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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2497-2504
Functional Analysis of NADPH Oxidase in Granulocytic Cells
Expressing a  488-497 gp91phox Deletion
Mutant
Lixin Yu,
Andrew R. Cross,
Ling Zhen, and
Mary C. Dinauer
From the Department of Pediatrics (Hematology-Oncology) and Medical
and Molecular Genetics, Herman B Wells Center for Pediatric Research,
Riley Hospital for Children, Indiana University School of Medicine,
Indianapolis, IN; and the Department of Molecular and Experiment
Medicine, The Scripps Research Institute, La Jolla, CA.
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 ( 488-497 gp91phox)
has previously been reported. In this study, we generated myeloid PLB-985 cells expressing the mutant 488-497
gp91phox to further characterize its functional
properties. These cells mimicked the phenotype of the patient's
neutrophils with normal expression of a nonfunctional 488-497
gp91phox flavocytochrome. Translocation of
p47phox and p67phox to
488-497 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 µmol/L), suggesting that
residues 488-497 of gp91phox are not directly
involved in oxidase assembly. Mutant 488-497 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 488-497 deletion in
gp91phox disrupts electron transfer to FAD, either
due to a defect in NADPH binding or to impaired delivery of electrons
from NADPH.
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