Blood, 15 April 2002, Vol. 99, No. 8, pp. 2635-2635
Reconstitution of active NADPH oxidase in
nonhematopoietic cells: a milestone in the study of a complex system
Price and colleagues (page 2653) have succeeded in
reconstituting NADPH oxidase, the enzyme responsible for respiratory
burst of phagocytes, by expressing its 4 main components in COS-7
cells, a cell line that is readily and efficiently transfected by a
variety of vectors. In activated phagocytes, membrane-associated NADPH oxidase (also referred to as phagocyte oxidase, or phox)
reduces molecular oxygen (O2) to superoxide
(O
), which is then converted into a variety of
potent microbicides. Although many cell types can divert trace amounts
of their metabolic oxygen to superoxide, respiratory burst is on a
different scale: the amounts of oxygen reduced to superoxide during
phagocytosis greatly exceed the normal metabolic oxygen consumption.
When the engineered COS cells were stimulated by appropriate agonists, the reconstituted components of the NADPH oxidase assembled in the
membrane and produced superoxide at rates comparable to professional phagocytes. The importance of this milestone is best appreciated in its
historical context. Since the 1950s, the molecular analysis of the
oxidase was propelled by studies of phagocytes from patients with
chronic granulomatous disease, a severe defect in the phagocytic production of superoxide and its products. In a tour de force of
biochemistry, analyses of several broken-cell and cell-free systems
were eventually combined with the genetic information to identify 5 proteins that were essential for the normal function and activation of
the oxidase. But the molecular dissection of the assembly and
regulation of the NADPH oxidase has been hampered by the lack of a
system that could be manipulated by such powerful techniques as
site-directed mutagenesis and the expression of modified components and
regulators of the oxidase.
Price and colleagues have provided initial evidence that the
reconstituted COS-phox system will be useful. They
identified specific molecular features of the 2 cytoplasmic components
p47phox and p67phox that
are required for enzyme assembly and activation, complementing previous
work done in the cell-free NADPH oxidase systems. By demonstrating the
inhibitory effects of the transfection of dominant-negative Rac mutants
and the Rac-antagonist RhoGDI, they confirmed the requirement of the
oxidase for a small G-protein Rac. (Rac1 is naturally present in COS-7
cells.) Looking ahead and linking to ongoing studies of reconstituted
Fc receptors that normally mediate phagocytosis of antibody-opsonized
particles, we may soon see the full reconstitution of the
phagocytic response from the afferent ligation of antibody-coated
targets to the respiratory burst.
Tomas Ganz
Washington University
School
