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

慢性肉芽肿病（CGD）是一种遗传性疾病，使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。该末端超氧化物生成酶（NADPH氧化酶）由以下5个组分组成：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）和p47-phox， p67-phox和未知的第三细胞溶胶因子。通过与神户大学Takai教授的合作，我们确定了这第三个细胞质因子是小G蛋白家族中的一个（rac1或2）。我们还发现NADPH氧化酶系统受racp21s的刺激和抑制GDP/GTP交换蛋白（smgGDS和rho GDI）的调节，并且racp21s的翻译后加工不仅对它们与调节蛋白的相互作用很重要，而且对它们的NADPH氧化酶活化也很重要。此外，我们报告了5例p67-phox缺乏症的CGD患者的分子分析。这些rna是由EB病毒转化的B细胞株制备的，这些B细胞株的细胞质中没有任何抗p67抗体反应蛋白。利用RT-PCR扩增的cDNA，采用直接测序法分析p67-phox cDNA的蛋白质编码序列。其中1例患者在423个核苷酸残基位置发现二核苷酸插入。在另一名患者中，从694到879个核苷酸残基中有186个被删除。由于这种大的缺失，大部分src同源位点3 （SH3）被认为是与小G蛋白相互作用的重要位点被截断。在其他3例患者中，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。

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.