Functional analysis of NADPH oxidase in granulocytic cells expressing a Δ488-497 gp91(phox) deletion mutant

Lixin Yu, Andrew R. Cross, Ling Zhen, Mary C. Dinauer

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34 Scopus citations

Abstract

Chronic granulomatous disease (CGD) is a group of inherited disorders in which phagocytes are unable to generate superoxide (O2-) due to genetic defects in any 1 of 4 essential NADPH oxidase components. Mutations in the X- linked gene for gp91(phox), the large subunit of the flavocytochrome b558 heterodimer, account for the majority of CGD. An X-CGD patient in which a splice junction mutation results in an in-frame deletion of 30 nucleotides encoding amino acids 488 to 497 of gp91(phox) (Δ488-497 gp91(phox)) has previously been reported. In this study, we generated myeloid PLB-985 cells expressing the mutant Δ488-497 gp91(phox) to further characterize its functional properties. These cells mimicked the phenotype of the patient's neutrophils with normal expression of a nonfunctional Δ488-497 gp91(phox) flavocytochrome. Translocation of p47(phox) and p67(phox) to Δ488-497 gp91(phox) PLB-985 plasma membranes was not affected, as determined both in activated intact cells and in the cell-free system. Furthermore, a synthetic peptide corresponding to residues 488-497 of gp91(phox) was relatively ineffective in inhibiting O2- production in the cell-free oxidase assay (IC50, ~500 μmol/L), suggesting that residues 488-497 of gp91(phox) are not directly involved in oxidase assembly. Mutant Δ488-497 gp91(phox) flavocytochrome failed to support iodonitrotetrazolium (INT) reduction, showing a disruption of electron transfer from NADPH to the FAD center of gp91(phox). However, the FAD binding capacity of the mutant flavocytochrome was normal, as measured by equilibrium dialysis. Taken together, these results suggest that the Δ488-497 deletion in gp91(phox) disrupts electron transfer to FAD, either due to a defect in NADPH binding or to impaired delivery of electrons from NADPH.

Original languageEnglish
Pages (from-to)2497-2504
Number of pages8
JournalBlood
Volume94
Issue number7
DOIs
StatePublished - Oct 1 1999

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