Structure of tetanus toxin. I. Breakdown of the toxin molecule and discrimination between polypeptide fragments.

Tetanus toxin was digested with papain, yielding one major polypeptide (Fragment C) with a molecular weight corresponding to 47,000 +/- 5%, thus comprising about one-third of the toxin molecule. Fragment C was antigenically active, atoxic, and stimulated the formation of antibodies neutralizing the lethal action of tetanus toxin in vivo. Furthermore, a second split product (Fragment B) was isolated from the papain digest, containing two polypeptide chains linked together via a disulfide bond. Fragment B (Mr = 95,000 +/- 5%) was atoxic and showed a reaction of nonidentity with Fragment C on immunodiffusion analysis against tetanus antitoxin. The basic two-chain structure (heavy and light chain polypeptide, cf. Matsuda, M., and Yoneda, M. (1975) Infect. Immun. 12, 1147-1153) of tetanus toxin has been confirmed and the relationship between Fragments B and C within this framework has been established. Fragment C was distinguished from the light chain by electrophoresis in sodium dodecyl sulfate and by immunodiffusion analysis, indicating that this fragment constitutes a portion of the heavy chain polypeptide. Fragment B showed a reaction of partial identity with the light as well as the heavy chain from tetanus toxin. Reduction of Fragment B with dithiothreitol followed by gel chromatography yielded a fraction which was indistinguishable from the light chain portion of the toxin molecule. It is concluded that Fragment B comprises the complementary portion of the heavy chain (remaining after scission of the polypeptide bond(s) releasing Fragment C) linked to the light chain by a disulfide bond.     

Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymatic subunits.

The A-subunit polypeptides of Shiga toxin, the Shiga-like toxins (SLTs), and the plant lectin ricin inactivate eucaryotic ribosomes by enzymatically depurinating 28S rRNA. Comparison of the amino acid sequences of the members of the Shiga toxin family and ricin revealed two regions of significant homology that lie within a proposed active-site cleft of the ricin A chain. In previous studies, these conserved sequences of the SLT-I and ricin A subunits have been implicated as active sites. To establish the importance of these regions of homology, we used site-directed mutagenesis to alter the A-subunit sequences of two members of the Shiga toxin family. Substitution of an aspartic acid for glutamic acid 166 of the Slt-IIA subunit decreased the capacity of the polypeptides to inhibit protein synthesis at least 100-fold in a cell-free translation system. However, this mutation did not prevent the expression of immunoreactive, full-length Slt-IIA. In addition, SLT-II holotoxin containing the mutated A subunit was 1,000-fold less toxic to Vero cells. Finally, site-directed mutagenesis was used to delete sequences encoding amino acids 202 through 213 of the Shiga toxin A subunit. Although this deletion did not prevent holotoxin assembly, it abolished cytotoxic activity.     

On the membrane translocation of diphtheria toxin: at low pH the toxin induces ion channels on cells.

Diphtheria toxin (DT) in acidic media forms ion-conducting channels across the plasma membrane and inhibits protein synthesis of both highly and poorly DT-sensitive cell lines. This results in loss of cell potassium and in entry of both sodium and protons with a concomitant rapid lowering of membrane potential. The pH dependency of the permeability changes is similar to that of the inhibition of cell protein synthesis. DT-induced ion channels close when the pH of the external medium is returned to neutrality and cells recover their normal monovalent cation content. Similar permeability changes were induced by two DT mutants defective either in enzymatic activity or in cell binding, but not with a mutant defective in membrane translocation. The implication of these findings for the mechanism of DT membrane translocation is discussed.     

Monoclonal antibody against diphtheria toxin. Effect on toxin binding and entry into cells

A number of monoclonal antibodies against diphtheria toxin were isolated. Some of their properties were determined. Antibody 2 reacts with the region of between 30 and 45 kDa from the NH2 terminus of toxin. Antibody 7 reacts with the COOH-terminal 17-kDa region of toxin. These two antibodies show sharp contrasts in their effects on toxin action in cultured cells. When antibody 2 or 7 and toxin were mixed, incubated at 37 degrees C, and then added to sensitive Vero cells, antibody 7 blocked toxin action, but antibody 2 did not. When antibody 2 or 7 was added to cells to which toxin had been prebound at 4 degrees C, and the cells were then shifted to 37 degrees C, antibody 7 did not block toxin action, but antibody 2 inhibited intoxication. Antibody 7 blocked binding of 125I-toxin to cells and did not block degradation of toxin associated with cells. Antibody 2 did not block binding of 125I-toxin to cells, and was able to bind to cells in the presence of toxin. The results obtained from the effect of antibody 2 on degradation of 125I-toxin associated with cells resemble those seen with amines, which block toxin action but do not inhibit binding of toxin to cells. These facts show that antibody 2 does not block binding of toxin to cell surfaces, but blocks the entry of toxin into the cytosol at a step after binding of toxin to the receptor. Antibodies 14 and 15 react with fragment A of diphtheria toxin, but have no effect on any activity of toxin. The other monoclonal antibodies have effects on toxin binding and entry intermediate between those of 2 and 7.     

Spider toxin and the glutamate receptors.

A neurotoxin (JSTX) was isolated from the venom of spider (Nephila clavata). JSTX blocked both the excitatory postsynaptic (EPSPs) and glutamate-induced potentials in lobster neuromuscular synapse and squid giant synapse. In mammalian central nervous system, JSTX blocked the EPSPs in CA1 pyramidal neurons resulting from stimulation of Schaffer collateral/commissure input. Pharmacological investigation showed that JSTX preferentially suppressed quisqualate/kainate receptor subtypes but was much less effective on NMDA receptor. Using synthesized spider toxins we studied the structure-activity relationship and found that the 2,4 dihydroxyphenylacetyl asparagine in the toxin structure was responsible for suppressive action, while the remaining part containing a polyamine was related to the agonist binding site with the polycationic part enhancing the toxic activity. Labeling of synthesized JSTX was used for histochemical as well as biochemical studies. Using autoradiography, 125I-JSTX-3 was found to bind at the lobster neuromuscular synapse. Histochemical study utilizing the interaction of biotinylated JSTX-3 with avidin showed specific binding of the toxin in rat cerebellum and hippocampus. JSTX-3-binding protein was purified from rat brain by affinity chromatography. SDS-PAGE of the affinity purified protein showed at least 4 bands ranging from 40 to 70 kDa.     

Crystallization of diphtheria toxin.

Two new crystal forms (forms III and IV) have been grown of diphtheria toxin (DT), which kills susceptible cells by catalyzing the ADP-ribosylation of elongation factor 2, thereby stopping protein synthesis. Forms III and IV diffract to 2.3 A and 2.7 A resolution, respectively. Both forms belong to space group C2; the unit cell parameters for form III are a = 107.3 A, b = 91.7 A, c = 66.3 A and beta = 94.7 degrees and those for form IV are a = 108.3 A, b = 92.3 A, c = 66.1 A and beta = 90.4 degrees. Both forms have one protein chain per asymmetric unit with the dimeric molecule on a twofold axis of symmetry. Form IV is exceptional among all crystal forms of DT in that it can be grown reproducibly. Thus the form IV crystals should yield a crystallographic structure giving insight into the catalytic, receptor-binding and membrane-insertion properties of DT.     

Transduction of the scorpion toxin maurocalcine into cells. Evidence that the toxin crosses the plasma membrane

Maurocalcine (MCa) is a 33-amino-acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. External application of MCa to cultured myotubes is known to produce Ca2+ release from intracellular stores. MCa binds directly to the skeletal muscle isoform of the ryanodine receptor, an intracellular channel target of the endoplasmic reticulum, and induces long lasting channel openings in a mode of smaller conductance. Here we investigated the way MCa proceeds to cross biological membranes to reach its target. A biotinylated derivative of MCa was produced (MCa(b)) and complexed with a fluorescent indicator (streptavidine-cyanine 3) to follow the cell penetration of the toxin. The toxin complex efficiently penetrated into various cell types without requiring metabolic energy (low temperature) or implicating an endocytosis mechanism. MCa appeared to share the same features as the so-called cell-penetrating peptides. Our results provide evidence that MCa has the ability to act as a molecular carrier and to cross cell membranes in a rapid manner (1-2 min), making this toxin the first demonstrated example of a scorpion toxin that translocates into cells.     

Binding of ATP by pertussis toxin and isolated toxin subunits.

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of [3H]ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP greater than ATP greater than GTP greater than CTP greater than TTP for pertussis toxin and ATP greater than GTP greater than TTP greater than CTP for the B oligomer. Phosphate ions inhibited the binding of [3H]ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of [3H]ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.