|Von Lohneysen, Katharina|
Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2006
Publication Date: 11/1/2006
Citation: Zhu, Y., Marchal, C., Casbon, A., Stull, N., Von Lohneysen, K., Knaus, U.G., Jesaitis, A.J., Mccormick, S., Nauseef, W.M., Dinauer, M. 2006. Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and nadph oxidase activity. Journal of Biological Chemistry. 281:30336-30346.
Interpretive Summary: Chronic granulomatous disease (CGD) is caused by genetic defect on several of a group of genes encoding a multi-component enzyme that catalyzes the transfer of electrons from NADPH to generate the superoxide radical (O2-) and other microbicidal oxidants. Although this disease has been study for many years and all the gene has been cloned for more than twenty years ago, the interaction between the subunits and inter-dependent nature of this enzyme complex are not fully understand. This study focused on in depth investigation of two interacting membrane protein: p22phox and gp91phox, looking into the molecular basis of their partnership. Results from this study have significant contribution on understanding the requirement of p22 for the maturation of gp91. This result can potentially impact the early diagnosis and better strategy for gene therapy for this severe infant genetic disease.
Technical Abstract: The phagocyte NADPH oxidase is a multicomponent enzyme that catalyzes the transfer of electrons from NADPH to generate the superoxide radical (O2-). The importance of this enzyme in innate immunity and inflammation is illustrated by chronic granulomatous disease (CGD), a syndrome characterized by absent NADPH oxidase activity, recurrent bacterial and fungal infections. The heterodimeric flavocytochrome b558, comprised of the two integral membrane proteins p22phox and gp91phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22phox required for maturation of gp91phox and for NADPH oxidase activity. N-terminal, C-terminal or internal deletions of human p22phox were generated and expressed in Chinese Hamster Ovary cells with transgenes for gp91phox and two other NADPH oxidase subunits, p47phox, and p67phox. The results demonstrate that p22phox-dependent maturation of gp91phox carbohydrate, cell surface expression of gp91phox, and the enzymatic function of flavocytochrome b558 are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22phox are required, as is a hydrophilic region between amino acids 65-90. Upon deletion of 54 residues at the C-terminus of p22phox (amino acids 142-195), maturation and cell surface expression of gp91phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47phox.