Capping and receptor-mediated endocytosis of cell-bound verotoxin (Shiga-like toxin). 1: Chemical identification of an amino acid in the B subunit necessary for efficient receptor glycolipid binding and cellular internalization.
The glycolipid globotriaosylceramide (Gb3) is the plasma membrane receptor that mediates the internalization of verotoxin (VT1) into susceptible cells by capping and receptor-mediated endocytosis (RME). Internalization of fluorescein isothiocyanate-conjugated holotoxin into Daudi lymphoma cells was found to be slower than the pentameric receptor binding B subunit alone, suggesting that the A subunit may interact with the membrane to compromise the lateral mobility of the receptor bound B subunit. 3-D reconstruction of fluorescent images by confocal microscopy confirmed the complete internalization of holotoxin. VT1 internalization and cytotoxicity was inhibited by monodansyl cadavarine, which supports a role for clathrin coated pits in the RME of VT1. Biotinylation of the B subunit (in contrast to fluorescein labelling) was found to prevent toxin internalization. This effect correlated with reduced binding of Gb3 and reduced cytotoxicity in vitro. By cleavage of the B subunit at the single tryptophan residue, the reduced Gb3 binding and lack of cellular internalization was shown to be due to the biotinylation of lysine 53 in the VT1 B subunit. This residue was not labelled with fluorescein isothiocyanate in the native protein. This conclusion was confirmed by the finding that biotinylation of VT2c (which contains lys 53) prevented glycolipid receptor binding, whereas biotinylation of VT2e (in which lys 53 is substituted by ile) had no effect.
Department of Microbiology, Hospital for Sick Children, Toronto, Ontario, Canada.
A A
AA
Khine
C A
CA
Lingwood
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism*; D001710:Biotin; D002103:Cadaverine / Q000031:analogs & derivatives / Q000494:pharmacology; D003603:Cytotoxins / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate; D006017:Glycolipids / Q000378:metabolism*; D007152:Immunologic Capping; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011971:Receptors, Immunologic / Q000502:physiology*; D022622:Shiga Toxin 1; D014407:Tumor Cells, Cultured
D001427:Bacterial Toxins; D003603:Cytotoxins; D006017:Glycolipids; D007166:Immunosuppressive Agents; D011971:Receptors, Immunologic; D022622:Shiga Toxin 1; D001710:Biotin; D016650:Fluorescein-5-isothiocyanate; C008542:monodansylcadaverine; D002103:Cadaverine