Functional epitope on human neutrophil flavocytochrome b558.
mAb NL7 was raised against purified flavocytochrome b(558), important in host defense and inflammation. NL7 recognized the gp91(phox) flavocytochrome b(558) subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the (498)EKDVITGLK(506) 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 (498)EKDVITGLK(506) segment resides on the cytosolic surface of gp91(phox) and represents a region important for oxidase function, but not substrate or cytosolic component binding.
Burritt
James B
JB
Department of Microbiology, Montana State University, Bozeman, MT 59717, USA. jburritt@montana.edu
Foubert
Thomas R
TR
Baniulis
Danas
D
Lord
Connie I
CI
Taylor
Ross M
RM
Mills
John S
JS
Baughan
Travis D
TD
Roos
Dirk
D
Parkos
Charles A
CA
Jesaitis
Algirdas J
AJ
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001666:Binding Sites, Antibody; D003573:Cytochrome b Group / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D004789:Enzyme Activation / Q000276:immunology; D000939:Epitopes / Q000276:immunology* / Q000378:metabolism; D006105:Granulomatous Disease, Chronic / Q000201:enzymology / Q000276:immunology; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology* / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D021381:Protein Transport / Q000276:immunology; D020744:rac GTP-Binding Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D003573:Cytochrome b Group; D000939:Epitopes; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; C036888:cytochrome b558; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D020744:rac GTP-Binding Proteins