Toxin a from Clostridium difficile binds to rabbit erythrocyte glycolipids with terminal Galα1–3Galβ1–4GlcNAc sequences

Abstract

The binding of Toxin A isolated from Clostridium difficile to rabbit erythrocyte glycolipids has been studied. Total lipid extracts from rabbit erythrocytes were subjected to thin-layer chromatography and toxin-binding glycolipids detected by using 125I-labeled Toxin A in a direct binding overlay technique. Two major and several minor toxinbinding glycolipids were detected in rabbit erythrocytes by this method. The results of structural analyses of the major toxin-binding glycolipids were consistent with a pentasaccharide-ceramide (Galα1–3Galβ1–4GlcNAcβ1–3Galβ1–4Glc-Cer) and a branched decasaccharide-ceramide (Galα1–3Gal,β1–4GlcNAcβ1–3[Galα1–3Galβ1–4GlcNAcβ1–6] Galβ1–4GlcNAcβ1–3Galβ1–4Glc-Cer) previously identified as the two most abundant glycolipids in rabbit erythrocytes. 125I-Toxin A binding to these glycolipids could be inhibited by bovine thyroglobulin, monospecific antiserum to the toxin, or by treatment of the glycolipids with α-galactosidase. The absence of toxin interaction with isoglobotriaosylceramide (Galα1–3Galβ1–4Glc-Cer) isolated from canine intestine suggested that the GlcNAc residue present in the terminal Galα1–3Galβ1–4GlcNAc sequence common to all known toxin binding glycoconjugates is required for carbohydrate-specific recognition by Toxin A. These observations are consistent with the proposed carbohydrate binding specificity of Toxin A for the nonreducing terminal sequence, Galα1–3Galβ1–4GlcNAc.