Differential contribution of the residues in C-terminal half of the heavy chain of botulinum neurotoxin type B to its binding to the ganglioside GT1b and the synaptotagmin 2/GT1b complex

Clostridium botulinum type B neurotoxin was effectively bound to synaptotagmin 2 (Stg2) associated with ganglioside GT1b, however, the molecular interaction between the neurotoxin and the Stg2/GT1b complex has not been identified. Previously, we found that infant botulism-related strain 111 generated a low activity of the neurotoxin (111/NT), which differed in some amino acid residues, especially in the carboxyl terminal half of the heavy chain (H(C)), from the original neurotoxin of strain Okra (Okra/NT) associated with a food-borne botulism. In this study, we evaluated the binding capabilities of site-directed mutants of Okra/H(C) to the Stg2/GT1b complex and to GT1b alone, and investigated the relationship between the toxic action and receptor binding. Replacement of K1187 and E1190 with glutamic acid and lysine, respectively, which substituted for the 111/NT residues, caused a reduction of binding affinity to the Stg2/GT1b complex, suggesting that both these residues contribute to the different binding affinity between Okra/NT and 111/NT. Substitution of four residues, H1240, S1259, W1261 and Y1262, which form a ganglioside pocket, drastically decreased the binding of H(C) to the Stg2/GT1b complex and to GT1b. Mutation in the residues, K1186, E1189, K1191 and K1260 reduced the binding of H(C) to GT1b alone, but not to the Stg2/GT1b complex. Analyses of effects of mutant toxins on toxicity of BoNT/B to cerebellar granule cells suggest the association of cell toxicity with binding to Stg2/GT1b complex but not that to GT1b alone.     

Endothelin receptor binding and cellular signal transduction in neurohybrid NG108-15 cells.

Endothelins are a novel group of potent vasoconstrictor peptides originally isolated from cultured porcine endothelial cells. We and others have previously reported the presence of endothelin receptors in the central nervous system, and this study was designed to further characterize endothelin receptors and their transduction mechanism in cultured neurohybrid NG108-15 cells. Specific binding of [125I]endothelin-1 to NG108-15 cells reached saturation within 60 min at 22 degrees C and was only partially reversible. Scatchard analysis of the saturation binding revealed the presence of one class of high-affinity binding sites with an apparent dissociation constant of 160 pM and a maximal binding capacity of 3.3 x 10(4) sites/cell. Unlabeled endothelin analogues competitively inhibited [125I]endothelin-1 binding to NG108-15 cells and the apparent dissociation constant values obtained from the competition curves correlated well with the EC50 values obtained for inducing elevation of intracellular free Ca2+ level. Endothelin stimulated phosphoinositide metabolism in a dose-dependent manner with an EC50 value of 5.4 nM for inositol trisphosphate formation. The protein kinase C-activator phorbol ester dose-dependently inhibited endothelin-induced phosphoinositide turnover and intracellular free Ca2+ increase, suggesting the involvement of protein kinase C in the regulation of endothelin-induced responses. Neither endothelin-induced phosphoinositide hydrolysis nor endothelin-induced increase in intracellular free Ca2+ were affected by pertussis toxin. These data indicate that endothelin receptors are present on NG108-15 cells and the G protein coupled to endothelin receptor for inducing activation of phospholipase C and increase of free intracellular Ca2+ is insensitive to pertussis toxin.     

Enkephalin induces Ca2+ mobilization in single cells of bradykinin-sensitized differentiated neuroblastoma hybridoma (NG108-15) cells.

A study of the intracellular Ca2+ ([Ca2+]i) response of differentiated neuroblastoma x glioma hybrid cells (NG108-15 cell) to enkephalin (EK) was carried out by fura-2 video-imaging. EK alone did not influence [Ca2+]i in single cells. The opioid did, however, induce a marked [Ca2+]i rise, when the cells were incubated with bradykinin (BK) prior to the EK treatment. Such BK-assisted stimulation of the differentiated hybridoma cells by EK was completely abolished by pertussis toxin treatment. These results suggest that in single NG108-15 cells, EK induces Ca2+ mobilization which is assisted by cross-talk between the EK and BK receptor systems via a pertussis toxin-sensitive G protein.     

Ganglioside GT1b as a complementary receptor component forClostridium botulinumneurotoxins

Clostridium botulinumtype B neurotoxin (BoNT/B) recognizes a complex of synaptotagmin II and ganglioside GT1b or GD1a as the high-affinity toxin binding site. Recombinant deletion mutants of synaptotagmin II allowed us to demonstrate that the N-terminal domain including the transmembrane region retains BoNT/B binding activity while the C-terminal domain is not involved in constituting the BoNT/B receptor. BoNT/B binding to reconstituted lipid vesicles containing synaptotagmin II and gangliosides showed that GT1b and GD1a confer the difference in the maximum binding capacity but not in the dissociation constant. The direct binding of GT1b to the deletion mutants revealed that the transmembrane region is required to bind GT1b, suggesting that synaptotagmin II binds to the ceramide portion of gangliosides within the plasma membrane. A monoclonal antibody against GT1b effectively inhibited not only BoNT/B binding to the reconstituted lipid vesicles and brain synaptosomes but also type A BoNT (BoNT/A) binding to brain synaptosomes. In addition, the monoclonal antibody antagonized the action of both BoNT/A and BoNT/B on synaptic transmission of rat superior cervical ganglion neurons. These results suggest that GT1b functions as a component of the receptor complex.     

Dissection of bradykinin-evoked responses by buffering intracellular Ca2+ in neuroblastoma x glioma hybrid NG108-15 cells.

Signal transduction pathways from bradykinin (BK) receptors were investigated in NG108-15 neuroblastoma x glioma hybrid cells by buffering the intracellular calcium (Ca2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a Ca2+ chelator. BK increased inositol-1,4,5-trisphosphate (Ins(1, 4,5)P3) formation at the same rate in the control and in BAPTA-acetoxy methyl ester (AM)-treated NG108-15 cells. However, a transient increase of intracellular Ca2+ concentrations in response to BK was significantly suppressed in Ca(2+)-buffered hybrid cells. Accordingly the BK-induced outward current was inhibited in BAPTA-AM-treated hybrid cells, while the subsequent inward current associated with a fall in membrane conductance was apparently increased. The initial phase of acetylcholine release from NG108-15 cells in response to BK was markedly inhibited in BAPTA-AM-treated coculture dishes when detected as miniature end-plate potentials of myotubes, though the late phase of acetylcholine secretion was observed. These results indicate that BK induces two distinct responses in NG108-15 cells: Ins(1,4,5)P3-dependent intracellular Ca2+ rise-sensitive and -insensitive components.     

The C-Terminal Heavy-Chain Domain of Botulinum Neurotoxin A Is Not the Only Site That Binds Neurons,as the N-Terminal Heavy-Chain Domain Also Plays a Very Active Role in Toxin-Cell Binding and Interactions

Botulinum neurotoxins (BoNTs) possess unique specificity for nerve terminals. They bind to the presynaptic membrane and then translocate intracellularly, where the light-chain endopeptidase cleaves the SNARE complex proteins, subverting the synaptic exocytosis responsible for acetylcholine release to the synaptic cleft. This inhibits acetylcholine binding to its receptor, causing paralysis. Binding, an obligate event for cell intoxication, is believed to occur through the heavy-chain C-terminal (H_C) domain. It is followed by toxin translocation and entry into the cell cytoplasm, which is thought to be mediated by the heavy-chain N-terminal (H_N) domain. Submolecular mapping analysis by using synthetic peptides spanning BoNT serotype A (BoNT/A) and mouse brain synaptosomes (SNPs) and protective antibodies against toxin from mice and cervical dystonia patients undergoing BoNT/A treatment revealed that not only regions of the H_C domain but also regions of the H_N domain are involved in the toxin binding process. Based on these findings, we expressed a peptide corresponding to the BoNT/A region comprising H_N domain residues 729 to 845 (H_N729–845). H_N729–845 bound directly to mouse brain SNPs and substantially inhibited BoNT/A binding to SNPs. The binding involved gangliosides GT1b and GD1a and a few membrane lipids. The peptide bound to human or mouse neuroblastoma cells within 1 min. Peptide H_N729–845 protected mice completely against a lethal BoNT/A dose (1.05 times the 100% lethal dose). This protective activity was obtained at a dose comparable to that of the peptide from positions 967 to 1296 in the H_C domain. These findings strongly indicate that H_N729–845 and, by extension, the H_N domain are fully programmed and equipped to bind to neuronal cells and in the free state can even inhibit the binding of the toxin.     

Sialic acid-binding immunoglobulin-like lectin 7 mediates selective recognition of sialylated glycans expressed on Campylobacter jejuni lipooligosaccharides

siglecs are a family of sialic-acid binding immunoglobulin-like lectins mostly expressed by cells of the immune system that have the potential to interact with sialylated glycans expressed not only on host cells but also on certain pathogens. Campylobacter jejuni is a common pathogen of humans that expresses surface lipooligosaccharides (LOS) that can be modified with ganglioside-like terminal structures in the core oligosaccharides. In this study, we examined the interaction of 10 siglecs with LOS purified from four different C. jejuni isolates expressing GM1-like, GD1a-like, GD3-like, and GT1a-like oligosaccharides. Of all siglecs examined, only Siglec-7 exhibited specific, sialic acid-dependent interactions with C. jejuni LOS in solid-phase binding assays. Binding was especially prominent with LOS from the HS:19(GM1(+) GT1a(+)) isolate, with weaker binding with LOS from the HS:19(GD3(+)) isolate. Binding of Siglec-7 was also observed with intact bacteria expressing these LOS structures. Specific binding of HS:19(GM1(+) GT1a(+)) bacteria was demonstrated with Siglec-7 expressed on transfected Chinese hamster ovary cells and with peripheral blood leukocytes, among which HS:19(GM1(+) GT1a(+)) bacteria bound selectively to both natural killer cells and monocytes which naturally express Siglec-7. These results raise the possibility that, in addition to their role in generating autoimmune antibody responses, C. jejuni LOS could interact with Siglec-7 expressed by leukocytes, modulate the host-pathogen interaction, and contribute to the clinical outcome and the development of secondary complications such as Guillain-Barré syndrome.     

Molecular properties of each subcomponent in Clostridium botulinum type B haemagglutinin complex

The role of each subcomponent of Clostridium botulinum serotype B haemagglutinin (HA), which is one component of 16S toxin, and consists of four subcomponents (HA1, 2, 3a, and 3b), was investigated. In order to identify the subcomponent contributing to the stability of a neurotoxin in the gastro-intestinal tract, each recombinant HA (rHA) subcomponent was incubated with gastro-intestinal proteases. Although rHA1 and rHA3 were stable to these proteases except for specific cleavage, rHA2 was not. Anti-free whole HA serum reacted with neither rHA2 nor HA2 in 16S toxin on both Western blot and ELISA, while anti-rHA2 serum reacted with both rHA2 and HA2 in 16S toxin on Western blots, although it did not react with 16S toxin in ELISA. Binding or haemagglutination activity against erythrocytes was found in rHA1 and rHA3, but not in rHA2. In addition, only HA1 bound to the intestinal section. These results indicate that the HA (and 16S toxin) complex is assembled in the way that HA1 and HA3 (HA3a plus HA3b) encase HA2, followed by modification with trypsin-like bacterial protease, leading to the conclusion that HA1 and HA3 act as protective factors for the neurotoxin and as attachment factors to host cells.     

Up-regulation of opiate receptors by opiate antagonists in neuroblastoma-glioma cell culture: The possibility of interaction with guanosine triphosphate-binding proteins

Neuroblastoma-glioma NG108-15 cells that were cultured for 48 h with the opiate antagonist, naloxone, respond to the guanosine 5′-triphosphate (GTP) analogue guanosine 5′-[β,γ-imido]-triphosphate (GMP-PNP) in the binding assay as the control, non-treated, cells. This was observed when the guanyl nucleotide was tested in the presence or absence of sodium chloride and also after subcellular fractionation of the membranes on a sucrose gradient which separated between two receptor-containing fractions. The findings suggest that the increase in δ type enkephalin receptors in naloxone-treated NG108-15 cells does not reflect an alteration in the interaction between the receptor and the adenylate cyclase-GTP-binding protein system.     

An assessment of the strength of NG108-15 cell adhesion to chemically modified surfaces

The strength of adhesion of NG108-15 cells to glass substrates modified with adsorbed proteins (laminin and poly-ornithine) or modified with covalently bound peptides (tri-ornithine and Tyr-Ile-Gly-Ser-Arg) was quantitatively assessed, by determining the shear stresses necessary to denude the cells from substrates using a spinning disk device. The shear stresses required to detach NG108-15 cells from glass modified with either adsorbed poly-ornithine or with both poly-ornithine and laminin were significantly (P < 0.05) higher than the shear stresses required to detach the cells from plain glass substrates. Covalent surface modifications resulted in higher strengths of NG108-15 adhesion than were exhibited on surfaces modified with adsorbed proteins. NG108-15 cell adhesion strength was maximal on surfaces covalently modified with only amine groups (without any peptides or proteins). These results indicate that general (i.e., not necessarily receptor-specific) surface modification strategies, which increase the net surface charge of a substrate, will elicit strong adhesion of NG108-15 cells.     

Tumorigenicity, major histocompatibility antigens, and karyotypes of interspecific hybrids between mouse neuroblastoma and rat glioma or liver cells

Five interspecific hybrids of mouse neuroblastoma with rat glioma (NG108-15, 140-3, and 141-B) or with nontransformed rat liver cells (NBr-10A and NBr-20A) were examined for major histocompatibility (MHC) antigens and tumorigenicity in comparison with their karyotypes. Both mouse and rat MHC antigens were present in each hybrid population, as determined by a simple cytotoxicity test. All five hybrid cell lines produced tumors in athymic nude mice with varied take incidences. Four hybrid cells, NG108-15, 140-3, NBr-10A, and NBr-20A, were highly tumorigenic. Their karyotypes were characterized by a higher modal chromosome numbers than would be expected from the fusion of parent cells in which at least one parent contained an increased number of chromosomes. In contrast, 141-B cells, with massive loss of chromosomes from both malignant parents, were weakly tumorigenic. The results suggest that the retention of marker chromosomes as well as double minutes (DMs) or microchromosomes of neuroblastoma origin may be required for expression of malignancy in these hybrid cells. The survival time of tumor-bearing mice also varied within the five cell lines, but it was significantly short in NG108-15, which yielded lung metastases in the host animals.     

Quantitation of binding and subcellular distribution of Clostridium perfringens enterotoxin in rat liver cells.

Binding of enterotoxin from Clostridium perfringens type A was studied in suspensions of parenchymal and nonparenchymal cells from rat liver. In hepatocytes, 1.5 X 10(6) specific binding sites per cell with an association constant of 3.2 X 10(6) M-1 were found. About 1% of the added toxin was nonspecifically bound to the hepatocytes. At concentrations of toxin below 0.1 micrograms/ml, 80% of the toxin density of 7 X 10(6) cells per ml. Binding did not increase after the cells became permeable to the toxin. Subcellular fractionation in a sucrose gradient produced no evidence for binding to parts of the cell other than the plasma membrane. The degree of binding to nonparenchymal cells was less than 10% of the binding to hepatocytes.     

The effect of neurotoxin on rabies virus binding to mouse neuroblastoma cells.

Mouse neuroblastoma cells were exposed to alpha bungarotoxin, a neurotoxin known to inhibit rabies virus binding to the nicotinic acetylcholine receptor located at the neuromuscular junction in muscle tissue. The total amount of 3H-CVS virus that bound to neurotoxin treated cells was separated into specific and non-specific binding using a cold competition assay. Comparison of untreated and neurotoxin treated cells demonstrated that the majority of cell-associated virus in untreated cells was of a specific nature whereas the majority of the cell-associated virus in neurotoxin treated cells was due to non-specific binding.     

Characterization of three monoclonal antibodies against C3 with selective specificities.

We raised 15 mouse monoclonal antibodies against human C3 from two separate fusions. Mouse plasmacytoma cells were fused with spleen cells from mice immunized either with a mixture of C3, C3b and C3c, or with a mixture of C3dg and C3d. Three of the 15 monoclonals were characterized in detail. N-7A reacted with native C3 as well as C3b and C3c. Two other monoclonals, C-5G and G-3E, recognized neoantigenic determinants on C3c and C3dg, respectively. In ELISA, C-5G reacted with C3b and C3c but not with native C3 nor with C3dg; and on the other hand G-3E reacted only with C3dg. The selective specificities of these monoclonals were further confirmed in a binding assay to C3 fragments formed on cellular intermediates. C-5G bound exclusively to EC3b, and G-3E bound to EiC3b, EC3dg and EC3d. N-7A bound only very poorly to EC3b. C-5G inhibited both the haemolytic activity of C5 convertase and also the CR1-mediated rosette formation of B-enriched peripheral mononuclear cells with EC3b. G-3E inhibited the CR2-mediated EC3dg-rosette formation of Raji cells. These monoclonals, with selective specificities, can distinguish the state of activation and degradation of the C3 molecule.     

Surface Patterning and Adhesion of Neuroblastoma X Glioma (NG108-15) Cells

A variable height flow cell was used to measure the adhesion properties of the neural cell line of neuroblastoma X glioma (NG108-15) cells cultured on substrates of organosilane self-assembled monolayers (SAMs). The SAMs tested in this study were 13F, 15F, PEG 550, OTS, DETA and APTS. Utilizing deep UV lithography, patterning of the SAMs create three regions for cell attachment; the original SAM, the backfilled SAM, and the interface between the two. Upon plating, the cell soma show no preference for any of the three regions. One exception was on PEG 550, which was found to resist cell adhesion upon normal plating conditions. The cell processes of the NG108-15 cells show a preference for growth at the interface between two patterned surfaces. A factor of three increase in adhesive properties was found for the patterned surfaces over an uncoated glass surface. Design rules of a single whole cell biosensor using the NG108-15 cells can be developed based on these findings.     

Unique biological activity of botulinum D/C mosaic neurotoxin in murine species

Clostridium botulinum types C and D cause animal botulism by the production of serotype-specific or mosaic botulinum neurotoxin (BoNT). The D/C mosaic BoNT (BoNT/DC), which is produced by the isolate from bovine botulism in Japan, exhibits the highest toxicity to mice among all BoNTs. In contrast, rats appeared to be very resistant to BoNT/DC in type C and D BoNTs and their mosaic BoNTs. We attempted to characterize the enzymatic and receptor-binding activities of BoNT/DC by comparison with those of type C and D BoNTs (BoNT/C and BoNT/D). BoNT/DC and D showed similar toxic effects on cerebellar granule cells (CGCs) derived from the mouse, but the former showed less toxicity to rat CGCs. In recombinant murine-derived vesicle-associated membrane protein (VAMP), the enzymatic activities of both BoNTs to rat isoform 1 VAMP (VAMP1) were lower than those to the other VAMP homologues. We then examined the physiological significance of gangliosides as the binding components for types C and D, and mosaic BoNTs. BoNT/DC and C were found to cleave an intracellular substrate of PC12 cells upon the exogenous addition of GM1a and GT1b gangliosides, respectively, suggesting that each BoNT recognizes a different ganglioside moiety. The effect of BoNT/DC on glutamate release from CGCs was prevented by cholera toxin B-subunit (CTB) but not by a site-directed mutant of CTB that did not bind to GM1a. Bovine adrenal chromaffin cells appeared to be more sensitive to BoNT/DC than to BoNT/C and D. These results suggest that a unique mechanism of receptor binding of BoNT/DC may differentially regulate its biological activities in animals.     

Clostridium botulinum type E toxins bind to Caco-2 cells by a different mechanism from that of type A toxins

Cultured Clostridium botulinum strains produce progenitor toxins designated as 12S, 16S, and 19S toxins. The 12S toxin consists of a neurotoxin (NTX, 7S) and a non-toxic non-hemagglutinin (NTNH). The 16S and 19S toxins are formed by conjugation of the 12S toxin with hemagglutinin (HA), and the 19S toxin is a dimer of the 16S toxin. Type A cultures produce all 3 of these progenitor toxins, while type E produces only the 12S toxin. The 7S toxin is cleaved into heavy (H) and light (L) chains by a protease(s) in some strains, and the H chain has 2 domains, the N-terminus (Hn) and C-terminus (Hc). It has been reported that type A toxins bind to the intestinal cells or cultured cells via either HA or Hc. In this study, we investigated the binding of type A and E toxins to Caco-2 cells using Western blot analysis. Both the type E 7S and 12S toxins bound to the cells, with the 7S toxin binding more strongly, whereas, in the type A strain, only the 16S/19S toxins showed obvious binding. Pre-incubation of the type E 7S toxin with IgG against recombinant type E Hc significantly inhibited the 7S toxin binding, indicating that Hc might be a main binding domain of the type E toxin.     

Regulation of stimulatory adenylyl cyclase signaling during forskolin-induced differentiation of mouse neuroblastoma × rat glioma (NG108–15) cells

Chronic exposure of neuroblastoma × glioma (NG108–15) cells to substances that elevate intracellular cAMP levels results in morphological differentiation into a more neuronal-like phenotype. Here we report that forskolin-induced differentiation is accompanied by a biphasic regulation of stimulatory adenylyl cyclase (AC) signaling. While 1 day of forskolin exposure produces an initial increase in basal, [AIF₄]⁻-, and prostaglandin E₁ (PGE₁)-stimulated AC activities, stimulatory signal transduction is substantially reduced after complete differentiation of the cells (6 days). Western blot analysis revealed that these functional changes correlate well with changes in the quantity of G_s, the stimulatory component of AC. Additional forskolin-induced adaptations were found for PGE₁ receptors, inhibitory G proteins and AC. These data demonstrate that neuronal differentiation of NG108–15 cells is associated with complex regulatory changes within the stimulatory PGE₁ receptor system.     

Pathogenesis of Shigella diarrhea: rabbit intestinal cell microvillus membrane binding site for Shigella toxin

This study examined the binding of purified 125I-labeled shigella toxin to rabbit jejunal microvillus membranes (MVMs). Toxin binding was concentration dependent, saturable, reversible, and specifically inhibited by unlabeled toxin. The calculated number of toxin molecules bound at 4 degrees C was 7.9 X 10(10) (3 X 10(10) to 2 X 10(11))/micrograms of MVM protein or 1.2 X 10(6) per enterocyte. Scatchard analysis showed the binding site to be of a single class with an equilibrium association constant, K, of 4.7 X 10(9) M-1 at 4 degrees C. Binding was inversely related to the temperature of incubation. A total of 80% of the labeled toxin binding at 4 degrees C dissociated from MVM when the temperature was raised to 37 degrees C, but reassociated when the temperature was again brought to 4 degrees C. There was no structural or functional change of MVM due to toxin as monitored by electron microscopy or assay of MVM sucrase activity. These studies demonstrate a specific binding site for shigella toxin on rabbit MVMs. The physiological relevance of this receptor remains to be determined.