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

Cytochrome b of human neutrophils is the central component of the micr obicidal NADPH-oxidase system. However, the folding topology of this i ntegral membrane protein remains undetermined. Two random-sequence bac teriophage peptide libraries were used to map structural features of c ytochrome 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 a ffinity 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 demonstra ted specific binding of each mAb to the respective cytochrome b subuni t and selected phage peptides. In flow cytometric analysis, mAb 44.1 b ound only permeabilized neutrophils, while 54.1 did not bind intact or permeabilized cells. However, mAb 54.1 immunosedimented detergent-sol ubilized cytochrome b in sucrose gradients. These results suggest the (181)GGPQVNPI(188) segment of p22(phox) is accessible on its intracell ular surface, but the (382)PKIAVDGP(389) region on gp91(phox) is not a ccessible to antibody, and probably not on the protein surface.