Internalization and degradation of cholera toxin by cultured cells: relationship to toxin action

Using anticholeragen antibodies and 125I-protein A, we developed a specific and quantitative assay for measuring choleragen on the surfaces of cultured cells. When neuroblastoma cells containing bound toxin were incubated at 37 degrees C, surface toxin disappeared with a half-life of approximately 2 h and a significant loss was detected by 10 min. When cells were incubated with 125I-choleragen in order to measure toxin degradation, cell-associated radioactivity disappeared with time and a corresponding amount of TCA-soluble label appeared in the culture medium with a half-life of 4-6 h. No degradation was detected until 45 min. Although there was a lag of 15 min before bound choleragen activated adenylate cyclase, the enzyme became maximally activated between 45 and 60 min. Similar results were obtained with Friend erythroleukemia cells. Internalization, degradation, and activation all were blocked when the cells were maintained at 4 degrees C. At 22 degrees C, internalization and activation occurred, albeit at a slower rate, whereas degradation was effectively inhibited. These results indicated that choleragen does not have to be degraded by intact cells in order for it to activate adenylate cyclase. Some internalization of the toxin, however, appears to precede the activation process.     

The mechanism of action of cholera toxin in pigeon erythrocyte lysates.

The adenylate cyclase activity of intact pigeon erythrocytes begins to rise after about 20 min of exposure to cholera toxin. The maximum rate at which the cyclase activity increases appears to be limited by the number of toxin molecules which can reach an intracellular target. If the erythrocytes are made permeable to the toxin by a bacterial hemolysin, no such limit exists, and adenylate cyclase activity starts to rise immediately upon the addition of toxin, and continues to rise to a maximum at an initially constant rate which is dependent upon the concentration of toxin. On lysed erythrocytes, the addition of cholera antitoxin immediately prevents any further rise in adenylate cyclase activity, but does not reverse any activation already achieved. Erythrocyte lysates may also be activated by isolated peptide A1 of cholera toxin, although activation of adenylate cyclase of intact erythrocytes requires the complete toxin molecule. In the intact cells, toxin first attaches by its Component B to surface receptors of which there are about 30 per erythrocyte. Subsequently, peptide A1 but not Component B is inserted into the erythrocyte. It takes only about 1 min at 37 degrees for peptide A1 to be sufficiently deep within the cell membrane to be inaccessible to extracellular antitoxin, but its complete transit through the membrane appears to take longer. The surface receptors are used only once, for they remain blocked by Component B. The number of receptors available on the surface may be increased by soaking cells in ganglioside GM1. Cholera toxin also decreases the rate of apparently spontaneous loss of adenylate cyclase activity and increases the response to epinephrine. Theophylline inhibits the action of cholera toxin.     

Lipoprotein lipases and stress hormones: studies with glucocorticoids and cholera toxin

The intravenous injection of cholera toxin in rats 17 h prior to experimentation results in increased levels of insulin and corticosterone in the blood. This is accompanied by a rise in lipoprotein lipase activity in muscle and a decrease in adipose tissue. Pre- and postheparin blood levels of the enzyme are increased, representing the higher overall muscle activity. Hepatic lipase is decreased by cholera toxin treatment. These enzyme changes are accompanied by increased levels of non-esterified fatty acids, ketone bodies and unesterified cholesterol in the blood, whereas triacylglycerol levels are lowered. The lipoprotein triacylglycerol secretion is not affected by cholera toxin, suggesting increased triacylglycerol removal from the blood. On the other hand the unesterified cholesterol removal may be decreased due to the decreased hepatic lipase activity. Administration of excess glucocorticoid 2 days prior to blood and tissue sampling also resulted in a rise in lipoprotein lipase, a decrease in hepatic lipase activity and an increase of non-esterified fatty acids. In contrast to the effect of cholera toxin, the triacylglycerol levels were increased. Adrenalectomy, whether by inhibition of 11-beta-steroid hydroxylase or by surgical intervention, did not abolish the choleratoxin effects. It is concluded that corticosterone increase is not essential to the cholera toxin effects. Corticosterone itself probably causes an increase of cyclic AMP and/or Ca2+ levels, as is discussed.     

Enzymic activity of cholera toxin. I. New method of assay and the mechanism of ADP-ribosyl transfer.

We tested various methods of assaying the ADP-ribosyltransferase activity of cholera toxin using artificial acceptors of the ADP-ribosyl group. Any of several proteins or poly(L-arginine) could be used with [adenine-14C]NAD+ as ADP-ribosyl donor, but this method was not ideal because of the heterogeneity of potential acceptor groups and the necessity of using costly labeled NAD+. We, therefore, developed an alternative assay using a synthetic low molecular weight acceptor, 125I-N-guanyltyramine (125I-GT). 125I-GT was specifically ADP-ribosylated by thiol-treated cholera toxin or its A1 peptide in the presence of beta-NAD. ADP-ribosyl-125I-GT was quantified after separation from unreacted 125I-GT by batch absorption of the latter to cation exchange resins. Analysis of the kinetics of ADP-ribosylation of 125I-GT indicated that the reaction proceeds by a sequential rather than a ping-pong mechanism. The Km values for NAD+ and 125I-GT were 3.6 mM and 44 microM, respectively. L-Arginine was a competitive inhibitor of 125I-GT (KI = 75 mM), but was at least 1000-fold less active than 125I-GT as an ADP-ribose acceptor.     

The primary structure of glycoprotein III from bovine adrenal medullary chromaffin granules. Sequence similarity with human serum protein-40,40 and rat Sertoli cell glycoprotein.

Glycoprotein III (GpIII) was purified from the soluble fraction of bovine chromaffin granules, the secretory vesicles of the adrenal medulla, by chromatography using wheat germ agglutinin-Sepharose followed by reverse-phase high performance liquid chromatography (HPLC). Characterization of this glycoprotein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse-phase HPLC, amino acid analysis and partial NH2-terminal sequence analysis indicated that GPIII was a disulfide-linked heterodimer with 37-kDa subunits. Analysis of in vitro translation products of adrenal medullary poly(A)+ RNA by immunoprecipitation using an anti-GpIII serum and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that both subunits are synthesized from a single precursor. Partial NH2-terminal sequence analysis allowed construction of oligonucleotides which were used as primers for a polymerase chain reaction to generate a GpIII-specific DNA probe. This probe was used to isolate a cDNA clone encoding the GpIII precursor from a bovine adrenal medullary cDNA library. The predicted amino acid sequence of GpIII has greater than 80% similarity to human serum protein-40,40, a protein implicated in the complement system, and to a major secretory product of Sertoli cells, glycoprotein 2, which is thought to play a role in spermatogenesis. Northern blot analysis confirmed that RNA encoding GpIII is also abundant in liver, testis, and brain.     

Mechanism of action of cholera toxin: Studies on the lag period

Summary The lag period for activation of adenylate cyclase by choleragen was shorter in mouse neuroblastoma N18 cells than in rat glial C6 cells. N18 cells have 500-fold more toxin receptors than C6 cells. Treatment of C6 cells with ganglioside G_M1 increased the number of toxin receptors and decreased the lag phase. Choleragen concentration also effected the lag phase, which increased as the toxin concentration and the amount of toxin bound decreased. The concentration, however, required for half-maximal activation of adenylate cyclase depended on the exposure time; at 1.5, 24, and 48 hr, the values were 200, 1.1., and 0.35pm, respectively. Under the latter conditions, each cell was exposed to 84 molecules of toxin.The length of the lag period was temperature-dependent. When exposed to choleragen at 37, 24, and 20 °C, C6 cells began to accumulate cyclic AMP after 50, 90, and 180 min, respectively. In G_M1-treated cells, the corresponding times were 35, 60, and 120 min. Cells treated with toxin at 15 °C for up to 22 hr did not accumulate cAMP, whereas above this temperature they did. Antiserum to choleragen, when added prior to choleragen, completely blocked the activation of adenylate cyclase. When added after the toxin, the antitoxin lost its inhibitory capability in a time and temperature-dependent manner. Cells, however, could be preincubated with toxin at 15 °C, and the antitoxin was completely effective when added before the cells were warmed up. Finally, cells exposed to choleragen for >10 min at 37 °C accumulated cyclic AMP when shifted to 15 °C. Under optimum conditions at 37°C, the minimum lag period for adenylate cyclase activation in these cells was 10 min. These findings suggest that the lag period for cholerage action represents a temperature-dependent transmembrane event, during which the toxin (or its active component) gains access to adenylate cyclase.Abbreviations used: ganglioside nomenclature according to Svennerholm [32] (see Table 1 for structures) cAMP adenosine 35-monophosphate - MIX 3-isobutyl-1-methylxanthine - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - PBS phosphate-buffered saline (pH 7.4)     

Gas phase characterization of the noncovalent quaternary structure of cholera toxin and the cholera toxin B subunit pentamer

Cholera toxin (CTx) is an AB5 cytotonic protein that has medical relevance in cholera and as a novel mucosal adjuvant. Here, we report an analysis of the noncovalent homopentameric complex of CTx B chain (CTx B5) using electrospray ionization triple quadrupole mass spectrometry and tandem mass spectrometry and the analysis of the noncovalent hexameric holotoxin usingelectrospray ionization time-of-flight mass spectrometry over a range of pH values that correlate with those encountered by this toxin after cellular uptake. We show that noncovalent interactions within the toxin assemblies were maintained under both acidic and neutral conditions in the gas phase. However, unlike the related Escherichia coli Shiga-like toxin B5 pentamer (SLTx B), the CTx B5 pentamer was stable at low pH, indicating that additional interactions must be present within the latter. Structural comparison of the CTx B monomer interface reveals an additional alpha-helix that is absent in the SLTx B monomer. In silico energy calculations support interactions between this helix and the adjacent monomer. These data provide insight into the apparent stabilization of CTx B relative to SLTx B.     

Evidence for a relationship between chloroquine and complement from studies with lymphocyte mitogens: possible implications for the mechanism of action of chloroquine in disease.

Chloroquine increases the inhibition of cultured lymphocytes by high concentrations of phytohemagglutinin (PHA) or concanavalin A (Con A). The inhibition is also increased by complement. Thus chloroquine and complement have similar effects. Time-course studies show that chloroquine increases the rate of onset of the complement-dependent inhibition. In serum preheated to inactivate complement, chloroquine can partially simulate the effect of complement. It is suggested that at certain stages in malaria or autoimmune disease the rate of clearance of parasitized erythrocytes or autoreactive lymphocytes is limited by the concentration of complement. Under these conditions a drug such as chloroquine, which could enhance or simulate the action of complement, might be of therapeutic value.     

An antipodean perception of the mode of action of glycoprotein hormones.

Continuance of vertebrate species and maintenance of metabolism have an absolute requirement for the glycoprotein hormones of the anterior pituitary gland. It is now firmly accepted that the N-glycans of these and the related placental hormone, chorionic gonadotropin, have essential if undefined roles in their mechanism of action. However, recent investigations by Weisshaar and his colleagues on the oligosaccharides of human luteinizing hormone and chorionic gonadotropin, do not support the generally accepted view of carbohydrates in hormone-receptor interactions and a further concept is proposed that invokes negative charges and changes in structured water.     

Sequence similarity between macrolide-resistance determinants and ATP-binding transport proteins.

The three macrolide-resistance-encoding genes, tlrC from Streptomyces fradiae, srmB from Streptomyces ambofaciens, and carA from Streptomyces thermotolerans, encode proteins that possess significant sequence similarity to ATP-dependent transport proteins. The N-terminal and C-terminal halves of these proteins are very similar to each other and contain highly conserved regions that resemble ATP-binding domains typically present within the superfamily of ATP-dependent transport proteins. These observations suggest that the mechanism by which these genes confer resistance to macrolides is due to export of the antibiotics, a process that is driven by energy derived from ATP hydrolysis.