40 soluble and NP40 insoluble fractions. Most of the mutant SOD1s were soluble in TBS, with only 10 to 30% as much protein in the NP40 fraction. TBS-soluble and NP40-soluble SOD1s from all animals were evenly distributed in the forebrain, spinal cord, cerebellum and brain stem. In contrast, the detergent insoluble mutant SOD1,both G93A and H46R, was isolated only from the spinal cord and brainstem, which are two regions of the central nervous system specifically affected by ALS. The activity of G93A in the NP40 insoluble fraction was significantly less than that in TBS soluble fraction. When mutant SOD1s in three fractions were subjected to immunoaffinity chromatography using a polyclonal antibody against SOD1,we found that mutant SOD1 in NP40 insoluble fraction had a higher affinity for a polyclonal antibody than that in TBS and NP40 soluble fractions. Mutant SOD1s from the three fractions purified on an affinity column had the same molecular mass as judged by mass spectrometry. Wild type and mutant SOD1(WT, A4V, G37R, H46R, G93A) were overproduced by sf21 insect cell system and were purified. Mutant SOD1s were barely detected by three monoclonal antibodies in Western blot analyses. Mutant SOD1s by DTT or heat treatment showed a lower immunoreactivity against the monoclonal antibodies. Because all the epitopes of these antibodies are mapped within the Greek key loop, these data suggest that different conformational changes occur in the loop between wild and mutant SOD1s during the unfolding process.

突变SOD1（G93A，H46R）转基因动物的大脑和脊髓是肌萎缩性侧索硬化症（ALS）的动物模型，分为三个馏分，例如Tris Buffered Saline（TBS）可溶性，NP40可溶性，NP40可溶性和NP40不溶性分裂。大多数突变体SOD1都可溶于TBS，在NP40馏分中只有10％至30％的蛋白质。来自所有动物的TBS溶解和NP40溶解的SOD1均匀分布在前脑，脊髓，小脑和脑干中。相反，仅从脊髓和脑干中分离出去污剂的不溶性突变体SOD1，G93A和H46R，这是中枢神经系统的两个区域，这些区域受ALS的特异性影响。 NP40不溶性级分中G93a的活性明显小于TBS可溶性部分的活性。当使用针对SOD1的多克隆抗体进行三个部分中的突变SOD1进行免疫亲和力色谱法时，我们发现NP40不溶性级分中的突变SOD1对多克隆抗体的亲和力比TBS和NP40可溶性馏分具有更高的亲和力。从亲和力柱上纯化的三个部分的突变SOD1具有与质谱法相同的分子质量。 SF21昆虫细胞系统过量生产野生型和突变体SOD1（WT，A4V，G37R，H46R，G93A）并被纯化。在蛋白质印迹分析中，几乎无法通过三种单克隆抗体检测到突变的SOD1。通过DTT或热处理进行突变的SOD1，对单克隆抗体的免疫反应性较低。由于这些抗体的所有表位都映射在希腊钥匙循环中，因此这些数据表明在展开过程中野生和突变体SOD1之间的循环中发生了不同的构象变化。

项目成果

Takamiya Rina: "Glycation proceeds faster in mutated Cu, Zn-superoxide dismutases related to familial amyotrophic lateral"FASEB Journal. 17.8. 938-940 (2003)

Takamiya Rina：“与家族性肌萎缩侧索相关的突变铜、锌超氧化物歧化酶中糖化进行得更快”FASEB 杂志。

Glycation proceeds faster in mutated Cu, Zn-superoxide dismutases related to familial amyotrophic lateral sclerosis.

与家族性肌萎缩侧索硬化症相关的突变铜、锌超氧化物歧化酶中糖化进行得更快。

• 发表时间: 2003
• 期刊: FASEB Journal 17
• 作者: Fujiwara N;Miyamoto Y;Ogasahara K;Takahashi M;Ikegami T;Takamiya R;Suzuki K;Taniguchi N.;Takamiya R
• 通讯作者: Takamiya R