Monoclonal antibody CL5 recognizes the amino terminal domain of human phagocyte flavocytochrome b₅₅₈ large subunit, gp91^phox

Abstract

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.

Monoclonal antibody 7D5 recognizes the R147 epitope on the gp91phox, phagocyte flavocytochrome b558 large subunit

“…Burritt and colleagues have previously reported that mAb CL5 is specific for the large subunit of b 558 and that the CL5 epitope contains the 135 ‐DPYSVALSELGDR region of gp91 phox . The CL5 epitope 135 ‐DPYSVALSELGDR overlaps with the 143 ‐ELGDR 147 part of 143 ELGDRQNES 151 , which we found to be an important region (EC2‐2) for FACS analysis using mAb 7D5 of gp91 phox .…”

“…2). We found that more than three amino acid substitutions in five amino acids approximate to the intensity of intact human gp91 phox (Table 4, numbers [19][20][21][22][23]. As the amino acid substitution rate in the four amino acids (E143, I144, Q148 and S151) increases, these chimeric expression proteins are recognized more strongly by mAb 7D5.…”

“…Studies characterizing the epitopes recognized by several mAbs against human Cyt b have reported that 54.1 (18), NL7 (19) and 48 (20,21) recognize the intracellular region of gp91 phox and that CL5 (22) and 7D5 (23)(24)(25) recognize the extracellular region of gp91 phox . Burritt and colleagues have reported that mAb 54.1 recognizes the 382 PKIAVDGP 389 region on gp91 phox and that mAb NL7 binds the 498 EKD-VITGLK 506 region on gp91 phox .…”