Insertion of ganglioside GM1 into rat glioma C6 cells renders them susceptible to growth inhibition by the B subunit of cholera toxin

The B subunit of cholera toxin does not affect the growth of rat glioma C6 cells which are deficient of its receptor, ganglioside G_M1. Insertion of ganglioside G_M1 into the plasma membrane of C6 cells renders them susceptible to inhibition of DNA synthesis by the B subunit. Exposure of C6 cells to butyrate induces an elevation of ganglioside G_M1 as measured by an increase in binding of iodinated cholera toxin and also results in an inhibition of DNA synthesis by the B subunit. The extent of inhibition of DNA synthesis correlated with the binding of B subunit and was independent of adenylate cyclase activation or increases in intracellular cAMP levels.     

Analysis of structure and function of the B subunit of cholera toxin by the use of site-directed mutagenesis

Oligonucleotide-directed mutagenesis of ctxB was used to produce mutants of cholera toxin B subunit (CT-B) altered at residues Cys-9, Gly-33, Lys-34, Arg-35, Cys-86 and Trp-88. Mutants were identified phenotypically by radial passive immune haemolysis assays and genotypically by colony hybridization with specific oligonucleotide probes. Mutant CT-B polypeptides were characterized for immunoreactivity, binding to ganglioside GM1, ability to associate with the A subunit, ability to form holotoxin, and biological activity. Amino acid substitutions that caused decreased binding of mutant CT-B to ganglioside GM1 and abolished toxicity included negatively charged or large hydrophobic residues for Gly-33 and negatively or positively charged residues for Trp-88. Substitution of lysine or arginine for Gly-33 did not affect immunoreactivity or GM1-binding activity of CT-B but abolished or reduced toxicity of the mutant holotoxins, respectively. Substitutions of Glu or Asp for Arg-35 interfered with formation of holotoxin, but none of the observed substitutions for Lys-34 or Arg-35 affected binding of CT-B to GM1. The Cys-9, Cys-86 and Trp-88 residues were important for establishing or maintaining the native conformation of CT-B or protecting the CT-B polypeptide from rapid degradation in vivo.     

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.     

Isolation ofEscherichia coli heat-labile enterotoxin by affinity chromatography: Characterization of subunits

This paper describes the isolation ofEscherichia coli heat-labile enterotoxin (LT) by affinity chromatography on an anti-cholera toxin immunoglobulin-Sepharose column, and the subunit composition of crude and affinity-isolated LT. LT and its subunits were assayed with ganglioside (G_M1)-ELISA, immunodiffusion, skin toxicity, and broken cell adenylate cyclase activation methods. The results show that the immunoaffinity method, applied to LT of different strains and batches, yielded about 100-fold purification with approximately 50% recovery of LT antigen. LT was shown to contain a G_M1-ganglioside binding subunit as well as another subunit which does not bind to G_M1 but activates adenylate cyclase. Immunodiffusion tests showed that the two LT subunits were immunologically related to but not identical with, respectively, the B and A subunits of cholera toxin. The LT “A” and “B” subunits were present in similar proportions in the affinity-isolated and crude LT preparations, but in the purified fraction they had only partially reassociated into holotoxin.     

Involvement of the acyl chain of ceramide in carbohydrate recognition by an anti-glycolipid monoclonal antibody: the case of an anti-melanoma antibody,M2590, to GM3-ganglioside

The effect of the chain length of the fatty acid residue of the ceramide moiety of ganglioside G_M3 on the binding ability of monoclonal antibody M2590, which is specific for the carbohydrate structure of G_M3-ganglioside, was examined by means of a direct binding assay on thin layer chromatography plates (TLC immunostaining) and a quantitative enzyme-linked immunosorbent assay (ELISA). Derivatives of G_M3 with a long fatty acid chain reacted with the M2590 antibody, but those with a short fatty acid chain showed no reaction in either assay system. These results suggested that the acyl fatty acid moiety of the ganglioside played an important role in the formation or maintenance of the antigenic structure of the carbohydrate moiety of the ganglioside.     

Mechanism of action of choleragen andE. coli heat-labile enterotoxin: activation of adenylate cyclase by ADP-ribosylation

Summary Choleragen exerts its effects on cells through the activation of adenylate cyclase. The initial event appears to be the binding of the B subunit of the toxin to ganglioside G_M1 on the cell surface, following which there is a delay prior to activation of adenylate cyclase. Patching and capping of the toxin on the cell surface, perhaps involved in the internalization of the enzymatically active subunit, may be occuring during this time. The activation of adenylate cyclase, which is catalyzed by the A₁ peptide of choleragen, does not require the B subunit or ganglioside G_M1. The A₁ peptide catalyzes the transfer of ADP-ribose from NAD to an amino acid, probably arginine, in a 42 000 dalton membrane protein. This protein appears to be the GTP-binding component (or G/F factor) of the adenylate cyclase system and is cruical to the regulation of cyclase activity by hormones such as epinephrine. ADP-ribosylation of the G/F factor is enhanced by GTP and, in some systems, by a cytosolic factor. GTP is also required for stabilization and optimal catalytic function of the choleragen-activated cyclase. Calmodulin, a calcium-binding protein, is necessary for expression of catalytic activity of the toxin-activated adenylate cyclase in brain and other tissues. The ADP-ribosyltransferase activity required for activation of the cyclase is an intrinsic property of the A₁ peptide of choleragen which is expressed only after the peptide is released from the holotoxin by reduction of a single disulfide bond. In the absence of cellular components, choleragen catalyzes the ADP-ribosylation of small guanidino compounds such as arginine as well as peptides and proteins that contain arginine. It is assumed, therefore, that the site of ADP-ribosylation in the natural acceptor protein is an arginine or similar amino acid. When guanidino compounds are not present as ADP-ribose acceptors, choleragen hydrolyzes NAD to ADP-ribose and nicotinamide at a considerably slower rate. E. coli heat-labile enterotoxin (LT) is very similar to choleragen in structure and function. It consists of two types of subunits, A and B, with sizes comparable to those of the A and B subunits of choleragen. Binding of LT to the cell surface is enhanced by prior incorporation of G_M1 but not other gangliosides; the oligosaccharide of G_M1 specifically interacts with LT and its B subunit. The A subunit of LT exhibits ADP-ribosyltransferase activity following activation by thiol to release the A₁ peptide. The A subunit of LT can be isolated in an ‘unnicked’ form and thus requires, in addition to reduction by a thiol, proteolytic cleavage to generate the active A₁ peptide. Like choleragen, LT uses guanidino compounds as model ADP-ribose acceptors and catalyzes the ADP-ribosylation of a 42 000 dalton protein in cell membrane prepatations. ADP-ribosyltransferases that use arginine as ADP-ribose acceptors are not restricted to bacterial systems; such an enzyme has been purified to apparent homogeneity (>500 000-fold) from turkey erythrocytes. Based on a subunit molecular weight of 28 000, its turnover number with arginine as the ADP-ribose acceptor is considerably higher than that of either toxin. Although with low molecular weight guanidino derivatives the substrate specificity of the enzyme is similar to that of choleragen, with protein substrates it clearly differs. The physiological role of the turkey erythrocyte transferase remains to be established.     

Immunochemistry of sea anemone toxins: structure-antigenicity relationships and toxin-receptor interactions probed by antibodies specific for one antigenic region

Two antibody subpopulations directed against Anemonia sulcata toxin I or II have been purified by immunoaffinity chromatography. These antibodies are specific for a single antigenic region and were used in a structure-antigenicity relationship study using homologous toxins and chemically modified derivatives of A. sulcata toxin II. Asp-7 and/or Asp-9 and Gln-47 of toxin II were found to be implicated in the antigenic region recognized by the two antibody subpopulations. On the contrary, Arg-14, Lys-35, -36, and -46, and alpha-NH2 of the glycine residue of A. sulcata toxin II are not involved in the corresponding antigenic region. When assayed for interaction with the sodium channel, the antigenic region of toxin II, including Asp-9 and Gln-47, appeared fully accessible to its specific antibodies, suggesting that it is not involved in the binding of the toxin to its receptor.     

Common antigenic determinant in the receptor binding sites of Escherichia coli enterotoxin and cholera toxin

Abstract A mutant (TUH No. 9) of a porcine strain of enterotoxigenic Escherichia coli (ETEC) produces as abnormal B subunit (B') of heat-labile enterotoxin (LT), which has aspartate instead of glycine at residue 33 from the N-terminus and does not bind to the receptor, GM1 ganglioside. The antigenicities of the receptor-binding site of LT were analyzed.
The antibody, which could not bind to the B' subunit in the anti-B subunit of porcine LT(LTp)-serum, could bind to cholera toxin (CT), LTp and LT produced by a human ETEC strain (LTh), suggesting that it recognizes a common epitope of LTp, LTh and CT. Thus glycine at residue 33 from the N-terminus in the B subunit of CT, LTh and LTp may be related to the common epitope of these three toxins. The bindings of CT, LTh and LTp to the antibody were inhibited by the GM1 ganglioside.
These data indicate that the antibody recognizes a common epitope in the receptor (GM1 ganglioside)-binding site of CT, LTh and LTp.     

Thermal stability and intersubunit interactions of cholera toxin in solution and in association with its cell-surface receptor ganglioside GM1

The thermal stability of cholera toxin free in solution and in association with its cell-surface receptor ganglioside GM1 has been studied by using high-sensitivity differential scanning calorimetry and differential solubility thermal gel analysis. In the absence of ganglioside GM1, cholera toxin undergoes two distinct thermally induced transitions centered at 51 and 74 degrees C, respectively. The low-temperature transition has been assigned to the irreversible thermal denaturation of the active A subunit. The second transition has been assigned to the reversible unfolding of the B subunit pentamer. The isolated B subunit pentamer exhibits a single transition also centered at 74 degrees C, suggesting that the attachment of the A subunit does not contribute to the stability of the pentamer. In the intact toxin, the A subunit dissociates from the B subunit pentamer at a temperature that coincides with the onset of the B subunit thermal unfolding. In aqueous solution, the denatured A subunit precipitates after dissociation from the B subunit pentamer. This phenomenon can be detected calorimetrically by the appearance of an exothermic heat effect. In the presence of ganglioside GM1, the B subunit is greatly stabilized as indicated by an increase of 20 degrees C in the transition temperature. In addition, ganglioside GM1 greatly enhances the cooperative interactions between B subunits. In the absence of ganglioside, each monomer within the B pentamer unfolds in an independent fashion whereas the fully ganglioside-bound pentamer behaves as a single cooperative unit. On the contrary, the thermotropic behavior of the A subunit is only slightly affected by the presence of increasing concentrations of ganglioside GM1.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel heptasialosyl c-series ganglioside in embryonic chicken brain: its structure and stage-specific expression

A ganglioside of unknown structure (ganglioside X) was purified from chicken brain at embryonic day 12 (E12) and characterized for its structure. Ganglioside X was reactive with a monoclonal antibody A2B5 and migrated below GH1c on thin-layer chromatography (TLC). Extensive treatment of ganglioside X with Clostridium perfringens sialidase produced a single ganglioside product. This ganglioside was identified as GM1 based upon its chromatographic mobility and reactivity to cholera toxin B subunit and anti-GM1 antibody. Partial hydrolysis of ganglioside X by sialidase generated several degradation products including GH1c, GP1c, and GQ1c. Electrospray ionization (ESI)-mass spectrometry (MS) of the permethylated derivative of ganglioside X produced a triple-charged parent ion peak at m/z 1355, which corresponded with the gangliotetraose oligosaccharide structure having seven sialic acids and ceramide with the molecular mass of 566 (as non-methylated form). Collision-induced dissociation (CID)-MS(2) showed fragment ions including those at m/z 1066 and 1931; these two ions matched the structures of (NeuAc)(3)-Gal-Glc-Cer and (NeuAc)(4)-Gal-GalNAc, respectively. These structures were confirmed by CID-MS(3) of the corresponding peaks. Based upon these findings, the structure of ganglioside X was identified as NeuAc-NeuAc-NeuAc-NeuAc-Galbeta1-3GalNAcbeta1-4(NeuAc-NeuAc-NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer. This ganglioside was designated as GS1c. A developmental study demonstrated that GS1c was expressed in chicken brain during a period from E6 to E13 and thereafter decreased rapidly in its concentration. The present study suggests that GS1c may play a specific role in early development of chicken brain.     

Fluorescently labeled liposomes for monitoring cholera toxin binding to epithelial cells

Vibrio cholerae, the causative agent for cholera, expresses a toxin required for virulence consisting of two subunits: the pentameric cholera toxin B (CTB) and cholera toxin A (CTA). CTB is frequently used as an indicator of the presence of pathogenic V. cholerae and binds to the G_M1 ganglioside on the surface of epithelial cells. To study V. cholerae virulence (CTB expression) in the presence of human epithelia, we devised an inexpensive, simple, and rapid method for quantifying CTB bound on epithelial surfaces in microtiter plates. G_M1 ganglioside was incorporated into the lipid bilayer of liposomes both encapsulating the fluorescent dye sulforhodamine B (SRB) and with SRB tagged to lipids in the bilayer (BEGs). In addition, G_M1-embedded liposomes encapsulating SRB only (EGs) and with SRB in their bilayers only (BGs) were synthesized. The three types of liposomes were compared with respect to their efficacy for both visualizing and quantifying CTB attached to the surface of Caco-2 cells. The BEGs were the most effective overall, providing both visualization under a fluorescence microscope and quantification after lysis in a microtiter plate reader. A limit of detection corresponding to 0.28 μg/ml applied CTB was attained for the on-cell assay using the microtiter plate reader approach, whereas as low as 2 μg/ml applied CTB could be observed under the fluorescence microscope.     

Variation in epitopes of the B subunit of Vibrio cholerae non-O1 and Vibrio mimicus cholera toxins.

Monoclonal antibodies reacting with the B subunit of Vibrio cholerae O1 strain 569B cholera toxin (CT-B) were used to identify unique and common epitopes of V. cholerae non-O1 and Vibrio mimicus CT-B. Vibrio cholerae non-O1 strains produced CT-B showing three monoclonal antibody reaction patterns (epitypes), which corresponded with epitypes described previously for V. cholerae O1 classical biotype CT-B (CT1), El Tor biotype CT-B (CT2), and a unique V. cholerae non-O1 CT-B (CT3), which lacked an epitope located in or near the GM1 ganglioside binding site of 569B CT-B. Vibrio mimicus CT-B was immunologically indistinguishable from 569B CT-B. These and previous results define six epitopes on 569B CT-B, and a fourth epitope in or near the GM1 ganglioside binding site.     

Genetic control of ganglioside biosynthesis in mice

The enzymatic basis for the differences in hepatic ganglioside patterns in the mouse strains C57Bl/6 and Swiss White (SW) was investigated. SW has a “Swiss-type” ganglioside profile, expressing G_M1 ^? and G_D1a ^? in addition to G_M2 ^? as major hepatic gangliosides, whereas C57Bl/6 shows a “G_M2-type” profile, expressing only G_M2 ^? as the major hepatic ganglioside. The enzyme UDP-galactose:G_M2 ganglioside galactosyltransferase (G_M2-GalT), which catalyzes the synthesis of G_M1 ganglioside, showed a four- to fivefold elevation in intact and solubilized liver Golgi membrane fractions of the SW strain compared to C57Bl/6. Crosses between C57Bl/6 and SW produced an F₁ generation with a hepatic ganglioside and enzymatic phenotype intermediate between those of the two parental strains. All three genotypic groups show two forms of the Golgi apparatus enzyme with isoelectric points of 6.5–6.8 and 8.3–9.0. The simplest mode of action of genes which control the enzymatic phenotype that would be consistent with these findings are one or two structural genes or one or two cis-regulatory genes affecting the rate of enzyme synthesis.     

Quartz crystal microbalance investigation of the interaction of bacterial toxins with ganglioside containing solid supported membranes

The binding of cholera toxin, tetanus toxin and pertussis toxin to ganglioside containing solid supported membranes has been investigated by quartz crystal microbalance measurements. The bilayers were prepared by fusion of phospholipid-vesicles on a hydrophobic monolayer of octanethiol chemisorbed on one gold electrode placed on the 5 MHz AT-cut quartz crystal. The ability of the gangliosides G_M1, G_M3, G_D1a, G_D1b, G_T1b and asialo-G_M1 to act as suitable receptors for the different toxins was tested by measuring the changes of quartz resonance frequencies. To obtain the binding constants of each ligand-receptor-couple Langmuir-isotherms were successfully fitted to the experimental adsorption isotherms. Cholera toxin shows a high affinity for G_M1 (K_a = 1.8 ⋅ 10⁸M^–1), a lower one for asialo-G_M1 (K_a = 1.0 ⋅ 10⁷ M^–1) and no affinity for G_M3. The C-fragment of tetanus toxin binds to ganglioside G_D1a, G_D1b and G_T1b containing membranes with similar affinity (K_a∼10⁶ M^–1), while no binding was observed with G_M3. Pertussis toxin binds to membranes containing the ganglioside G_D1a with a binding constant of K_a = 1.6 ⋅ 10⁶ M^–1, but only if large amounts (40 mol%) of G_D1a are present. The maximum frequency shift caused by the protein adsorption depends strongly on the molecular structure of the receptor. This is clearly demonstrated by an observed maximum frequency decrease of 99 Hz for the adsorption of the C-fragment of tetanus toxin to G_D1b. In contrast to this large frequency decrease, which was unexpectedly high with respect to Sauerbrey's equation, implying pure mass loading, a maximum shift of only 28 Hz was detected after adsorption of the C-fragment of tetanus toxin to G_D1a. Received: 14 January 1997 / Accepted: 15 April 1997     

Mechanism of action of choleragen

Choleragen exerts its effect on cells through activation of adenylate cyclase. Choleragen initially interacts with cells through binding of the B subunit of the toxin to the ganglioside G_M1 on the cell surface. Subsequent events are less clear. Patching or capping of toxin on the cell surface may be an obligatory step in choleragen action. Studies in cell-free systems have demonstrated that activation of adenylate cyclase by choleragen requires NAD. In addition to NAD, requirements have been observed for ATP, GTP, and calcium-dependent regulatory protein. GTP also is required for the expression of choleragen-activated adenylate cyclase. In preparations from turkey erythrocytes, choleragen appears to inhibit an isoproterenol-stimulated GTPase. It has been postulated that by decreasing the activity of a specific GTPase, choleragen would stabilize a GTP-adenylate cyclase complex and maintain the cyclase in an activated state. Although the holotoxin is most effective in intact cells, with the A subunit having 1/20th of its activity and the B subunit (choleragenoid) being inactive, in cell-free systems the A subunit, specifically the A₁ fragment, is required for adenylate cyclase activation. The B protomer is inactive. Choleragen, the A subunit, or A₁ fragment under suitable conditions hydrolyzes NAD to ADP-ribose and nicotinamide (NAD glycohydrolase activity) and catalyzes the transfer of the ADP-ribose moiety of NAD to the guandino group of arginine (ADP-ribosyltransferase activity). The NAD glycohydrolase activity is similar to that exhibited by other NAD-dependent bacterial toxins (diphtheria toxin, Pseudomonas exotoxin A), which act by catalyzing the ADP-ribosylation of a specific acceptor protein. If the ADP-ribosylation of arginine is a model for the reaction catalyzed by choleragen in vivo, then arginine is presumably an analog of the amino acid which is ADP-ribosylated in the acceptor protein. It is postulated that choleragen exerts its effects on cells through the NAD-dependent ADP-ribosylation of an arginine or similar amino acid in either the cyclase itself or a regulatory protein of the cyclase system.     

Environmental and nutritional factors which regulate population dynamics and toxin production in the dinoflagellate Alexandrium catenella

The effects of environmental and nutritional factors on population dynamics and toxin production were examined in Alexandrium catenella, maintained in enriched K media in laboratory cultures. Starting with a density of 50 cell ml⁻¹, the dinoflagellate population typically showed a lag phase and an exponential growth phase which lasted 14 days each, and then entered the stationary phase, with a maximal capacity of 12–18,000 cell ml⁻¹-. Population densities showed distinct diurnal patterns, with population growth beginning 2–4 hours in darkness. The optimal physical conditions for growth were pH 8.5,salinity of 30–35‰, temperature of 20–25°C, and photoperiod of 14//10D to 16L/8D. The cell cycle was determined by flow cytometry on synchronized batch cultures maintained at optimal pH, salinity, temperature and under 5 different photoperiod regimes. It was found that the G₁ phase was timed to end at approximately 3 h after onset of darkness, and the G₂/M phase had begun at 4 hours. Nutrient supply markedly affected population growth. Under optimal physical conditions, the optimal concentrations for macronutrients and micronutrients were: NH⁺⁻⁴- 0.025–0.2 mM,NO⁻³ 0.22–8.83 mM, glycerophosphate0.04–0.06 mM, silicate 0.1–0.54 mM; FeEDTA 0.07–0.11 mM;Co 0.1 μM, Cu 0.005–0.04 μM; Mn 0.22–7.2 μM;Mo 0.03–0.6 μM; Se 0.02–0.1 μM; Zn 0.04–1.6μM; thiamin 0.075–6 μM; vitamin B₁₂0.0004–0.004 μM; biotin 0.007–0.015 μM; EDTA5–40 μM. The toxin profile of A. catenella was determined by HPLC and found to include in descending order: GTX-4, GTX-3, GTX-1, B2, neosaxitoxin, saxitoxin. Toxin content per cell was highest in cell populations in the early exponential phase. The highest toxin per litre medium was recorded at 20°C at the beginning of the stationary phase,when cell density was highest and toxin/cell was still relatively high. At10°C, the cell density was low while the amount of toxin/cell was high;while at 30°C, the population at full capacity was low and the toxin/cell was also low. The population and toxin data thus provided an explanation for the peak level of PSP contamination in shellfish during the months of March–April around the eastern and southern side of Hong Kong and a minor peak extending to the western side in September–October, when the physical conditions of the seawater provided the right environment for toxin accumulation. Toxin content in the dinoflagellate reached its maximum during the S-phase of the cell cycle. Nitrogen restriction in the medium reduced population growth and toxin production, while phosphorus restriction reduced only population growth but enhanced toxin accumulation in the cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Metabolism of GM1 ganglioside in cultured skin fibroblasts: anomalies in gangliosidoses,sialidoses, and sphingolipid activator protein (SAP,saposin) 1 and prosaposin deficient disorders

Summary Cultured skin fibroblasts from controls and patients with lysosomal storage diseases were loaded with G_M1 ganglioside that had been labelled with tritium in its ceramide moiety. After a 65-h or 240-h incubation, a large percentage of this ganglioside remained undegraded in G_M1 gangliosidoses, whereas in the other storage diseases studied, one of its metabolites accumulated by 2–4 fold relative to controls. Labelled G_M2 ganglioside accumulated in 4 variants of G_M2 gangliosidosis, whereas labelled G_M3 ganglioside accumulated in sialidosis, galactosialidoses and sphingolipid activator protein 1 (SAP-1, saposin B) and prosaposin (saposin A, B, C an D) deficient lipidoses. The reduced degradation of G_M3 ganglioside in the SAP-1 and prosaposin deficiencies was attributed to the deficient function of SAP-1. The prosaposin deficient cells also showed a reduced re-utilization of radioactive metabolites from G_M1 ganglioside (i.e. sphingosine and fatty acid) for phospholipid biosynthesis compared with fibroblasts from the SAP-1 deficient patient or normal controls. This anomaly was ascribed to the previously shown defect in ceramide degradation in prosaposin deficiency.     

Purification of El Tor cholera enterotoxins and comparisons with classical toxin.

In 55 clinical isolates of Vibrio cholerae biotype El Tor, cholera toxin (CT) production was higher after growth in liquid medium first under relatively anaerobic conditions followed by excessive aeration (AKI conditions) as compared with growth under the optimal conditions for CT production from V. cholerae of classical biotype (median toxin level being 400 ng ml-1 and 1 ng ml-1 respectively, for the two different growth conditions). Large growth volumes further enhanced El Tor toxin production to levels at or above 3-5 micrograms ml-1 from several strains, which allowed for easy purification of toxin by salt precipitation, aluminium hydroxide adsorption and/or GM1 ganglioside affinity chromatography. However, such purified El Tor CT completely lacked the A subunit when examined by SDS-PAGE or by monoclonal anti-A subunit antibody GM1-ELISA. In contrast, when El Tor CT was prepared from bacteria grown in the presence of specific antiserum against soluble haemagglutinin/protease it contained the A subunit (unnicked) in the same proportion to the B subunit (1A:5B) as classical CT. Immunodiffusion-in-gel tests revealed that the B subunits of El Tor and classical CTs share major epitopes but also have one or more weaker biotype-specific epitopes. The two types of toxin were practically indistinguishable in various GM1-ELISA tests, and antisera raised against El Tor and classical CT, respectively, could also completely neutralize the heterologous as well as the homologous toxin activity in vivo. The results indicate that CTs from El Tor and classical V. cholerae, despite demonstrable epitope differences, are predominantly cross-reactive and give rise to antisera with strong cross-neutralizing activity.     

Synthesis and characterization of N-parinaroyl ganglioside GM1: effect of choleragen binding on fluorescence anisotropy in model membranes

N-cis-Parinaroyl ganglioside GM1 and N-trans-parinaroyl ganglioside GM1 were synthesized and characterized by HPLC, TLC, component analysis, absorbance spectroscopy, and proton NMR spectroscopy. Steady-state fluorescence anisotropy of the purified compounds, incorporated into phosphatidylcholine liposomes, was measured in the presence and absence of choleragen (cholera toxin) and choleragenoid (cholera toxin B subunit). In gel-phase liposomes, anisotropy measurements indicated that the motion of the parinaroyl ganglioside was not affected by addition of choleragen or choleragenoid. In fluid-phase liposomes, however, addition of toxin resulted in increased anisotropy (decreased rotational motion) of the fluorescent gangliosides. This decreased motion was not observed with other parinaroyl lipid probes, such as phosphatidylcholine, glucosylceramide, or free fatty acids, indicating that the effect was due to specific ganglioside/toxin interactions. Varying the amount of ganglioside or the amount of toxin suggested that the effect of toxin on probe motion was saturable at approximately 1 choleragen (or choleragenoid) molecule/5 ganglioside molecules. These results are consistent with previous hypotheses regarding the ganglioside/choleragen interaction and indicate that parinaroyl ganglioside probes will be useful in elucidation of the molecular details of this interaction.     

Secretory and GM1 receptor binding role of N-terminal region of LTB in Vibrio cholerae

Heat labile enterotoxin from enterotoxigenic Escherichia coli is similar to cholera toxin (CT) and is a leading cause of diarrhea in developing countries. It consists of an enzymatically active A subunit (LTA) and a carrier pentameric B subunit (LTB). In the current study, we evaluated the importance of the N-terminal region of LTB by mutation analysis. Deletion of the glutamine (ΔQ3) residue and a substitution mutation E7G in the α1 helix region led to defects in LTB protein secretion. Deletion of the proline residue (ΔP2) caused a decrease in α helicity. The ΔP2 mutant affected GM₁ ganglioside receptor binding activity without affecting LTB pentamer formation. Upon refolding/reassembly, the ΔP2 mutant showed defective biological activity. The single substitution mutation (E7D) strengthened the helix, imparting structural stability and thereby improved the GM₁ ganglioside receptor binding activity. Our results demonstrate the important role of N-terminal α1 helix in maintaining the structural stability and the integrity of GM₁ ganglioside receptor binding activity.