PHAGOCYTES Functional Analysis of NADPH Oxidase in Granulocytic Cells Expressing a D488-497 gp91phox Deletion Mutant

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 gp91phox, 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 gp91phox (D488-497 gp91phox) has previously been reported. In this study, we generated myeloid PLB-985 cells expressing the mutant D488-497 gp91phox to further characterize its functional properties. These cells mimicked the phenotype of the patient’s neutrophils with normal expression of a nonfunctional D488-497 gp91phox flavocytochrome. Translocation of p47phox and p67phox to D488-497 gp91phox 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 gp91phox was relatively ineffective in inhibiting O2 production in the cell-free oxidase assay (IC50, ,500 mmol/L), suggesting that residues 488-497 of gp91phox are not directly involved in oxidase assembly. Mutant D488497 gp91phox flavocytochrome failed to support iodonitrotetrazolium (INT) reduction, showing a disruption of electron transfer from NADPH to the FAD center of gp91phox. However, the FAD binding capacity of the mutant flavocytochrome was normal, as measured by equilibrium dialysis. Taken together, these results suggest that the D488-497 deletion in gp91phox disrupts electron transfer to FAD, either due to a defect in NADPH binding or to impaired delivery of electrons from NADPH. r 1999 by The American Society of Hematology.