title: Differential intracellular transport and binding of verotoxin 1 and verotoxin 2 to globotriaosylceramide-containing lipid assemblies.
abstract: Although verotoxin-1 (VT1) and verotoxin-2 (VT2) share a common receptor, globotriaosyl ceramide (Gb(3)), VT2 induces distinct animal pathology and is preferentially associated with human disease. Moreover VT2 cytotoxicity in vitro is less than VT1. We therefore investigated whether these toxins similarly traffic within cells via similar Gb(3) assemblies. At 4 degrees C, fluorescent-VT1 and VT2 bound both coincident and distinct punctate surface Gb(3) microdomains. After 10 min at 37 degrees C, similar distinct/coincident micropunctate intracellular localization was observed. Most internalized VT2, but not VT1, colocalized with transferrin. After 1 h, VT1 and VT2 coalesced during retrograde transport to the Golgi. During prolonged incubation (3-6 h), VT1, and VT2 (more slowly), exited the Golgi to reach the ER/nuclear envelope. At this time, VT2 induced a previously unreported, retrograde transport-dependent vacuolation. Cell surface and intracellular VT1 showed greater detergent resistance than VT2, suggesting differential 'raft' association. >90% (125)I-VT1 cell surface bound, or added to detergent-resistant cell membrane extracts (DRM), was in the Gb(3)-containing sucrose gradient 'insoluble' fraction, whereas only 30% (125)I-VT2 was similarly DRM-associated. VT1 bound more efficiently to Gb(3)/cholesterol DRMs generated in vitro. Only VT1 binding was inhibited by high cholesterol/Gb(3) ratios. VT2 competed less effectively for (125)I-VT1/Gb(3) DRM-binding but only VT2-Gb(3)/cholesterol DRM-binding was augmented by sphingomyelin. Differential VT1/VT2 Gb(3) raft-binding may mediate differential cell binding/intracellular trafficking and cytopathology.
authors.affiliation: Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Patty
authors.initials: P
authors.lastname: Tam
authors.forename: Radhia
authors.initials: R
authors.lastname: Mahfoud
authors.forename: Anita
authors.initials: A
authors.lastname: Nutikka
authors.forename: Aye Aye
authors.initials: AA
authors.lastname: Khine
authors.forename: Beth
authors.initials: B
authors.lastname: Binnington
authors.forename: Paul
authors.initials: P
authors.lastname: Paroutis
authors.forename: Clifford
authors.initials: C
authors.lastname: Lingwood
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D011485:Protein Binding; D022622:Shiga Toxin 1 / Q000378:metabolism*; D022641:Shiga Toxin 2 / Q000378:metabolism*; D014281:Trihexosylceramides / Q000737:chemistry / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism
chemical_list: D008055:Lipids; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D014281:Trihexosylceramides; C018549:globotriaosylceramide