Complementation of NADPH oxidase in p67-phox-deficient CGD patients - p67-phox/p40-phox interaction

Chronic granulomatous disease (CGD) is due to a functional defect of the O- 2(-) generating NADPH oxidase of phagocytes. Epstein-Barr-virus-immortalize d B lymphocytes express all the constituents of oxidase with activity 100 t imes less than that of neutrophils. As in neutrophils, oxidase activity of Epstein-Barr-virus-immortalized B lymphocytes was shown to be defective in the different forms of CGD; these cells were used as a model for the comple mentation studies of two p67-phox-deficient CGD patients. Reconstitution of oxidase activity was performed in vitro by using a heterologous cell-free assay consisting of membrane-suspended or solubilized and purified cytochro me b(558) that was associated with cytosol or with the isolated cytosolic-a ctivating factors (p67-phox, p47-phox, p40-phox) from healthy or CGD patien ts. In p67-phox-deficient CGD patients, two cytosolic factors are deficient or missing: p67-phox and p40-phox. Not more than 20% of oxidase activity w as recovered by complementing the cytosol of p67-phox-deficient patients wi th recombinant p67-phox. On the contrary, a complete restoration of oxidase activity was observed when, instead of cytosol, the cytosolic factors were added in the cell-free assay after isolation in combination with cytochrom e b(558) purified from neutrophil membrane. Moreover, the simultaneous addi tion of recombinant p67-phox and recombinant p40-phox reversed the previous complementation in a p40-phox dose-dependent process. These results suggest that in the reconstitution of oxidase activity, p67-p hox is the limiting factor; the efficiency of complementation depends on th e membrane tissue and the cytosolic environment. In vitro, the transition f rom the resting to the activated state of oxidase, which results from assem bling, requires the dissociation of p40-phox from p67-phox for efficient ox idase activity. In the process, p40-phox could function as a negative regul atory factor and stabilize the resting state.