Interaction of the verotoxin 1B subunit with soluble aminodeoxy analogues of globotriaosyl ceramides

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Abstract
Specific hydroxy groups of the terminal disaccharide unit of globotriaosyl ceramide (Gb3Cer) were identified from binding studies with deoxyGb3Cer and verotoxins (VTs) [Nyholm, Magnusson, Zheng, Norel, Binnington-Boyd and Lingwood (1996) Chem. Biol. 3, 263—275]. Four such hydroxy groups (2′′, 4′′, 6′′ and 6′) were each substituted with an amino group and the corresponding deoxyamino globotrioses were conjugated to a ceramide-like aglycone which contained an adamantyl group instead of an acyl chain. Such aglycone modification significantly enhanced the water-solubility of the glycoconjugates [Mylvaganam and Lingwood (1999) Biochem. Biophys. Res. Commun. 257, 391—394]. The inhibitory potential of these soluble aminodeoxy conjugates on the binding of VT1 to Gb3Cer immobilized on an ELISA plate was evaluated. Only the 2′′ and the 6′ deoxyamino conjugates were effective inhibitors (IC50 10μM); the 4′′ and 6′′ conjugates were ineffective up to 10mM. To evaluate the importance of incorporating a rigid adamantyl hydrocarbon group into the ceramide aglycone, globotriaose was conjugated to a t-butylacetamido or an adamantaneacetamido aglycone. By similar ELISAs, only the adamantaneacetamido conjugate inhibited the binding of VT1 to Gb3Cer. When deoxyamino conjugates were adsorbed to silica on TLC plates, only the 2′′ and 6′′ conjugates bound VT1 and VT2. By a similar TLC assay, acetamido derivatives of 2′′ and 6′ deoxyamino conjugates showed less binding to VT1 and VT2. Neither the crystallographically determined structure of the VT1—globotriaose complex nor modelling studies fully explain the binding patterns shown by these deoxyamino glycoconjugates. Enhanced solvation of the ammonium group of the deoxyamino conjugate could enforce greater constraints in the binding interactions.