The Bacillus thuringiensis insecticidal toxin binds biotin-containing proteins.

Brush border membrane vesicles from larvae of the tobacco hornworm, Manduca sexta, contain protein bands of 85 and 120 kDa which react directly with streptavidin conjugated to alkaline phosphatase. The binding could be prevented either by including 10 microM biotin in the reaction mixture or by prior incubation of the brush border membrane vesicles with an activated 60- to 65-kDa toxin from Bacillus thuringiensis HD-73. The ability of B. thuringiensis toxins to recognize biotin-containing proteins was confirmed by their binding to pyruvate carboxylase, a biotin-containing enzyme, as well as to biotinylated ovalbumin and biotinylated bovine serum albumin but not to their nonbiotinylated counterparts. Activated HD-73 toxin also inhibited the enzymatic activity of pyruvate carboxylase. The biotin binding site is likely contained in domain III of the toxin. Two highly conserved regions within domain III are similar in sequence to the biotin binding sites of avidin, streptavidin, and a biotin-specific monoclonal antibody. In particular, block 4 of the B. thuringiensis toxin contains the YAS biotin-specific motif. On the basis of its N-terminal amino acid sequence, the 120-kDa biotin-containing protein is totally distinct from the 120-kDa aminopeptidase N reported to be a receptor for Cry1Ac toxin.     

Cholera toxin modifies diverse GTP-modulated regulatory proteins

Using thin layer chromatography (TLC) and various colorimetric procedures, the exometabolite of $\text{Leishmania}\text{donovani}$ was shown to be a novel glycopeptidophosphosphingolipid. In aqueous medium the exometabolite aggregated to form micellar structures of high molecular weight. Purity of the various preparations and the novel nature of the micellar structures was demonstrated by TLC. These micelles are unique because they do not break up upon solvation in organic solvents. This indicates that once the supramolecular structure is established, its integrity is maintained by forces other than the apolar ones involved in its formation.     

Phosphorylation of cellular proteins in response to treatment with Clostridium difficile toxin B and Clostridium sordellii toxin L.

Toxin B from Clostridium difficile induces typical morphological changes of cultured cells consisting of rounding up and arborization, which are associated with a dramatic disruption of microfilaments. In this study, we show that toxin L, a cytotoxin produced by bacterial strain Clostridium sordellii, has similar effects on cultured cells including the redistribution of F-actin and of the adhesion plaque protein vinculin. It has been assumed that the mechanisms involved in cytopathic effects of toxin B are related to the function of an unidentified component that regulates the organization of the actin cytoskeleton. We demonstrate that the treatment of cultured astrocytes with toxin B or toxin L alters the incorporation of inorganic phosphate into several proteins. Immunoblot analysis revealed that one of these proteins is tropomyosin. Since tropomyosin stabilizes microfilaments and inhibits the severing activity of gelsolin, the toxin-induced phosphorylation may counteract this inhibition resulting in severing of microfilaments and capping of short filaments. A decrease in the radioactivity associated with intermediate filament protein vimentin was also detected using a monoclonal antibody which specifically recognizes a phosphorylated epitope of vimentin. Since vimentin is an in vivo substrate for various protein kinases, these data are in favor of broad effects of these toxins. Direct measurement of protein kinase C in cells exposed to toxin B or to toxin L did not reveal a significant change in protein kinase C activity. Furthermore, treatments with toxins do not increase cAMP levels, suggesting that toxins do not activate protein kinase A. Although further studies are required to determine the primary target site for the clostridial cytotoxin B and L, our results show that they provoke the alteration in the phosphorylation of cellular proteins.     

Cholera toxin up-regulates endoplasmic reticulum proteins that correlate with sensitivity to the toxin

Cholera toxin (CT) contains one A chain and five B chains. The A chain is an enzyme that covalently modifies a trimeric G protein in the cytoplasm, resulting in the overproduction of cAMP. The B chain binds the glycosphingolipid G(M1), the cell surface receptor for CT, which initiates receptor-mediated endocytosis of the toxin. After endocytosis, CT enters the endoplasmic reticulum (ER) via retrograde vesicular traffic where the A chain retro-translocates through the ER membrane to reach the cytoplasm. The retro-translocation mechanism is poorly understood, but may involve proteins of the ER stress response, including the ER associated degradation (ERAD) pathway. We report here that treating cells with CT or CTB quickly up-regulates the levels of BiP, Derlin-1, and Derlin-2, known participants in the ER stress response and ERAD. CT did not induce calnexin, another known responder to ER stress, indicating that the CT-mediated induction of ER proteins is selective in this time frame. These data suggest that CT may promote retro-translocation of the A chain to the cytoplasm by rapidly up-regulating a set of ER proteins involved in the retro-translocation process. In support of this idea, a variety of conditions that induced BiP, Derlin-1, and Derlin-2 sensitized cells to CT and conditions that inhibited their induction de-sensitized cells to CT. Moreover, specifically suppressing Derlin-1 with siRNA protected cells from CT. In addition, Derlin-1 co-immunoprecipitated with CTA or CTB from CT-treated cells using anti-CTA or anti-CTB antibodies. Altogether, the results are consistent with the hypothesis that the B chain of CT up-regulates ER proteins that may assist in the retro-translocation of the A chain across the ER membrane.     

Immunological relationships among proteins making up the Bacillus thuringiensis subsp. israelensis crystalline toxin.

The immunological relationships among the proteins of the mosquito larvicidal toxin produced by Bacillus thuringiensis subsp. israelensis have been investigated by using polyclonal antisera specific for the 28-, 70-, and 135-kilodalton proteins. Each of these proteins was immunologically distinct. There was no cross-reaction among the three proteins and the two non-homologous antisera. Treatment of toxin proteins with larval gut enzymes for 20 h identified protease-resistant domains at approximately 65, 38, and 22 kilodaltons. Similar domains were generated by treatment with trypsin and chymotrypsin. Our immunological and kinetic data indicate that the 28-kilodalton protein is degraded successively to protein bands at 26, 25, 23, and 22 kilodaltons, the 70-kilodalton protein is degraded to a protein at 38 kilodaltons, and the 135-kilodalton protein is degraded successively to protein bands at 94, 72, and, probably, 65 kilodaltons. Solubilized toxin possesses two biological activities, larvicidal and general cytolytic (hemolytic). We used nondenaturing gel electrophoresis to show that the hemolytic activity resides in the 28-kilodalton protein. However, higher-molecular-weight proteins are required to achieve the level of toxicity observed in intact toxin.     

Self-protection of Pseudomonas syringae pv. "tabaci" from its toxin, tabtoxinine-beta-lactam

An extracellular toxin, tabtoxinine-beta-lactam (T beta L), is produced by Pseudomonas syringae pv. "tabaci." This toxin irreversibly inhibits its target, glutamine synthetase; yet P. syringae pv. "tabaci" retains significant amounts of glutamine synthetase activity during toxin production in culture. As part of our investigation of the self-protection of P. syringae pv. "tabaci," we compared the effects of T beta L on Tox+ (T beta L-producing, insensitive to T beta L) and Tox- (T beta L nonproducing, sensitive to T beta L) strains. The extent of protection afforded to the Tox- strain when induced to adenylylate glutamine synthetase was tested. We concluded that an additional protection mechanism was required. A detoxification activity was found in the Tox+ strain which opens the beta-lactam ring of T beta L to produce the inactive, open-chain form, tabtoxinine. Whole cells of the Tox+ strain incubated for 24 h with [14C]T beta L (0.276 mumol/3 X 10(10) cells) contained [14C]tabtoxinine (0.056 mumol), and the medium contained T beta L (0.226 mumol). Extracts of spheroplasts of the Tox+ stain also converted T beta L to tabtoxinine, whereas extracts of the Tox- strain did not alter T beta L. The conversion was time dependent and stoichiometric and was destroyed by boiling for 30 min or by the addition of 5 mM EDTA. Penicillin, a possible substrate and competitive inhibitor of this lactamase activity, inhibited the conversion of T beta L to tabtoxinine. Periplasmic fluid did not catalyze the conversion of T beta L.     

Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins

Cholera toxin catalyzes transfer of radiolabel from [32P]NAD+ to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of Mr = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (Mr = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and [32P]NAD+ caused radiolabeling of purified microtubule and intermediate filament proteins.     

Carbon distribution to toxic effect in toxin proteins

The role of hydrophobic force in biological function through the formation of several local macro-molecular structures is evident. Carbon is the element that contributes to biological function in living systems. We show that carbon distribution is related to protein activity using an example. The carbon distribution profile is foreseen to help undestand unfolded and misfolded regions of protein structures. The carbon distribution profile in a toxin protein that is found associated with the toxic shock syndrome is described in this study. The carbon profile provides insight to the association of specific residues responsible for toxicity.     

Cytolethal distending toxin B as a cell-killing component of tumor-targeted anthrax toxin fusion proteins

Cytolethal distending toxin (Cdt) is produced by Gram-negative bacteria of several species. It is composed of three subunits, CdtA, CdtB, and CdtC, with CdtB being the catalytic subunit. We fused CdtB from Haemophilus ducreyi to the N-terminal 255 amino acids of Bacillus anthracis toxin lethal factor (LFn) to design a novel, potentially potent antitumor drug. As a result of this fusion, CdtB was transported into the cytosol of targeted cells via the efficient delivery mechanism of anthrax toxin. The fusion protein efficiently killed various human tumor cell lines by first inducing a complete cell cycle arrest in the G2/M phase, followed by induction of apoptosis. The fusion protein showed very low toxicity in mouse experiments and impressive antitumor effects in a Lewis Lung carcinoma model, with a 90% cure rate. This study demonstrates that efficient drug delivery by a modified anthrax toxin system combined with the enzymatic activity of CdtB has great potential as anticancer treatment and should be considered for the development of novel anticancer drugs.     

Evolution of the "titin epitope" in neurofilament proteins.

1. The specificity of a monoclonal antibody raised to human titin was characterized. The antibody reacts with an epitope which is common to titin and the high mol. wt subunits NF-H and NF-M of mammalian neurofilaments. 2. Mapping of the epitope indicated that it is located in the carboxyterminal extension of NF-H and NF-M, and that its reactivity does not depend on the phosphorylation state of the molecule. 3. A comparative study on neurofilament protein of lower vertebrates revealed that this epitope has been conserved during vertebrate evolution.     

"Subunit-exchange" chromatography of proteins: behavior of alpha-chymotrypsin.

Protein subunits attached covalently to a solid matrix bind free subunits under conditions where an association-dissociation equilibrium exists in the soluble protein. This is the basis of a process which may be called “subunit-exchange chromatography”. The process, which may be applied to any self-associating protein, is exemplified here with α-chymotrypsin. The behavior of the system has been studied in detail especially with respect to the conditions of immobilization of the monomer. The results indicate the potentialities of the method for analytical and preparative purposes.     

Functional homology between signal-coupling proteins. Cholera toxin inactivates the GTPase activity of transducin

Both the light-stimulated cGMP phosphodiesterase of retinal rod outer segments (ROS) and hormone-stimulated adenylate cyclase are regulated by guanine nucleotide-binding regulatory proteins (N). Transducin serves as the signal-carrying regulatory protein in ROS, and the N protein (also called G or G/F) performs this role in the adenylate cyclase system. The GTP form of these regulatory proteins activates the corresponding enzyme, whereas the GDP form does not. Both transducin and the N protein possess a GTPase activity that restores the regulatory protein to the unstimulated state. Cholera enterotoxin catalyzes the transfer of ADP-ribose from NAD+ to the N protein, which inhibits its GTPase activity and activates adenylate cyclase. We report here that the toxin also catalyzes ADP-ribosylation of the alpha-subunit of transducin in ROS membranes. This modification of the guanine nucleotide-binding subunit of transducin is markedly enhanced by the bleaching of rhodopsin and by the addition of guanosine-5'-(beta, gamma-imino)triphosphate. In contrast, GDP, GTP, and guanosine-5'-(3-O)thiotriphosphate inhibit the reaction, while GMP and ATP have no effect. Under optimal conditions, toxin catalyzes labeling of 0.7 mol of the alpha-subunit of transducin/mol of bound [3H]guanosine-5'-(beta, gamma-imido)triphosphate and causes 70% inhibition of the light-dependent GTPase activity of transducin in ROS. These results indicate close functional homology between transducin of ROS and the N protein of adenylate cyclase.     

The HA proteins of botulinum toxin disrupt intestinal epithelial intercellular junctions to increase toxin absorption

The type B botulinum neurotoxin (BoNT) elicits flaccid paralysis and death in humans by intoxicating peripheral nerves after oral absorption. Here, we examine the function of the haemagglutinin (HA), a non-toxic component of the large 16S BoNT complex. We find that the HA acts in the intestine to disrupt epithelial barrier function by opening intercellular tight and adherens junctions. This allows transport of BoNT and other large solutes into the systemic circulation and explains how the type B BoNT complexes are efficiently absorbed. In vitro , HA appears to act on the epithelial cell via the basolateral membrane only, suggesting the possibility of another step in the absorptive process. These studies show that the 16S BoNT complex is a multifunctional protein assembly equipped with the machinery to efficiently breach the intestinal barrier and act systemically on peripheral nerves.     

Immunological relationships among proteins making up the Bacillus thuringiensis subsp. israelensis crystalline toxin

The immunological relationships among the proteins of the mosquito larvicidal toxin produced by Bacillus thuringiensis subsp. israelensis have been investigated by using polyclonal antisera specific for the 28-, 70-, and 135-kilodalton proteins. Each of these proteins was immunologically distinct. There was no cross-reaction among the three proteins and the two non-homologous antisera. Treatment of toxin proteins with larval gut enzymes for 20 h identified protease-resistant domains at approximately 65, 38, and 22 kilodaltons. Similar domains were generated by treatment with trypsin and chymotrypsin. Our immunological and kinetic data indicate that the 28-kilodalton protein is degraded successively to protein bands at 26, 25, 23, and 22 kilodaltons, the 70-kilodalton protein is degraded to a protein at 38 kilodaltons, and the 135-kilodalton protein is degraded successively to protein bands at 94, 72, and, probably, 65 kilodaltons. Solubilized toxin possesses two biological activities, larvicidal and general cytolytic (hemolytic). We used nondenaturing gel electrophoresis to show that the hemolytic activity resides in the 28-kilodalton protein. However, higher-molecular-weight proteins are required to achieve the level of toxicity observed in intact toxin.