Identification of a thermolabile component of the human neutrophil NADPH oxidase. A model for chronic granulomatous disease caused by deficiency of the p67-phox cytosolic component

Abstract

Mild heating of human neutrophils inactivates the respiratory burst oxidase, producing a defect in Superoxide production and bacterial killing comparable to that seen in patients afflicted with chronic granulomatous disease (CGD). We have now investigated the mechanism and specificity of this inactivation by examining the effect of mild heating on the known oxidase components: the membrane-bound subunits of the cytochrome b558 (gp91-phox and p22-phox) and the two cytosolic oxidase factors (p41-phox and p61-phox). Heating (46°C for 7.5 min) caused intact neutrophils to lose > 85% of their capacity to produce Superoxide, a defect which was localized to the cytosolic, but not the membrane, fraction. Complementation studies with CGD cytosols deficient in either p41-phox or p67-phox suggested that the defective component of heat-inactivated cytosol was p61-phox. This was confirmed by experiments showing that recombinant p61-phox, but not p47-phox, exhibited lability at 46°C and completely reconstituted oxidase activity of heat-treated cytosol. These studies indicate that mild heating of either intact neutrophils or normal neutrophil cytosol results in a selective inactivation of p61-phox, providing a model oxidase system for the extremely rare p67-phox-deficient form of CGD.