Analysis of glycosylation sites on gp91phox, the flavocytochrome of the NADPH oxidase, by site-directed mutagenesis and translation in vitro

Flavocytochrome b(558) of the NADPH oxidase which generates superoxide in phagocytic cells, is a alpha(1) beta(1) heterodimer of gp91phox and p22phox, which together form a membrane-spanning electron-transport chain that transfers electrons from NADPH in the cytosol to oxygen. The C-terminal portion of gp91phox is a member of the ferredoxin-NADP(+) reductase family of reductases. Little is known of the organization of the N-terminal section of this molecule, which is associated with the two haem structures. It is N-glycosylated, and site-directed mutagenesis has been used to eliminate the five potential N-linked glycosylation consensus sites. Mutated cDNAs were expressed in vitro. This approach provided evidence for glycosylation of residues Asn(131), Asn(148) and Asn(239), but not of Asn(96) and Asn(429).