title: Comparison of binding platforms yields insights into receptor binding differences between shiga toxins 1 and 2.
abstract: Protein-glycan interactions are typically very weak, and avid binding is achieved when proteins express multiple glycan binding sites. Shiga toxin (Stx) uses glycan receptors to enter cells. Stx has five identical binding subunits, each with three nonidentical glycan binding sites. Previous studies examined binding to biantennary glycans expressing Pk trisaccharide mimics immobilized on streptavidin, resulting in display of four trisaccharides per streptavidin face. Stx1 preferred the Pk trisaccharide of its native receptor, globotriaosylceramide (Gb3), while the more potent and clinically relevant variant, Stx2, preferred the Pk trisaccharide with the terminal galactose replaced with N-acetylgalactosamine (NHAc-Pk). In the present study, binding of Stxs to Pk analogues was examined using two experimental platforms, ELISA and surface plasmon resonance (SPR). ELISA was more sensitive than SPR. Sensitivity in the ELISA was due to high streptavidin density, suggesting that avid binding may require engagement of more than four trisaccharides. Selectivity for the Pk analogues was maintained in both experimental platforms. Glycan preference was mapped to binding site 2, since reciprocal mutation of a single amino acid (asparagine 32 of Stx1 B-subunit/serine 31 of Stx2 B-subunit) reversed binding preference. However, native Stx1 bound well to plates loaded with a 50:50 mixture of Pk-NHAc-Pk, while Stx2 bound less efficiently, suggesting that one of the Stx1 binding sites may only engage Pk, while another may tolerate either Pk or NHAc-Pk. Varying glycan structure and density across different in vitro binding platforms revealed important differences in receptor binding properties between Stx1 and Stx2.
authors.affiliation: Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Flagler
authors.forename: Sujit S
authors.initials: SS
authors.lastname: Mahajan
authors.forename: Ashish A
authors.initials: AA
authors.lastname: Kulkarni
authors.forename: Suri S
authors.initials: SS
authors.lastname: Iyer
authors.forename: Alison A
authors.initials: AA
authors.lastname: Weiss
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D022622:Shiga Toxin 1 / Q000737:chemistry* / Q000378:metabolism*; D022641:Shiga Toxin 2 / Q000737:chemistry* / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D014281:Trihexosylceramides / Q000737:chemistry / Q000378:metabolism*; D014312:Trisaccharides / Q000737:chemistry / Q000378:metabolism
chemical_list: D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D014281:Trihexosylceramides; D014312:Trisaccharides; C018549:globotriaosylceramide