Solution structure and lipid membrane partitioning of VSTx1, an inhibitor of the KvAP potassium channel

VSTx1 is a voltage sensor toxin from the spider Grammostola spatulata that inhibits KvAP, an archeabacterial voltage-activated K(+) channel whose X-ray structure has been reported. Although the receptor for VSTx1 and the mechanism of inhibition are unknown, the sequence of the toxin is related to hanatoxin (HaTx) and SGTx, two toxins that inhibit eukaryotic voltage-activated K(+) channels by binding to voltage sensors. VSTx1 has been recently shown to interact equally well with lipid membranes that contain zwitterionic or acidic phospholipids, and it has been proposed that the toxin receptor is located within a region of the channel that is submerged in the membrane. As a first step toward understanding the inhibitory mechanism of VSTx1, we determined the three-dimensional solution structure of the toxin using NMR. Although the structure of VSTx1 is similar to HaTx and SGTx in terms of molecular fold and amphipathic character, the detailed positions of hydrophobic and surrounding charged residues in VSTx1 are very different than what is seen in the other toxins. The amphipathic character of VSTx1, notably the close apposition of basic and hydrophobic residues on one face of the toxin, raises the possibility that the toxin interacts with interfacial regions of the membrane. We reinvestigated the partitioning of VSTx1 into lipid membranes and find that VSTx1 partitioning requires negatively charged phospholipids. Intrinsic tryptophan fluorescence and acrylamide quenching experiments suggest that tryptophan residues on the hydrophobic surface of VSTx1 have a diminished exposure to water when the toxin interacts with membranes. The present results suggest that if membrane partitioning is involved in the mechanism by which VSTx1 inhibits voltage-activated K(+) channels, then binding of the toxin to the channel would likely occur at the interface between the polar headgroups and the hydrophobic phase of the membrane.     

Comparison of Clostridium botulinum toxins type D and C1 in molecular property, antigenicity and binding ability to rat-brain synaptosomes

Botulinum type D neurotoxin was purified 950-fold from the culture supernatant with an overall yield of 32%. The purified toxin had a specific toxicity of 5.8 X 10(7) mouse minimal lethal dose per mg of protein and a relative molecular mass of 140000. The purified toxin had a di-chain structure consisting of heavy and light chains with relative molecular masses of 85000 and 55000, respectively, linked by one disulfide bond. These subunits had different amino acid compositions and antigenicities. A similarity in molecular constructions and amino acid compositions was observed between type D and type C1 toxins as well as between their subunits. Among the seven kinds of monoclonal antibodies against type D toxin, six reacted with the heavy chain of type D toxin, while one of the six also reacted with the heavy chain of type C1 toxin and neutralized the toxicities of the two toxins. The other one of monoclonal antibodies reacted with the light chains of both toxins. This evidence indicates that both toxins have common antigenic sites on their heavy and light chains and that the antigenic site on the heavy chain may contribute to the neutralization of both toxins by antibody. The binding of type D toxin to rat brain synaptosomes was examined by use of 125I-labelled type D toxin. The binding was competitively inhibited not only by unlabelled type D and C1 toxins, but also by the heavy chains of both toxins, however, it was not inhibited by the light chain of type D toxin. These results suggest that the toxin receptors on synaptosomal membrane are common for type D and C1 toxins, and that the heavy chain contributes to the binding of toxin to synaptosomes and the structure of the binding sites on the heavy chains of both toxins is quite similar.     

Structure and Orientation of a Voltage-Sensor Toxin in Lipid Membranes

Amphipathic protein toxins from tarantula venom inhibit voltage-activated potassium (Kv) channels by binding to a critical helix-turn-helix motif termed the voltage sensor paddle. Although these toxins partition into membranes to bind the paddle motif, their structure and orientation within the membrane are unknown. We investigated the interaction of a tarantula toxin named SGTx with membranes using both fluorescence and NMR spectroscopy. Depth-dependent fluorescence-quenching experiments with brominated lipids suggest that Trp³⁰ in SGTx is positioned ∼9 Å from the center of the bilayer. NMR spectra reveal that the inhibitor cystine knot structure of the toxin does not radically change upon membrane partitioning. Transferred cross-saturation NMR experiments indicate that the toxin's hydrophobic protrusion contacts the hydrophobic core of the membrane, whereas most surrounding polar residues remain at interfacial regions of the bilayer. The inferred orientation of the toxin reveals a twofold symmetry in the arrangement of basic and hydrophobic residues, a feature that is conserved among tarantula toxins. These results have important implications for regions of the toxin involved in recognizing membranes and voltage-sensor paddles, and for the mechanisms by which tarantula toxins alter the activity of different types of ion channels.     

The modulation of thermal properties of vinblastine by cholesterol in membrane bilayers

It has been shown that the partitioning of vinblastine in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) single and multiple bilayer dispersions induces partial interdigitation of the lipid alkyl chains. Similar behavior has been observed for abietic and ursodeoxycholic acids and may well be generalized for the partitioning of bulky amphoteric molecules, which tend to localize in the vicinity of the polar heads. For the present study, differential scanning calorimetry (DSC) has been employed to investigate the role of lipid molecular characteristics such as the alkyl chain length and the polarity of the head-group, as well as the impact of cholesterol upon vinblastine-induced interdigitation. It is found that vinblastine does not induce interdigitation in lipids with either shorter or longer alkyl chains than DPPC, or having head-groups of different polarity. In addition, it is shown that the presence of cholesterol in the lipid bilayer tends to modulate the phase behavior of the lipid/vinblastine bilayer system. Preliminary studies show that such properties directly affect the encapsulation efficiency and the pharmacokinetics of liposomes.     

The modulation of thermal properties of vinblastine by cholesterol in membrane bilayers

It has been shown that the partitioning of vinblastine in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) single and multiple bilayer dispersions induces partial interdigitation of the lipid alkyl chains. Similar behavior has been observed for abietic and ursodeoxycholic acids and may well be generalized for the partitioning of bulky amphoteric molecules, which tend to localize in the vicinity of the polar heads. For the present study, differential scanning calorimetry (DSC) has been employed to investigate the role of lipid molecular characteristics such as the alkyl chain length and the polarity of the head-group, as well as the impact of cholesterol upon vinblastine-induced interdigitation. It is found that vinblastine does not induce interdigitation in lipids with either shorter or longer alkyl chains than DPPC, or having head-groups of different polarity. In addition, it is shown that the presence of cholesterol in the lipid bilayer tends to modulate the phase behavior of the lipid/vinblastine bilayer system. Preliminary studies show that such properties directly affect the encapsulation efficiency and the pharmacokinetics of liposomes.     

Comparison of three classes of snake neurotoxins by homology modeling and computer simulation graphics

We present a systematic structure comparison of three major classes of postsynaptic snake toxins, which include short and long chain alpha-type neurotoxins plus one angusticeps-type toxin of black mamba snake family. Two novel alpha-type neurotoxins isolated from Taiwan cobra (Naja naja atra) possessing distinct primary sequences and different postsynaptic neurotoxicities were taken as exemplars for short and long chain neurotoxins and compared with the major lethal short-chain neurotoxin in the same venom, i.e., cobrotoxin, based on the derived three-dimensional structure of this toxin in solution by NMR spectroscopy. A structure comparison among these two alpha-neurotoxins and angusticeps-type toxin (denoted as FS2) was carried out by the secondary-structure prediction together with computer homology-modeling based on multiple sequence alignment of their primary sequences and established NMR structures of cobrotoxin and FS2. It is of interest to find that upon pairwise superpositions of these modeled three-dimensional polypeptide chains, distinct differences in the overall peptide flexibility and interior microenvironment between these toxins can be detected along the three constituting polypeptide loops, which may reflect some intrinsic differences in the surface hydrophobicity of several hydrophobic peptide segments present on the surface loops of these toxin molecules as revealed by hydropathy profiles. Construction of a phylogenetic tree for these structurally related and functionally distinct toxins corroborates that all long and short toxins present in diverse snake families are evolutionarily related to each other, supposedly derived from an ancestral polypeptide by gene duplication and subsequent mutational substitutions leading to divergence of multiple three-loop toxin peptides.     

Mutation of the toxin binding site of PP-1c: comparison with PP-2B

The catalytic cores of PP-1c and PP-2B (calcineurin) are structurally conserved. However, PP-2B is resistant to inhibition by toxins of the okadaic acid and cyclic peptide classes, while PP-1c is potently inhibited. Molecular docking of the structure of microcystin-LR onto the catalytic core of PP-2B identified residues that may be responsible for blocking access of toxins to the catalytic site. Amino acids in PP-1c were substituted with these PP-2B residues to investigate their contribution to PP-2B toxin resistance. Mutants of PP-1c were also produced to test the importance of hydrophobic interactions to toxin binding. Our results suggest that different classes of toxin inhibitors interact with the same hydrophobic side chains of PP-1c through different mechanisms. Substitution of amino acids in PP-1c with PP-2B residues demonstrated no highly significant changes in toxin inhibition. We hypothesize that an interaction outside the catalytic core causing the L7 loop of PP-2B to block the catalytic site may be responsible for PP-2B resistance to toxins.     

Analysis of the physicochemical interactions between Clostridium difficile toxins and cholestyramine using liquid chromatography with post-column derivatization

A potential therapy for antibiotic-associated pseudomembranous colitis is to bind Clostridium difficile toxins A and B using cholestyramine, a hydrophobic anion exchange medium. Frontal analysis in isotonic phosphate buffer was studied using post-column derivatization with o-phthalaldehyde, which gave a highly sensitive (> or =30 ng) flow-through analysis. Following load (1.5-3.0 microg toxin/3.6 mg), toxin A was bound at a slightly higher capacity than B, due to slower kinetics. A salt gradient eluted roughly 20% of bound toxin A with 0.6 M NaCl and toxin B with 1.1 M NaCl, hence toxin A showed weaker electrostatic affinity. The remainder of toxin A (65%) and some of toxin B (10% out of 50%) were eluted using a subsequent gradient to 60% acetonitrile in normal saline, which measured predominantly hydrophobic binding. Low and high affinity populations of both toxins were observed. Glycocholic acid or amino acids were competitive binders, although these components had little effect on the toxin A population bound primarily through ionic interactions. Competitive protein constituents in hamster cecal contents were also profiled. These results help to explain the variable clinical response in using cholestyramine to treat colitis. Using quaternary amine-polyhydroxymethacrylate (PHM) ion exchange chromatography, a trend for increased binding at higher pH was observed, especially for toxin A. Binding to strong cation exchange resins (sulfonate-PHM) was not observed. A range of reversed phase media retained both toxins, although recovery was very poor relative to protein standards. Size exclusion chromatography with light scattering detection showed that toxin B exists in different aggregation states, while toxin A remains monomeric.     

Cytotoxic properties of DAB486EGF and DAB389EGF, epidermal growth factor (EGF) receptor-targeted fusion toxins

Elevated expression of the receptor for epidermal growth factor (EGF) is a characteristic of several malignancies including those of the breast, bladder, prostate, lung, and neuroglia. To therapeutically target the cytotoxic action of diphtheria toxin to EGF receptor-expressing tumor cells, we have constructed a hybrid gene in which the sequences for the binding domain of diphtheria toxin have been replaced by those for human EGF. The resulting fusion toxins, DAB486EGF and DAB389EGF, bind specifically to the EGF receptor and inhibit protein synthesis in a variety of EGF receptor expressing human tumor cell lines with an IC50 as low as 0.1 pM. Comparisons of DAB486EGF and DAB389EGF showed that DAB389EGF was consistently 10- to 100-fold more cytotoxic than DAB486EGF. Like diphtheria toxin, the cytotoxic action of DAB389EGF results from ADP-ribosylation of elongation factor-2 and is sensitive to the action of chloroquine. Studies of the kinetics of cellular intoxication showed that a 15-min exposure of EGF receptor-expressing A431 cells to DAB389EGF results in complete protein synthesis inhibition within 4 h. Furthermore, inhibition of protein synthesis results in elimination of human tumor cell colonies. These findings show that DAB389EGF is a potential therapeutic agent for a wide variety of EGF receptor-expressing solid tumors.     

Enzymatic activation of hydrophobic self-immolative dendrimers: The effect of reporters with ionizable functional groups

Self-immolative dendrimers are uniquely structured molecules that release multiple tail units through a chain fragmentation initiated by a single cleavage at the dendrimer’s core. Although bioactivation of self-immolative dendritic molecules with only two reporter groups was demonstrated, enzymatic activation failed for self-immolative dendrimers with more reporters. These large and hydrophobic dendrimers aggregated under aqueous conditions and enzyme did not efficiently trigger chain fragmentation. Here we demonstrate a simple solution to the problem of enzymatic activation of hydrophobic self-immolative dendrimers. The reporter units on the dendritic platform were equipped with ionizable functional group. Polar interactions with water significantly decreased hydrophobicity of the dendrimers and prevented aggregate formation. Consequently, hydrophobic self-immolative dendrons were effectively activated.     

Modification of cellulose fiber surfaces by use of a lipase and a xyloglucan endotransglycosylase

A strategy for the modification of cellulose fiber surfaces was developed that used the ability of Candida antarctica lipase B (CALB) to acylate carbohydrates with high regioselectivity, combined with the transglycosylating activity of the Populus tremula x P. tremuloides xyloglucan endotransglycosylase 16A (PttXET16A). Xyloglucan oligosaccharides (XGOs) prepared from tamarind xyloglucan were acylated with CALB as a catalyst and vinyl stearate or gamma-thiobutyrolactone as acyl donors to produce carbohydrate molecules with hydrophobic alkyl chains or reactive sulfhydryl groups, respectively. The modified XGOs were shown to act as glycosyl acceptors in the transglycosylation reaction catalyzed by PttXET16A and could therefore be incorporated into high M(r) xyloglucan chains. The resulting xyloglucan molecules exhibited a high affinity for cellulose surfaces, which enabled the essentially irreversible introduction of fatty acid esters or thiol groups to cellulose fibers.     

Peptides of arachnid venoms with insecticidal activity targeting sodium channels

Arachnids have a venom apparatus and secrete a complex chemical mixture of low molecular mass organic molecules, enzymes and polypeptide neurotoxins designed to paralyze or kill their prey. Most of these toxins are specific for membrane voltage-gated sodium channels, although some may also target calcium or potassium channels and other membrane receptors. Scorpions and spiders have provided the greatest number of the neurotoxins studied so far, for which, a good number of primary and 3D structures have been obtained. Structural features, comprising a folding that determines a similar spatial distribution of charged and hydrophobic side chains of specific amino acids, are strikingly common among the toxins from spider and scorpion venoms. Such similarities are, in turn, the key feature to target and bind these proteins to ionic channels. The search for new insecticidal compounds, as well as the study of their modes of action, constitutes a current approach to rationally design novel insecticides. This goal tends to be more relevant if the resistance to the conventional chemical products is considered. A promising alternative seems to be the biotechnological approach using toxin-expressing recombinant baculovirus. Spider and scorpion toxins having insecticidal activity are reviewed here considering their structures, toxicities and action mechanisms in sodium channels of excitable membranes.     

树状分子在提高免疫分析性能方面的应用进展

基于抗原-抗体识别的免疫分析技术在小分子化学性污染物监测领域占有重要地位,已成功应用于农药、兽药、生物毒素等的快速检测,为保障食品安全发挥了重要作用.但是,如何提高小分子半抗原的免疫原性及抗体的亲和力仍然是制约该领域发展的关键技术瓶颈.树状分子作为一类新型的高分子化合物,具有分子组成明确、结构规整、高度支化、纳米尺寸、单分散性以及表面呈现高密度功能团等众多优良的结构特性和良好的组织相容性,在小分子免疫分析领域具有潜在的应用优势.本文主要综述了树状分子作为载体在抗体制备及免疫分析方面的应用,重点介绍了树状分子作为载体在免疫原及包被原制备、作为免疫佐剂提高抗原免疫原性以及作为信号放大载体在提高免疫分析灵敏度等方面的研究现状,最后对其在小分子化学性污染物免疫检测领域的应用前景进行了评述,期望能为本领域研究人员提供借鉴.     

Molecular surface of tarantula toxins interacting with voltage sensors in K(v) channels

The venom from spiders, scorpions, and sea anemone contain a rich diversity of protein toxins that interact with ion channel voltage sensors. Although atomic structures have been solved for many of these toxins, the surfaces that are critical for interacting with voltage sensors are poorly defined. Hanatoxin and SGTx are tarantula toxins that inhibit activation of K(v) channels by interacting with each of the four voltage sensors. In this study we set out to identify the active surface of these toxins by alanine-scanning SGTx and characterizing the interaction of each mutant with the K(v)2.1 channel. Examination of the concentration dependence for inhibition identified 15 mutants with little effect on the concentration dependence for toxin inhibition of the K(v)2.1 channel, and 11 mutants that display moderate to dramatic perturbations. Mapping of these results onto the structure of SGTx identifies one face of the toxin where mutations with pronounced perturbations cluster together, and a backside of the toxin where mutations are well tolerated. The active surface of SGTx contains a ring-like assembly of highly polar residues, with two basic residues that are particularly critical, concentrically arranged around a hydrophobic protrusion containing critical aliphatic and aromatic residues. These results identify the active surface of the toxin and reveal the types of side chains that are important for interacting with voltage sensors.     

Three-dimensional solution structure of a curaremimetic toxin from Naja nigricollis venom: a proton NMR and molecular modeling study.

The solution conformation of toxin alpha from Naja nigricollis (61 amino acids and four disulfides), a snake toxin which specifically blocks the activity of the nicotinic acetylcholine receptor (AcChoR), has been determined using nuclear magnetic resonance spectroscopy and molecular modeling. The solution structures were calculated using 409 distance and 73 dihedral angle restraints. The average atomic rms deviation between the eight refined structures and the mean structure is approximately 0.5 A for the backbone atoms. The overall folding of toxin alpha consists of three major loops which are stabilized by three disulfide bridges and one short C terminal loop stabilized by a fourth disulfide bridge. All the disulfides are grouped in the same region of the molecule, forming a highly constrained structure from which the loops protrude. As predicted, this structure appears to be very similar to the 1.4-A resolution crystal structure of another snake neurotoxin, namely, erabutoxin b from Laticauda semifasciata. The atomic rms deviation for the backbone atoms between the solution and crystal structures is approximately 1.7 A. The minor differences which are observed between the two structures are partly related to the deletion of one residue from the chain of toxin alpha. It is notable that, although the two toxins differ from each other by 16 amino acid substitutions, their side chains have an essentially similar spatial organization. However, most of the side chains which constitute the presumed AcChoR binding site for the curaremimetic toxins are poorly resolved in toxin alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein engineering of diphtheria-toxin-related interleukin-2 fusion toxins to increase cytotoxic potency for high-affinity IL-2-receptor-bearing target cells

We have used site-directed insertion and point mutagenesis in an attempt to increase the cytotoxic potency and receptor-binding affinity of the diphtheria-toxin-related interleukin-2 (IL-2) fusion toxins. Previous studies have demonstrated that both the DAB486-IL-2 and DAB389-IL-2 forms of the fusion toxin consist of three functional domains: the N-terminal fragment-A-associated ADP-ribosyltransferase, the hydrophobic-membrane-associating domains, and the C-terminal receptor-binding domain of human IL-2. By insertion mutagenesis we have increased the apparent flexibility of the polypeptide chain between the membrane-associating domains and the receptor-binding domain of this fusion toxin. In comparison to DAB486-IL-2, the cytotoxic potency of the insertion mutants was increased by approximately 17-fold for high-affinity IL-2-receptor-bearing cell lines in vitro. Moreover, competitive displacement experiments using [125I]rIL-2 demonstrate that the increase in cytotoxic potency correlates with an increase in receptor-binding affinity for both the high and intermediate forms of the IL-2 receptor.     

Mechanisms of plasmid stable maintenance with special focus on plasmid addiction systems

The stable inheritance of bacterial plasmids is achieved by a number of different mechanisms. Among them are resolution of plasmid oligomers into monomers, active plasmid partitioning into dividing cells and selective killing of plasmid-free segregants. A special focus is given to the last mechanism. It involves a stable toxin and an unstable antidote. The antidotes neutralize their cognate toxins or prevent their synthesis. The different decay rates of the toxins and the antidotes underlie molecular mechanisms of toxin activation in plasmid-free cells. By eliminating of plasmid-free cells from the population of plasmid-bearing ones the toxin-antidote couples therefore act as plasmid addiction systems.     

The influence of alkyl pyridinium sponge toxins on membrane properties, cytotoxicity, transfection and protein expression in mammalian cells

The ability of two alkyl pyridinium sponge toxin preparations (poly-APS and halitoxin) to form transient pores/lesions in cell membranes and allow transfection of plasmid cDNA have been investigated using HEK 293 cells. Poly-APS and halitoxin preparations caused a collapse in membrane potential, reductions in input resistance and increased Ca²⁺ permeability. At least partial recovery was observed after poly-APS application but recovery was more rarely seen with halitoxin. The transfection with plasmid cDNAs for an enhanced green fluorescent protein (EGFP) and human tumour necrosis factor receptor 2 (TNFR2) was assessed for both toxin preparations and compared with lipofectamine. Stable transfection was achieved with poly-APS although it was less efficient than lipofectamine. These results show that viable cells transfected with alien cDNA can be obtained using novel transient pore-forming alkyl pyridinium sponge toxins and a simple pre-incubation protocol. This provides the first proof of principle that pore-forming alkyl pyridinium compounds can be used to deliver cDNA to the intracellular environment without permanently compromising the plasma membrane.     

The influence of alkyl pyridinium sponge toxins on membrane properties,cytotoxicity, transfection and protein expression in mammalian cells

The ability of two alkyl pyridinium sponge toxin preparations (poly-APS and halitoxin) to form transient pores/lesions in cell membranes and allow transfection of plasmid cDNA have been investigated using HEK 293 cells. Poly-APS and halitoxin preparations caused a collapse in membrane potential, reductions in input resistance and increased Ca2+ permeability. At least partial recovery was observed after poly-APS application but recovery was more rarely seen with halitoxin. The transfection with plasmid cDNAs for an enhanced green fluorescent protein (EGFP) and human tumour necrosis factor receptor 2 (TNFR2) was assessed for both toxin preparations and compared with lipofectamine. Stable transfection was achieved with poly-APS although it was less efficient than lipofectamine. These results show that viable cells transfected with alien cDNA can be obtained using novel transient pore-forming alkyl pyridinium sponge toxins and a simple pre-incubation protocol. This provides the first proof of principle that pore-forming alkyl pyridinium compounds can be used to deliver cDNA to the intracellular environment without permanently compromising the plasma membrane.     

Lachesis muta (Viperidae) cDNAs reveal diverging pit viper molecules and scaffolds typical of cobra (Elapidae) venoms: implications for snake toxin repertoire evolution

Efforts to describe toxins from the two major families of venomous snakes (Viperidae and Elapidae) usually reveal proteins belonging to few structural types, particular of each family. Here we carried on an effort to determine uncommon cDNAs that represent possible new toxins from Lachesis muta (Viperidae). In addition to nine classes of typical toxins, atypical molecules never observed in the hundreds of Viperidae snakes studied so far are highly expressed: a diverging C-type lectin that is related to Viperidae toxins but appears to be independently originated; an ohanin-like toxin, which would be the third member of the most recently described class of Elapidae toxins, related to human butyrophilin and B30.2 proteins; and a 3FTx-like toxin, a new member of the widely studied three-finger family of proteins, which includes major Elapidae neurotoxins and CD59 antigen. The presence of these common and uncommon molecules suggests that the repertoire of toxins could be more conserved between families than has been considered, and their features indicate a dynamic process of venom evolution through molecular mechanisms, such as multiple recruitments of important scaffolds and domain exchange between paralogs, always keeping a minimalist nature in most toxin structures in opposition to their nontoxin counterparts.