Monoclonal antibody CL5 recognizes the amino terminal domain of human phagocyte flavocytochrome b558 large subunit, gp91phox.
Human phagocyte flavocytochrome b558 (Cytb) is a heterodimeric integral membrane protein that serves as the electron transferase of the beta-nicotinamide adenine dinucleotidephosphate, reduced (NADPH)-oxidase, an enzyme complex important in the host defense function of phagocytic cells. In this study, we report the characterization of monoclonal antibody (mAb) CL5 that is specific for the large subunit, gp91phox, of the oxidase protein. This antibody recognizes gp91phox by immunoblot analysis of membrane extracts and samples of the immunopurified gp91phox/p22phox heterodimer, prepared on anti-p22phox affinity matrices. Phage display analysis confirmed this specificity, indicating that the CL5 epitope contains the region 135-DPYSVALSELGDR of gp91phox. The antibody was used to probe for the presence of gp91phox in membrane preparations from neutrophils of patients with nine genetically distinct forms of X-linked chronic granulomatous disease (CGD). The causative mutations included missense errors as well as nonsense errors that result in premature termination of gp91phox synthesis. Analysis of the CGD samples by immunoblotting indicated that CL5 recognizes only the full-length wild-type and two missense mutations, consistent with the absence of stable short gp91phox peptide expression in CGD neutrophils. Interestingly, CL5 was also shown to be cross-reactive with cytosolic and membrane-bound gelsolin, identified by purification, mass spectrometry and immunoblot analysis. CL5 probably cross-reacts with the sequence 771-DPLDRAMAEL in the C-terminus of gelsolin. We conclude that mAb CL5 is a useful probe for detection of full length and possibly truncated N-terminal fragments of gp91phox from membranes of Cytb-producing cells.
Baniulis
Danas
D
Department of Microbiology, Montana State University, Bozeman, MT 59717, USA.
Burritt
James B
JB
Taylor
Ross M
RM
Dinauer
Mary C
MC
Heyworth
Paul G
PG
Parkos
Charles A
CA
Magnusson
Karl-Eric
KE
Jesaitis
Algirdas J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001483:Base Sequence; D003429:Cross Reactions; D018076:DNA, Complementary / Q000235:genetics; D018604:Epitope Mapping; D018260:Gelsolin / Q000276:immunology; D015870:Gene Expression; D006105:Granulomatous Disease, Chronic / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D009504:Neutrophils / Q000737:chemistry / Q000276:immunology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D010586:Phagocytes / Q000737:chemistry* / Q000276:immunology; D017434:Protein Structure, Tertiary
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D018260:Gelsolin; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D019151:Peptide Library; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases