THE HEAT-LABILE ENTEROTOXIN OF ESCHERICHIA-COLI BINDS TO POLYLACTOSAMINOGLYCAN-CONTAINING RECEPTORS IN CACO-2 HUMAN INTESTINAL EPITHELIAL-CELLS

The E. coli type I heat-labile enterotoxin (LT-I) shares considerable functional, structural, and immunological homology with cholera toxin (CT). Although the ganglioside G(M1) is the sole receptor for CT, LT-I also appears to utilize additional, unique receptors on intestinal cells not recognized by CT. We characterized this second class of LT-I receptors using the human intestinal epithelial cell line, CaCo-2. CaCo-2 cells bound 8-fold more LT-I than CT, and some of these additional LT-I receptors appeared to be functional, as CT-B only partially inhibited LT-I activity at concentrations that completely inhibited CT activity. Membranes from unlabeled or [H-3] galactose-labeled cells were incubated with toxin B subunits and extracted with Triton X-100, and the solubilized toxin B-receptor complexes were immunoabsorbed with anti-B bound to protein A-Sepharose. When organic extracts of the complexes were separated by thin-layer chromatography and overlayed with [I-125] toxin, both toxins were found to bind only G(M1). Separation of the complexes from [H-3] galactose-labeled membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a series of galactoproteins specifically recognized by LT-I but not by CT. Similar proteins were detected on Western blots probed with [I-125]toxin. LT-I activity on intact cells and binding to membranes and the above galactoproteins were enhanced by neuraminidase treatment even in the presence of CT-B. beta-1,4-Galactosidase and endo-beta-1,4-galactosidase, but not beta-1,3-galactosidase, significantly reduced LT-I binding. LT-I binding to fetuin and transferrin exhibited a similar glycosidase sensitivity. When analyzed by Western blotting with [I-125]toxin, LT-I cross-reacted with glycoproteins immunoprecipitated from membrane extracts with antibodies specific for Le(x) and the I/i blood groups and for the lysosomal membrane glycoproteins LAMP 1 and 2. These results indicated that polylactosylaminylated membrane glycoproteins represented an alternate class of receptors for LT-I in CaCo-2 cells.