Differential intracellular transport and binding of verotoxin 1 and verotoxin 2 to globotriaosylceramide-containing lipid assemblies

Although verotoxin‐1 (VT1) and verotoxin‐2 (VT2) share a common receptor, globotriaosyl ceramide (Gb3), 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 Gb3 assemblies. At 4°C, fluorescent‐VT1 and VT2 bound both coincident and distinct punctate surface Gb3 microdomains. After 10 min at 37°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% 125I‐VT1 cell surface bound, or added to detergent‐resistant cell membrane extracts (DRM), was in the Gb3‐containing sucrose gradient ‘insoluble’ fraction, whereas only 30% 125I‐VT2 was similarly DRM‐associated. VT1 bound more efficiently to Gb3/cholesterol DRMs generated in vitro. Only VT1 binding was inhibited by high cholesterol/Gb3 ratios. VT2 competed less effectively for 125I‐VT1/Gb3 DRM‐binding but only VT2‐Gb3/cholesterol DRM‐binding was augmented by sphingomyelin. Differential VT1/VT2 Gb3 raft‐binding may mediate differential cell binding/intracellular trafficking and cytopathology. J. Cell. Physiol. 216: 750–763, 2008, © 2008 Wiley‐Liss, Inc.