Liposome fluidity alters interactions between the ganglioside GM1 and cholera toxin B subunit

Cholera toxin is a complex protein with a biologically active protein (A subunit) and a cell targeting portion (B subunit). The B subunit is responsible for specific cell binding and entry of the A subunit. One way to limit potential toxicity of the toxin after exposure is to introduce cellular decoys to bind the toxin before it can enter cells. In this study the ganglioside GM1, a natural ligand for cholera toxin, was incorporated into liposomes and the interaction between fluorescent B subunit and the liposome determined. Liposome membrane fluidity was determined to play a major role in the binding between liposomes and the cholera toxin B subunit. Liposomes with lower fluidity demonstrated greater binding with the B subunit. The findings from this study could have important implications on formulation strategies for liposome decoys of toxins.     

Effect of covalent modification on the binding of cholera toxin B subunit to ileal brush border surfaces.

A competitive binding assay has been developed to determine how modifications to the B subunit of cholera toxin affect the binding affinity of the subunit for an ileal brush border membrane surface. The Ricinus communis120 agglutinin (RCA120) specifically binds to terminal beta-D-galactosyl residues such as those found in oligosaccharide side chains of glycoproteins and ganglioside GM1. Conditions were designed to produce binding competition between the B subunit of cholera toxin and the RCA120 agglutinin. Displacement of RCA120 from brush border surfaces was proportional to the concentration of B subunit added. This assay was used to study the effect of modification of B subunit on competitive binding affinity for the ileal brush border surface. The B subunit of cholera toxin was modified by coupling an average of five sulfhydryl groups to each B subunit molecule and by reaction of the SH-modified B subunit with liposomes containing a surface maleimide group attached to phosphatidylethanolamine. SH-modified B subunit was approximately 200-fold more effective than native B subunit in displacing lectin from brush border surfaces in the competitive binding assay. The enhanced binding activity was retained on covalent attachment of the modified B subunit to the liposome surface. We conclude that the B subunit of cholera toxin may be a useful targeting agent for directing liposomes to cell surfaces that contain a ganglioside GM1 ligand.     

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.     

CHOLERA TOXIN

1. Death in several infectious diseases is caused by protein toxins secreted by invading bacteria. Cholera toxin is a simple protein secreted by Vibrio cholerae colonizing the gut; it is responsible for the massive diarrhoea that is cholera. 2. The primary action of cholera toxin is an activation of adenylate cyclase, an enzyme found on the inner membrane of eukaryotic cells that catalyses the conversion of ATP to cyclic AMP. Consequent increases in the intracellular concentration of cyclic AMP are responsible for other manifestations of cholera toxin including the diarrhoea. The toxin is active on almost all eukaryotic cells. 3. The toxin can be purified from culture filtrates of V. cholera. It has a molecular weight of 82000; and is composed of one subunit A (itself two polypeptide chains joined by a disulphide bond: AI (22000) and A2 (5000)) and five subunits B (11500). These can be separated in dissociating solvents such as detergents or 6 M guanidine hydrochloride. An amino-acid sequence of subunit B has been published. The five B subunits (sometimes found by themselves in the filtrate and known as ‘choleragenoid’) are probably arranged in a ring with the subunit A in the middle joined to them non-covalently by peptide A2. 4. The first action of cholera toxin on a cell is to bind to the membrane strongly and irreversibly. Several thousand molecules of toxin bind to each cell and the binding constants are of the order of 10^-10 M. The binding is rapid, but is followed by a lag phase of about an hour before the intracellular cyclic AMP concentration begins to increase. 5. Ganglioside G_M1, a complex amphiphilic lipid found in cell membranes, binds tightly to the toxin which shows an enzyme-like specificity for this particular ganglioside. Toxin that has already bound ganglioside can no longer bind to cells and is therefore inactive. This and other experiments using cells depleted of endogenous ganglioside suggest that ganglioside G_M1 is the natural receptor of the toxin on the cell surface. The binding is followed by a lateral movement of the toxin-ganglioside complex in the cell surface forming a ‘cap’ at one pole of the cell. 6. The binding of ganglioside by toxin is a function exclusively of subunit B; Subunit A does not bind and can be eluted with 8 M urea from an insolubilized toxin-ganglioside complex. Subunit B is not by itself active, and so preincubation with B can protect cells or even whole gut from the action of toxin by occupying all the ganglioside binding sites. 7. Subunit A is responsible for activation of adenylate cyclase. Purified subunit A or just peptide AI is active by itself and this activity is not inhibited by ganglioside or by antisera to subunit B. In intact cells the activity is low and shows the characteristic lag phase but in lysed cells the subunit (or the whole toxin) is much more active and there is no lag phase. This suggests that the lag phase represents the time that subunit A takes to cross the cell membrane and get to its target. 8. Several cofactors are needed for toxin activity in lysed cells: NAD+, ATP, sulphydryl compounds and another unidentified cytoplasmic component. The activity of the cyclase is altered in a complex way generally rather similarly to the action of hormones such as adrenalin, but it is difficult to draw any general conclusions. 9. There are two chief theories of how cholera toxin acts. The first is that subunit A (or just peptide AI) enters the cell and there catalyses some reaction leading to activation of the cyclase. The cleavage of NAD+ into nicotinamide and adenosine diphosphoribose could be such a reaction; it is catalysed by high concentrations of cholera toxin. 10. The other theory is that part of the toxin binds directly to the adenylate cyclase or to some other molecule that can then interact with the cyclase, perhaps after the lateral movement of the toxin-ganglioside complex in the cell surface. This binding may be related to the known action of guanyl nucleotides on the cell surface. 11. The entry of peptide AI into the cell and its transport through the membrane is mediated by the binding of subunits B to the cell surface, perhaps just because the binding increases the local concentration of subunit A, or perhaps following specific conformational changes in the subunits and the formation of a tunnel of B subunits through the membrane. An experiment showing that the toxin remains active when the subunits are covalently bonded together suggests that peptide AI does not separate completely from the rest of the molecule. 12. There are several other proteins that resemble cholera toxin in structure and function. For example, glycoprotein hormones such as thyrotrophin also activate adenylate cyclase and have an apparently similar subunit structure with one type of subunit that binds to a ganglioside. There may also be analogies between the amino-acid sequences of toxin and hormones. 13. The enterotoxin made by some strains of Escherichia coli produces a similar diarrhoea to that of cholera. Several different toxic proteins have been prepared but they all seem to activate adenylate cyclase in the same sort of way as cholera toxin does and also to cross-react immunologically with it. The E. coli toxin also reacts with ganglioside G, but the reaction is weak and probably physiologically insignificant. Salmonella typhimurium secretes a similar toxin. 14. Tetanus toxin also reacts with a ganglioside receptor. This protein has two polypeptide chains of which only one reacts with the ganglioside; but the molecular activity is not yet known. 15. Diphtheria toxin has an A fragment that is directly responsible for the toxicity (by catalysing an NAD+ cleavage reaction leading to the total inhibition of protein synthesis) and a B fragment that gets the A fragment into the cells. This structure of active and binding components therefore seems to be common to many toxins. 16. The ability to produce toxin may confer some selective advantage on V. cholerae. The toxin may originate from accidental incorporation of DNA from an eukaryotic host, or alternatively from some material involved with the cyclic AMP metabolism of the bacterium.     

Short communications. Subunit A from cholera toxin is an activator of adenylate cyclase in pigeon erythrocytes.

Intact cholera toxin and its purified subunit A both activate the adenylate cyclase of pigeon erythrocyte membranes, but subunit B does not. The activation by subunit A is unaffected by treatments that inhibit whole toxin by interfering with the binding of subunit B to cell membranes.     

Chemical and immunochemical studies on the receptor binding domain of cholera toxin B subunit

The contributions of various amino acids to the structure and function of cholera toxin B subunit were assessed with quantifiable, chemically conservative, reversible derivatizations, and sensitive assays of activity. A panel of monoclonal antibodies was employed to monitor the conformational integrity of modified protein and help distinguish the direct from indirect effects of chemical derivatization. We describe a novel monoclonal antibody, which competes with the receptor GM1 for binding to cholera toxin B subunit, and use this reagent to help identify critically located residues. Our data support the hypothesis that tryptophan participates directly in binding GM1. In addition, we propose a dual role for lysine: first, these basic residues maintain an electrostatic attraction vital to receptor recognition; second, at least 1 lysine resides near the receptor binding domain and may interact with GM1. The influence of arginyl and tyrosyl residues upon activity is re-examined. Finally, we present data which suggest, in variance with previous studies, that the intramolecular disulfide bond is vital to the structure and function of cholera toxin B subunit.     

Studies on the time course and rate-limiting steps in the activation of adenylate cyclase in rat liver by cholera toxin.

Cholera toxin stimulates adenylate cyclase in rat liver after intravenous injection. The stimulation follows a short latent period of 10min, and maximum stimulation was attained at 120min. Half-maximal stimulation was achieved at 35min. In contrast with this lengthy time course in the intact cell, adenylate cyclase in broken-cell preparations of rat liver in vitro were maximally stimulated by cholera toxin (in the presence of NAD+) in 20min with half-maximal stimulation in 8min. Binding of cholera toxin to cell membranes by the B subunits is followed by translocation of the A subunit into the cell or cell membrane, and separation of the A1 polypeptide chain from the A2 chain by disulphide-bond reduction, and finally activation of adenylate cyclase by the A1 chain and NAD+. As the binding of cholera toxin is rapid, two possible rate-limiting steps could be the determinants of the long time course of action. These are translocation of the A1 chain from the outside of the cell membrane to its site of action (this includes the time required for separation from the whole toxin) or the availability of NAD+ for activation. When NAD+ concentrations in rat liver were elevated 4-fold, by the administration of nicotinamide, no change in the rate of activation of adenylate cyclase by cholera toxin was observed. Thus the intracellular concentration of NAD+ is not rate-limiting and the major rate-limiting determinant in intact cells must be between the time of toxin binding to the cell membrane and the appearance of subunit A1 at the enzyme site.     

cAMP-independent effects of cholera toxin on B cell activation. I. A possible role for cell surface ganglioside GM1 in B cell activation

Cholera toxin has been used as a tool to study the effects of cAMP on the activation of B cells but may have effects independent of its ability to elevate cAMP. We found five lines of evidence which suggested that cholera toxin suppressed mitogen-stimulated B cell activation through a cAMP-independent pathway. 1) Cholera toxin (1 microgram/ml) was consistently more suppressive than forskolin (100 microM) despite the induction of higher intracellular cAMP levels by forskolin. 2) Cholera toxin was more suppressive at 1 microgram/ml than at 0.1 microgram/ml despite equivalent elevations of cAMP. 3) Washing B cells following their incubation with cholera toxin reversed much of the inhibition without altering intracellular cAMP levels. 4) The A subunit of cholera toxin, which at high concentrations (10 micrograms/ml) induced levels of cAMP comparable to those induced by cholera toxin (1 and 0.1 microgram/ml), did not inhibit B cell activation. 5) cAMP derivatives at high concentrations were much less effective than was cholera toxin in suppressing B cell activation. Although the elevation of cAMP may cause a mild inhibition of B cell proliferation, we found that even a marked elevation of cAMP did not suppress B cell proliferation, unless the elevation was persistent. We did, however, observe that the degree of toxin inhibition more closely paralleled binding of the toxin to B cells than toxin stimulation of cAMP. This result raised the possibility that binding of cholera toxin to its ganglioside GM1 receptor mediated an inhibitory signal which suppressed B cell proliferation.     

Inhibition of protein kinase C-dependent cellular proliferation by interaction of endogenous ganglioside GM1 with the B subunit of cholera toxin

In quiescent Swiss 3T3 fibroblasts, the B subunit of cholera toxin, a protein which binds specifically to ganglioside GM1 on the cell surface, stimulates DNA synthesis and potentiates the effects of several other growth factors such as insulin, epidermal growth factor, bombesin, and even unfractionated serum. In contrast to its synergistic effect with other known growth factors, the B subunit markedly inhibited DNA synthesis induced by the phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA). The inhibitory effect of the B subunit was observed even in the presence of insulin, which greatly potentiates the mitogenic response to TPA or the B subunit. In contrast to the effect of the B subunit, calcium ionophores and cholera toxin stimulated DNA synthesis induced by TPA. The antagonism between the B subunit and TPA is not simply due to their abilities to modify their mutual binding sites or known effector systems. TPA did not block the early rise in cytosolic free calcium in response to the B subunit, and conversely, the B subunit did not modify the ability of TPA to activate protein kinase C. However, in protein kinase C-deficient cells, the antagonistic effect between TPA and the B subunit was abolished. In addition, there was no indication for the involvement of a pertussis toxin-sensitive G protein in the antagonism. Maximum inhibition was found when the B subunit was added 2 h after the addition of TPA. Significant inhibition was still evident when the time of addition of the B subunit was delayed until 6 h after the addition of TPA. This suggests that the cross-talk between signal transduction induced through endogenous gangliosides and protein kinase C is a late step in mitogenesis.     

Actions of cholera toxin on dispersed Chief cells from guinea pig stomach

Although much is known about the actions of cholera toxin on intestinal and extra-gastrointestinal tissues, almost nothing is known about the interaction of this toxin with cells in the stomach. In the present study, we prepared 125I-labeled cholera toxin (1900 Ci/mmol) and examined the binding of this radioligand to dispersed Chief cells from guinea pig stomach. Moreover, we examined the actions of cholera toxin on cellular cAMP and pepsinogen secretion from Chief cells. Binding of 125I-labeled cholera toxin could be detected within 5 min, was maximal by 60 min, and was increased by increasing the radioligand or cell concentrations. Inhibition of binding by unlabeled toxin indicated a dissociation constant of 3 nM and 8.7 X 10(5) cholera toxin receptors per Chief cell. In contrast to the rapidity of binding, a cholera toxin-induced increase in cAMP and pepsinogen secretion was not detected until 30-45 min of incubation. A 3 to 6-fold increase in cAMP and pepsinogen secretion was observed with maximal concentrations of cholera toxin. Binding of 125I-labeled cholera toxin and the toxin's actions on cAMP and pepsinogen secretion were inhibited by the B subunit of the toxin. Binding was not altered by other agents that have been shown to stimulate pepsinogen secretion (carbachol, CCK-8, secretin, vasoactive intestinal peptide, prostaglandin E1, or forskolin). These data indicate that Chief cells from guinea pig stomach possess a specific class of cholera toxin receptors. Binding of cholera toxin to these receptors causes an increase in cellular cAMP that stimulates pepsinogen secretion.     

Human platelets are defective in processing of cholera toxin.

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.     

Analysis of receptor-binding site in Escherichia coli enterotoxin

Heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and cholera enterotoxin are both composed of A and B subunits. The A subunit is an enzymatically active ADP-ribosylating subunit, while the B subunit, consisting of 103 amino acids, binds the toxin to a receptor, GM1-ganglioside, on the cell surface. A mutant isolated after treatment of E. coli producing heat-labile enterotoxin with N-methyl-N'-nitro-N-nitrosoguanidine produces a B subunit that is unable to bind to ganglioside. This subunit was purified and its primary amino acid sequence was determined. It differed from the native B subunit in only one amino acid at position 33; namely it had aspartate instead of glycine at position 33 from the N terminus. Thus glycine at position 33 from the N terminus of the B subunit is important for binding the B subunit to the ganglioside receptor.     

Cautionary note on the use of the B subunit of cholera toxin as a ganglioside GM1 probe: detection of cholera toxin A subunit in B subunit preparations by a sensitive adenylate cyclase assay

The use of the B subunit of cholera toxin, a protein that binds specifically to ganglioside GM1, has provided a new paradigm for studying physiological functions of ganglioside GM1. The B subunit inhibited the growth of rat glioma C6 cells that had been pretreated with ganglioside GM1. In some preparations of the B subunit, the inhibition was independent of adenylate cyclase activation and was due to the binding of the B subunit to ganglioside GM1 inserted onto the cell surface. However, in other preparations of the B subunit, there was an additional inhibitory effect due to small contaminations with the A subunit, which caused increases in intracellular cyclic adenosine monophosphate (cAMP) levels and concomitant growth inhibition. This vanishingly small contamination with the A subunit could not be detected by conventional protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis but could be measured utilizing a sensitive adenylate cyclase activation assay. Thus caution must be used to ensure that any biological effects of the B subunit are not due to contaminating A subunit and are due solely to the binding of the B subunit to ganglioside GM1 exposed on the cell surface. This is especially important in cyclic nucleotide-sensitive systems.     

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)     

Conformations of higher gangliosides and their binding with cholera toxin - investigation by molecular modeling, molecular mechanics, and molecular dynamics

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface higher gangliosides (GT1A and GT1B) and their interaction with Cholera Toxin. The water mediated hydrogen bonding network exists between sugar residues in gangliosides. An integrated molecular modeling, molecular mechanics, and molecular dynamics calculation of cholera toxin complexed with GT1A and GT1B reveal that, the active site of cholera toxin can accommodate these higher gangliosides. Direct and water mediated hydrogen bonding interactions stabilize these binding modes and play an essential role in defining the order of specificity for different higher ganglioside towards cholera toxin. This study identifies that the binding site of cholera toxin is shallow and can accommodate a maximum of two NeuNAc residues. The NeuNAc binding site of cholera toxin may be crucial for the design of inhibitors that can prevent the infection of cholera.     

Homologous sequences in cholera toxin A and B subunits to peptide domains in myelin basic protein

Recent reports that myelin basic protein (MBP) can be ADP-ribosylated and contains specific sites that bind GTP and GM1 ganglioside, have suggested an analogy to the properties of cholera toxin. Comparisons of pairs of sequences between these two proteins yielded two regions of homology between MBP and the cholera toxin B (chol B) subunit, and one region of homology with the cholera toxin A (chol A) subunit. The matching sites within chol B consisted of a 17 amino acid residue sequence (residues 30-46 in chol B and residues 102-118 in human-MBP, hMBP, p less than 0.0007) and an 11 residue span (residues 31-41 in chol B and sequence 29-39 in hMBP, p less than 0.0004). The homologous site within chol A corresponded to an 11 residue span (residues 130-140 in chol A and 67-77 in hMBP sequence, p less than 0.00007). Since portions of the cholera toxin sequence are virtually identical to sections of the sequence in E. coli toxin, the homology is also valid for the same sequences in this toxin. The highly antigenic behavior of MBP that is related to the induction of experimental allergic encephalomyelitis may be paralleled by comparable neural pathology from the homologous regions of cholera toxin.     

Structural characterization of pertussis toxin A subunit

The relationship between the structure of the A subunit of pertussis toxin and its function was analyzed. Limited tryptic digestion of the A subunit converted the protein to two stable fragments (Mr = 20,000 and 18,000). Antibodies raised to synthetic peptides homologous to regions in the A subunit were used to map these fragments. Both fragments were shown to contain the NH2-terminal portion but not the COOH-terminal portion of the A subunit. While these fragments exhibited NAD glycohydrolase activity, they were unable to reassociate with the B oligomer of the toxin. Thus the COOH-terminal portion of the A subunit does not contain the residues which are required for the NAD glycohydrolase activity of the toxin. However, this region of the molecule may be important for maintaining the oligomeric structure of the toxin. These results suggest that the A subunit of pertussis toxin is similar in structure to the A subunit of cholera toxin. In addition, antibodies raised to a synthetic peptide identical to residues 6-17 of the A subunit of pertussis toxin will bind to the A subunit of cholera toxin.     

Possible involvement of a GTP-binding protein in a late event during endogenous ganglioside-modulated cellular proliferation

The B subunit of cholera toxin, a protein which binds specifically to ganglioside GM1 on the cell surface, stimulates DNA synthesis in quiescent Swiss 3T3 fibroblasts as measured by an increase in [3H]thymidine incorporation. Pertussis toxin pretreatment markedly inhibits B subunit-induced DNA synthesis. The inhibitory effects of pertussis toxin were observed even in the presence of insulin which greatly potentiates the mitogenic response to the B subunit. Treatment with either pertussis toxin or insulin did not alter the binding of the B subunit to the cells. The dose-response for pertussis toxin-induced inhibition of DNA synthesis correlated closely with the dose-response for ADP-ribosylation of a 41-kDa membrane protein, suggesting the involvement of a GTP-binding protein that is a substrate for pertussis toxin (Gi) in mitogenesis induced via cross-linking of endogenous gangliosides. Pertussis toxin, in a similar concentration-dependent manner, also inhibited the mitogenic response to unfractionated fetal calf serum and to bombesin in the absence or presence of insulin. The inhibitory effect of pertussis toxin was clearly unrelated to any effects on known G proteins coupled to adenylate cyclase or phospholipase C. In addition, pertussis toxin did not impair the early increase in cytosolic free Ca2+ induced by the B subunit or bombesin. Pertussis toxin-induced inhibition of DNA synthesis could still be observed even when the toxin was added as late as 6 h after addition of the growth-promoting agents. This suggests the involvement of a GTP-binding protein in a late step of the B subunit- and bombesin-mediated pathways of mitogenesis. The possibility that other growth factors bypass this pathway is shown by their lack of sensitivity to pertussis toxin.     

Conformations of Higher Gangliosides and Their Binding with Cholera Toxin—Investigation by Molecular Modeling,Molecular Mechanics,and Molecular Dynamics

Abstract

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface higher gangliosides (GT1A and GT1B) and their interaction with Cholera Toxin. The water mediated hydrogen bonding network exists between sugar residues in gangliosides. An integrated molecular modeling, molecular mechanics, and molecular dynamics calculation of cholera toxin complexed with GT1A and GT1B reveal that, the active site of cholera toxin can accommodate these higher gangliosides. Direct and water mediated hydrogen bonding interactions stabilize these binding modes and play an essential role in defining the order of specificity for different higher ganglioside towards cholera toxin. This study identifies that the binding site of cholera toxin is shallow and can accommodate a maximum of two NeuNAc residues. The NeuNAc binding site of cholera toxin may be crucial for the design of inhibitors that can prevent the infection of cholera.