Functional Epitope on Human Neutrophil Flavocytochrome b558

Abstract

mAb NL7 was raised against purified flavocytochrome b558, important in host defense and inflammation. NL7 recognized the gp91phox flavocytochrome b558 subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the 498EKDVITGLK506 region of gp91phox. In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (Km). mAb NL7 did not inhibit translocation of p47phox, p67phox, or Rac to the plasma membrane, and bound its epitope on gp91phox independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91phox on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47phox binding sites. We conclude that the 498EKDVITGLK506 segment resides on the cytosolic surface of gp91phox and represents a region important for oxidase function, but not substrate or cytosolic component binding.

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

“…3, curve c), the gp91 phox -specific mAb NL7 (data not shown), or an irrelevant, anti-rhodopsin mAb K16 (data not shown). Similar to mAb 44.1, mAbs 54.1 and NL7 recognize the cytoplasmic surface of Cyt b (31,33) and have been shown to efficiently bind detergent-solubilized Cyt b by fluorescence resonance energy transfer under these experimental conditions (R. M. Taylor and A. J. Jesaitis, unpublished data). Additional control experiments demonstrated that preincubation of DDM-solubilized Cyt b with unlabeled mAb 44.1 does not interfere with the binding of CCB-mAb 54.1 (data not shown).…”

“…FACS analysis evaluating the binding of mAbs to intact human neutrophils was conducted as previously described (31).…”

“…phox -specific mAbs on catalytic activity of the NADPH oxidase complex A recent study by our group reported the inhibition of NADPH oxidase activity by the Cyt b, large subunit-specific Ab mAb NL7 (31). In the interest of characterizing additional inhibitors of NADPH oxidase function, the p22 phox -specific mAbs were preincubated with isolated neutrophil membrane fractions (the source of Cyt b) and then evaluated for effects on superoxide generation in (9,42,43).…”

“…All mAbs reported in this study were produced and purified using standard hybridoma technology as previously described (31). The mAbs NS1, NS2, and NS5 were generated in normal mice, while mAbs CS6, CS8, and CS9 were generated in an X-linked CGD mouse strain.…”

Phospholipase D1 Regulates Phagocyte Adhesion

“…DMF-differentiated PLB cells are a well-characterized model of PMN that have been extensively used for studies of biochemical activation and functional responses (e.g., respiratory burst, granule exocytosis) (55,56,73,74). Incubation of differentiated PLB cells in FN-coated wells was associated with rapid and sustained production of PA and PEt (Fig.…”