Fine definition of the epitopes on the human gp91phox/NOX2 for the monoclonal antibodies CL-5 and 48

Abstract

Superoxide-producing NADPH oxidase, gp91^phox/NOX2, in phagocytes plays a critical role in the host defenses against pathogens. Moreover, gp91^phox/NOX2 contributes to the oxidative stress in endothelial cells. Therefore, investigating the level of gp91^phox/NOX2 with immunoblotting is important for estimating the amount of superoxide produced. Here, we showed that the epitopes in human gp91^phox/NOX2 recognized by monoclonal antibodies (mAbs) CL-5 and 48 were in amino acids 132-147 and 136-144, respectively. Although the epitopes were close to the N-glycosylation sites, N-glycan maturation did not affect mAbs recognition. When Pro-136 was substituted with Arg, the corresponding mouse residue, human gp91^phox/NOX2 was not recognized by mAbs CL-5 and 48; however, the substitution did not affect gp91^phox/NOX2-based oxidase activity. This finding explains why these mAbs specifically recognize the human but not mouse gp91^phox/NOX2. Hence, these mAbs are useful for investigating the level of gp91^phox/NOX2 without amino acid substitutions in the epitopes.

Keywords: Chronic granulomatous disease (CGD); Endothelial cell; Monoclonal antibody; Phagocyte; gp91(phox)/NOX2.