TOPOLOGICAL MAPPING OF NEUTROPHIL CYTOCHROME-B EPITOPES WITH PHAGE-DISPLAY LIBRARIES

Cytochrome b of human neutrophils is the central component of the microbicidal NADPH-oxidase system. However, the folding topology of this integral membrane protein remains undetermined. Two random-sequence bacteriophage peptide libraries were used to map structural features of cytochrome b by determining the epitopes of monoclonal antibodies (mAbs) 44.1 and 54.1, specific for the p22(phox) and gp91(phox) cytochrome b chains, respectively. The unique peptides of phage selected by mAb affinity purification were deduced from the phage DNA sequences. Phage selected by mAb 44.1 displayed the consensus peptide sequence GGPQVXPI, which is nearly identical to (181)GGPQVNPI(188) of p22(phox). Phage selected by mAb 54.1 displayed the consensus sequence PKXAVDGP, which resembles (382)pKIAVDGP(389) of gp91(phox). Western blotting demonstrated specific binding of each mAb to the respective cytochrome b subunit and selected phage peptides. In flow cytometric analysis, mAb 44.1 bound only permeabilized neutrophils, while 54.1 did not bind intact or permeabilized cells. However, mAb 54.1 immunosedimented detergent-solubilized cytochrome b in sucrose gradients. These results suggest the (181)GGPQVNPI(188) segment of p22(phox) is accessible on its intracellular surface, but the (382)PKIAVDGP(389) region on gp91(phox) is not accessible to antibody, and probably not on the protein surface.