Epitope mapping of the Pseudomonas aeruginosa major outer membrane porin protein OprF

Abstract

Pseudomonas aeruginosa major outer membrane protein OprF has been proposed for use as a vaccine and as a target for immunotherapeutic and diagnostic monoclonal antibodies. The well-conserved epitopes for 10 surface-reactive, OprF-specific monoclonal antibodies were localized by both overlapping peptide analysis and immunodetection of OprF peptides generated by cyanogen bromide and the protease papain. Three of the monoclonal antibodies bound to specific overlapping octapeptides, which had been synthesized on 160 pins to cover the entire 326 amino acids of OprF. The highest reactivities were as follows: MA7-1 to the pin with attached peptide GTYETGNK (amino acids 55 to 62), MA7-2 to NLADFMKQ (amino acids 237 to 244), and MA5-8 to TAEGRAIN (amino acids 307 to 314). The other monoclonal antibodies showed no reactivity, indicating that they do not recognize linear epitopes. Two polyclonal sera were also tested and demonstrated weak reactivity with discrete regions of OprF, suggesting that the majority of antibodies produced might recognize conformational epitopes. Utilizing defined peptides generated with cyanogen bromide and papain, the conformational epitopes recognized by the seven monoclonal antibodies were localized to regions that were 42 to 90 amino acids long. These regions were located on two adjacent loops in the middle of an amended structural model of OprF.

Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli

“…The similar ion channel activity of the three N-terminal domains observed in the present study suggests that the pores of OprF 175 and OprF 177 are very likely constituted of eight L-strands as OmpA 171 . This would con¢rm the previous structural predictions made for the N-terminal domain of OprF [11,13,20]. Thus, ion channel activity data and structural models would suggest similar function for the amino-terminal ends of OprFs and OmpA.…”

“…Without crystallographic data to date, the functional tertiary structure of OprF is not well established. Nikaido's group agrees with a periplasmic C-terminal part [12], whilst other authors hypothesise that OprF is constituted of 16 L-strands, suggesting that the structure of the C-terminal part is not globular [11,13]. Nevertheless, there is no consensus to propose the number of L-strands necessary for building the ion channel in native OprF.…”

Insights into the structure and assembly of Escherichia coli outer membrane protein A

“…In this structure, the C290-C302 disulfide bond is part of a large extracellular loop of 20 amino acids that includes six charged residues -three aspartates, two arginines and a lysine -all of which must cross the bilayer. The segment contains sites homologus to OprF sites that are proposed to be on the extracellular side by peptide-insertion studies [87]. Molecular modeling of this segment indicates that the disulfide bond facilitates the formation of a more compact structure with ion pair formation between the three positive and three negative amino acid residues that would expedite the transport of this segment across the outer membrane bilayer (Fig.…”

Alternative folding pathways of the major porin OprF of Pseudomonas aeruginosa

“…Interestingly, before our studies, OprF protein was often thought to fold as a single-domain conformer. For example, some monoclonal anti-OprF Igs were shown to react with sequences in the C-terminal domain [24], and, even more importantly, a four-residue malarial epitope inserted into the C-terminal half (e.g. at position 310) was shown to react with the specific monoclonal antibody in intact cells [15].…”