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Functional reconstitution of the phagocyte NADPH oxidase by transfection of
its multiple components in a heterologous system
I de Mendez and TL Leto
Laboratory of Host Defenses, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD 20892.
The phagocyte NADPH oxidase system, as previously defined by cell-free
reconstitution, is comprised of five essential components, three of which
are produced during late phagocytic differentiation--namely, two cytosolic
proteins, p47- and p67-phox--and the large subunit of cytochrome b558,
gp91-phox. To confirm that these are the only phagocyte-specific components
necessary for oxidase activity in whole cells, the recombinant NADPH
oxidase was reconstituted in a heterologous cell line. An undifferentiated
multipotent leukemic cell line, K562, which expresses endogenous Rac and
the small subunit of the flavocytochrome b558 (p22-phox), was cotransfected
with episomal expression vectors containing cDNAs for the three other
oxidase components. After 4 days of selection, the complete oxidase system
was functionally reconstituted in transfected cells stimulated with phorbol
myristate acetate or calcium ionophore. These easily transfected cells
provide an ideal model system in which several oxidase components can be
genetically manipulated and readily expressed. This system can be used to
test the effects of mutations associated with any of the genes affected in
chronic granulomatous disease and will facilitate studies on
structure-function relationships within several oxidase components. This
system will also aid in delineation of upstream regulators functioning
through various signaling pathways.
Volume 85,
Issue 4,
pp. 1104-1110,
02/15/1995
Copyright © 1995 by The American Society of Hematology
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