title: Differential carbohydrate epitope recognition of globotriaosyl ceramide by verotoxins and a monoclonal antibody.
abstract: The role of renal expression of the glycosphingolipid verotoxin receptor, globotriaosylceramide, in susceptibility to verotoxin-induced hemolytic uremic syndrome is unclear. We show that a single glycosphingolipid can discriminate multiple specific ligands. Antibody detection of globotriaosylceramide in renal sections does not necessarily predict verotoxin binding. The deoxyglobotriaosylceramide binding profile for verotoxin 1, verotoxin 2 and monoclonal anti-globotriaosylceramide are distinct. Anti-globotriaosylceramide had greater dependency on the intact alpha-galactose and reducing glucose of globotriaosylceramide than verotoxin 1, while verotoxin 2 was intermediate. These ligands differentially stained human kidney sections. Glomerulopathy is the primary verotoxin-associated pathology in hemolytic uremic syndrome. For most samples, verotoxin 1 immunostaining within adult glomeruli was observed (type A). Some samples, however, lacked glomerular binding (type B). Anti-globotriaosylceramide (and less effectively, verotoxin 2) stained all glomeruli. Verotoxin 1/anti-globotriaosylceramide tubular staining was comparable. Type B glomerular/tubular globotriaosylceramide showed minor, but significant, fatty acid compositional differences. Verotoxin 1 type B glomerular binding became evident following pretreatment with cold acetone, or methyl-beta-cyclodextrin, used to deplete cholesterol. Direct visualization, using fluorescein isothiocyanate-verotoxin 1B, showed paediatric, but no adult glomerular staining; this was confirmed by anti-fluorescein isothiocyanate immunostaining. Acetone induced fluorescein isothiocyanate-verotoxin 1B glomerular staining in type A, but poorly in type B samples. Comparison of fluorescein isothiocyanate-verotoxin 1B and native verotoxin 1B deoxyglobotriaosylceramide analogue binding showed an alteration in subspecificity. These studies indicate a marked heterogeneity of globotriaosylceramide expression within renal glomeruli and differential binding of verotoxin 1/verotoxin 2/anti-globotriaosylceramide to the same glycosphingolipid. Verotoxin 1 derivatization can induce subtle changes in globotriaosylceramide binding to significantly affect tissue binding. Heterogeneity in glomerular globotriaosylceramide expression may play a significant (cholesterol-dependent?) role in determining renal pathology following verotoxemia.
authors.affiliation: Research Institute, Division of Infection, Immunity, Injury and Repair, The Hospital for Sick Children, Ontario, Canada.
authors.forename: Davin
authors.initials: D
authors.lastname: Chark
authors.forename: Anita
authors.initials: A
authors.lastname: Nutikka
authors.forename: Natasha
authors.initials: N
authors.lastname: Trusevych
authors.forename: Julia
authors.initials: J
authors.lastname: Kuzmina
authors.forename: Clifford
authors.initials: C
authors.lastname: Lingwood
mesh_terms: D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D002784:Cholesterol / Q000172:deficiency; D000939:Epitopes; D006801:Humans; D007674:Kidney Diseases / Q000378:metabolism* / Q000473:pathology; D007678:Kidney Glomerulus / Q000378:metabolism*; D008024:Ligands; D013058:Mass Spectrometry; D011485:Protein Binding; D022622:Shiga Toxin 1 / Q000378:metabolism*; D022641:Shiga Toxin 2 / Q000378:metabolism*; D014281:Trihexosylceramides / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D000939:Epitopes; D008024:Ligands; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D014281:Trihexosylceramides; C018549:globotriaosylceramide; D002784:Cholesterol