title: The 13C4 monoclonal antibody that neutralizes Shiga toxin Type 1 (Stx1) recognizes three regions on the Stx1 B subunit and prevents Stx1 from binding to its eukaryotic receptor globotriaosylceramide.
abstract: The 13C4 monoclonal antibody (MAb) recognizes the B subunit of Stx1 (StxB1) and neutralizes the cytotoxic and lethal activities of Stx1. However, this MAb does not bind to the B polypeptide of Stx2, despite the 73% amino acid sequence similarity between StxB1 and StxB2. When we compared the amino acid sequences of StxB1 and StxB2, we noted three regions of dissimilarity (amino acids 1 to 6, 25 to 32, and 54 to 61) located near each other on the crystal structure of StxB1. To identify the 13C4 epitope, we generated seven Stx1/Stx2 B chimeric polypeptides that contained one, two, or three of the dissimilar StxB1 regions. The 13C4 MAb reacted strongly with StxB1 and the triple-chimeric B subunit but not with the other chimeras. Mice immunized with the triple-chimeric B subunit survived a lethal challenge with Stx1 but not Stx2, substantiating the identified regions as the 13C4 MAb epitope and suggesting that the incorporation of this epitope into StxB2 altered sites necessary for anti-Stx2-neutralizing Ab production. Next, single amino acid substitutions were made in StxB1 to mimic Stx1d, a variant not recognized by the 13C4 MAb. The 13C4 MAb reacted strongly to StxB1 with the T1A or G25A mutations but not with the N55T change. Finally, we found that the 13C4 MAb blocked the binding of Stx1 to its receptor, globotriaosyl ceramide. Taken together, these results indicate that the 13C4 MAb prevents the interaction of Stx1 with its receptor by binding three nonlinear regions of the molecule that span receptor recognition sites on StxB1, one of which includes the essential residue 55N.
authors.affiliation: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Room B4052, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Smith
authors.forename: Humberto M
authors.initials: HM
authors.lastname: Carvalho
authors.forename: Angela R
authors.initials: AR
authors.lastname: Melton-Celsa
authors.forename: Alison D
authors.initials: AD
authors.lastname: O'Brien
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D018360:Crystallography, X-Ray; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011956:Receptors, Cell Surface / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D016415:Sequence Alignment; D022622:Shiga Toxin 1 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology*; D022641:Shiga Toxin 2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology; D014281:Trihexosylceramides / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D021122:Protein Subunits; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D014281:Trihexosylceramides; C018549:globotriaosylceramide