The role of zinc binding in the biological activity of botulinum toxin.

Botulinum toxin is a zinc-dependent endoprotease that acts on vulnerable cells to cleave polypeptides that are essential for exocytosis. To exert this poisoning effect, the toxin must proceed through a complex sequence of events that involves binding, productive internalization, and intracellular expression of catalytic activity. Results presented in this study show that soluble chelators rapidly strip Zn(2+) from its binding site in botulinum toxin, and this stripping of cation results in the loss of catalytic activity in cell-free or broken cell preparations. Stripped toxin is still active against intact neuromuscular junctions, presumably because internalized toxin binds cytosolic Zn(2+). In contrast to soluble chelators, immobilized chelators have no effect on bound Zn(2+), nor do they alter toxin activity. The latter finding is because of the fact that the spontaneous loss of Zn(2+) from its coordination site in botulinum toxin is relatively slow. When exogenous Zn(2+) is added to toxin that has been stripped by soluble chelators, the molecule rebinds cation and regains catalytic and neuromuscular blocking activity. Exogenous Zn(2+) can restore toxin activity either when the toxin is free in solution on the cell exterior or when it has been internalized and is in the cytosol. The fact that stripped toxin can reach the cytosol means that the loss of bound Zn(2+) does not produce conformational changes that block internalization. Similarly, the fact that stripped toxin in the cytosol can be reactivated by ambient Zn(2+) or exogenous Zn(2+) means that productive internalization does not produce conformational changes that block rebinding of cation.

Pure botulinum neurotoxin is absorbed from the stomach and small intestine and produces peripheral neuromuscular blockade.

Clostridium botulinum serotype A produces a neurotoxin composed of a 100-kDa heavy chain and a 50-kDa light chain linked by a disulfide bond. This neurotoxin is part of a ca. 900-kDa complex, formed by noncovalent association with a single nontoxin, nonhemagglutinin subunit and a family of hemagglutinating proteins. Previous work has suggested, although never conclusively demonstrated, that neurotoxin alone cannot survive passage through the stomach and/or cannot be absorbed from the gut without the involvement of auxiliary proteins in the complex. Therefore, this study compared the relative absorption and toxicity of three preparations of neurotoxin in an in vivo mouse model. Equimolar amounts of serotype A complex with hemagglutinins, complex without hemagglutinins, and purified neurotoxin were surgically introduced into the stomach or into the small intestine. In some experiments, movement of neurotoxin from the site of administration was restricted by ligation of the pylorus. Comparison of relative toxicities demonstrated that at adequate doses, complex with hemagglutinins, complex without hemagglutinins, and pure neurotoxin can be absorbed from the stomach. The potency of neurotoxin in complex was greater than that of pure neurotoxin, but the magnitude of this difference diminished as the dosage of neurotoxin increased. Qualitatively similar results were obtained when complex with hemagglutinins, complex without hemagglutinins, and pure neurotoxin were placed directly into the intestine. This work establishes that pure botulinum neurotoxin serotype A is toxic when administered orally. This means that pure neurotoxin does not require hemagglutinins or other auxiliary proteins for absorption from the gastrointestinal system into the general circulation.

Characterization of a vertebrate neuromuscular junction that demonstrates selective resistance to botulinum toxin.

Botulinum toxin blocks transmitter release by proceeding through a series of four steps: binding to cell surface receptors, penetration of the cell membrane by receptor-mediated endocytosis, penetration of the endosome membrane by pH-induced translocation, and intracellular proteolysis of substrates that govern exocytosis. Each of these steps is essential for toxin action on intact cells. Therefore, alterations in cell structure or cell function that impede any of these steps should confer resistance to toxin. In the present study, screening for susceptibility to four serotypes of botulinum toxin revealed that the cutaneous-pectoris nerve-muscle preparation of Rana pipiens is resistant to type B botulinum toxin. Resistance was demonstrated both by electrophysiologic techniques and by dye-staining techniques. In addition, resistance to serotype B was demonstrated at toxin concentrations that were 2 orders of magnitude higher than those associated with blockade produced by other serotypes. In experiments on broken cell preparations, type B toxin cleaved synaptobrevin from frog brain synaptosomes. However, the toxin did not bind to frog nerve membranes. These findings suggest that resistance is due to an absence of cell surface receptors for botulinum toxin type B. The fact that cutaneous-pectoris preparations were sensitive to other botulinum toxin serotypes (A, C, and D), as well as other neuromuscular blocking agents (alpha-latrotoxin, beta-bungarotoxin), indicates that botulinum toxin type B receptors are distinct.

Botulinum toxin as a carrier for oral vaccines.

Botulinum toxin is an unusually potent substance that acts on the nervous system to produce the clinical outcome of flaccid paralysis. To produce this effect, the toxin ordinarily proceeds through two separate but essential sequences of events. During the first, the toxin is ingested, it traverses a portion of the gastrointestinal system and then it is transcytosed from the lumen of the gut to the general circulation. During the second, circulating toxin binds to peripheral cholinergic nerve endings, it is endocytosed and then it acts as a metalloendoprotease to cleave polypeptides that are essential for exocytosis. Although botulinum toxin is antigenic, it ordinarily does not evoke an immune response during or after cases of oral poisoning. This is due to the fact that the dose of toxin that produces flaccid paralysis-and potentially death-is less than the dose needed to evoke an antibody response. In the recent past, the techniques of molecular biology have been used to generate an expression product of botulinum toxin that retains the ability to escape the gut and reach the general circulation, retains the ability to evoke an immune response, but has lost the ability to produce neurotoxicity. This modified toxin may have two clinical applications. The expression product itself may have utility as an oral vaccine against botulism. Beyond this, the modified toxin, or a truncation mutant of the toxin, may have utility as a carrier in the construction of other oral vaccines. Both potential applications could lead to the expression of oral vaccines in common foods.

Binding and transcytosis of botulinum neurotoxin by polarized human colon carcinoma cells.

T-84 and Caco-2 human colon carcinoma cells and Madin-Darby canine kidney (MDCK) cells were used to study binding and transcytosis of iodinated Clostridium botulinum neurotoxin serotypes A, B, and C, as well as tetanus toxin. Specific binding and transcytosis were demonstrated for serotypes A and B in intestinal cells. Using serotype A as an example, the rate of transcytosis by T-84 cells was determined in both apical to basolateral (11.34 fmol/h/cm2) as well as basolateral to apical (8.98 fmol/h/cm2) directions, and by Caco-2 cells in the apical to basolateral (8.42 fmol/h/cm2) direction. Serotype A retained intact di-chain structure during transit through T-84 or Caco-2 cells, and when released on the basolateral side was toxic in vivo to mice and in vitro on mouse phrenic nerve-hemidiaphragm preparations. Serotype C and tetanus toxin did not bind effectively to T-84 cells, nor were they efficiently transcytosed (8-10% of serotype A). MDCK cells did not bind or efficiently transcytose (0.32 fmol/h/cm2) botulinum toxin. Further characterization demonstrated that the rate of transcytosis for serotype A in T-84 cells was increased 66% when vesicle sorting was disrupted by 5 microM brefeldin A, decreased 42% when microtubules were disrupted by 10 microM nocodazole, and decreased 74% at 18 degreesC. Drugs that antagonize toxin action at the nerve terminal, such as bafilomycin A1 (which prevents acidification of endosomes) and methylamine HCl (which neutralizes acidification of endosomes), produced only a modest inhibitory effect on the rate of transcytosis (17-22%). These results may provide an explanation for the mechanism by which botulinum toxin escapes the human gastrointestinal tract, and they may also explain why specific serotypes cause human disease and others do not.

Induction of an immune response by oral administration of recombinant botulinum toxin.

A gene encoding the full-size botulinum neurotoxin serotype C was reconstructed in vector pQE-30 and expressed at high levels in Escherichia coli. Three amino acid mutations (H229-->G, E230-->T, and H233-->N) were generated in the zinc-binding motif, resulting in complete detoxification of the modified recombinant holotoxin. The PCR-amplified wild-type light chain of botulinum neurotoxin serotype C was also expressed in E. coli and used as a control in all experiments. Modified recombinant holotoxin and light chain contained a histidine affinity tag at the amino terminus, which was used for detection and purification. Recombinant proteins were purified on nickel affinity resin and analyzed by Western blotting with the anti-histidine tag and anti-neurotoxin C antibodies. The results indicated that the 150-kDa molecule of modified recombinant holotoxin and the 50-kDa recombinant light chain were synthesized without degradation; however, E. coli did not provide for efficient nicking of modified recombinant toxin. Modified recombinant holotoxin was not toxic to mice, had no effect on nerve-evoked muscle twitch in vitro, and was not able to cleave syntaxin in crude synaptosome preparations. The recombinant light chain was also nontoxic in vivo, had no effect on evoked muscle twitch, but was able to cleave syntaxin. Modified recombinant neurotoxin and light chain were administered to animals either orally or subcutaneously. Both oral administration and subcutaneous administration of modified recombinant neurotoxin evoked high levels of serum antibodies and protective immunity. Oral administration of recombinant light chain evoked no systemic response, whereas subcutaneous administration evoked antibody production and immunity.

Cloning and sequence analysis of a cDNA encoding human syntaxin 1A, a polypeptide essential for exocytosis.

A clone encoding human syntaxin 1A was isolated and sequenced from a human brain library. The deduced sequence encodes a 288-amino-acid (aa) protein that presumably plays a critical role in neurotransmitter exocytosis and is 98.0% identical to the aa sequence of rat syntaxin 1A.

The role of transglutaminase in the mechanism of action of tetanus toxin.

Tetanus toxin is a zinc-dependent metalloendoprotease that cleaves synaptobrevin, a polypeptide found in the membranes of synaptic vesicles. This action is thought to account for toxin-induced blockade of transmitter release. However, Facchiano and Luini (Fachiano, F., and Luini, A. (1992) J. Biol Chem. 267, 13267-13271) have proposed that tetanus toxin can stimulate transglutaminase, and Facchiano et al. (Facchiano, F., Benfenati, F., Valtorta, F., and Luini, A. (1993) J. Biol Chem. 268, 4588-4591) have further proposed that the stimulated enzyme produces cross-linking of synapsin. These actions might also account for toxin-induced blockade of exocytosis. Therefore, a series of experiments were performed to evaluate the possibility that tetanus toxin exerts its effects via transglutaminase. The results indicated that clostridial neurotoxins were poor substrates for the cross-linking effects of transglutaminase, and transglutaminase was a poor substrate for the proteolytic actions of tetanus toxin. In addition, at concentrations relevant to blockade of exocytosis, clostridial neurotoxins did not act on intact cells to stimulate transglutaminase, nor did they act on the isolated enzyme to stimulate cross-linking of putrescine and dimethylcasein. When used as competitive inhibitors of endogenous transglutaminase substrates, glycine methyl ester and monodansylcadaverine did not block toxin action. Furthermore, concentrations of calcium that were too low to support transglutaminase activity did not prevent toxin action. The data suggest that stimulation of transglutaminase is not the principal mechanism by which tetanus toxin blocks exocytosis in nerve cells.

Assembly of glucocorticoid receptor and c-JUN homodimer on the promoter of mouse mammary tumor virus-long terminal repeat is influenced by order of addition.

Interactions between the glucocorticoid receptor (GR) and c-Jun/c-Jun homodimer (JUN) on the promoter DNA of mouse mammary tumor virus-long terminal repeat (MMTV-LTR) are reported here using the electrophoretic mobility shift assay (EMSA). Both GR and JUN are capable of independently binding to their respective response elements, including glucocorticoid response element (GRE) and phorbol ester response element (TRE), on MMTV-LTR promoter. The protein-DNA complex, assembled by pre-incubating JUN and DNA before the addition of GR, migrates slower (supershift) on gel electrophoresis than do the complexes formed by the other orders of addition. The formation of the supershifted complex is GR and JUN dose-dependent. The supershift is not detected with the cleaved fragments of MMTV-LTR promoter that separate GRE from TRE, indicating that the integrity of the promoter and possibly the spacing between GRE and TRE are important. The interaction of GR and JUN on the MMTV-LTR promoter appears to be more complex than simple protein-protein interaction.

Efficacy of pyridoxal treatment in controlling the growth of melanomas in cell culture and an animal pilot study.

We have demonstrated, using confocal laser scanning microscopy, that pyridoxal treatment of B16C3 murine melanoma cells inhibits triamcinolone acetonide induced translocation of the glucocorticoid receptor to the nucleus of intact cells. In addition to inhibiting glucocorticoid receptor nuclear translocation, pyridoxal kills B16C3 murine melanoma cells and WM983A human melanoma cells in culture. Cortexolone, a glucocorticoid antagonist, also kills cells in culture. This mechanism, however, appears to initiate in the glucocorticoid receptor signal transducing cascade at a point prior to the impact of pyridoxal treatment alone. The glucocorticoid antagonist RU486 has no detrimental effect on melanoma cell viability, however, in combination with pyridoxal, RU486 extends cell viability. Since pyridoxal kills melanoma cells in culture, a pilot study was carried out examining the efficacy of topical application of a pyridoxal cream to inhibit the growth and/or cause regression of (B16C3) xenograft melanoma tumors in an immunocompetent (Hairless Rhino-J3) and an immunocompromised (Crl: nu/nu (CD1)BR) murine animal model. The results of the study with immunocompetent animals are encouraging. While tumors are brought under control by pyridoxal treatment, further work is needed to determine the most efficacious treatment regimen and to establish formal concentrations for pyridoxal in topical ointments. Trials using immunocompromised animals indicated that although some qualitative differences may be detected between the control and experimental animals, tumor growth in these animals is so aggressive that multiple applications or higher concentrations of pyridoxal may be needed to obtain useful data.

A novel, highly conserved structural motif is present in all members of the steroid receptor superfamily.

Steroid and thyroid hormone receptor superfamily members are ligand potentiated transcription factors. Recent evidence indicates that one aspect of steroid receptor action is an interaction with other trans-acting factors, such as the glucocorticoid receptor with the AP1 transcription factor, for example. Using a structural approach to identify domains of the glucocorticoid receptor responsible for interactions with affiliated transacting factors and DNA, we have identified a putative helix-turn-zipper motif that is conserved in all steroid, thyroid hormone, retinoic acid, and vitamin-D3 receptors. This structural motif is also conserved among new members of the family, the peroxisome proliferator-activated receptors and the retinoid-X receptors. This structural domain is characterized by a pair of amino acids (I,L,V)P that is conserved in all superfamily members. Additional characteristics include six heptad repeats of hydrophobic amino acids, four of which form a canonical leucine zipper in the rat glucocorticoid receptor. Although this leucine repeat is not absolutely conserved among superfamily members, the periodicity of hydrophobic residues is conserved throughout. Based on sequence analyses from the GenEMBL and SwissProt databases using the Genetics Computer Group and MacVector sequence analysis software packages, and the Brookhaven structural database, we present evidence for a novel structural domain, a helix-turn-zipper that is conserved in all superfamily members, and may function in transactivation of cognate genes.

Characterization of in vitro translated human mineralocorticoid receptor. Structure and activation.

The structures of the unactivated and activated mineralocorticoid receptors have been difficult to characterize because of receptor lability and steroid dissociation. Therefore, human mineralocorticoid receptor mRNA was translated in rabbit reticulocyte lysate in the presence and absence of [35S]methionine to compare the structure of [3H]aldosterone-bound and [35S]labeled receptor. In vitro synthesized receptor was able to specifically bind [3H]aldosterone. Unactivated [3H]aldosterone-bound and 60% of unactivated [35S]labeled receptor eluted from DEAE-cellulose with 250 mM salt and had a Rs of 72A. Forty percent of unactivated [35S]labeled receptor eluted from DEAE-cellulose with 100 mM salt and had a Rs of 54A. SDSPAGE showed intact hMR was present in both DEAE-cellulose eluates as three bands between M(r) 110,000-120,000. However, the low salt eluate contained less intact receptor and more lower MW bands. Neither [3H]aldosterone-bound nor [35S]labeled receptor was activated by incubation at 25 degrees C as readily as glucocorticoid receptor studied under identical conditions. Activated [3H]aldosterone-bound receptor eluted from DEAE-cellulose at 100 mM salt and had a Rs of 37A. After activation, 60% of [35S]labeled receptor eluted from DEAE-cellulose with 100 mM salt and had a Rs of 91A. SDS-PAGE of the high and low salt DEAE-cellulose eluates showed that 50% of intact receptor eluted in the low salt peak after activation. These data indicate that: 1. Some in vitro synthesized mineralocorticoid receptor assembles into the heteromeric unactivated form; 2. The remaining intact receptor remains monomeric and unable to bind steroid; 3. Activation causes dissociation of intact receptor from a larger complex; and 4. Activated receptor tends to aggregate.

Overexpression and characterization of the human mineralocorticoid receptor.

The full-length human renal mineralocorticoid receptor (hMR) has been overproduced in Spodoptera frugiperda (Sf9) insect cells using baculovirus-mediated expression. The overproduced hMR binds aldosterone with high affinity (Kd = 1.36 nM) and has high affinity for cortisol, cortexolone, and progesterone. Immunoprecipitation and immunoblot analysis of the recombinant hMR with MR-specific antibodies reveal three major protein bands with molecular masses of 115, 119, and 125 kDa. hMR isoforms show maximal accumulation at 48 h post-infection with the recombinant baculovirus. Maximal aldosterone binding was detected at 24 h rather than at 48 h post-infection, suggesting that the assembly of hMR monomers into the nonactivated steroid-binding receptor complexes and/or their stability deteriorates after 24 h post-infection. It is estimated by specific aldosterone binding that 1.2 x 10(6) hMR molecules are expressed per Sf9 cell (equivalent to 7 pmol/mg of cytosolic protein) at 24 h post-infection. 5-Fold more receptor molecules/cell are expressed but not detected by steroid binding at 48 h post-infection as determined by immunoblot analysis. Using the MR-specific H10E anti-idiotypic monoclonal antibody, immunoprecipitation of cytosol from recombinant baculovirus-infected Sf9 cells pulse-labeled with 32Pi demonstrated for the first time that the recombinant hMR is highly phosphorylated. The hMR is expressed as 9-10 S oligomeric complexes (Stokes radii approximately 67-85 A) that are slightly heavier than the unactivated glucocorticoid receptor and can be converted to smaller 4 S receptor monomers (Stokes radii approximately 25-55 A) by elevated temperature, pH, and ionic strength. Unlike the glucocorticoid receptor, the oligomeric hMR complex can bind DNA-cellulose without prior activation. Finally, indirect immunofluorescence demonstrated that the hMR is expressed primarily as a cytoplasmic protein that can be induced to translocate to the nucleus upon treatment with hormone.

Characterization and purification of a functional rat glucocorticoid receptor overexpressed in a baculovirus system.

The structure-function relationship of the oligomeric unactivated glucocorticoid receptor is not fully understood. An essential step in the process of understanding such a relationship involves the production of large quantities of the receptor. Using a baculovirus expression system we have been able to overproduce a recombinant rat glucocorticoid receptor (rGR). A cDNA coding for the entire rGR was introduced into the genome of the wild type baculovirus, Autographa californica nuclear polyhedrosis virus, by an in vivo recombination event. Based on specific steroid binding, insect cells infected with the recombinant baculovirus expressed 1-3 x 10(6) receptor molecules/cell which is 15-45 times more than that expressed normally in a hepatocyte. The recombinant rGR expressed in insect cells is indistinguishable from the bona fide rGR with respect to immunogenic reactivity, cytoplasmic localization, sedimentation, chromatographic and electrophoretic mobility, and hormone and DNA binding. Furthermore, the recombinant rGR is expressed as a functional protein as demonstrated by its ability to specifically bind a glucocorticoid agonist, to translocate from the cytoplasm to the nucleus upon hormone-binding, and to act as a transcriptional enhancer. Pulse labeling of recombinant baculovirus-infected insect cells with 32Pi and isolation of the labeled products by immunoprecipitation demonstrated that the recombinant rGR is a phosphoprotein. Thus, the recombinant rGR expressed in insect cells is biologically active and is suitable for structural and functional analysis. A simple three-step purification procedure of the unactivated recombinant rGR is described.

Cell-free synthesis of rat glucocorticoid receptor in rabbit reticulocyte lysate. In vitro synthesis of receptor in Mr 90,000 heat shock protein-depleted lysate.

The glucocorticoid receptor is present in the cytosol of cell extracts as a large nonactivated (i.e. non-DNA-binding) approximately 9 S (Mr 300,000) complex. Experimental evidence indicates that the purified nonactivated glucocorticoid receptor contains a single steroid-binding protein and two approximately 90-kDa nonsteroid-binding subunits identified as heat shock protein (hsp) 90. Translation of the glucocorticoid receptor mRNA in vitro in reticulocyte lysates produces a large nonactivated glucocorticoid receptor complex similar to that found in cytosols. The cell-free synthesized glucocorticoid receptor is able to bind steroid and can be activated further to the DNA-binding form. To test the hypothesis of an active role played by hsp90 in the stabilization of a competent steroid-binding conformation of the glucocorticoid receptor, we have synthesized the receptor in a reticulocyte lysate that has been depleted of hsp90 by immunoadsorption with AC88 anti-hsp90. Although the translation capacity of the reticulocyte system was reduced considerably upon hsp90 removal, the glucocorticoid receptor was synthesized, and a significant number of molecules were found to bind [3H]triamcinolone acetonide. Chromatography on DEAE-cellulose showed that most of the receptor molecules synthesized in hsp90-depleted lysate had lost the capacity to form an oligomeric receptor complex. Addition of purified rat liver hsp90 to the hsp90-depleted lysate before translation did not increase steroid binding nor did it restore formation of the heteromeric receptor complex. Analysis of [35S] methionine-labeled glucocorticoid receptor molecules synthesized in the hsp90-depleted lysate showed the production of polypeptides differing from the expected chromatographic pattern on DEAE-cellulose. Upon addition of purified hsp90 to the hsp90-depleted lysate, before translation, the 35S-labeled synthesized receptor fractionated on DEAE-cellulose as an intermediate peak between activated and nonactivated receptor forms. The data suggest that hsp90 alone may not be sufficient for the formation of the nonactivated steroid receptor complex.