An association of 27- and 40-kDa molecules with glycolipids that bind A-B bacterial enterotoxins to cultured cells.
It is well recognized that the Shiga-like toxins (Stxs) preferentially bind to Gb3 glycolipids and the cholera toxin (CT) and heat-labile enterotoxin (LTp) bind to GM1 gangliosides. After binding to the cell surface, A-B bacterial enterotoxins have to be internalized by endocytosis. The transport of the toxin-glycolipid complex has been documented in several manners but the actual mechanisms are yet to be clarified. We applied a heterobifunctional cross-linker, sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropionate (SASD), to detect the membrane proteins involved in the binding and the transport of A-B bacterial enterotoxins in cultured cells. Both Stx1 and Stx2 bound to the detergent-insoluble microdomain (DIM) of Vero cells and Caco-2 cells, which were susceptible to the toxin, but neither was bound to insusceptible CHO-K1 cells. Both CT and LTp bound to the DIM of Vero cells, Caco-2 cells, and CHO-K1 cells. In a cross-linking experiment, Stx1 cross-linked only with a 27-kDa molecule, while Stx2, which was more potently toxic than Stx1, cross-linked with 27- and 40-kDa molecules of Vero cells as well as of Caco-2 cells; moreover, no molecules were cross-linked with the insusceptible CHO-K1 cells. LTp was cross-linked only to the 27-kDa molecule of these three cell types but the CT, which was more toxic than LTp, was also cross-linked with 27- and 40-kDa molecules of Vero cells, Caco-2 cells, and CHO-K1 cells. The 27- and the 40-kDa molecules might play a role in the endocytosis and retrograde transport of A-B bacterial enterotoxins.
Shimizu
Takeshi
T
Department of Microbiology, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Ten-nohdai, Tsukuba 305-8575, Japan. kshimizu@md.tsukuba.ac.jp
Hamabata
Takashi
T
Yoshiki
Akemi
A
Hori
Takeya
T
Ito
Satoru
S
Takeda
Yoshifumi
Y
Hayashi
Hideo
H
D000818:Animals; D001386:Azides / Q000737:chemistry / Q000378:metabolism; D001427:Bacterial Toxins / Q000378:metabolism; D002460:Cell Line; D048429:Cell Size; D002772:Cholera Toxin / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D004768:Enterotoxins / Q000378:metabolism; D029968:Escherichia coli Proteins; D006017:Glycolipids / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D008970:Molecular Weight; D011485:Protein Binding; D022622:Shiga Toxin 1 / Q000737:chemistry / Q000378:metabolism*; D022641:Shiga Toxin 2 / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013388:Succinimides / Q000737:chemistry / Q000378:metabolism
D001386:Azides; D001427:Bacterial Toxins; D003432:Cross-Linking Reagents; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D006017:Glycolipids; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D013388:Succinimides; D002772:Cholera Toxin; C047257:sulfosuccinimidyl 2-(4-azidosalicylamido)ethyl-1,3'-dithiopropionate; C027538:heat-labile enterotoxin, E coli