Binding of Escherichia coli heat-stable enterotoxin to rat intestinal cells and brush border membranes

Abstract

The association of heat-stable enterotoxin (ST(a)) produced by enterotoxigenic Escherichia coli 431 with isolated rat intestinal epithelial cells and brush border membranes was characterized. Specific binding of strain 431 125I-ST(a) to a single class of specific high-affinity receptors was saturable and temperature dependent and reached a maximum between 5 and 10 min. A 1,000-fold excess of unlabeled 431 ST(a) competitively displaced 90 to 95% of radiolabeled enterotoxin bound to brush border membranes. In contrast, specific binding of 431 125I-ST(a) to intestinal cells ranged from 40 to 65%. The number of ST(a)-specific receptors on rat intestinal cells determined by Scatchard analysis was 47,520 ± 14,352 (mean ± standard error of the mean) per cell, with affinity constants (K(a)s) of 2.55 x 1011 and 4.32 x 1011 liters/mol determined for intestinal cells and brush border membranes, respectively. Villus intestinal cells appeared to possess about twice as many ST(a) receptors as did crypt cells. Dissociation of specifically bound 431 125I-ST(a) from intestinal cells and brush border membranes was minimal (2 to 5%). In addition, neither the rate nor the extent of dissociation was increased by a 1,000-fold excess of unlabeled homologous 431 ST(a). Binding experiments with 431 125I-ST(a) and brush border membranes showed that purified unlabeled ST(a)s from enterotoxigenic E. coli strains 667 (class 1 porcine eneteropathogen), B-41 (bovine enteropathogen), and human strains 213C2 (Mexico) and 153961-2 (Dacca, Bangladesh) exhibited patterns of competitive inhibition similar to those of homologous unlabeled 431 ST(a) (class 2 enteropathogen). A lipid extract which contained gangliosides and glycolipids exhibited dose-dependent competitive inhibition of heat-labile enterotoxin binding to brush border membranes but did not inhibit binding of 431 125I-ST(a). Purified heat-labile enterotoxin from strain 286C2 did not inhibit binding of 431 ST(a) to brush border membranes. Pronase treatment of brush border membranes reduced binding of 431 125I-ST(a) by about 30%, suggesting that the ST(a) receptor was a protein or a glycoprotein. The putative ST(a) receptor was radiolabeled with 431 125I-ST(a) and solubilized with sodium deoxycholate. One major radioactive band was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by radioautography. These data suggested that ST(a)s bind essentially irreversibly to a specific receptor on the cell surface of intestinal cells before activation of guanylate cyclase.