Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase.
Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase.
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DOI：
10.1073/pnas.86.18.7195
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发表时间：
1989-09
影响因子：
11.1
通讯作者：
B. Volpp;W. Nauseef;J. Donelson;D. Moser;R. Clark
中科院分区：
文献类型：
--
作者：
B. Volpp;W. Nauseef;J. Donelson;D. Moser;R. Clark
中性粒细胞 NADPH 氧化酶是一种多组分酶，被激活可产生超氧阴离子，并且在慢性肉芽肿病患者的细胞中存在缺陷。它需要膜和胞质成分，后者包括多克隆抗血清 B-1 识别的 47-和 67-kDa 蛋白质。对诱导的 HL-60 lambda ZAP cDNA 文库进行免疫筛选，产生了 7 个编码 47 kDa 成分的交叉杂交 cDNA。 B-1 识别 22-50 kDa 的融合蛋白。针对融合蛋白的抗血清可识别正常中性粒细胞中的 47-kDa 蛋白，但不能识别来自缺乏 47-kDa 胞质氧化酶成分的常染色体慢性肉芽肿病患者的中性粒细胞。在无细胞 NADPH 氧化酶系统中，全长和 C 端融合蛋白增强了超氧化物的产生，并重建了缺失 47 kDa 蛋白的患者的胞质缺陷。该 cDNA 与来自诱导的 HL-60 细胞的 1.4 KB mRNA 杂交。最长的 cDNA 包含一个开放阅读框，编码 41,440 Da 的蛋白质（计算的等电点为 10.4）、一个 N 末端甘氨酸、有利于磷酸化的位点、一个核苷酸结合域以及一个与 src 蛋白激酶、磷脂酶 C 和 α-fodrin 同源的区域。这些结构特征与该蛋白质在呼吸爆发氧化酶中的功能作用相关。
Neutrophil NADPH oxidase is a multicomponent enzyme that is activated to generate superoxide anion and is defective in the cells of patients with chronic granulomatous disease. It requires both membrane and cytosolic components, the latter including 47- and 67-kDa proteins recognized by the polyclonal antiserum B-1. Immunoscreening of an induced HL-60 lambda ZAP cDNA library yielded seven cross-hybridizing cDNAs encoding the 47-kDa component. Fusion proteins of 22-50 kDa were recognized by B-1. Antiserum against a fusion protein recognized a 47-kDa protein in normal neutrophils but not in those from patients with autosomal chronic granulomatous disease who lack the 47-kDa cytosolic oxidase component. In a cell-free NADPH oxidase system full-length and C-terminal fusion proteins augmented superoxide generation and reconstituted the cytosolic defect of a patient missing the 47-kDa protein. The cDNA hybridized with a 1.4-kilobase mRNA from induced HL-60 cells. The longest cDNA contained an open reading frame encoding a protein of 41,440 Da with a calculated pI of 10.4, an N-terminal glycine, sites favorable for phosphorylation, a nucleotide binding domain, and a region of homology to the src protein kinases, phospholipase C, and alpha-fodrin. These structural features are pertinent to proposed functional roles of the protein in the respiratory burst oxidase.