Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase.
Open Access
- 1 September 1989
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 86 (18) , 7195-7199
- https://doi.org/10.1073/pnas.86.18.7195
Abstract
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.Keywords
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