Site-Specific Inhibitors of NADPH Oxidase Activity and Structural Probes of Flavocytochrome b: Characterization of Six Monoclonal Antibodies to the p22phox Subunit

Abstract

The integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the human phagocyte NADPH oxidase, an enzyme complex that initiates a cascade of reactive oxygen species important in the elimination of infectious agents. This study reports the generation and characterization of six mAbs (NS1, NS2, NS5, CS6, CS8, and CS9) that recognize the p22phox subunit of the Cyt b heterodimer. Each of the mAbs specifically detected p22phox by Western blot analysis but did not react with intact neutrophils in FACS studies. Phage display mapping identified core epitope regions recognized by mAbs NS2, NS5, CS6, CS8, and CS9. Fluorescence resonance energy transfer experiments indicated that mAbs CS6 and CS8 efficiently compete with Cascade Blue-labeled mAb 44.1 (a previously characterized, p22phox-specific mAb) for binding to Cyt b, supporting phage display results suggesting that all three Abs recognize a common region of p22phox. Energy transfer experiments also suggested the spatial proximity of the mAb CS9 and mAb NS1 binding sites to the mAb 44.1 epitope, while indicating a more distant proximity between the mAb NS5 and mAb 44.1 epitopes. Cell-free oxidase assays demonstrated the ability of mAb CS9 to markedly inhibit superoxide production in a concentration-dependent manner, with more moderate levels of inhibition observed for mAbs NS1, NS5, CS6, and CS8. A combination of computational predictions, available experimental data, and results obtained with the mAbs reported in this study was used to generate a novel topology model of p22phox.

Analysis of Human Phagocyte Flavocytochrome b558 by Mass Spectrometry

“…The p22 phox subunit is required for biosynthesis of the mature Cyt b heterodimer in human phagocytes (28), and current models suggest that p22 phox contains 2-4 membrane-spanning domains (12,29,27). Concerning oxidase function, p22 phox has been shown to be phosphorylated in activated neutrophils (30), and several regions in the primary sequence appear to be involved in binding the cytosolic oxidase subunits p47 phox and p67 phox during oxidase assembly (17,31).…”

“…The ability of this domain to bind redox cofactors (NADPH and FAD) has been demonstrated by biochemical labeling experiments (22,24), whereas a variety of methods have been used to identify residues in the gp91 phox C-terminal domain that are critical for assembly of the NADPH oxidase complex (17,18,25,26). Current models localize the gp91 phox C-terminal domain to the cytoplasmic aspect of the plasma or phagosomal membrane (4,12,27).…”

NADPH Oxidases as Novel Pharmacologic Targets against Influenza A Virus Infection

“…It possesses 195 amino acids, has a molecular weight of ∼21 kDa, and hydrophobicity analysis of its amino acid structure indicates that p22phox most likely possesses a cytoplasmic N terminus (residues 1-90), two transmembrane a-helices (residues 91-106 and 112-127) connected by a short extracellular loop (residues 107-111) (Taylor et al, 2004;Zhu et al, 2006) and a C terminus that extends back into the cytoplasm (residues . The N terminus contains two regions (residues 6-11 and 65-90), which are essential for maturation of NOX2 into a fully glycosylated protein and its expression at the membrane.…”

Redox Control of Renal Function and Hypertension

“…However, the development of antibodies that are active in vivo against NAD(P)H oxidase is lagging far behind. Several mouse antibodies against gp91 phox and p22 phox have been developed in individual laboratories with variable inhibition of NAD(P)H oxidase activity in vitro (321). Polyclonal and monoclonal antibodies against these and other subunits are commercially available from Upstate Signaling (now Millipore, Billerica, MA) and Santa Cruz Biotechnology, Santa Cruz, CA.…”