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Injection of a crude preparation of cholera enterotoxin into the superior mesenteric artery caused isotonic fluid secretion by the canine small bowel. In dogs previously exposed to cellular antigens of Vibrio cholerae, the time course of the intestinal response to intra-arterial injection of crude enterotoxin was similar to that observed after intraluminal exterotoxin challenge.
An increase in content of cAMP in the rabbit jejunum mucosa after treatment with cholerae toxin was due to adenylate cyclase activation. The maximal activation of adenylate cyclase (AC) and increase in the cAMP content in the mucosa were observed within 4 hrs after administration of the cholerae toxin in situ into the lumen of an isolated strip of the rabbit jejunum. Cholerae enterotoxin did not affect the activity of phosphodiesterase (PDE). It was shown that AC activation did not correlate with an increase in the cAMP content in rabbit jejunum mucosa exposed to cholerae enterotoxin. Absence of the correlation was demonstrated by kinetic characteristics of PDE and by regulatory effect of Ca2+ on the activity of AC and PDE.
Certain enteropathogenic bacteria, including strains of Escherichia coli, Klebsiella pneumoniae and Yersinia enterocolitica, elicit their diarrhoeagenic effects by elaborating small molecular weight, heat-stable enterotoxins (STs). Structural and functional characteristics indicate that ST peptides are heterogeneous and two major subtypes, STa and STb, have been identified. Molecules of STa, unlike those of STb, are methanol-soluble and elicit their pathogenic effects by activating host cell guanylate cyclase activity and thereby increasing tissue cyclic GMP content: this increase in cyclic GMP causes fluid secretion. STa binds to specific proteinaceous receptors on intestinal cells but the nature of STa-receptor coupling to guanylate cyclase is poorly understood. The actions of STa, including binding to its receptor, activation of guanylate cyclase and stimulation of electrolyte transport, are rapid, reversible and tissue-specific. STa activates only particulate and not soluble guanylate cyclase. It alters the Vmax but not the apparent Km of this enzyme for Mg-GTP or Mn-GTP. At concentrations above 0.5-1 mM, calcium inhibits the STa activation of guanylate cyclase. The effects of calmodulin antagonists such as chlorpromazine on the activation of guanylate cyclase by STa are less clear. Inhibitors of phospholipid and arachidonic acid cascade pathways interfere with both basal and STa-stimulated guanylate cyclase. Membrane integrity is essential for STa activation of guanylate cyclase and the STa-receptor complex may activate the enzyme by intramembrane protein-protein interactions and/or perturbations. Interference with membrane phospholipid could alter such coupling.
Escherichia coli heat stable enterotoxin (STa) and the newly identified endogenous ligand guanylin bind to an intestinal receptor and activate membrane bound guanylate cyclase. We compared STa binding and affinity crosslinking of STa receptors in human small intestine to those in the Caco-2 human colon carcinoma cell line. STa had similar kinetics of binding in human intestinal and Caco-2 brush border membranes. In both human intestine and Caco-2 brush border membranes, multiple specifically radiolabeled bands, including a 140-165 kDa band, were identified by affinity crosslinking. However, in human intestine the most prominent autoradiographic species was a 60 kDa band. A 60 kDa protein was also specifically immunoprecipitated from solubilized human brush border membranes using antisera raised against a cloned STa receptor fusion protein. Our observations of multiple crosslinked proteins in human intestine and Caco-2 cells could be explained by the existence of several members of a family of STa receptors and/or the existence of smaller STa binding proteins generated by the protease cleavage of a larger complete STa receptor.
Heat-stable enterotoxin (ST(a)) elaborated by E. coli is a major cause of diarrhea. The transmembrane protein guanylyl cyclase C (GC-C) is the acknowledged receptor for ST(a) and for the mammalian peptides guanylin and uroguanylin. Binding to GC-C results in generation of cGMP, activation of type II cGMP-dependent protein kinase, phosphorylation of CFTR and increased chloride and bicarbonate secretion. We had previously shown that ST(a) receptors (GC-C) are found on the brush border membranes of small intestinal enterocytes and of colonocytes. However, since it has subsequently been shown that the endogenous ligands for these receptors, guanylin and uroguanylin, circulate in blood, we proposed the existence of ST(a) binding sites on the basolateral membranes (BLM) of colonocytes. Specific binding of 125I-ST(a) to rat colonocyte BLM was seen. The kinetics of binding to the BLM were similar to binding to BBM. The nature of the BLM receptor is unknown. This suggests that circulating guanylin and uroguanylin, analogues of ST(a), may also function via the basolateral surface.
A fragment of guanylate cyclase C (GC-C) of about 1.7 k bp corresponding to amino acids 1-553 spanning the extracellular and transmembrane domains and a portion of the intracellular region was amplified using template cDNA prepared from rat intestinal cells by the polymerase chain reaction method. The cloned 1.7 k bp fragment was inserted into the mammalian expression vector pCGUT and the truncated GC-C expressed on the surface of COS-7 cells was demonstrated to bind heat-stable enterotoxin by photo affinity labeling with 125I-N-5-azidonitrobenzoyl-STh[5-19]. Analysis by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis showed that the truncated GC-C formed dimers on the surface of COS-7 cells. The intracellular region of GC-C was found not to be necessary for dimer formation by the GC-C. Comparison of the molecular weights of the truncated GC-C expressed in COS-7 cells and Escherichia coli suggested that the truncated GC-C was glycosylated in the mammalian expression system.
The entire extracellular domain of the human heat-stable enterotoxin (ST) receptor as well as a truncated N-terminal domain were cloned as glutathione S-transferase fusion proteins and expressed in Escherichia coli. The recombinant fusion proteins were purified from both the cytosol and the inclusion body fractions by selective detergent extraction followed by glutathione-agarose affinity chromatography. The purified protein, corresponding to the entire extracellular domain, bound the stable toxin peptide with an affinity comparable to that of the native receptor characterized from the human colonic T84 cell line. No binding was observed with the N-terminal truncated fragment of the receptor under similar conditions. Polyclonal antibodies were raised to the entire extracellular domain fusion protein as well as the truncated extracellular domain fusion protein, and the antibodies were purified by affinity chromatography. Addition of the purified antibodies to T84 cells inhibited ST binding and abolished ST-mediated cGMP production, indicating that critical epitopes involved in ligand interaction are present in the N-terminal fragment of the receptor. Purified antibodies recognized a single protein of Mr 160,000 Da on Western blotting with T84 membranes, corresponding to a size of the native glycosylated receptor in T84 cells. These studies are the first report of the expression, purification, and characterization of any member of the guanylyl cyclase family of receptors in E. coli and show that binding of the toxin to the extracellular domain of the receptor is possible in the absence of any posttranslational modifications such as glycosylation. The recombinant fusion proteins as well as the antibodies that we have generated could serve as useful tools in the identification of critical residues of the extracellular domain involved in ligand interaction.
Enterotoxigenic strains of Escherichia coli that produce heat-stable enterotoxin (STa), are a major cause of diarrheal disease worldwide. Resistance to diarrheal disease in human infants and newborn animals has been attributed to a gradual turnover in the intestinal brush border membrane receptors to bacterial pili. In this study, we demonstrated age-dependent variation in the density and affinity of the mouse enterocyte receptors specific for STa. Flow cytometry and radiolabeled-STa (125I-STa) assays were used as more reliable quantitative measures for the characterization of STa-enterocyte receptor interaction. These assays indicated a stronger interaction of STa with its putative receptor on the enterocytes of the 2-day-old suckling mice than with enterocytes from 1-week, 2-week and 2-month-old mice. Scatchard plot analysis of 125I-STa-receptor interaction suggested that STa-receptors exist at a higher number on enterocytes from the 2-day-old mice than enterocytes of the older mice. Additionally, receptors from the 2-day-old mice had a greater affinity for STa ligand than receptors from the older mice. Density of STa receptors on enterocytes and their affinity to STa may determine the extent of binding and severity of secretory response. This may further explain the increased susceptibility of newborn animals and human infants to STa-mediated diarrheal disease.
Effect of insulin on the response of suckling mice to the enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) was studied. Four groups (8-10 in each group) of two day old Swiss Webster suckling mice were used. Five, 10, 25, and 50 micrograms of insulin were given orally to half the mice in each group respectively. The rest of the mice in each group were given normal saline as intra-litter controls. After 7 days, the suckling mouse assay for STa was performed on three mice from each insulin-treated and control groups. Enterocyte suspensions were prepared from mice in all groups. Intestinal tissue samples were taken for electron microscopy. Interaction of STa with its putative receptor on the enterocytes was evaluated using indirect immunofluorescence and flow cytometry. The suckling mouse assay revealed a significant increase in the gut weight to body weight ratio in all mice in the insulin treated groups compared to control mice (p < 0.05). Flow cytometry and indirect immunofluorescence analyses suggested that insulin had an upregulatory effect on the STa receptor level. Similarly, insulin was found to increase intestinal brush border membrane differentiation as indicated by the increase in the inward movement of milk particles through the intestinal mucosa. Insulin seems to modify the structure-function of the brush border membrane including the response of suckling mice to STa. This study may provide further insights into the mechanism of STa/receptor interaction in diarrhea in newborn animals and human infants.
We studied the binding of E. coli heat-stable enterotoxin (STa) to rat brush borders (BB) and to basolateral membranes (BLM) using a biologically active monoiodinated radioligand [( 125I]STa) and highly enriched BB and BLM preparations free of other significant organelle contamination. Binding of [125I]STa to BB was specific; time-, temperature-, and pH-dependent; saturable; and partially reversible. Nonlabeled toxin competitively inhibited the binding of radioligand to BB in a dose-related manner. Scatchard analysis revealed a single class of receptors with an apparent affinity constant of 8.7 +/- 1.5 X 10(8) l/mol. Binding was not affected by amino acids, sugars, and lectins. Proteolytic enzymes significantly decreased binding, although several did so by modifying the radioligand. Trypsin inhibited binding without modifying the radioligand thus supporting the proteinaceous nature of the receptor. Since the enrichment in binding activity in the BB over the homogenate was significantly lower than the enrichment in sucrase activity, we concluded that binding activity is probably associated with other membranous domains, but direct examination revealed no binding activity on basolateral membranes.
Extraction of pig intestinal brush border membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (Chaps) in the presence of 0.5 M KCl yielded a solution which contained 60-70% of the receptor for the Escherichia coli heat-stable enterotoxin (STa) and of the Lubrol PX-activated guanylate cyclase activity present in the membrane. When the supernatant solution was diluted fivefold with 10 mM Hepes buffer (pH 7.4) and kept at 4 degrees C overnight, a precipitate formed. Centrifugation yielded a pellet (P2) which contained 25-30% of both the cyclase and the receptor in the original membranes, with a 2.5- to 3-fold enrichment of both. The process could be repeated for further enrichment (P4). The addition of MgCl2 to the diluted extract affected both basal and STa-stimulated activity of P2; 1 mM was optimal. P2 resembled membranes with respect to competitive inhibition of 125I-STa binding by STa, and the concentration-dependent activation of cyclase by STa. Guanylate cyclase in resolubilized P2 was also activated by STa. Most of the enzymes interfering with guanylate cyclase determinations were removed, as were the brush border marker enzymes sucrase and gamma-glutamyltransferase, and a GTP-binding protein that is a pertussis toxin substrate. Specific cross-linking of 125I-STa to receptors in the membrane was preserved in P2 and P4, the three proteins showing the strongest radioactivity having relative molecular masses of 55,000-60,000, 70,000-80,000, and 135,000-140,000. P2 and P4 appear to contain a complex of membrane proteins with certain functional properties intact.
To study the predominant binding substance for the heat-labile enterotoxin (LTc) isolated from chicken enterotoxigenic Escherichia coli, competitive binding assays were performed with neuraminidase-treated human type B erythrocytes and 125I-labeled B subunit of LTc (LTc-B). Of all inhibitors used, the ganglioside GM1 was the most effective in inhibiting the binding of 125I-labeled LTc-B to the erythrocytes. The other gangliosides used as inhibitors, gangliosides GD1b, GD1a, GM2, GT1b and GM3, were about 24, 166, 250, 440 and at least 440 times less reactive than ganglioside GM1, respectively. With glycoproteins as inhibitors, on the other hand, hog A + H, porcine thyroglobulin and bovine salivary mucin were over 10(4) times less potent. No inhibition was obtained by other mono-, di- and polysaccharides at the highest concentrations used. These findings suggest that the predominant binding substance on neuraminidase-treated human type B erythrocytes for the LTc-B is ganglioside GM1 and that the combining site of LTc-B may be specific for the terminal disaccharide (galactose-N-acetyl-D-galactosamine)-linked portion of ganglioside GM1.
Proteins binding Escherichia coli heat-stable enterotoxin were isolated from the cytoskeleton of intestinal membranes using an affinity matrix of biotinylated ST immobilized on monomeric avidin-agarose. ST binding proteins were purified 343-fold using this affinity technique, with 7% of the initial binding activity recovered in these preparations. ST binding proteins isolated by affinity chromatography possessed a native and subunit molecular mass of 56 kDa. These preparations exhibited both high- and low-affinity binding sites for ST. Guanylate cyclase in extracts of the intestinal membrane cytoskeleton was completely recovered in fractions which did not associate with the affinity matrix. In addition, ST binding proteins isolated by affinity chromatography were devoid of guanylate cyclase activity. These data, taken together with those obtained previously with crude and partially purified receptors, suggest that ST binds to different proteins in intestinal membranes, some of which do not possess guanylate cyclase activity.
A heat-stable enterotoxin (STp) consisting of 18 amino acid residues including 6 half-cystine residues is produced by a porcine strain of enterotoxigenic Escherichia coli. Analogs of STp with replacements of single residues at each from positions 5 to 17 by the corresponding D-amino acid residue were synthesized by a solid-phase method. Of these analogs, [D-Cys5]-STp[5-17] showed the same biological properties as STp[5-17]. Moreover, its activity to cause fluid accumulation in suckling mouse lasts more than 24 hours, whereas the activity of STp[5-17] decreases after 6-10 hours. These results indicate that the action of the analog [D-Cys5]-STp[5-17] is strongly agonistic to that of the native ST.
The Escherichia coli heat-stable enterotoxin (ST1 or STa) binds to specific receptors on mammalian intestinal brush border membranes, and stimulates guanylate cyclase in those membranes. We have found a similar signal transduction system in brush border membranes prepared from kidney cortex of the American opossum (Didelphis virginiana, and in a cell line (OK cell) derived from that tissue. Activation of guanylate cyclase by ST1 is therefore not limited to intestinal cells. Furthermore, since it is unlikely that ST1 which is produced in the intestinal lumen, would have access to kidney receptors, this suggests the existence of an endogenous peptide resembling ST1, at least in marsupials.
Analogs of a heat-stable enterotoxin (ST) that have a CH2-S linkage instead of an S-S linkage in the molecule were synthesized by conventional methods. The synthetic peptides showed toxicity, assayed as induction of fluid secretion in suckling mice, although their toxicities were hundredth that of native ST. This finding implies that ST is not recognized by its receptor protein through an exchange reaction between its disulfide linkages and thiol-groups of its receptor protein(s), but through hydrophobic or electrostatic interactions.
1. Escherichia coli heat-stable enterotoxin (ST) induces a secretory diarrhea by binding to receptors on brush borders of intestinal villus cells, activating particulate guanylate cyclase and increasing intracellular concentrations of guanosine 3',5'-cyclic monophosphate (cyclic GMP). 2. However, little is known concerning coupling of receptor-ligand interaction to enzyme activation. 3. This study compares the kinetics of toxin-receptor binding and enzyme activation to better understand this transmembrane signal cascade. 4. Toxin receptor binding was linear and saturable with 50% of maximum displacement of [125I]ST by unlabeled toxin observed at 1.1 x 10(-7) M. ST increased the maximum velocity (Vmax) of guanylate cyclase with magnesium or manganese as the cation substrate without altering the affinity of the enzyme for its substrate or its positive cooperativity. 5. The concentration of toxin yielding half-maximum stimulation of guanylate cyclase was 1.2 x 10(-6) M, 10-fold higher than the affinity of the ligand for its receptor. 6. These data are consistent with the suggestion that ST-receptor interaction is coupled to activation of particulate guanylate cyclase. 7. However, the discrepancy between the affinity of ST for its receptor and its efficacy in activating the enzyme suggests that this coupling is complex. 8. Possible mechanisms underlying this coupling are discussed.
Heat-stable enterotoxins (ST) activate guanylyl cyclase in T84 cells, rapidly and specifically. Activation is monitored by cGMP production and occurs at lower concentrations of ST than required for eliciting fluid accumulation in suckling mice. Atrial natriuretic factor (ANF) neither activates guanylyl cyclase nor modulates the response to ST in T84 cells, indicating the absence of receptors for ANF on T84 cells. Monitoring the production of cGMP under conditions known to alter fluid accumulation in suckling mice is an accurate and quantifiable assay of ST activity and its interaction with the receptor. STs produced by Escherichia coli, Vibrio cholerae non-01 and Yersinia enterocolitica individually produce elevated levels of cGMP in T84 cells, but to differing extents, suggesting that this model system can be used to elucidate the different events of ST-receptor interactions at the molecular level.