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Functional Epitope on Human Neutrophil Flavocytochrome b₅₅₈¹

James B. Burritt^2,*, Thomas R. Foubert^*, Danas Baniulis^*, Connie I. Lord^*, Ross M. Taylor^*, John S. Mills^*, Travis D. Baughan^*, Dirk Roos, Charles A. Parkos and Algirdas J. Jesaitis^*

^* Department of Microbiology, Montana State University, Bozeman, MT 59717; Sanquin Research and Landsteiner Laboratory of the Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; and Division of Gastrointestinal Pathology, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322

mAb NL7 was raised against purified flavocytochrome b₅₅₈, important in host defense and inflammation. NL7 recognized the gp91^phox 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 gp91^phox. 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 (K_m). mAb NL7 did not inhibit translocation of p47^phox, p67^phox, or Rac to the plasma membrane, and bound its epitope on gp91^phox 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 gp91^phox 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 p47^phox binding sites. We conclude that the ⁴⁹⁸EKDVITGLK⁵⁰⁶ segment resides on the cytosolic surface of gp91^phox and represents a region important for oxidase function, but not substrate or cytosolic component binding.

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