Cholera toxin entry into pig enterocytes occurs via a lipid raft- and clathrin-dependent mechanism.
The small intestinal brush border is composed of lipid raft microdomains, but little is known about their role in the mechanism whereby cholera toxin gains entry into the enterocyte. The present work characterized the binding of cholera toxin B subunit (CTB) to the brush border and its internalization. CTB binding and endocytosis were performed in organ-cultured pig mucosal explants and studied by fluorescence microscopy, immunogold electron microscopy, and biochemical fractionation. By fluorescence microscopy CTB, bound to the microvillar membrane at 4 degrees C, was rapidly internalized after the temperature was raised to 37 degrees C. By immunogold electron microscopy CTB was seen within 5 min at 37 degrees C to induce the formation of numerous clathrin-coated pits and vesicles between adjacent microvilli and to appear in an endosomal subapical compartment. A marked shortening of the microvilli accompanied the toxin internalization whereas no formation of caveolae was observed. CTB was strongly associated with the buoyant, detergent-insoluble fraction of microvillar membranes. Neither CTB's raft association nor uptake via clathrin-coated pits was affected by methyl-beta-cyclodextrin, indicating that membrane cholesterol is not required for toxin binding and entry. The ganglioside GM(1) is known as the receptor for CTB, but surprisingly the toxin also bound to sucrase-isomaltase and coclustered with this glycosidase in apical membrane pits. CTB binds to lipid rafts of the brush border and is internalized by a cholesterol-independent but clathrin-dependent endocytosis. In addition to GM(1), sucrase-isomaltase may act as a receptor for CTB.
Hansen
Gert H
GH
Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
Dalskov
Stine-Mathilde
SM
Rasmussen
Christina Rehné
CR
Immerdal
Lissi
L
Niels-Christiansen
Lise-Lotte
LL
Danielsen
E Michael
EM
D000818:Animals; D002451:Cell Compartmentation; D002772:Cholera Toxin / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D007421:Intestine, Small / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D050356:Lipid Metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009924:Organ Culture Techniques; D013394:Sucrase-Isomaltase Complex / Q000378:metabolism; D013552:Swine
D002966:Clathrin; D002772:Cholera Toxin; D013394:Sucrase-Isomaltase Complex