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PubMed2005

Guanylyl cyclase C as a reliable immunohistochemical marker and its ligand Escherichia coli heat-stable enterotoxin as a potential protein-delivering vehicle for colorectal cancer cells.

mRNA-based technologies and preclinical research in a variety of animal models have shown that guanylyl cyclase C (GCC) is a highly sensitive and specific molecular marker for the diagnosis of colorectal cancer (CRC). GCC is also a receptor for Escherichia coli (E. coli) heat-stable enterotoxin (STa) and can be used for STa-directed delivery of small-sized imaging agents to human CRC tumours. In this study, we have evaluated GCC as a new immunohistochemical (IHC) marker for CRC tissues and STa as a suitable vector for delivering high-sized protein molecules to CRC cells. Firstly, we have developed a highly sensitive EnVision(+)-based IHC staining method for detecting GCC in serial paraffin-embedded sections of primary and metastatic CRC (38 cases) or non-CRC (14 cases) adenocarcinomas. Carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20) were chosen as controls. Our results indicate that GCC staining was positive in 100% of CRC tumours and was comparable to CEA (95%) or CK20 (92%). In contrast to CEA and CK20, GCC was negative in all of the extra-intestinal non-CRC tumours examined. GCC appears to display higher specificity than either CEA or CK20 while retaining high sensitivity, suggesting that it is a better CRC marker than CEA or CK20. Secondly, STa was genetically coupled to green fluorescent protein (GFP) and the resulting GFP-tagged STa was characterized for expression in E. coli and enterotoxicity in mouse. The binding characteristics of GFP-STa in CRC Caco-2 cells were followed by immunofluorescence microscopy. In this work we show that GFP-tagged STa is biologically active and has retained its ability to internalise into Caco-2 cells making it a potential vehicle for the delivery of anticancer therapeutic protein agents.

Adult↗

PubMed1999

Use of flow cytometry to measure the interaction between Escherichia coli heat-stable enterotoxin and its intestinal receptor in mice.

Binding of Escherichia coli heat-stable enterotoxin (STa) to its putative receptor on the brush border membrane of enterocytes is a prerequisite for the induction of diarrhea in infected humans and animals. Humans and animals of different ages vary in their susceptibility to the effect STa, perhaps due to the difference in STa interaction with its intestinal receptor. Flow cytometry was compared to indirect immunofluorescence and 125I-STa binding assays to measure the STa-enterocytes receptor interaction in different age groups of Swiss Webster mice (2-, 7-, 14-day-old). Flow cytometry indicated stronger interaction between STa and its putative receptor on enterocytes from the 2-day-old mice than enterocytes from older mice. 125I-STa-binding assay suggested that the stronger fluorescence intensity on enterocytes from younger mice is due to higher STa receptor density and higher receptor affinity to STa. Flow cytometry is more sensitive quantitative assay to measure the interaction between STa and its intestinal receptor than indirect immunofluorescence microscopy.

Aging↗

PubMed2003

Selective induction of CD8+CD4- thymocyte apoptosis mediated by the B-subunit of Escherichia coli heat-labile enterotoxin.

Receptor-binding by the B-subunit of Escherichia coli heat-labile enterotoxin (EtxB) induces apoptosis of peripheral CD8(+), but not CD4(+) T-cells. Given that peripheral CD8(+) and CD4(+) T cells arise from a common developmental pathway in the thymus, we investigated the effects of EtxB on different thymocyte populations. We show that the acquisition of sensitivity to EtxB-mediated cell death arises following transition of CD4(+)CD8(+) double positive cells into the CD4(-)CD8(+) pathway. Maturation of T cells into CD4(-)CD8(+) single positive cells is associated with upregulated expression of receptors for EtxB.

Animals↗

PubMed1996

A conformational epitope in the N-terminus of the Escherichia coli heat-stable enterotoxins is involved in receptor-ligand interactions.

The heat-stable enterotoxins are a family of low molecular weight, cysteine rich peptide toxins which are one of the major causes of watery diarrhea in children and adults. These toxins bind to a cell surface receptor in intestinal cells and mediate their action through elevation of intracellular cyclic GMP. We have generated a monoclonal antibody to these peptide toxins which is able to neutralise the activity of the peptides in a human colonic cell line, the T84 cell line. The monoclonal antibody, ST:G8, appears to be directed to an epitope distinct from antibodies previously generated, and prior incubation of this antibody, or Fab generated from this antibody, with full length STh and STp peptides prevents cGMP accumulation in T84 cells. This inhibition is a direct result of the antibody preventing binding of the peptides to the receptor. ST:G8 Mab does not recognize a 13-mer biologically active analog of STp, comprising the core sequence of STp peptide, suggesting that it is directed to a region in the N-terminus of the peptides, which may modulate receptor interaction/activation. The antibody recognizes a conformational epitope in the ST peptides, since reduction and carboxyamidation of ST abolishes antibody cross-reactivity. Differential cross-reactivity of the Mab with STh and STp peptides which differ markedly only in their N-termini, suggests that this antibody recognizes a distinct conformation in the two peptides, which is essential for receptor interaction.

Adult↗

PubMed1995

Rat guanylyl cyclase C expressed in COS-7 cells exhibits multiple affinities for Escherichia coli heat-stable enterotoxin.

Intestinal cells exhibit binding sites with different affinities for Escherichia coli heat-stable enterotoxin (ST) and guanylin, suggesting the existence of different receptors for these peptides. Guanylyl cyclase C from intestinal cells has been identified as one receptor for these peptides. Equilibrium and kinetic binding characteristics of rat guanylyl cyclase C expressed in COS-7 cells were examined, employing ST, to determine if this receptor exhibited multiple affinities. Scatchard analysis of equilibrium binding yielded curvilinear isotherms consistent with the presence of high (pM) and low (nM) affinity sites. Kinetic analysis of binding demonstrated that these sites exhibited similar dissociation but different association kinetics. In addition, two distinct affinity states of low affinity sites were identified with dissociation constants of 0.15 and 5.85 nM. Association of ST and low affinity sites was biphasic, while dissociation from these sites was unimodal. Close agreement of equilibrium and kinetic dissociation constants suggested that low affinity sites were in the lowest affinity state at equilibrium. Comparison of the ligand dependence of guanylyl cyclase activity (EC50 = 110 nM) with receptor occupancy revealed that binding of ST to the lowest affinity state of low affinity sites (EC50 = 80 nM) is directly coupled to catalytic activation. These studies suggest that binding sites with different affinities for ST exhibited by intestinal cells reflect the expression of a single gene product, guanylyl cyclase C, rather than different receptors for the ligand. The shift in affinity state of low affinity sites and its correlation with catalytic activation suggest a central role for this phenomenon in mechanisms mediating receptor-effector coupling of membrane guanylyl cyclases.

Animals↗

PubMed1992

Affinity purification of functional receptors for Escherichia coli heat-stable enterotoxin from rat intestine.

Active receptors for Escherichia coli heat-stable enterotoxin (ST) were partially purified by ligand-affinity chromatography. The affinity column was prepared by coupling ST to biotin derivatized with an extended N-hydroxysuccinylated spacer arm prior to binding to monomeric avidin immobilized on agarose. Detergent extracts of rat intestinal mucosa membranes were quantitatively depleted of ST binding activity when chromatographed on this affinity matrix. Biotinylated ST-receptor complexes were eluted from affinity columns with 2 mM biotin and these complexes quantitatively dissociated with bile salts. Using this technique, functional ST receptors were purified maximally about 2000-fold, with about 3% of the total activity in crude extracts recovered in these purified preparations. Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis and silver staining demonstrated a major protein subunit of 74 kDa. Affinity cross-linking of these preparations to 125I-ST demonstrated specific labeling predominantly of the 74-kDa subunit. In addition, lower amounts of labeled ST were incorporated into subunits of 164 and 45 kDa, confirming the heterogeneous nature of ST receptors. Purified receptors bound ST in a concentration-dependent fashion, with an IC50 of 10(-9) M. These studies demonstrate that ligand-affinity chromatography can be employed to purify ST receptors. The availability of purified receptors will facilitate further studies of mechanisms underlying ST-induced intestinal secretion.

Animals↗

PubMed1994

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 GM1 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 [3H]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 [125I]toxin, both toxins were found to bind only GM1. Separation of the complexes from [3H]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 [125I]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.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Sugars↗

PubMed2002

A comparative molecular field analysis (COMFA) of the structural determinants of heat-stable enterotoxins mediating activation of guanylyl cyclase C.

The heat-stable enterotoxin binds to and activates guanylyl cyclase C (GC-C), regulating fluid and electrolyte secretion in intestinal epithelial cells. A COMFA model was developed to predict the primary interactions between GC-C agonists and their receptor. This model predicts that the amide backbone of Cys(5)-Cys(6)-Glu(7)-Leu(8), the beta carbon atoms of Cys(5)-Cys(6), and the side chains of Pro(12), Ala(13), and Ala(15) comprise the primary interactions of GC-C agonists with the receptor surface.

Amino Acid Sequence↗

PubMed1997

Modulation of B-cell activation by the B subunit of Escherichia coli enterotoxin: receptor interaction up-regulates MHC class II, B7, CD40, CD25 and ICAM-1.

The B subunits of cholera toxin (CtxB) and Escherichia coli heat-labile enterotoxin (EtxB) are non-toxic lectins that bind and cross-link a ubiquitous cell glycolipid receptor, ganglioside GM1, and are recognized as potent mucosal and systemic immunogens. Here we examine the role of EtxB receptor occupancy in modulating the activation of B cells, in vitro, in primary lymphocyte cultures containing B and T cells. When 48-hr spleen cell cultures containing EtxB were compared with those in the presence of a non-receptor binding mutant, EtxB(G33D), a marked shift in the ratio of CD4+ T cells: B cells was noted. Evidence suggested that this was the result of either enhanced survival or proliferation of B cells associated with receptor occupancy by EtxB. Investigation revealed that EtxB induced only a minimal increase in proliferation above that of EtxB(G33D), in mixed cell cultures, and failed to induce any cell division of purified B cells or T cells. In contrast, receptor-binding by EtxB markedly up-regulated the expression of major histocompatability complex (MHC) class II, B7, intracellular adhesion molecule-1 (ICAM-1), CD40 and CD25 on the B-cell surface. These results indicate that the polyclonal effects of EtxB on B cells are not associated with wide-scale proliferation, but more likely with maintenance of B-cell survival by activation of molecules essential for B-cell differentiation. The findings also highlight the essential role of GM1-interaction with EtxB in the regulation of lymphocyte responses.

Animals↗

PubMed2003

Cellular refractoriness to the heat-stable enterotoxin peptide is associated with alterations in levels of the differentially glycosylated forms of guanylyl cyclase C.

The heat-stable enterotoxin peptides (ST) produced by enterotoxigenic Escherichia coli are one of the major causes of transitory diarrhea in the developing world. Toxin binding to its receptor, guanylyl cyclase C (GC-C), results in receptor activation and the production of high intracellular levels of cGMP. GC-C is expressed in two differentially glycosylated forms in intestinal epithelial cells. Prolonged exposure of human colonic cell lines to ST peptides induces cellular refractoriness to the ST peptide, in terms of intracellular cGMP accumulation. We have investigated the mechanism of cellular desensitization in human colonic Caco2 cells, and observe that exposure of cells to ST leads to a time and dose-dependent inability of cells to respond to the peptide in terms of GC-C stimulation, both in whole cells and membranes prepared from desensitized cells. This is concomitant with a 50% reduction in ST-binding activity in desensitized cells. Desensitization was correlated with a loss of the plasma membrane-associated, hyperglycosylated 145 kDa form of GC-C, while the predominant 130 kDa form, localized both on the plasma membrane and the endoplasmic reticulum, continued to be present in ST-treated cells. Desensitized cells recovered ST-responsiveness on removal of the ST peptide, which was correlated with a reappearance of the 145 kDa form on the cell surface, following processing of the endoplasmic reticulum-associated pool of the 130 kDa form. Selective internalization of the 145 kDa form of the receptor was required for cellular desensitization, as ST-treatment of cells at 4 degrees C did not lead to refractoriness. We therefore show a novel means of regulation of cellular responsiveness to the ST peptide, whereby altering cellular levels of the differentially glycosylated forms of GC-C can lead to differential ligand-mediated activation of the receptor.

Bacterial Toxins↗

PubMed1995

Regulation of cell signaling by the cytoplasmic domains of the heat-stable enterotoxin receptor: identification of autoinhibitory and activating motifs.

Infection with enterotoxigenic Escherichia coli is a leading cause of traveler's diarrhea. Many enterotoxigenic E. coli strains produce heat-stable enterotoxin (ST), a peptide that binds to the intestinal receptor guanylyl cyclase C known as STaR. The toxin-receptor interaction elevates intracellular cGMP, which then activates apical chloride secretion, resulting in secretory diarrhea. In this report, we examine how the intracellular domains of STaR participate in the propagation and regulation of signaling. We show that STaR exists as an oligomer in both the presence and the absence of toxin. We also demonstrate that deletion of the intracellular kinase-homology domain produces a constitutively active mutant, suggesting that this domain subserves an autoinhibitory function. Finally, we constructed a point mutant within a highly conserved region of the cyclase domain that completely inactivates the catalytic activity of guanylyl cyclase. Cotransfection of this point mutant with wild-type receptor causes a dominant-negative effect on receptor activation. This suggests that interaction of receptor subunits is required for toxin-induced activation and that the cyclase domain is involved in this essential interaction. We propose that the binding of ST to STaR promotes a conformational change across the cell membrane. This removes the inhibitory effects of the kinase-homology domain and promotes an interaction between cyclase domains that leads to receptor activation. The data suggest a paradigm of signal transduction that may also be relevant to other members of the guanylyl cyclase receptor family.

Amino Acid Sequence↗

PubMed2004

Ability of blood group A-active glycosphingolipids to act as Escherichia coli heat-labile enterotoxin receptors in HT-29 cells.

We examined the ability of blood group A-active glycoconjugates to act as receptors for Escherichia coli heat-labile type I enterotoxin (LT-I) in HT-29 cells. These cells contained ~4 times more specific binding sites for LT-I than for cholera toxin (CT). Binding of LT-I could not be blocked by the B subunit of CT (CT-B), indicating the existence of LT-I receptors in addition to the glycosphingolipid GM1. LT-I was able to increase levels of cyclic adenosine monophosphate (AMP), even in the presence of CT-B. Helix pomatia and anti-blood group A antibody caused a dose-dependent inhibition of binding of LT-I to cells and production of cyclic AMP. LT-I recognized several complex blood group A-active glycosphingolipids from cells, and this interaction was also interfered with by H. pomatia. Treatment of cells with D,L-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol diminished surface expression of blood group A-active glycosphingolipids and binding of LT-I to non-GM1 receptors. These observations suggest that blood group A-active glycosphingolipids can function as alternative receptors for LT-I in HT-29 cells.

ABO Blood-Group System↗

PubMed1994

Intestinal kinetics and dynamics of Escherichia coli heat-stabile enterotoxin in suckling mice.

The heat-stabile enterotoxin produced by Escherichia coli (ST) induces diarrhea by binding to receptors on intestinal cells, activating guanylyl cyclase, and increasing cyclic GMP. High- and low-affinity receptors for this toxin have been identified previously. ST induces intestinal secretion in suckling mice in picomole doses, suggesting a role for high-affinity receptors in this process. The present studies examine the relative roles of high- and low-affinity receptors in this process. The time course of changes in free ST concentrations in suckling mouse intestine was determined after intragastric inoculation. Also, binding characteristics of high- and low-affinity receptors and their coupling to guanylyl cyclase were defined in intestinal membranes from suckling mice. Intestinal concentrations of toxin and receptor binding characteristics empirically determined were used in a dynamic model correlating fractional occupancy of high- and low-affinity receptors with intestinal secretion to estimate their relative contributions to ST-induced diarrhea.

Animals↗

PubMed1994

Pig intestinal membrane-bound receptor (guanylyl cyclase) for heat-stable enterotoxin: cDNA cloning, functional expression, and characterization.

A cDNA encoding the receptor protein for a heat-stable enterotoxin (STa) produced by enterotoxigenic Escherichia coli was cloned from intestinal epithelial cells of a 10-week-old pig. The cDNA had an open reading frame of 3,219 base pairs and coded for a protein with 1,073 amino acid residues. The mature protein consisted of 1,050 amino acid residues with a molecular mass of ca. 121 kDa and was 87% and 82% identical with the human and rat protein, respectively. The CHO cell line overexpressing the pig recombinant STa receptor specifically bound to a photoaffinity-labeled analog of STa and showed marked elevation of the cellular content of cGMP in response to STa.

Amino Acid Sequence↗

PubMed1993

Ligand-based histochemical localization and capture of cells expressing heat-stable enterotoxin receptors.

The heat stable enterotoxins (ST) of enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by binding specific intestinal receptors. Precise histochemical localization of ST receptors could provide more information about the pathophysiology of secretory diarrhoea and the role of ST receptors in normal biology. To accomplish this, we quantitatively coupled biotin to the N-terminus of ST1b using biotin-X-X-N-hydroxysuccinimide ester. The derivatized toxin (BST) has an apparent Kd of 11.7 +/- 10 nM for rat brush border receptors. We used BST in an affinity panning cell-capture system, to validate its ability to discriminate between receptor-positive and receptor-negative cells. Cell lines expressing ST receptors (human colon carcinoma T84, and COS cells transfected with guanylyl cyclase-C (GC-C) ST receptor cDNA) were captured to streptavidin and anti-biotin-coated plates with high efficiency and specificity. This system provides a novel approach to screening cells for the presence of unique ST-binding proteins. BST was then used with streptavidin-gold to demonstrate the cellular topography of ST receptors at the light microscopic level. Villus enterocytes were intensely stained, but only a faint signal was observed in upper crypts of rat small intestine. Thus, a gradient of increasing receptor density was seen as upper crypt cells matured into villus enterocytes. Higher magnification revealed that ST receptors are concentrated at the apical aspect of villus enterocytes. Recently, guanylin, a putative endogenous ligand for ST receptors, has been localized to Paneth cells, at the base of intestinal crypts. Thus, ST receptors are concentrated in villus enterocytes, while guanylin appears to be produced at the base of the crypts.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2000

Characterization of the interaction of Escherichia coli heat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn calves.

Enterotoxigenic Escherichia coli (ETEC) induces severe diarrhea in newborn calves through the elaboration of heat-stable enterotoxin (STa). We investigated the distribution and characteristics of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from anterior jejunum, posterior jejunum, ileum and colon of newborn calves. We found that density of the receptors and their affinity to STa were higher on enterocytes and BBMVs that were derived from the ileum than enterocytes and BBMVs prepared from other segments of the calf intestine. This study suggests that, in newborn calves, the ileum is the major part of the intestinal tract that is affected in the course of secretory diarrhea caused by STa-producing ETEC strains.

Animals↗

PubMed2006

Interaction of Escherichia coli heat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn kids.

The elaboration of heat stable enterotoxin (STa) is an important step in the pathogenesis of enterotoxigenic Escherichia coli (ETEC), which causes severe diarrhea in newborn animals. In this study, the distribution of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from the anterior jejunum, posterior jejunum, ileum and colon of newborn kids was investigated. The density of STa-receptors on enterocytes and BBMVs was higher in the posterior jejunum than that in other segments of the kids' intestines. Additionally, the affinity of the posterior jejunum STa-receptors was higher than the affinity of receptors present on the epithelium of other intestinal segments. Our findings suggest that the posterior jejunum is a major target for STa within the intestinal tract of newborn kids.

Animals↗