Epitope mapping by cysteine mutagenesis: identification of residues involved in recognition by three monoclonal antibodies directed against LamB glycoporin in the outer membrane of Escherichia coli.
Site-directed mutagenesis of the lamB gene was used to introduce individual cysteine substitutions at 20 sites in two regions (surface loops L7 and L8) of LamB protein significant in antibody recognition. Characterisation of cysteine mutants involved immunoblotting with three surface-specific monoclonal antibodies (mAb72, mAb302, mAb347) before and after incubation with thiol-specific reagents. In contrast to an earlier study that showed no amino acid changes affecting recognition by all three antibodies, changes at six amino acids were found to influence a common core epitope. These core sites included one residue (T336) in the predicted loop L7 containing amino acids 329-342 and four (Y379, N387, N389, K392, F398) in the large surface loop involving residues 370-412. Individual antibodies made additional but distinct contacts within the two studied regions, with mAb347 binding the most different and affected by seven substitutions in the 328-338 regions. The lamB mutants were also tested for phage lambda receptor activity and starch binding before and after thiol modification and were useful in extending previous maps of these ligand binding sites.
Notley
L
L
Department of Microbiology, University of Sydney, NSW, Australia.
Hillier
C
C
Ferenci
T
T
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology*; D010582:Bacteriophage lambda / Q000378:metabolism; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D000939:Epitopes / Q000032:analysis* / Q000235:genetics; D004926:Escherichia coli / Q000276:immunology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D018272:Porins; D011991:Receptors, Virus / Q000276:immunology*
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D018272:Porins; D011991:Receptors, Virus; C016532:maltoporins; D003545:Cysteine