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.
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