Determinants of OmpF porin antigenicity and structure.
Sixty-six murine hybridomas raised to Escherichia coli B/r porin were used to identify and differentiate the epitopes of this outer membrane protein. Anti-porin monoclonal antibodies (mAb) were raised against outer membrane fragments, purified native trimeric porin (trimer), and purified sodium dodecyl sulfate-denatured monomeric porin (monomer). Immunochemical and flow cytometric methods identified five distinct cell surface-exposed determinants on OmpF. The peptide composition of porin epitopes was determined by analysis of mAb reactivity with cyanogen bromide-generated peptide fragments. Four of 43 anti-monomer mAb reacted with surface exposed sites on OmpF, defining epitopes that consist of residues within CNBr peptides d2, d3, and B. The anti-porin mAb panel was also used to evaluate changes in porin antigenic structure in strains with short ompF deletions. Flow cytometric experiments indicated that despite changes in porin permeability, little if any alteration of surface epitopes occurred in these strains. Western immunoblot analysis of the mutant porins showed loss of reactivity with numerous mAb, which was caused by changes in three spatially distinct epitopes at residues 108-111, 118-123, and 124-129. Our findings indicate that in these ompF mutants the residues responsible for altering porin permeability are not exposed on the cell surface, but are buried within the tertiary structure of the protein. One of these regions, which is apparently involved in the determination of channel permeability characteristics, is conserved among 15 of 16 different porin molecules which were screened with the anti-OmpF mAb panel.
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
P E
PE
Klebba
S A
SA
Benson
S
S
Bala
T
T
Abdullah
J
J
Reid
S P
SP
Singh
H
H
Nikaido
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology*; D003488:Cyanogen Bromide; D004755:Enterobacteriaceae / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000032:analysis*; D005434:Flow Cytometry; D015151:Immunoblotting; D007143:Immunoglobulin Heavy Chains; D008961:Models, Structural; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D018272:Porins; D011487:Protein Conformation; D011863:Radioimmunoassay; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D010446:Peptide Fragments; D018272:Porins; D003488:Cyanogen Bromide