P22-PHOX-DEFICIENT CHRONIC GRANULOMATOUS-DISEASE - RECONSTITUTION BY RETROVIRUS-MEDIATED EXPRESSION AND IDENTIFICATION OF A BIOSYNTHETIC INTERMEDIATE OF GP91-PHOX

Chronic granulomatous disease (CGD) results from defects in the phagoc yte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, centr al to which is the membrane-bound cytochrome b(-245). The cytochrome i s composed of two protein subunits. the larger (gp91-phox) being defic ient in X-linked CGD. In this study, we have analyzed expression of th e cytochrome subunits in B-cell lines from two autosomal CGD patients for whom the disease is caused by deficiency of p22-phox, the smaller subunit. We report the presence of a 65-kD precursor of gp91-phox in t he membrane fraction of both p22-phox-deficient cell lines, correspond ing to the core protein with N-linked carbohydrate side chains in the high mannose form. Expression of p22-phox in these cells resulted in f unctional correction of NADPH oxidase. In addition, gp91-phoxin the re constituted cells was processed to its terminally glycosylated form. T hese data suggest that the association of the 65-kD gp98-phox precurso r with p22-phox is a prerequisite for processing of the carbohydrate s ide chains to the complex form in the Golgi. The detection of this pre cursor will enable characterization of mutations disrupting the subuni t interaction (either naturally occurring or derived by in vitro mutag enesis) and so aid in structure-function analysis of cytochrome b(-245 ). Reconstitution of p22-phox-deficient cells shows the potential of g ene therapy for this autosomal form of CGD. (C) 1994 by The American S ociety of Hematology.