Surface localization of wheat germ agglutinin and concanavalin A binding sites on normal human blood cells: an ultrastructural histochemical study

In order to determine the extent and variations in surface concavanalin A (CON A) and wheat germ agglutinin (WGA) labeling of different varieties of normal blood cells, gluraraldehyde-fixed human blood cells were exposed to CON A-gold labeled horseradish peroxidase (CON A-HRP-G) and WGA-gold labeled ovomucoid (WGA-OVO-G) histochemical methods. The resultant particulate reaction product permitted assessment of binding and number of gold particles per micrometer of cell surface. Particle counts and data were subjected to statistical analysis. Six subjects (three female and three male) were used and compared in this study. In spite of moderate variations in surface labeling of the various types of leukocytes, erythrocytes and platelets within a given subject, determinations of mean labeling values for similar cell varieties proved quite similar between subjects with the given lectin. WGA and CON A had substantially different labeling densities on the various hemic cells. WGA surface labeling of all types of hemic cells, with the exception of platelets, showed far more labeling than was found with CON A. WGA mean labeling of the grouped subjects was significantly higher for each variety of leukocyte than for either erythrocytes or platelets although this distinction was not always evident in an individual subject. With CON A, mean labeling density for each of hemic cell types showed significant differences between each of the hemic cell varieties. Erythrocytes had only minimal CON A binding while monocyte and platelet populations represented the most reactive of the hemic cells. No difference was noted between corresponding cell varieties in the female vs. male subjects.     

Loss of GM1 surface expression precedes annexin V-phycoerythrin binding of neutrophils undergoing spontaneous apoptosis during in vitro aging.

BACKGROUND: Apoptosis of neutrophil granulocytes is an important determinant of the resolution of inflammation. Apoptotic neutrophils undergo specific alterations in their receptor profiles. These alterations are likely to contribute to the characteristic functional silencing of the dying cells. METHODS: By flow cytometry and fluorescence microscopy, we analyzed the ganglioside GM1, a lipid raft marker, with respect to its surface expression on neutrophil and eosinophil granulocytes. Apoptosis was monitored by morphological changes and by the binding of annexin V-phycoerythrin (AxV-PE). RESULTS: GM1, which was stained by the cholera toxin subunit B, was found only on neutrophil granulocytes; eosinophil granulocytes did not bind cholera toxin subunit B. GM1 was lost from the surfaces of neutrophils before AxV-PE binding (early apoptosis). Surprisingly, GM1 reappeared during the late stages of apoptosis, although without functional consequences. GM1 was found on the cell surface and in intracellular membranes, whereas CD16 was found only at the cell surface. CONCLUSIONS: Loss of surface GM1 is a new marker for the detection of the aging of neutrophils. Its loss precedes the binding of AxV-PE of neutrophils.     

Intoxication of cultured cells by cholera toxin: evidence for different pathways when bound to ganglioside GM1 or neoganglioproteins.

We previously reported that when the oligosaccharide of ganglioside GM1 is covalently attached to cell surface proteins of GM1-deficient rat glioma C6 cells, the cells bind large amounts of cholera toxin (CT) but their cAMP response to CT is not enhanced [Pacuszka, T., & Fishman, P. H. (1990) J. Biol. Chem. 265, 7673-7668]. We now report that when such cells were exposed to CT in the presence of chloroquine, an acidotropic agent, they accumulated cAMP. This raised the possibility that CT bound to cell surface "neoganglioproteins" may be entering the cells through a different pathway from that of CT-bound GM1. To further explore this phenomenon, we covalently attached GM1 oligosaccharide to human transferrin (Tf). The modified protein (GM1OS-Tf) bound with high affinity to Tf receptors on HeLa cells and increased the binding of CT to the cells. The bound CT, however, was unable to activate adenylyl cyclase as measured by cyclic AMP accumulation. By contrast, treatment of HeLa cells with GM1 increased both CT binding and stimulation of cyclic AMP accumulation. Control cells and cells treated with either GM1 or GM1OS-Tf were exposed to CT in the presence of chloroquine. Whereas chloroquine had little or no effect on the response of control or GM1-treated cells to CT, it made the cells treated with GM1OS-Tf responsive to the toxin. Our results indicate that CT bound to its natural receptor GM1 enters the cells through a pathway different from that of toxin bound to neoganglioproteins.     

Endocytosis of exogenous GM1 ganglioside and cholera toxin by neuroblastoma cells.

Cholera toxin (CT) covalently linked to horseradish peroxidase (HRP) is a specific cytochemical marker for its receptor, the monosialoganglioside GM1. The binding and endocytosis of exogenous [3H]GM1 by cultured murine neuroblastoma cells (line 2A [CCl-131] ), which contain predominantly GM3, was examined by quantitative electron microscope autoradiography. The relationship between exogenous receptor, [3H]GM1, and CT HRP was studied in double labeling experiments consisting of autoradiographic demonstration of [3H]GM1 and cytochemical visualization of HRP. Exogenous [3H]GM1 was not degraded after its endocytosis by cells for 2 h at 37 degrees C. Quantitative studies showed similar grain density distributions in cells treated with [3H]GM1 alone and in cells treated with [3H]GM1 followed by CT-HRP. Qualitative studies conducted in double labeling experiments showed autoradiographic grains over the peroxidase-stained plasma membrane, lysosomes, and vesicles at the trans aspect of the Golgi apparatus. The findings indicate that exogenous glycolipid is associated with the plasmid membrane of deficient cells and undergoes endocytosis. The quantitative ultra-structural autoradiographic studies are consistent with the hypothesis that the spontaneous endocytosis of exogenous [3H]GM1 controls the subsequent uptake of CT-HRP.     

Spontaneous curvature of ganglioside GM1--effect of cross-linking

The membrane-curvature dependent lateral distribution of outer leaflet ganglioside GM1 (GM1) and the influence of GM1 cross-linking induced by fluorophore-tagged cholera toxin subunit B (CTB) plus anti-CTB was analysed in cell membranes by fluorescence microscopy. Data are presented indicating that cross-linked GM1-ligand patches accumulated at the tips of human erythrocyte echinocytic spiculae induced by Ca(2+)/ionophore A23187. However, when lipid fixative osmium tetroxide was added prior to the ligand no accumulation in spiculae occurred. GM1-staining remained here distributed over the spheroid cell body and in spiculae. Similarly, osmium tetroxide completely prohibited CTB plus anti-CTB-induced GM1 patching in representatives for flat membrane, i.e. discoid erythrocytes and K562 cells. Our results demonstrate that GM1 per se shows low membrane curvature dependent distribution and therefore holds flexible spontaneous curvature. In contrast, the cross-linked GM1-ligand complex has a strong preference for highly outward curved membrane and possesses overall positive spontaneous curvature. Osmium tetroxide efficiently immobilises GM1.     

Damaging effects of Clostridium perfringens delta toxin on blood platelets and their relevance to ganglioside GM2

The lytic effect of Clostridium perfringens delta toxin was investigated on goat, human, rabbit, and guinea pig platelets. In contrast to erythrocytes from the latter three species, which are insensitive to the toxin, the platelets were equally lysed by the same amount of toxin. These results suggest the presence of GM2 or GM2-like ganglioside(s) as a specific recognition site of the toxin on platelet plasmic membrane as previously established for sensitive erythrocytes. Plasmic membrane damage of human platelets was evidenced by the release of entrapped alpha-[14C]aminoisobutyric acid used as a cytoplasmic marker. The specific binding of hemolytically active 125I-delta toxin by human and rabbit platelets was practically identical, dose dependent, and inhibitable by GM2. Labeled toxin was also bound by various subcellular organelles separated from rabbit platelets except the 5-hydroxytryptamine (5-HT)-containing dense bodies, suggesting the absence or inaccessibility of GM2 on the surface of the latter organelles. This result correlates with the low amounts of 5-[3H]HT liberated after platelet challenge with delta toxin whereas this mediator was massively liberated upon lysis by the sulfhydryl-activated toxin alveolysin. The levels of M and P forms of phenol sulfotransferase (PST), involved in 5-HT catabolism, were determined in human platelet lysates after challenge with delta toxin, alveolysin, and other disruptive treatments. The low PST-M activities detected after lysis by delta toxin suggest that this isoenzyme is very likely associated to dense bodies in contrast to PST-P which is cytoplasmic. Platelet lysis by the toxin allows easy separation of these organelles.     

Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide.

Cholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B-pentamer complexed with the GM1 pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue. The binding of GM1 to cholera toxin thus resembles a 2-fingered grip: the Gal(beta 1-3)GalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue. The CTB5:GM1 pentasaccharide complex described here provides a detailed view of a protein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in GM1-mediated signal transduction.     

Generation of cell surface neoganglioproteins. GM1-neoganglioproteins are non-functional receptors for cholera toxin

GM1 (II3Neu5Ac-GgOse4Cer)-oligosaccharide was prepared from the ganglioside by ozonolysis and alkaline fragmentation, reductively aminated and coupled to the heterobifunctional cross-linker succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate. The resulting derivative reacted with free sulfhydryl groups and readily cross-linked to cell surface components on rat glioma C6 cells which are GM1-deficient. Attachment of the GM1-oligosaccharide derivative, which was monitored by increased binding of 125I-cholera toxin to the cells, was both time- and concentration-dependent. Prior treatment of the cells with dithiothreitol enhanced the attachment by generating additional free sulfhydryl groups. The affinity of cholera toxin for cells treated with the GM1-oligosaccharide derivative or with GM1 was similar. The nature of the newly generated toxin receptors was determined by Western blotting. Membranes from derivatized cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved components were electrophoretically transferred to a nitrocellulose sheet which was overlain with 125I-cholera toxin. The toxin bound to a wide variety of membrane proteins, most of which were trypsin-sensitive. No such binding was observed using membranes from control cells. Although the GM1-neoganglioproteins newly generated on the surface of rat glioma C6 cells readily bound cholera toxin, the cells did not become more responsive to the toxin as measured by increased production of cyclic AMP or activation of adenylate cyclase. In contrast, cells exposed to GM1 became highly responsive to the toxin. Thus, neoganglioproteins on the cell surface appear to behave as nonfunctional receptors for cholera toxin.     

Binding of Vibrio cholera toxin and the heat-labile enterotoxin of Escherichia coli to GM1, derivatives of GM1, and nonlipid oligosaccharide polyvalent ligands

Vibrio cholera toxin and the heat-labile enterotoxin of Escherichia coli have been shown to differ somewhat in their ligand specificity and in the antigenicity of their binding sites. Therefore, the components of the oligosaccharide portion of GM1 bound by cholera toxin and the heat-labile enterotoxin of E. coli were identified by determining the concentration of GM1, derivatives of GM1, oligosaccharide isolated from GM1, or clustered oligosaccharide needed to inhibit toxin binding to GM1-coated plastic wells. The KIs for GM1, the C(7) sialosyl alcohol [corrected] of GM1, and ethanolamine-sialosyl-GM1 were similar (approximately 30-50 nM) for both toxins. N-Deacetylation of GM1 resulted in a small increase in KI; formation of the sialosyl methyl ester increased the KI 2-5 fold; loss of the terminal galactosyl residue (GM2) increased the KI by 10-15-fold; and removal of the sialosyl moiety (asialo-GM1) resulted in loss of inhibition of both toxins. Oligosaccharide isolated from GM1 had a KI for both toxins that was approximately 100-fold greater than that obtained for GM1 and approximately 1000-fold greater than that for a clustered oligosaccharide derivative having an average of 8 oligosaccharide residues (isolated from GM1) per molecule of poly-L-lysine. These results indicate that both toxins are functionally quite similar in their recognition of GM1 as a ligand in that each requires the free carboxyl group of sialic acid for optimum binding, does not need carbons 8 and 9 of the sialosyl moiety nor the acetyl groups associated with the sialic acid and galactosamine residues, and can have its binding to GM1 blocked by a nonlipid compound, i.e. oligo-GM1-poly-L-lysine.     

Endocytosis of cholera toxin in GERL-like structures of murine neuroblastoma cells pretreated with GM1 ganglioside. Cholera toxin internalization into Neuroblastoma GERL

Cholera toxin (CT), covalently attached to horseradish peroxidase (HRP), is a specific cytochemical marker for GM1 ganglioside (GM1) and retains the ability of the native toxin to raise levels of cyclic AMP in avian erythrocytes. Using a cytochemical stain for HRP, we found that 9% of control cultured murine neuroblastoma cells bound cholera toxin-horseradish peroxidase conjugates (CT-HRP) on their surfaces after incubations for 1 h at 4 degrees C. Exogenous GM1, the natural receptor of CT, becomes associated in the culture medium with the plasma membranes of these cells so that 96% of cells are stained. Cells preincubated with GM1 at 4 degrees C were exposed to CT-HRP for 1 h at 4 degrees C. After washing, cells were incubated at 37 degrees C for 30 min-24 h. Endocytosis of CT-HRP occurred within 30 min and CT-HRP remained, throughout the 24-h period, in tubules, vesicles, and cisternae often found near the Golgi apparatus; this aggregate of peroxidase-positive elements probably corresponds to Golgi apparatus-endoplasmic reticulum-lysosomes (GERL) of neurons. In metaphase cells, CT-HRP was observed in aggregates of vesicles and tubules clustered near the centriole. Conjugates of HRP with subunit B, the GM1 binding component of CT, were internalized by cells pretreated with GM1 as was CT-HRP. The 9% of neuroblastoma cells binding CT-HRP in the absence of exogenous GM1 internalized the ligand in a manner indistinguishable from that of the treated cells. These findings indicate that, in neuroblastoma cells, a system of vesicles, tubules, and cisternae, analogous to GERL of neurons, is the primary recipient of adsorptive endocytosis of CT bound to endogenous or exogenously introduced GM1.     

Association of Vibrio cholerae 569B outer membrane vesicles with host cells occurs in a GM1‐independent manner

The primary virulence factor of Vibrio cholerae, cholera toxin (CT), initiates a pathway in epithelial cells that leads to the severe diarrhoea characteristic of cholera. Secreted CT binds to GM1 on the surface of host cells to facilitate internalisation. Many bacterial toxins, including CT, have been shown to be additionally delivered via outer membrane vesicles (OMVs). A fraction of the closely related heat labile toxin produced by enterotoxigenic Escherichia coli has been demonstrated to reside on the surface of OMVs, where it binds GM1 to facilitate OMV internalisation by host cells. In this work, we investigated whether OMV‐associated CT is likewise trafficked to host cells in a GM1‐dependent mechanism. We demonstrated that a majority of CT is secreted in its OMV‐associated form and is located exclusively inside the vesicle. Therefore, the toxin is unable to bind GM1 on the host cell surface, and the OMVs are trafficked to the host cells in a GM1‐independent mechanism. These findings point to a secondary, noncompeting mechanism for secretion and delivery of CT, beyond its well‐studied secretion via a Type II secretion system and underscore the importance of focusing future studies on understanding this GM1‐independent delivery mechanism to fully understand Vibrio cholerae pathogenesis.     

Cholera toxin binds to lipid rafts but has a limited specificity for ganglioside GM1

Lipid rafts and the formation of an immunological synapse are crucial for T-cell activation. Binding of cholera toxin B subunit (CTB) to ganglioside GM1 is a marker to identify lipid rafts. Primary human T cells were isolated from healthy donors and were stimulated with superantigen staphylococcus enterotoxin B (SEB) and stained with cholera toxin B-fluorescein isothiocyanate (CTB-FITC). An optimized staining procedure is required to stain lipid rafts exclusively on the cell surface. Unstimulated T cells show a few CTB binding spots on the cell surface. The size and number of CTB-binding lipid rafts are strongly upregulated during T-cell activation in SEB-stimulated CD4(+) T cells. However, our data show that the specificity of CTB for GM1 ganglioside is limited, because the binding capacity is partly resistant to inhibition of ganglioside synthesis and sensitive to trypsin digestion. Our results indicate that the binding of FITC-labeled CTB can be divided into at least three different categories: a specific binding of CTB to ganglioside GM1, a nonspecific binding of CTB probably to glycosylated surface proteins and a nonspecific binding of FITC to the cell surface.     

Specific binding of cholera toxin to rat erythrocytes revealed by analysis with a fluorescence-activated cell sorter

The direct binding of cholera toxin to the receptor on the native cell surface was analyzed with a fluorescence-activated cell sorter (FACS) by the direct membrane immunofluorescence technique using FITC-conjugated cholera toxin B subunit as a ligand and erythrocytes, but the binding was significantly affected by a change in pH, showing optimum pH of 7.2. The optimum conditions for analysis of the cholera toxin-binding with a FACS were reaction of the target cells with 0.2 M phosphate-buffer (pH 7.2) containing 0.025% of BSA and 0.175 M of NaCl at 4 degrees C for 40 min. The binding of cholera toxin B subunit to rat erythrocytes was linear in the range of 1.2 ng to 80 ng, which corresponded to 2,469 to 163,500 molecules of toxin per cell, and the latter was almost the saturated level of binding. although erythrocytes from different strains of rats possessed equal binding ability for the cholera toxin, no binding was observed with erythrocytes from mouse, guinea pig, cow, pig, man, or rabbit, indicating that the cholera-toxin binding occurs specifically on rat erythrocytes. This is in accord with our previous analytical deta on the absence of GM1 in erythrocytes of these animals except rat, of which erythrocytes contain GM1. Also, the structural specificity of the receptor for cholera toxin was assessed by a binding inhibition experiment using glycolipid-containing liposomes as inhibitors and GM1 was found to be the most potent inhibitor, showing complete inhibition of toxin (40 ng) binding to 5 x 10(6) erythrocytes at 505.6 pmol of GM1.     

GM1-ganglioside-induced Abeta assembly on synaptic membranes of cultured neurons

The cell-surface expression of GM1 ganglioside was studied using various cultured cells, including brain-derived endothelial cells, astrocytes, neuroblastoma cells (SH-SY5Y), and pheochromocytoma cells (PC12). GM1 ganglioside was detected only on the surface of native and nerve-growth-factor (NGF)-treated PC12 cells. We investigated whether GM1 ganglioside on the surface of these cells is sufficiently potent to induce the assembly of an exogenous soluble amyloid beta-protein (Abeta). A marked Abeta assembly was observed in the culture of NGF-treated PC12 cells. Notably, immunocytochemical study revealed that, despite the ubiquitous surface expression of GM1 ganglioside throughout cell bodies and neurites, Abeta assembly initially occurred at the terminals of SNAP25-immunopositive neurites. Abeta assembly in the culture was completely suppressed by the coincubation of Abeta with the subunit B of cholera toxin, a natural ligand for GM1 ganglioside, or 4396C, a monoclonal antibody specific to GM1-ganglioside-bound Abeta (GAbeta). In primary neuronal cultures, Abeta assembly initially occurred at synaptophysin-positive sites. These results suggest that the cell-surface expression of GM1 ganglioside is strictly cell-type-specific, and that expression of GM1 ganglioside on synaptic membranes is unique in terms of its high potency to induce Abeta assembly through the generation of GAbeta, which is an endogenous seed for Abeta assembly in Alzheimer brain.     

Histochemical evidence for the differential surface labeling, uptake, and intracellular transport of a colloidal gold-labeled insulin complex by normal human blood cells

A colloidal gold-labeled insulin-bovine serum albumin (GIA) reagent has been developed for the ultrastructural visualization of insulin binding sites on the cell surface and for tracing the pathway of intracellular insulin translocation. When applied to normal human blood cells, it was demonstrated by both visual inspection and quantitative analysis that the extent of surface labeling, as well as the rate and degree of internalization of the insulin complex, was directly related to cell type. Further, the pathway of insulin (GIA) transport via round vesicles and by tubulo-vesicles and saccules and its subsequent fate in the hemic cells was also related to cell variety. Monocytes followed by neutrophils bound the greatest amount of labeled insulin. The majority of lymphocytes bound and internalized little GIA, however, between 5-10% of the lymphocytes were found to bind considerable quantities of GIA. Erythrocytes rarely bound the labeled insulin complex, while platelets were noted to sequester large quantities of the GIA within their extracellular canalicular system. GIA uptake by the various types of leukocytic cells appeared to occur primarily by micropinocytosis and by the direct opening of cytoplasmic tubulo-vesicles and saccules onto the cell surface in regions directly underlying surface-bound GIA. Control procedures, viz., competitive inhibition of GIA labeling using an excess of unlabeled insulin in the incubation medium, preincubation of the GIA reagent with an antibody directed toward porcine insulin, and the incorporation of 125I-insulin into the GIA reagent, indicated the specificity and selectivity of the GIA histochemical procedure for the localization of insulin binding sites.     

Interleukin 3-dependent mouse mast cells express the cholera toxin-binding acidic glycosphingolipid, ganglioside GM1, and increase their histamine content in response to toxin

The acidic glycosphingolipid, ganglioside GM1, which is the binding site for cholera toxin on many cell types, was identified by chemical and by flow cytometric analyses of mouse interleukin 3-dependent, bone marrow culture-derived mast cells (BMMC). Ganglioside GM1 and other acidic glycosphingolipids were isolated from BMMC by chloroform/methanol extraction and chromatography on DEAE-Sephadex and were analyzed by thin layer chromatography. The presence of ganglioside GM1 in the BMMC extract was demonstrated by its co-migration with ganglioside GM1 standard in thin layer chromatography and by the binding of peroxidase-labeled cholera toxin B subunit to both molecules. As assessed by fluorescence flow cytometric analysis of the binding of fluorescein-conjugated cholera toxin B subunit, the majority of BMMC expressed ganglioside GM1 on their surface, and the total presentation per cell increased as cells progressed from the G1 to S to G2 + M phases of the cell cycle. The addition of increasing amounts of cholera toxin starting with 0.08 microgram/ml to BMMC cultured in 50% WEHI 3-conditioned medium containing IL 3 for 48 hr caused the adhesion of BMMC to the tissue culture flasks to increase in a dose-related manner, from less than 1% adherent cells in cultures without toxin to a plateau value of approximately 17% adherent in the presence of 1.25 micrograms/ml of toxin. The histamine content of BMMC increased from 26.7 +/- 3.59 ng/10(6) cells (mean +/- SD, n = 4) for control cultures to 201 +/- 17.4 ng/10(6) cells (mean +/- SD, n = 4) for nonadherent cells and to 588 +/- 89.4 ng/10(6) cells (mean +/- SD, n = 4) for adherent cells after 48 hr of culture in 0.31 microgram/ml cholera toxin, which was the optimal dose for nonadherent and adherent populations. The content of another preformed intragranular mediator, beta-hexosaminidase, did not increase appreciably in the presence of cholera toxin (n = 3). The increase in the histamine content of BMMC after the addition of 0.31 microgram/ml cholera toxin was detectable at 4 hr, plateaued by 24 to 48 hr, and gradually declined over the next 6 days. Cholera toxin also augmented the histamine content of BMMC in the presence of purified synthetic IL 3. Preincubation of whole cholera toxin with purified ganglioside GM1 inhibited the histamine-augmenting effects of cholera toxin on BMMC, indicating that the effect was not due to a contaminant, and neither the A nor B subunit of cholera toxin alone increased the histamine content of BMMC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Demonstration of corticotropin-releasing factor binding sites on human and rat erythrocyte membranes and their modulation by chronic ethanol treatment in rats

In a previous study we reported the presence of specific corticotropin-releasing factor (CRF) binding sites in peripheral tissues of the rat (Endocrinology, 116, 2152, 1985). Using 125I-labeled rat or human CRF, specific CRF binding sites were identified on rat and human erythrocytes, but not on lymphocytes or platelets. Furthermore, identical CRF binding was observed in the presence of intact erythrocytes or lysed erythrocyte membranes. Maximal binding of 125I-CRF occurred within 25 min at 4 degrees C and was saturable. Scatchard analysis of CRF binding to erythrocyte membranes revealed the existence of a single class of binding site. Chronic exposure of rats to ethanol vapor, known to lower specific CRF binding to pituitary tissue by 35%, also decreased 125I-rat CRF binding to erythrocyte membranes by approximately 45%, which was due to a decrease in the number of CRF binding sites. The parallel decrease of CRF binding to rat-erythrocyte and pituitary membranes following chronic ethanol treatment suggests that CRF binding to erythrocyte and pituitary membranes is modulated in a similar direction, which further suggests that the determination of CRF binding to erythrocytes may provide an important clinical tool to indirectly assess CRF-receptor levels in the pituitary gland and thereby enhance our understanding of ethanol-induced disorders of the hypothalamic-pituitary-adrenal axis in patients.     

Comparison of water exposed area of cholera toxin when free in solution and bound to liposomes containing the ganglioside GM1

Membrane impermeable diazocoupling reagents were used for studying the water exposure of subunits (alpha, beta, gamma) of cholera toxin (CT) when bound to liposomes containing the ganglioside GM1 (Lip-GM1). The interaction between CT with Lip-GM1 shielded the binding region in particular, since a maximum of one amino acid residue on each beta subunit was modifiable. When CT was labeled free in solution five residues of each beta subunit can be coupled, but it produced loss of binding ability. New area of beta subunit was exposed to reagents after having removed alpha subunit. This labeling may serve as a tool to assess the topology of CT upon binding with Lip-GM1.     

The role of gangliosides in the interaction of human chorionic gonadotropin and cholera toxin with murine Leydig tumor cells

A clonal line of murine Leydig tumor cells (MLTC-1) bound both human chorionic gonadotropin (hCG) and cholera toxin (CT) with high affinity and accumulated cyclic AMP in response to either effector. The major cellular ganglioside was GM3 with small amounts of GM2, GM1, and GD1a. The gangliosides became labeled when the cells were grown in medium containing [3H] galactose or were exposed to galactose oxidase or NaIO4 followed by NaB3H4. CT specifically protected GM1 from surface labeling whereas hCG did not protect any gangliosides from being labeled. When the cells were exposed to sialidase, surface GD1a was eliminated, and GM1 increased with a corresponding increase in CT binding. When sialidase-treated cells were first incubated with the B component of CT, binding and action of CT was blocked. The cells, however, retained their ability to bind and respond to hCG. Addition of purified gangliosides to the medium effectively inhibited the binding and action of CT but not hCG. The cells incorporated the exogenous gangliosides and exhibited increased binding of and responsiveness to CT but not hCG. Both hCG- and CT-receptor complexes were extracted from the cells with nonionic detergent and analyzed by sucrose gradient centrifugation. The hCG-receptor complex had an apparent molecular weight of 190,000 whereas the CT-receptor complex sedimented only slightly faster than CT itself. MLTC-1 gangliosides were separated on thin layer chromatograms which were overlayed with either iodinated CT or hCG. The toxin bound to a ganglioside corresponding to GM1 whereas the hormone did not bind to any of the gangliosides. When the cells were incubated overnight with hCG, they lost their hCG receptors but exhibited an increase in CT binding and gangliosides. Our results indicate that GM1 is the specific receptor for CT whereas gangliosides are not involved in the binding and action of hCG.     

Bacterial-associated cholera toxin and GM1 binding are required for transcytosis of classical biotype Vibrio cholerae through an in vitro M cell model system

To elucidate mechanisms involved in M cell uptake and transcytosis of Vibrio cholerae, we used an in vitro model of human M-like cells in a Caco-2 monolayer. Interspersed among the epithelial monolayer of Caco-2 cells we detect cells that display M-like features with or without prior lymphocyte treatment and we have established key parameters for V. cholerae transcytosis in this model. Cholera toxin (CT) mutants lacking the A subunit alone or both the A and B subunits were deficient for transcytosis. We explored this finding further and showed that expression of both subunits is required for binding by whole V. cholerae to immobilized CT receptor, the glycosphingolipid GM1. Confocal microscopy showed CT associated with transcytosing bacteria, and transcytosis was inhibited by pre-incubation with GM1 before infection. Finally, heat treatment of the bacterial cells caused a loss of binding to GM1 that was correlated with a significant decrease in uptake and transcytosis by the monolayer. Our data support a model in which the ability of bacteria to interact with GM1 in a CT-dependent fashion plays a critical role in transcytosis of V. cholerae by M cells.