Preparation of affinity-purified,biotinylated tetanus toxin,and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 11 to 201 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60–80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using¹²⁵I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2–3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these primed cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.Abbreviations PBS phosphate-buffered saline - STS sucrose-Tris-serum solution - NGF nerve growth factor - C collagen - PL polylysine - BBG bovine brain ganglioside mixture - GM₁ gafactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1a [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1a [N-aceylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1b galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1b [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl] galactosylglucosyl ceramide - NANA N-acetylneuraminic acid     

The development of oral vaccines based on live attenuated Salmonella strains

Abstract Safe, live attenuated Salmonella strains can be produced by introducing defined non-reverting mutations into the chromosome. Such rationally attenuated strains have proved to be excellent oral vaccines in several animal species and can therefore be considered as candidate vaccines against invasive salmonellosis in both animals and man. A panel of attenuating lesions is now available from which it is possible to tailor the level of attenuation and hence produce strains with different immunogenic properties. Because of the spectrum of immune responses produced by such Salmonella vaccine strains they have been utilised extensively as vectors for delivering heterologous antigens to the mammalian immune system. We have focussed on the development of a single dose oral tetanus vaccine based on attenuated Salmonella strains expressing a non-toxic, immunogenic protein derived from tetanus toxin (fragment C). Several different expression systems have been used for fragment C and candidate vaccine strains have been constructed that are capable of protecting orally immunised mice against a lethal challenge with tetanus toxin. An oral tetanus vaccine may help to reduce the mortality rate from tetanus in the developing world by overcoming the problems associated with the implementation of vaccine programmes using the current parenteral vaccine.     

The detection of picomolar quantities of GD1b,GT1b and GQ1b on thin-layer chromatograms by the direct binding of125I-labeled tetanus toxin,fragment C

The¹²⁵I-labeled fragment C of tetanus toxin was found to bind specifically to the gangliosides G_D1b, G_T1b, and G_Q1b when applied to thin-layer chromatograms on which a mixture of gangliosides had been resolved. As little as 2.5 pmoles of these gangliosides could be detected by this method. In addition to factors determined by the sample, namely the amount and species of gangliosides present, optimal binding of the¹²⁵I-labeled fragment C also depended upon the iodination procedure used to generate the probe, the toxin concentration, and the concentration, buffer type, pH, and ionic strength of the binding solution. This new technique was shown to be a sensitive method for the detection and identification of specific gangliosides originating from extraneural or neural cells.Nomenclature: The gangliosides follow the nomenclature system of Svennerholm [Eur J Biochem (1977) 79:11–21] G_M3 II³NeuAc-LacCer - G_D3 II³(NeuAc)₂-LacCer - G_M1 II³NeuAc-GgOse₄Cer - G_D1a IV³NeuAc, II³NeuAc-GgOse₄Cer - G_D1b II³(NeuAc)₂-GgOse₄Cer - G_T1b IV³NeuAc, II³(NeuAc)²-GgOse₄Cer - G_Q1b IV³(Neu-Ac)₂, II³(NeuAc)₂-GgOse₄Cer - G_P1b IV³(NeuAc)₃, II³(NeuAc)₂-GgOse₄Cer     

Molecular dynamics simulations of alpha2 --> 8-linked disialoside: conformational analysis and implications for binding to proteins.

Computational methods have played a key role in elucidating the various three-dimensional structures of oligosaccharides. Such structural information, together with other experimental data, leads to a better understanding of the role of oligosaccharide in various biological processes. The disialoside Neu5Ac-alpha2-->8-Neu5Ac appears as the terminal glycan in glycoproteins and glycolipids, and is known to play an important role in various events of cellular communication. Neurotoxins such as botulinum and tetanus require Neu5Ac-alpha2 --> 8-Neu5Ac for infecting the host. Glycoconjugates containing this disialoside and the enzymes catalyzing their biosynthesis are also regulated during cell growth, development, and differentiation. Unlike other biologically relevant disaccharides that have only two linkage bonds, the alpha2-->8-linked disialoside has four: C2-O, O-C8', C8'-C7', and C7'-C6'. The present report describes the results from nine 1 ns MD simulations of alpha2-->8-linked disialoside (Neu5Ac-alpha2-->8-Neu5Ac); simulations were run using GROMOS96 by explicitly considering the solvent molecules. Conformations around the O-C8' bond are restricted to the +sc/+ap regions due to stereochemical reasons. In contrast, conformations around the C2-O and C8'-C7' bonds were found to be largely unrestricted and all the three staggered regions are accessible. The conformations around the C7'-C6' bond were found to be in either the -sc or the anti region. These results are in excellent agreement with the available NMR and potential energy calculation studies. Overall, the disaccharide is flexible and adopts mainly two ensembles of conformations differing in the conformation around the C7'-C6' bond. The flexibility associated with this disaccharide allows for better optimization of intermolecular contacts while binding to proteins and this may partially compensate for the loss of conformational entropy that may be incurred due to disaccharide's flexibility.     

Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope “tags”

Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system's ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.     

Induction of antibody responses in mice immunized intranasally with Type I interferon as adjuvant and synergistic effect of chitosan

Type I IFNs are a range of host-derived molecules with adjuvant potential; they have been used for many years in the treatment of cancer and viral hepatitis. Therefore, the safety of IFNs for human use has been established. In this study, we evaluated the mucosal adjuvanticity of IFN-β administered intranasally to mice with diphtheria toxoid, and suggested a method to improve its adjuvanticity. When IFN-β alone was used as a mucosal adjuvant, no clear results were obtained. However, simultaneous administration of IFN-β and chitosan resulted in an enhancement of the specific serum immunoglobulin G (IgG) and IgA antibody responses, the mucosal IgA antibody response, and antitoxin titers. Furthermore, the intranasal administration of IFN-α alone resulted in a greater increase in antibody titer than IFN-β, and a synergistic effect with chitosan was also observed. These findings suggest that intranasal administration of chitosan and Type I IFNs may display an effective synergistic mucosal adjuvant activity.     

Endobrevin/VAMP8 mediates exocytotic release of hexosaminidase from rat basophilic leukaemia cells

Mast cells are important players in innate immunity and mediate allergic responses. Upon stimulation, they release biologically active mediators including histamine, cytokines and lysosomal hydrolases. We used permeabilized rat basophilic leukaemia cells as model to identify R-SNAREs (soluble NSF (N-ethylmaleimide-sensitive fusion protein)) mediating exocytosis of hexosaminidase from mast cells. Of a complete set of recombinant mammalian R-SNAREs, only vesicle associated membrane protein (VAMP8)/endobrevin consistently blocked hexosaminidase release, which was also insensitive to treatment with clostridial neurotoxins. Thus, VAMP8, which also mediates fusion of late endosomes and lysosomes, plays a major role in hexosaminidase release, strengthening the view that mast cell granules share properties of both secretory granules and lysosomes.     

Lactobacilli as live vaccine delivery vectors: progress and prospects

Evidence is accumulating that lactobacilli influence the immune response in a strain-dependent manner. This immunomodulatory capacity is important for the development of the immune response, and also identifies Lactobacillus as a potent oral vaccine carrier. Most of our current knowledge of the use of lactobacilli for vaccination purposes has been obtained with tetanus toxin fragment C (TTFC) as the model antigen. This knowledge, together with our ever-increasing understanding of the immune system and recent developments in cloning and expression techniques, should enable the utilisation of antigens other than TTFC and has made the development of lactobacilli as live vaccines a realistic prospect.     

The HC fragment of tetanus toxin forms stable, concentration-dependent dimers via an intermolecular disulphide bond

Protein oligomerisation is a prerequisite for the toxicity of a number of bacterial toxins. Examples include the pore-forming cytotoxin streptolysin O, which oligomerises to form large pores in the membrane and the protective antigen of anthrax toxin, where a heptameric complex is essential for the delivery of lethal factor and edema factor to the cell cytosol. Binding of the clostridial neurotoxins to receptors on neuronal cells is well characterised, but little is known regarding the quaternary structure of these toxins and the role of oligomerisation in the intoxication process. We have investigated the oligomerisation of the receptor binding domain (H(C)) of tetanus toxin, which retains the binding and trafficking properties of the full-length toxin. Electrophoresis, size exclusion chromatography and mass spectrometry were used to demonstrate that H(C) undergoes concentration-dependent oligomerisation in solution. Reducing agents were found to affect H(C) oligomerisation and, using mutagenesis, Cys869 was shown to be essential for this process. Furthermore, the oligomeric state and quaternary structure of H(C) in solution was assessed using synchrotron small-angle X-ray scattering. Ab initio shape analysis and rigid body modelling coupled with mutagenesis data allowed the construction of an unequivocal model of dimeric H(C) in solution. We propose a possible mechanism for H(C) oligomerisation and discuss how this may relate to toxicity.