title: Topological analysis of the Escherichia coli ferrichrome-iron receptor by using monoclonal antibodies.
abstract: Ferrichrome-iron transport in Escherichia coli is initiated by the outer membrane receptor FhuA. Thirty-five anti-FhuA monoclonal antibodies (MAbs) were isolated to examine the surface accessibility of FhuA sequences and their contribution to ligand binding. The determinants of 32 of the MAbs were mapped to eight distinct regions in the primary sequence of FhuA by immunoblotting against (i) five internal deletion FhuA proteins and (ii) four FhuA peptides generated by cyanogen bromide cleavage. Two groups of MAbs bound to FhuA in outer membrane vesicles but not to intact cells, indicating that their determinants, located between residues 1 and 20 and 21 and 59, are exposed to the periplasm. One of the 28 strongly immunoblot-reactive MAbs bound to FhuA on intact cells in flow cytometry, indicating that its determinant, located between amino acids 321 and 381, is cell surface exposed. This MAb and four others which in flow cytometry bound to cells expressing FhuA were tested for the ability to block ligand binding. While no MAb inhibited growth promotion by ferrichrome or cell killing by microcin 25, some prevented killing by colicin M and were partially able to inhibit the inactivation of T5 phage. These data provide evidence for spatially distinct ligand binding sites on FhuA. The lack of surface reactivity of most of the immunoblot-reactive MAbs suggests that the majority of FhuA sequences which lie external to the outer membrane may adopt a tightly ordered organization with little accessible linear sequence.
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Canada.
authors.forename: G S
authors.initials: GS
authors.lastname: Moeck
authors.forename: M J
authors.initials: MJ
authors.lastname: Ratcliffe
authors.forename: J W
authors.initials: JW
authors.lastname: Coulton
mesh_terms: D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001412:Bacillus subtilis / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000276:immunology*; D001483:Base Sequence; D001667:Binding, Competitive; D003087:Colicins / Q000494:pharmacology; D018604:Epitope Mapping; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005291:Ferrichrome; D005434:Flow Cytometry; D006825:Hybridomas; D015151:Immunoblotting; D007501:Iron; D008024:Ligands; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011991:Receptors, Virus / Q000737:chemistry* / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D013604:T-Phages
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D008024:Ligands; D011991:Receptors, Virus; D011994:Recombinant Proteins; D005291:Ferrichrome; D007501:Iron