Direct expression in E.coli of a functionally active protein A-snake toxin fusion protein

We constructed a recombinant expression plasmid encoding a proteinA–neurotoxin fusion protein. The fused toxin is directlyexpressed in the periplasmic space of Escherichia coli and canbe purified in the milligram range by a single immuno-affinitystep. The LD₅₀ values of the fused toxin and native toxin are130 and 20 nmol/kg mouse respectively. The K_d values characterizingtheir binding to the nicotinic acetylcholine receptor (AcChoR)are respectively 4.8 ± 0.8 and 0.07 ± 0.03 nM.In contrast, the fused and native toxins are equally well recognizedby a toxin-specific monoclonal antibody which recognizes theAcChoR binding site. The lower toxicity of the fused toxin mightresult, therefore, from a steric hindrance, due to the presenceof the bulky protein A moiety (mol. wt = 31 kd) rather thanto a direct alteration of the ‘toxic’ site. Thefused toxin is more immunogenic than native toxin, since 1 nmolof hybrid toxin and 14 nmol of native toxin give rise to comparabletiters of antitoxin antibodies which, furthermore, are equallypotent at neutralizing neurotoxicity. The work described inthis paper shows that the use of fused toxins may be of paramountimportance for future development of serotherapy against envenomationby snake bites.     

Gut Microbiota as a Source of Uremic Toxins

Uremic retention solutes are the compounds that accumulate in the blood when kidney excretory function is impaired. Some of these compounds are toxic at high concentrations and are usually known as “uremic toxins”. The cumulative detrimental effect of uremic toxins results in numerous health problems and eventually mortality during acute or chronic uremia, especially in end-stage renal disease. More than 100 different solutes increase during uremia; however, the exact origin for most of them is still debatable. There are three main sources for such compounds: exogenous ones are consumed with food, whereas endogenous ones are produced by the host metabolism or by symbiotic microbiota metabolism. In this article, we identify uremic retention solutes presumably of gut microbiota origin. We used database analysis to obtain data on the enzymatic reactions in bacteria and human organisms that potentially yield uremic retention solutes and hence to determine what toxins could be synthesized in bacteria residing in the human gut. We selected biochemical pathways resulting in uremic retention solutes synthesis related to specific bacterial strains and revealed links between toxin concentration in uremia and the proportion of different bacteria species which can synthesize the toxin. The detected bacterial species essential for the synthesis of uremic retention solutes were then verified using the Human Microbiome Project database. Moreover, we defined the relative abundance of human toxin-generating enzymes as well as the possibility of the synthesis of a particular toxin by the human metabolism. Our study presents a novel bioinformatics approach for the elucidation of the origin of both uremic retention solutes and uremic toxins and for searching for the most likely human microbiome producers of toxins that can be targeted and used for the therapy of adverse consequences of uremia.     

Characterization of a ricin fusion toxin targeted to the interleukin-2 receptor

Fusion toxins are hybrid proteins consisting of peptide ligandslinked through amide bonds to polypeptide toxins. The liganddirects the molecule to the surface of target cells and thetoxin enters the cytosol and induces cell death. Ricin is anexcellent candidate for use in fusion toxins because of itsextreme potency, the extensive knowledge of its atomic structureand the lack of prior immunological exposure in patients. Wesynthesized a baculovirus transfer vector with the polyhedrinpromoter followed sequentially from the 5' end with DNA encodingthe gp67A leader sequence, the tripeptide ADP, IL-2 (interleukin-2),another ADP tripeptide and RTB (ricin toxin B chain) with lectinsitemutations W37S and Y248H. Recombinant baculovirus was generatedin Sf9 insect cells and used to infect Sf9 cells. RecombinantIL-2-RTB[W37S/Y248H] protein (fusion protein of IL-2 with modificationsW37S and Y248H) was recovered at high yields from day 6 insectcell supernatants, partially purified by affinity chromatographyand reassociated with RTA (ricin toxin A chain). The fusiontoxin was soluble, immunoreactive with antibodies to RTB, LL-2and RTA and had a molecular weight of 80 kDa by SDS-PAGE. Themolecule reacted poorly with asialofetuin, but bound stronglyto IL-2 receptor based on selective cytotoxicity to IL-2 receptorbearing cells. The specific cytotoxicity could be blocked withIL-2 but not lactose. Thus, we report a novel targeted fusiontoxin protein with full biological activity.     

Stepwise transplantation of an active site loop between heat-labile enterotoxins LT-II and LT-I and characterization of the obtained hybrid toxins

Members of the cholera toxin family, including Escherichia coli heat- labile enterotoxins LT-I and LT-II, catalyze the covalent modification of intracellular proteins by transfer of ADP-ribose from NAD to a specific arginine of the target protein. The ADP-ribosylating activity of these toxins is located in the A-subunit, for which LT-I and LT-II share a 63% sequence identity. The flexible loop in LT-I, ranging from residue 47 to 56, closes over the active site cleft. Previous studies have shown that point mutations in this loop have dramatic effects on the activity of LT-I. Yet, in LT-II the sequence of the equivalent loop differs at four positions from LT-I. Therefore five mutants of the active site loop were created by a stepwise replacement of the loop sequence in LT-I with virtually all the corresponding residues in LT- II. Since we discovered that LT-II had no activity versus the artificial substrate diethylamino-benzylidine-aminoguanidine (DEABAG) while LT-I does, our active site mutants most likely probe the NAD binding, not the arginine binding region of the active site. The five hybrid toxins obtained (Q49A, F52N, V53T, Q49V/F52N and Q49V/F52N/V53T) show (i) great differences in holotoxin assembly efficiency; (ii) decreased cytotoxicity in Chinese hamster ovary cells; and (iii) increased in vitro enzymatic activity compared with wild type LT-I. Specifically, the three mutants containing the F52N substitution display a greater Vmax for NAD than wild type LT-I. The enzymatic activity of the V53T mutant is significantly higher than that of wild type LT-I. Apparently this subtle variation at position 53 is beneficial, in contrast to several other substitutions at position 53 which previously had been shown to be deleterious for activity. The most striking result of this study is that the active site loop of LT- I, despite great sensitivity for point mutations, can essentially be replaced by the active site loop of LT-II, yielding an active 'hybrid enzyme' as well as 'hybrid toxin'.      

Role of Toxins in Intake of Varied Diets by Sheep

Herbivores foraging on toxic plants may consume a variety of foods that contain different toxins to increase food intake and to avoid toxicosis. We studied whether lambs offered two foods, each containing a different toxin, could ingest more food than lambs offered one food with a single toxin. Thirty-two lambs were allotted to four groups that received: (1) a ration with toxin A, (2) a ration with toxin B, (3) two rations, one with toxin A and the other with toxin B, and (4) a ration with no toxins. Toxin pairs used in the study were (1) amygdalin and lithium chloride (LiCl), (2) LiCl and LiCl, (3) sparteine and saponin, (4) oxalate and nitrate, and (5) tannin and saponin. For an hour each morning, lambs were offered their ration(s) and intakes were measured. Lambs were maintained on an alfalfa pellet or grass hay diet. Each trial lasted either five or six days. Whether or not lambs ate more when offered foods with different toxins depended on the kind and amount of toxin in the food. Lambs offered rations with amygdalin and LiCl or oxalate and nitrate consumed more food than lambs offered a ration with only one of these toxins. Lambs offered rations with sparteine and saponin or tannin and saponin did not eat more food than lambs offered a ration with either saponin or sparteine or tannin alone. Nor did lambs eat more when offered two rations both containing LiCl. In all trials, lambs offered toxins showed no signs of toxicosis, and they ate less food than lambs offered rations without toxins. Our results indicate that in some cases ruminants can increase intake of toxic foods by consuming foods containing different toxins. However, currently the only way to determine how specific toxins may interact in the body to influence intake would be to conduct feeding trials using plants or ground diets that contain toxins.     

Minigene as a Novel Regulatory Element in Toxin-Antitoxin Systems

The axe-txe type II toxin-antitoxin (TA) system is characterized by a complex and multilayered mode of gene expression regulation. Precise and tight control of this process is crucial to keep the toxin in an appropriate balance with the cognate antitoxin until its activation is needed for the cell. In this report, we provide evidence that a minigene encoded within the axe-txe operon influences translation of the Txe toxin. This is the first example to date of such a regulatory mechanism identified in the TA modules. Here, in a series of genetic studies, we employed translational reporter gene fusions to establish the molecular basis of this phenomenon. Our results show that translation of the two-codon mini-ORF displays an in cis mode of action, and positively affects the expression of txe, possibly by increasing its mRNA stability through protection from an endonuclease attack. Moreover, we established that the reading frame in which the two cistrons are encoded, as well as the distance between them, are critical parameters that affect the level of such regulation. In addition, by searching for two-codon ORFs we found sequences of several potential minigenes in the leader sequences of several other toxins belonging to the type II TA family. These findings suggest that this type of gene regulation may not only apply for the axe-txe cassette, but could be more widespread among other TA systems.     

Chemical engineering of a three-fingered toxin with anti-{alpha}7 neuronal acetylcholine receptor activity

Though it possesses four disulfide bonds the three-fingeredfold is amenable to chemical synthesis, using a Fmoc-based method.Thus, we synthesized a three-fingered curaremimetic toxin fromsnake with high yield and showed that the synthetic and nativetoxins have the same structural and biological properties. Bothwere characterized by the same 2D NMR spectra, identical highbinding affinity (K_d = 22 ± 5 pM) for the muscular acetylcholinereceptor (AChR) and identical low affinity (K_d = 2.0 ±0.4 µM) for     

A recombinant immunotoxin engineered for increased stability by adding a disulfide bond has decreased immunogenicity

Recombinant immunotoxins (RITs) are anti-cancer agents that combine the Fv of an antibody against cancer cells with a protein toxin from bacteria or plants. Since RITs contain a non-human protein, immunogenicity can be an obstacle in their development. In this study, we have explored the hypothesis that increasing stability can reduce the immunogenicity of a RIT using HA22-LR, which is composed of an anti-CD22 Fv fused to domain III of Pseudomonas exotoxin A. We introduced a disulfide bond into domain III by identifying and mutating two structurally adjacent residues to cysteines at sites suggested by computer modeling. This RIT, HA22-LR-DB, displays a remarkable increase in thermal stability and an enhanced resistance to trypsin degradation. In addition, HA22-LR-DB retains cytotoxic and anti-tumor activity, while exhibiting significantly lower immunogenicity in mice. This study demonstrates that it is possible to design mutations in a protein molecule that will increase the stability of the protein and thereby reduce its immunogenicity.     

The effects of helix breaking mutations in the diphtheria toxin transmembrane domain helix layers of the fusion toxin DAB389IL-2

The fusion protein toxin DAB389IL-2 is composed of the catalytic and transmembrane domains of diphtheria toxin genetically linked to human interleukin 2 (IL-2). This fusion toxin is selectively toxic for eukaryotic cells which express the high-affinity form of the IL-2 receptor and the mechanism of intoxication parallels that of native diphtheria toxin. We used site-directed mutagenesis to introduce Pro residues into each of the three helical layers of the transmembrane domain. Although each of the mutations results in the complete loss of cytotoxic activity, individual mutants were found to vary with respect to channel formation in planar lipid bilayers, binding affinity and melting temperature. We propose that each of the three helix layers plays a critical role in the productive delivery of the catalytic domain to the cell cytosol.      

Mechanism of Ganglioside Receptor Recognition by Botulinum Neurotoxin Serotype E

The botulinum neurotoxins are potent molecules that are not only responsible for the lethal paralytic disease botulism, but have also been harnessed for therapeutic uses in the treatment of an increasing number of chronic neurological and neuromuscular disorders, in addition to cosmetic applications. The toxins act at the cholinergic nerve terminals thanks to an efficient and specific mechanism of cell recognition which is based on a dual receptor system that involves gangliosides and protein receptors. Binding to surface-anchored gangliosides is the first essential step in this process. Here, we determined the X-ray crystal structure of the binding domain of BoNT/E, a toxin of clinical interest, in complex with its GD1a oligosaccharide receptor. Beyond confirmation of the conserved ganglioside binding site, we identified key interacting residues that are unique to BoNT/E and a significant rearrangement of loop 1228–1237 upon carbohydrate binding. These observations were also supported by thermodynamic measurements of the binding reaction and assessment of ganglioside selectivity by immobilised-receptor binding assays. These results provide a structural basis to understand the specificity of BoNT/E for complex gangliosides.     

Special Delivery: Potential Mechanisms of Botulinum Neurotoxin Uptake and Trafficking within Motor Nerve Terminals

Botulinum neurotoxins (BoNTs) are highly potent, neuroparalytic protein toxins that block the release of acetylcholine from motor neurons and autonomic synapses. The unparalleled toxicity of BoNTs results from the highly specific and localized cleavage of presynaptic proteins required for nerve transmission. Currently, the only pharmacotherapy for botulism is prophylaxis with antitoxin, which becomes progressively less effective as symptoms develop. Treatment for symptomatic botulism is limited to supportive care and artificial ventilation until respiratory function spontaneously recovers, which can take weeks or longer. Mechanistic insights into intracellular toxin behavior have progressed significantly since it was shown that toxins exploit synaptic endocytosis for entry into the nerve terminal, but fundamental questions about host-toxin interactions remain unanswered. Chief among these are mechanisms by which BoNT is internalized into neurons and trafficked to sites of molecular toxicity. Elucidating how receptor-bound toxin is internalized and conditions under which the toxin light chain engages with target SNARE proteins is critical for understanding the dynamics of intoxication and identifying novel therapeutics. Here, we discuss the implications of newly discovered modes of synaptic vesicle recycling on BoNT uptake and intraneuronal trafficking.     

N-Terminal Modification of Gly-His-Tagged Proteins with Azidogluconolactone

Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective in vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pH 7.5 in 1 h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.     

Validation of a mutant of the pore-forming toxin sticholysin-I for the construction of proteinase-activated immunotoxins

The use of pore-forming toxins from sea anemones (actinoporins) in the construction of immunotoxins (ITs) against tumour cells is an alternative for cancer therapy. However, the main disadvantage of actinoporin-based ITs obtained so far has been the poor cellular specificity associated with the toxin's ability to bind and exert its activity in almost any cell membrane. Our final goal is the construction of tumour proteinase-activated ITs using a cysteine mutant at the membrane binding region of sticholysin-I (StI), a cytolysin isolated from the sea anemone Stichodactyla helianthus. The mutant and the ligand moiety would be linked by proteinase-sensitive peptides through the StI cysteine residue blocking the toxin binding region and hence the IT non-specific killing activity. To accomplish this objective the first step was to obtain the mutant StI W111C, and to evaluate the impact of mutating tryptophan 111 by cysteine on the toxin pore-forming capacity. After proteolysis of the cleavage sequence, a short peptide would remain attached to the toxin. The next step was to evaluate whether this mutant is able to form pores even with a residual peptide linked to cysteine 111. In this work we demonstrated that (i) StI W111C shows pore-forming capacity in a nanomolar range, although it is 8-fold less active than the wild-type recombinant StI, corroborating the previously reported importance of residue 111 for the binding of StI to membranes, and (ii) the mutant is able to form pores even with a residual seven-residue peptide linked to cysteine 111. In addition, it was demonstrated that binding of a large molecule to cysteine 111 renders an inactive toxin that is no longer able to bind to the membrane. These results validate the mutant StI W111C for its use in the construction of tumour proteinase-activated ITs.     

Structure activity relationships of monocyte chemoattractant proteins in complex with a blocking antibody

Monocyte chemoattractant proteins (MCPs) are cytokines that direct immune cells bearing appropriate receptors to sites of inflammation or injury and are therefore attractive therapeutic targets for inhibitory molecules. 11K2 is a blocking mouse monoclonal antibody active against several human and murine MCPs. A 2.5 A structure of the Fab fragment of this antibody in complex with human MCP-1 has been solved. The Fab blocks CCR2 receptor binding to MCP-1 through an adjacent but distinct binding site. The orientation of the Fab indicates that a single MCP-1 dimer will bind two 11K2 antibodies. Several key residues on the antibody and on human MCPs were predicted to be involved in antibody selectivity. Mutational analysis of these residues confirms their involvement in the antibody-chemokine interaction. In addition to mutations that decreased or disrupted binding, one antibody mutation resulted in a 70-fold increase in affinity for human MCP-2. A key residue missing in human MCP-3, a chemokine not recognized by the antibody, was identified and engineering the preferred residue into the chemokine conferred binding to the antibody.     

Antibody engineering reveals the important role of J segments in the production efficiency of llama single-domain antibodies in Saccharomyces cerevisiae

Variable domains of llama heavy-chain antibodies (VHH) are becoming a potent tool for a wide range of biotechnological and medical applications. Because of structural features typical of their single-domain nature, they are relatively easy to produce in lower eukaryotes, but it is not uncommon that some molecules have poor secretion efficiency. We therefore set out to study the production yield of VHH. We computationally identified five key residues that are crucial for folding and secretion, and we validated their importance with systematic site-directed mutations. The observation that all key residues were localised in the V segment, in proximity of the J segment of VHH, led us to study the importance of J segment in secretion efficiency. Intriguingly, we found that the use of specific J segments in VHH could strongly influence the production yield. Sequence analysis and expression experiments strongly suggested that interactions with chaperones, especially with the J segment, are a crucial aspect of the production yield of VHH.     

Structure–Activity Relationship Study of Spider Polyamine Toxins as Inhibitors of Ionotropic Glutamate Receptors

The spider polyamine toxins Joro spider toxin‐3 (JSTX‐3) and Nephila polyamine toxins‐1 and ‐8 (NPTX‐1 and NPTX‐8) are isolated from the venom of the orb‐weaver spider Nephila clavata (Joro spider). They share a high degree of structural resemblance, their aromatic head groups being the only difference, and were recently found to be very potent open‐channel blockers of ionotropic glutamate (iGlu) receptors. In this study we designed and synthesized a collection of 24 analogues of these toxins using a recently developed solid‐phase synthetic methodology. Systematic variation in two regions of the toxins and subsequent evaluation of biological activity at AMPA and NMDA subtypes of iGlu receptors provided succinct information on structure–activity relationships. In particular, one set of analogues were found to display exquisite selectivity and potency for AMPA receptors relative to the natural products. Thus, this systematic SAR study has provided new pharmacological tools for studies of iGlu receptors.     

Heavy chain CDR3 optimization of a germline encoded recombinant antibody fragment predisposed to bind the U1A protein

Previously, we described a DP-65 encoded heavy chain variable (VH) gene restriction in anti-U1A antibodies. The U1A protein (a component of the U1 ribonucleoprotein particle) is an important autoantigenic target in certain systemic lupus erythematosus (SLE) patients. Here we examined the effect of randomizing amino acids in the heavy chain complementarity determining region 3 (CDR3) of this germline encoded recombinant antibody fragment on binding to the U1A protein. A phage display library was constructed using the DP-65 VH domain with four randomized CDR3 residues and our results showed that a high frequency (10%) of the randomized mutants in the unselected library were able to bind the U1A protein. This corroborates our previous finding that this VH domain provides an appropriate structure for U1A binding, although the nature of the CDR3 residues appears crucial in determining whether or not this VH domain binds U1A. After two rounds of selection U1A binders show a consensus sequence in their randomized CDR3 residues i.e. S(K,R,S)XG, in which X is an uncharged residue. This consensus is partially present in an antibody which was derived from an SLE patient indicating that this consensus, to some extent, is also followed in vivo. Clones which match the consensus sequence obtained up to 25-fold higher affinities compared with the original clones, illustrating the importance of the VH CDR3 residues in determining the affinity of these antibodies.      

Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody

Tetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are not detectable, nor is detection of their VAMP substrate sensitive enough. The immune detection of the cleaved substrate is much more sensitive, as we have previously shown for botulinum neurotoxin type A. Here, we describe the production in rabbit of a polyclonal antibody raised versus a peptide encompassing the 13 residues C-terminal with respect to the neurotoxin cleavage site. The antibody was affinity purified and found to recognize, with high specificity and selectivity, the novel N-terminus of VAMP that becomes exposed after cleavage by tetanus toxin and botulinum toxin type B. This antibody recognizes the neoepitope not only in native and denatured VAMP but also in cultured neurons and in neurons in vivo in neurotoxin-treated mice or rats, suggesting the great potential of this novel tool to elucidate tetanus and botulinum B toxin activity in vivo.     

Variation in Chemical Defense Among Natural Populations of Common Toad, <Emphasis Type="Italic">Bufo bufo</Emphasis>, Tadpoles: the Role of Environmental Factors

Defensive toxins are widespread in nature, yet we know little about how various environmental factors shape the evolution of chemical defense, especially in vertebrates. In this study we investigated the natural variation in the amount and composition of bufadienolide toxins, and the relative importance of ecological factors in predicting that variation, in larvae of the common toad, Bufo bufo, an amphibian that produces toxins de novo. We found that tadpoles’ toxin content varied markedly among populations, and the number of compounds per tadpole also differed between two geographical regions. The most consistent predictor of toxicity was the strength of competition, indicating that tadpoles produced more compounds and larger amounts of toxins when coexisting with more competitors. Additionally, tadpoles tended to contain larger concentrations of bufadienolides in ponds that were less prone to desiccation, suggesting that the costs of toxin production can only be afforded by tadpoles that do not need to drastically speed up their development. Interestingly, this trade-off was not alleviated by higher food abundance, as periphyton biomass had negligible effect on chemical defense. Even more surprisingly, we found no evidence that higher predation risk enhances chemical defenses, suggesting that low predictability of predation risk and high mortality cost of low toxicity might select for constitutive expression of chemical defense irrespective of the actual level of predation risk. Our findings highlight that the variation in chemical defense may be influenced by environmental heterogeneity in both the need for, and constraints on, toxicity as predicted by optimal defense theory.     

Synthesis,Chiral Separation,Absolute Configuration Assignment,and Biological Activity of Enantiomers of Retro‐1 as Potent Inhibitors of Shiga Toxin

The Shiga toxin (Stx) family is composed of related protein toxins produced by the bacteria Shigella dysenteriae and certain pathogenic strains of E. coli. No effective therapies for Stx intoxication have been developed yet. However, inhibitors that act on the intracellular trafficking of these toxins may provide new options for the development of therapeutic strategies. This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of Retro‐1, a compound active against Stx and other such protein toxins. Retro‐1 works by inhibiting retrograde transport of these toxins inside cells. In vitro experiments proved that the configuration of the stereocenter at position 5 is not crucial for the activity of this compound. X‐ray diffraction data revealed (S)‐Retro‐1 to be slightly more active than (R)‐Retro‐1.