Effect of Gb3 in lipid rafts in resistance to Shiga-like toxin of mutant Vero cells.
Shiga-like toxin 1 (Stx1), produced by enterohemorrhagic Escherichia coli, binds to its receptor, globotriaosylceramide (Gb3), on target cell membranes, as a prerequisite for inducing host cell intoxication. To examine further toxin-receptor interactions, we established an Stx1-resistant clone of Vero cells by chemical mutagenesis. The mutant cells, expressed Gb3, but did not bind Stx1. These mutant cells were larger and had more Gb3 per cell than wild-type cells. Gb3 from both wild-type and mutant Vero cells was recovered in lipid rafts, isolated from cell lysates as detergent resistant membranes (DRMs); the DRMs derived from mutant cells had a lower density of Gb3 than did those from wild-type cells. Stx1 did not bind to the DRMs of mutant cells, both by ELISA and surface plasmon resonance. However, Stx1 bound to Gb3 separated by thin-layer chromatograms from the DRMs of mutant cells. The results indicate that not only presence of Gb3 but also Gb3 density on lipid rafts were important for Stx binding.
Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba 260-8670, Japan.
Tatsuo
T
Hanashima
Masami
M
Miyake
Kinnosuke
K
Yahiro
Yoshifumi
Y
Iwamaru
Akikazu
A
Ando
Naoko
N
Morinaga
Masatoshi
M
Noda
D000818:Animals; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000494:pharmacology* / Q000502:physiology; D002522:Chlorocebus aethiops; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000737:chemistry*; D011485:Protein Binding / Q000187:drug effects*; D011956:Receptors, Cell Surface / Q000032:analysis / Q000378:metabolism*; D022603:Shiga Toxins / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000633:toxicity; D014281:Trihexosylceramides; D014709:Vero Cells / Q000382:microbiology
D015295:Antigens, Tumor-Associated, Carbohydrate; C472766:Gb3 antigen; D011956:Receptors, Cell Surface; D022603:Shiga Toxins; D014281:Trihexosylceramides; C018549:globotriaosylceramide