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Analysis of the third cytosol factor involving in the neutrophil NADPH oxidase

中性粒细胞NADPH氧化酶第三胞质因子的分析

基本信息

批准号：
03671086
负责人：
NUNOI Hiroyuki
金额：
$ 1.28万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03671086/
关键词：
Chronic Granulomatous Disease (CGD)NADPH oxidase Small G Protein (rac1 2)p47-phox p67-phox

项目摘要

Chronic Granulomatous Disease (CGD) is an inherited condition rendering neutrophils incapable of killing invading pathogens. The neutrophils from the patients with CGD fail to produce the superoxide. This terminal superoxide generating enzyme (NADPH oxidase) had been found to be consisted of the following 5 components ; gp91 phagocyte oxidase(phox) and p22-phox (membrane-bound heterodimer proteins of cytochrome b558) and p47-phox, p67-phox and the unknown third cytsol factor.By the collaboration with professor Takai in Kobe University, we identified this third cytosol factor as one of the family of small G proteins (rac1 or 2). We also found NADPH oxidase system was regulated by the stimulatory and inhibitory GDP/GTP exchange proteins for racp21s (smgGDS and rho GDI) and the posttranslational processing of racp21s is important not only for their interaction with regulatory proteins but also for their NADPH oxidase activation.In addition, we report the molecular analysis of five CGD patients with the p67-phox deficiency. The RNAs were prepared from EB virus transformed B cell lines from the patients who had not any anti-p67 antibody reactive protein in the B cell cytosol. Protein coding sequences of p67-phox cDNA were analyzed by the direct sequence method using RT-PCR amplified cDNA. One of the patients, dinucleotides(AG) insertion at the position of 423 nucleotide residue was found. In another patient, 186 nucleotide residues from 694 to 879 residues were deleted. By this large deletion, most of src homology locus 3 (SH3) which was thought to be important for interacting with small G protein was truncated. In the other 3 patients, two other point mutations at the 566 and 1007 residues were found. However, the relationships of the mutation and the occurrence of CGD has not been established yet.By these analysis, we have been revealing the genetic background of CGD patients in Japan.

慢性肉芽肿病 (CGD) 是一种遗传性疾病，导致中性粒细胞无法杀死入侵的病原体。 CGD 患者的中性粒细胞不能产生超氧化物。这种末端超氧化物生成酶（NADPH氧化酶）被发现由以下5种成分组成； gp91吞噬细胞氧化酶(phox)和p22-phox(细胞色素b558的膜结合异二聚体蛋白)以及p47-phox、p67-phox和未知的第三细胞质因子。通过与神户大学高井教授的合作，我们鉴定出该第三细胞质因子是小G蛋白家族(rac1或2)之一。我们还发现 NADPH 氧化酶系统受到 racp21s 的刺激性和抑制性 GDP/GTP 交换蛋白（smgGDS 和 rho GDI）的调节，racp21s 的翻译后加工不仅对于它们与调节蛋白的相互作用很重要，而且对于它们的 NADPH 氧化酶激活也很重要。此外，我们报告了 5 名患有 p67-phox 的 CGD 患者的分子分析不足。 RNA是从B细胞胞浆中没有任何抗p67抗体反应蛋白的患者的EB病毒转化的B细胞系中制备的。使用RT-PCR扩增的cDNA通过直接测序方法分析p67-phox cDNA的蛋白质编码序列。其中一名患者在423个核苷酸残基位置发现了二核苷酸（AG）插入。在另一名患者中，694至879个残基中的186个核苷酸残基被删除。通过这种大的缺失，大部分被认为对于与小G蛋白相互作用很重要的src同源基因座3（SH3）被截断。在另外 3 名患者中，在 566 和 1007 残基处发现了另外两个点突变。然而，该突变与CGD发生的关系尚未确定。通过这些分析，我们揭示了日本CGD患者的遗传背景。