Partial reconstitution of the respiratory burst oxidase in lymphoblastoid B cell lines lacking p67-phox after transfection with an expression vector containing wild-type and mutant p67-phox cDNAs: Deletions of the carboxy and amino terminal residues of 67-phox are not required for activity

The respiratory burst oxidase of phagocytes and B lymphocytes is a multicomponent enzyme that catalyzes the reduction of oxygen by NADPH. It is responsible for O₂^- production in response to stimulation with phorbol 12-myristate 13-acetate (PMA). The study of patients with chronic granulomatous disease (CGD), an inherited disorder characterized by deficient or absent respiratory burst activity, has contributed greatly to our understanding of the NADPH-oxidase. The absence of any one of four components results in the clinical expression of CGD: the two membrane-bound components of the cytochrome b-558, gp91-phox and p22-phox, or the cytosolic factors, p47-phox and p67-phox. We used a system to investigate the activity of mutant p67-phox proteins expressed in a reconstitution assay. This system is characterized by the partial reconstitution of O₂^- production in an Epstein-Barr virus (EBV)-transformed lymphoblastoid B cell line from a patient with p67-phox-deficient CGD by transfection with an expression plasmid containing the p67-phox cDNA in the sense orientation. No O₂^- production was detectable in p67-phox-deficient lymphoblastoid B cell lines transfected with an antisense plasmid or in untransfected p67-phox lymphoblastoid cells stimulated by PMA. We tested two mutants, pEBOp67Δ1-22 and pEBOp67Δ512-526, and found that both recombinant proteins are active in our system. Thus, we conclude that the first 22 amino acid residues and the last 14 amino acid residues are not critical for initiation of O₂^- production.