title: Carbohydrate-binding specificity of the Escherichia coli cytolethal distending toxin CdtA-II and CdtC-II subunits.
abstract: Intoxication by cytolethal distending toxin depends on assembly of CdtB, the active A component of this AB toxin, with the cell surface-binding (B) component, composed of the CdtA-CdtC heterodimer, to form the active holotoxin. Here we examine the cell surface binding properties of Escherichia coli-derived CdtA-II (CdtA-II(Ec)) and CdtC-II(Ec) and their capacity to provide a binding platform for CdtB-II(Ec). Using a flow cytometry-based binding assay, we demonstrate that CdtB-II(Ec) binds to the HeLa cell surface in a CdtA-II(Ec)- and CdtC-II(Ec)-dependent manner and that CdtA-II(Ec) and CdtC-II(Ec) compete for the same structure on the HeLa cell surface. Preincubation of cells with glycoproteins (thyroglobulin and fetuin), but not simple sugars, blocks surface binding of CdtA-II(Ec) and CdtC-II(Ec). Moreover, CdtA-II(Ec) and CdtC-II(Ec) bind immobilized fetuin and thyroglobulin as well as fucose and to a lesser degree N-acetylgalactoseamine and N-acetylglucoseamine. Removal of N- but not O-linked carbohydrates from fetuin and thyroglobulin prevents binding of CdtA-II(Ec) and CdtC-II(Ec) to these glycoproteins. In addition, removal of N- but not O-linked surface sugar attachments prevents CDT-II(Ec) intoxication. To characterize the cell surface ligand recognized by CdtA-II(Ec) and CdtC-II(Ec), lectins having various carbohydrate specificities were used to block CDT activity and the cell surface binding of CdtA-II(Ec) and CdtC-II(Ec). Pretreatment of cells with AAA, SNA-I, STA, UEA-I, GNA, and NPA partially or completely blocked CDT activity. AAA, EEA, and UEA-I lectins, all having specificity for fucose, blocked surface binding of CdtA-II(Ec) and CdtC-II(Ec). Together, our data indicate that CdtA-II(Ec) and CdtC-II(Ec) bind an N-linked fucose-containing structure on HeLa cells.
authors.affiliation: Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 5007 Rockhill Rd., Kansas City, MO 64110, USA.
authors.forename: Leslie A
authors.initials: LA
authors.lastname: McSweeney
authors.forename: Lawrence A
authors.initials: LA
authors.lastname: Dreyfus
mesh_terms: D001427:Bacterial Toxins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000506:poisoning; D001667:Binding, Competitive; D050260:Carbohydrate Metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006023:Glycoproteins / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D037102:Lectins / Q000494:pharmacology; D021122:Protein Subunits
chemical_list: D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D006023:Glycoproteins; D037102:Lectins; D021122:Protein Subunits; C067044:cytolethal distending toxin