New p22-phox monoclonal antibodies: identification of a conformational probe for cytochrome b 558.
The phagocyte NADPH oxidase, belonging to the NADPH oxidase family (Nox), is dedicated to the production of bactericidal reactive oxygen species. The enzyme catalytic center is the cytochrome b(558), formed by 2 subunits, Nox2 (gp91-phox) and p22-phox. Cytochrome b(558) activation results from a conformational change induced by cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac). The catalytic subunit is Nox2, while p22-phox is essential for both Nox2 maturation and the membrane anchorage of regulatory proteins. Moreover, it has been shown to be necessary for novel Nox activity. In order to characterize both p22-phox topology and cytochrome b(558) conformational change, 6 monoclonal antibodies were produced against purified cytochrome b(558). Phage display epitope mapping combined with a truncation analysis of recombinant p22-phox allowed the identification of epitope regions. Some of these antibodies almost completely inhibited in vitro reconstituted NADPH oxidase activity. Data analysis identified antibodies that recognized epitopes involved in either Nox2 maturation or Nox2 activation. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils showed that the monoclonal antibody 12E6 bound preferentially active cytochrome b(558). These monoclonal antibodies provided novel and unique probes to investigate maturation, activation and activity, not only of Nox2 but also of novel Nox.
Campion
Yannick
Y
GREPI, TIMC-Imag UMR 5525 CNRS/Université Joseph Fourier, Laboratoire d'Enzymologie, CHU Grenoble, Grenoble, France.
Jesaitis
Algirdas J
AJ
Nguyen
Minh Vu Chuong
MV
Grichine
Alexei
A
Herenger
Yvan
Y
Baillet
Athan
A
Berthier
Sylvie
S
Morel
Françoise
F
Paclet
Marie-Hélène
MH
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015153:Blotting, Western; D002469:Cell Separation; D003573:Cytochrome b Group / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D004789:Enzyme Activation / Q000276:immunology; D018985:Epitopes, B-Lymphocyte / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D047468:Immunoprecipitation; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D009504:Neutrophils / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D019151:Peptide Library; D020836:Protein Structure, Quaternary
D000911:Antibodies, Monoclonal; D003573:Cytochrome b Group; D018985:Epitopes, B-Lymphocyte; D008562:Membrane Glycoproteins; D019151:Peptide Library; C036888:cytochrome b558; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human