Solubilization and reprecipitation from intestinal brush border membranes of a complex containing guanylate cyclase activatable by the heat-stable enterotoxin.

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.     

Regulation of particulate guanylate cyclase by Escherichia coli heat-stable enterotoxin: receptor binding and enzyme kinetics

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.     

Identification of a binding region on Escherichia coli heat-stable enterotoxin to intestinal guanylyl cyclase C

The heat-stable enterotoxin (ST), produced by Escherichia coli, causes acute diarrhea in infants and domestic animals by activation of the intestinal membrane-bound receptor, guanylyl cyclase C. We have investigated a region on the ST molecule, which is recognized by the receptor, by introducing a photochromophore, p-azidophenylalanine (Pap), into three different regions of STp(4–17), which has the full toxic activity. Each ST analog bound to the receptor, but only STp(4–17) containing a Pap residue at position 11 in the central portion of the ST molecule, showed a high efficiency in the reaction which cross- linked with the receptor by UV radiation. These data clearly demonstrate that the region of the ST molecule encompassing the Asn11 residue directly interacts with the receptor.     

Identification of a binding region on Escherichia coli heat-stable enterotoxin to intestinal guanylyl cyclase C

Summary The heat-stable enterotoxin (ST), produced byEscherichia coli, causes acute diarrhea in infants and domestic animals by activation of the intestinal membrane-bound receptor, guanylyl cyclase C. We have investigated a region on the ST molecule, which is recognized by the receptor, by introducing a photochromophore,p-azidophenylalanine (Pap), into three different regions of STp(4–17), which has the full toxic activity. Each ST analog bound to the receptor, but only STp(4–17) containing a Pap residue at position 11 in the central portion of the ST molecule, showed a high efficiency in the reaction which cross-linked with the receptor by UV radiation. These data clearly demonstrate that the region of the ST molecule encompassing the Asn¹¹ residue directly interacts with the receptor.     

Recognition and signal transduction mechanism of Escherichia coli heat-stable enterotoxin and its receptor, guanylate cyclase C.

Guanylate cyclase C (GC-C), a member of the membrane-bound GC family, consists of an extracellular domain (ECD) and an intracellular domain, which are connected by a single-transmembrane region. GC-C is a receptor protein, i.e. specifically stimulated by the endogenous peptides guanylin, uroguanylin, lymphoguanylin, and the exogenous peptide heat-stable enterotoxin (ST(a)), secreted by pathogenic Escherichia coli and acting on the intestinal brush border membranes. The binding of these peptide ligands to the ECD of GC-C results in the synthesis of cyclic GMP in cells, which, in turn, regulates a variety of intracellular physiologic processes. As the cloning of GC-C, its physiologic functions of each domain have been vigorously investigated. The structural characterization of the ligand-binding domain of the receptor promises to provide important clues for better understanding of the mechanisms of receptor recognition and activation. Recently, structural data for each domain of membrane-bound GCs and related proteins has become available. Coupling information obtained from such work and validation of structure-function relationships of GC-C and its ligands should allow for three-dimensional mapping of their interaction site in detail. Our approach to this issue involved designing photoaffinity-labeling ST(a) analogs, capable of binding covalently to the ligand-binding region of the ECD of GC-C. The photoaffinity-labeling ligand was used to covalently label a soluble form of the recombinant ECD protein. Mass spectrometric analyses of an endoproteinase digest of the ECD revealed that the ligand specifically bound to a narrow region contained in the membrane-proximal subdomain of the ECD of GC-C. These results will enable us to identify the possible binding motifs within the ligand-binding domain by computer modeling. In this review, we summarize the available data on the recognition mechanism between ST(a) and GC-C at the molecular level.     

Effect of heat-stable and heat-labile enterotoxins of Escherichia coli on intestinal brush border membrane enzymes of mice

The activities of intestinal brush border membrane (BBM) enzymes alkaline phosphatase, maltase, lactase, sucrase, gamma-glutamyl transpeptidase and leucine aminopeptidase were determined in intestinal homogenates and purified BBMs from control, heat-stable and heat-labile enterotoxin treated mice. The activities of all the enzymes except lactase were decreased significantly (p less than 0.01) in homogenates while increased significantly (p less than 0.001) in BBMs of experimental groups as compared to controls. Calmodulin activities were increased significantly (p less than 0.01) as compared to control in heat-stable enterotoxin treated mice but remained unaltered in heat-labile enterotoxin treated mice. DNA contents of intestinal homogenates were decreased in experimental groups demonstrating the decrease in cell number in these groups. The altered BBM enzyme activities could not be attributed to changes in calmodulin activities. The increase in enzyme activities in BBMs may reflect a compensatory phenomenon in the remaining cells.     

Activation of guanylate cyclase by E. coli heat-stable enterotoxin (STa). Modulation by NAD and pertussis toxin

The heat-stable enterotoxin (STa) of E. coli activates intestinal guanylate cyclase and leads to increased cGMP levels by an as yet undetermined mechanism. In comparing this cGMP system to other known toxin-mediated alterations in cAMP metabolism, we observed that pertussis toxin caused lower levels of intestinal cGMP synthesis in response to purified STa. Another participant in ADP-ribosylation reactions, NAD, enhanced the ability of STa to activate guanylate cyclase, yet had no effect on basal enzyme activity. Niacinamide and isoniacinamide also had no effect on basal activity, but attenuated the STa activation. These results are discussed in relation to current models of hormone/toxin-sensitive adenylate cyclase, and may suggest an involvement of guanine-nucleotide-binding proteins in intestinal cGMP metabolism.     

Structural features of Escherichia coli heat-stable enterotoxin that activates membrane-associated guanylyl cyclase.

Heat-stable enterotoxin (ST), a small peptide of 18 or 19 amino acid residues produced by enterotoxigenic Escherichia coli, is the cause of acute diarrhea in infants and travelers in developing countries. ST triggers a biological response by binding to a membrane-associated guanylyl cyclase C (GC-C) which is located on intestinal epithelial cell membranes. This binding causes an increase in the concentration of cGMP as a second messenger in cells and activates protein kinase A and cystic fibrosis transmembrane conductance regulator. Here we describe the crystal structure of an ST at 0.89 A resolution. The molecule has a ring-shaped molecular architecture consisting of six peptide molecules with external and internal diameters of approximately 35 and 7 A, respectively and a thickness of approximately 11 A. The conserved residues at the central portion of ST are distributed on the outer surface of the ring-shaped peptide hexamer, suggesting that the hexamer may be implicated in the association with GC-C through these invariant residues.     

Preparative purification of Escherichia coli heat-stable enterotoxin

Heat-stable enterotoxin (STa) isolated from bovine Escherichia coli strains was purified to homogeneity by growing the bacterial strains in a chemically defined medium, desalting, and concentrating the culture filtrate by batch adsorption chromatography on Amberlite XAD-2 resin, batch adsorption chromatography on reversed-phase silica, and preparative reversed-phase high-performance liquid chromatography. This rapid preparative purification scheme gave high recovery yields of pure STa which exhibited biochemical homology to STa purified by more complicated procedures.     

Phosphorylation and activation of the intestinal guanylyl cyclase receptor for Escherichia coli heat-stable toxin by protein kinase C

The heat-stable enterotoxin STa of E. coli causes diarrhea by binding to and stimulating intestinal membrane-bound guanylyl cyclase, triggering production of cyclic GMP. Agents which stimulate protein kinase C (PKC), including phorbol esters, synergistically enhance STa effects on cGMP and secretion. We investigated whether PKC causes phosphorylation of the STa receptor in vivo and in vitro.Immunoprecipitation of the STa receptor-guanylyl cyclase was carried out from extracts of T84 colon cells metabolically labelled with [³²P]-phosphate using polyclonal anti-STa receptor antibody. The STa receptor was phosphorylated in its basal state, and ³²P content in the 150 kDa holoreceptor band increased 2-fold in cells exposed to phorbol ester for 1 h. In vitro, immunopurified STa receptor was readily phosphorylated by purified rat brain PKC. Phosphorylation was inhibited 40% by 5 M of a synthetic peptide corresponding to the sequence around Ser¹⁰²⁹ of the STa receptor, a site previously proposed as a potential PKC phosphorylation site. Treatment of the immunopurified STaR/GC with purified PKC increased STa-stimulated guanylyl cyclase activity 2-fold. We conclude that PKC phosphorylates and activates the STa receptor/guanylyl cyclase in vitro and in vivo; Ser¹⁰²⁹ of the STaR/GC remains a candidate phosphorylation site by PKC.Abbreviations STa the heat-stable enterotoxin of E. coli, which has also been called ST-I and STp. The 18 amino acid variant was used throughout - PBS phosphate-buffered saline - PDB 4--12, 13-phorbol dibutyrate - ANP atrial natriuretic peptide - STaR/GC STa receptor/guanylyl cyclase, also called GC-C - PKC protein kinase C     

Binding protein for Escherichia coli heat-stable enterotoxin II in mouse intestinal membrane

Abstract The protein binding Escherichia coli heat-stable enterotoxin II (STII) was isolated from cell membranes of mouse intestine. The binding of ¹²⁵I-labeled STII to the proteins was inhibited by unlabeled STII, showing that it is specific. Proteins cross-linked with ¹²⁵I-STII were purified by column chromatography on hydroxyapatite and TSK gel. Analyses of the purified protein by SDS-polyacrylamide gel electrophorosis and gel filtration showed that the molecular mass was 25 kDa.     

Characterization of the recombinant human receptor for Escherichia coli heat-stable enterotoxin.

We report here the molecular characterization of a recombinant cell line (293-STaR) expressing the heat-stable enterotoxin receptor (STaR) from human intestine. We have compared the 293-STaR cell line with the human colonic cell line T84 that endogenously expresses STa binding sites. Scatchard analysis of displacement binding studies revealed a single STa binding site with an affinity (Ki) of 97 pM in 293-STaR compared with 55 pM in T84 cells. Saturation isotherms of STa binding gave a Kd of 94 pM for the cloned receptor expressed in 293 cells and 166 pM for the receptor present in T84 cells. Kinetic measurements of STa binding to 293-STaR gave an association rate constant, K1, of 2.4 x 10(8) M-1 min-1 and a dissociation rate constant, K2, of 0.016 min-1. The half-time of dissociation was 43 min, and the Kd calculated from the ratio of the kinetic constants was 67 pM. The pH profile of STa binding showed that the number of STa binding sites is increased 3-fold at pH 4.0 compared with pH 7.0, with no effect on binding affinity. A polyclonal antibody directed against the extracellular domain of STaR immunoprecipitated two proteins of approximately 140 and 160 kDa from both 293-STaR and T84 cells. Cross-linking of 125I-STa to 293-STaR cells resulted in the labeling of proteins with a molecular mass of approximately 153, 133, 81, 68, 56, and 49 kDa, the two smallest being the more abundant. Similar results have been reported for the STaR present on rat brush border membranes. These data suggest that the STaR-guanylyl cyclase identified by molecular cloning is the only receptor for STa present in T84 cells.     

Characterization of the receptor for heat-stable enterotoxin from Escherichia coli in rat intestine

The receptor for the heat-stable enterotoxin (ST) from Escherichia coli was solubilized with Lubrol-PX from rat intestinal brush-border membranes and characterized. The binding kinetics and analog specificity of the solubilized receptor were virtually identical to those obtained with the membrane-bound receptor. Furthermore, the regulation of the receptor's affinity by cations was also maintained after solubilization, indicating a conservation of the toxin-binding site after removal of the receptor from its membrane environment. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 5.5 nm and a sedimentation coefficient of 7.0 S for the solubilized receptor. The isoelectric point of the receptor was determined as 5.5 using Sephadex isoelectric focusing electrophoresis. In all of these separation techniques, the ST receptor showed a single peak of activity that was clearly separated from that of guanylate cyclase. When 125I-ST was cross-linked to brush-border membranes with disuccinimidyl suberate, the affinity-labeled receptor solubilized with 0.1% Lubrol-PX eluted at a similar position as the native receptor on gel filtration chromatography. Analysis of the affinity-labeled receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent and by autoradiography revealed the presence of three specifically labeled polypeptides with apparent molecular weights of 80,000, 68,000, and 60,000. These results suggest that the ST receptor is solubilized by Lubrol-PX in an active form with preservation of its regulation by cations. Also, the ST receptor is separable from particulate guanylate cyclase indicating that the receptor is coupled to the activation of guanylate cyclase by an as yet undefined mechanism. Three subunit peptides may constitute a binding region of the receptor.     

Catabolite repression of Escherichia coli heat-stable enterotoxin activity.

The Escherichia coli heat-stable enterotoxin (ST) coded for by plasmid pYK007 (Apr ST+) showed a dependence for cyclic adenosine 3',5'-monophosphate (cAMP) to express ST activity in an adenyl cyclase (cya) deletion mutant; no ST activity was detected in the presence of cAMP in a cAMP receptor protein (crp) deletion mutant or in a double deletion mutant (delta cya delta crp). The cya-crp effect on ST activity could not be accounted for by a modification of the copy number of plasmid deoxyribonucleic acid per chromosome equivalent or by an alteration in the secretion of an active intracellular enterotoxin.     

Colonocyte basolateral membranes contain Escherichia coli heat-stable enterotoxin receptors

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.     

Interaction of Escherichia coli heat-stable enterotoxin B with rat intestinal epithelial cells and membrane lipids

The binding of 125I-labeled Escherichia coli heat-stable enterotoxin B to rat intestinal epithelial cells was unsaturable and nonspecific, at concentrations well above that required to mediate biological events. Following its interaction with intestinal cells, approximately 50-80% of heat-stable enterotoxin B remained stably associated with the cells, implying that it was partitioned into the membrane and/or internalized by the cell. The toxin bound with different affinities to lipids isolated from intestinal epithelial cells, phospholipids, glycolipids, neutral lipids and to model membrane vesicles containing negatively charged lipids. These results indicate that heat-stable enterotoxin B utilizes the membrane bilayer, rather than a surface protein or glycoprotein in modulating toxin-induced enterotoxicity.     

Amino-acid sequence of a heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli

A heat-stable enterotoxin produced by a human strain of enterotoxigenic Escherichia coli was extensively purified by reverse-phase high-performance liquid chromatography. The minimum effective dose of the purified toxin to cause fluid accumulation in suckling mice was 2.5 ng. The amino acid sequence of the purified toxin was determined by Edman degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn-Ser-Ser-Asn-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. This sequence was identical to that deduced from the nucleotide sequence encoding a human heat-stable enterotoxin, reported by Moseley et al., except for the C-terminal Tyr residue.