Biochemical,pharmacological and genomic characterisation of Ts IV,an α-toxin from the venom of the South American scorpion Tityus serrulatus

The venom of the scorpion, Tityus serrulatus, was fractionated to investigate the chemical and pharmacological properties of its α-toxin content. Three α-toxins (Ts III, Ts IV and Ts V) were purified by conventional chromatography (gel filtration and ion-exchange chromatography), followed by immunoaffinity chromatography. Competition experiments using reference α- and β-toxins suggested that these α-toxins were contaminated with around 0.01% of β-toxin. The sequence of the first 30 amino acids of Ts IV was established. Using an oligonucleotide probe, a cDNA encoding its precursor was cloned from a venom gland cDNA library. The primary structure deduced from the cDNA nucleotide sequence provides possible explanations for the polymorphism of these three molecules.     

Evolutionary diversification of Mesobuthus α-scorpion toxins affecting sodium channels

α-Scorpion toxins constitute a family of peptide modulators that induce a prolongation of the action potential of excitable cells by inhibiting voltage-gated sodium channel inactivation. Although they all adopt a conserved structural scaffold, the potency and phylogentic preference of these toxins largely vary, which render them an intriguing model for studying evolutionary diversification among family members. Here, we report molecular characterization of a new multigene family of α-toxins comprising 13 members (named MeuNaTxα-1 to MeuNaTxα-13) from the scorpion Mesobuthus eupeus. Of them, five native toxins (MeuNaTxα-1 to -5) were purified to homogeneity from the venom and the solution structure of MeuNaTxα-5 was solved by nuclear magnetic resonance. A systematic functional evaluation of MeuNaTxα-1, -2, -4, and -5 was conducted by two-electrode voltage-clamp recordings on seven cloned mammalian voltage-gated sodium channels (Na(v)1.2 to Na(v)1.8) and the insect counterpart DmNa(v)1 expressed in Xenopus oocytes. Results show that all these four peptides slow inactivation of DmNa(v)1 and are inactive on Na(v)1.8 at micromolar concentrations. However, they exhibit differential specificity for the other six channel isoforms (Na(v)1.2 to Na(v)1.7), in which MeuNaTxα-4 shows no activity on these isoforms and thus represents the first Mesobuthus-derived insect-selective α-toxin identified so far with a half maximal effective concentration of 130 ± 2 nm on DmNa(v)1 and a half maximal lethal dose of about 200 pmol g(-1) on the insect Musca domestica; MeuNaTxα-2 only affects Na(v)1.4; MeuNaTxα-1 and MeuNaTxα-5 have a wider range of channel spectrum, the former active on Na(v)1.2, Na(v)1.3, Na(v)1.6, and Na(v)1.7, whereas the latter acting on Na(v)1.3-Na(v)1.7. Remarkably, MeuNaTxα-4 and MeuNaTxα-5 are two nearly identical peptides differing by only one point mutation at site 50 (A50V) but exhibit rather different channel subtype selectivity, highlighting a switch role of this site in altering the target specificity. By the maximum likelihood models of codon substitution, we detected nine positively selected sites (PSSs) that could be involved in functional diversification of Mesobuthus α-toxins. The PSSs include site 50 and other seven sites located in functional surfaces of α-toxins. This work represents the first thorough investigation of evolutionary diversification of α-toxins derived from a specific scorpion lineage from the perspectives of sequence, structure, function, and evolution.     

To4, the first Tityus obscurus β-toxin fully electrophysiologically characterized on human sodium channel isoforms

Many scorpion toxins that act on sodium channels (NaScTxs) have been characterized till date. These toxins may act modulating the inactivation or the activation of sodium channels and are named α- or β-types, respectively. Some venom toxins from Tityus obscurus (Buthidae), a scorpion widely distributed in the Brazilian Amazon, have been partially characterized in previous studies; however, little information about their electrophysiological role on sodium ion channels has been published. In the present study, we describe the purification, identification and electrophysiological characterization of a NaScTx, which was first described as Tc54 and further fully sequenced and renamed To4. This toxin shows a marked β-type effect on different sodium channel subtypes (hNa_v1.1–hNa_v1.7) at low concentrations, and has more pronounced activity on hNa_v1.1, hNa_v1.2 and hNa_v1.4. By comparing To4 primary structure with other Tityus β-toxins which have already been electrophysiologically tested, it is possible to establish some key amino acid residues for the sodium channel activity. Thus, To4 is the first toxin from T. obscurus fully electrophysiologically characterized on different human sodium channel isoforms.     

Anti-influenza active and low toxic N-phenyl-substituted β-amidoamidines structurally related to natural antibiotic amidinomycin

A set of racemic N-phenyl-substituted β-amidoamidines hydrochlorides 4, which are structurally related to natural antiviral agent amidinomycin (1), was synthesized in four steps starting from methacryloyl anilide (5). In the final step of the synthetic route, an uncommon monoacylation of β-aminoamidine 8 at the less reactive β-phenylamino-group took place. To rationalize this result, a mechanism which involves initial acylation at the more active amidine-function followed by intramolecular acyl-group transfer to β-phenylamino-group was suggested. All three β-amidoamidines 4d–f bearing long linear aliphatic chain (from n-C₈H₁₇ to n-C₁₂H₂₅) revealed significant in vitro activity against influenza A virus (H3N2) and modest cytotoxicity. The in vitro antiviral potency of 4d,e is 6–20 times greater than that of commercial rimantadine with lower EC₅₀ values and higher therapeutic index. The non-toxic in vivo compounds 4d–f showed a beneficial protective effect in influenza A (H3N2) infected mice.     

Membrane damage by staphylococcal α-toxin to different types of cultured mammalian cell

Staphylococcal α-toxin was shown to be more membrane-damaging to epithelial-like cells than to neuroblasts or normal fibroblasts. Mouse adrenal cortex tumor (Y₁Ac) epithelioid cells and human embryonal lung (MRC-5) fibroblasts were used for further comparison. Alpha-toxin was considerably more cytotoxic to adrenal cells than to fibroblasts. This difference did not depend on the presence of fibronectin on the fibroblast surface, or on a general difference in the response to other membrane-damaging hemolytic toxins and detergents. Incubation of adrenal cells at 0°C with α-toxin induced some irreversible change, and membrane damage and a cytotoxic effect developed upon further incubation in toxin-free growth medium. In fibroblasts the membrane damage progressed slowly and only in the continued presence of the toxin. Toxin-induced damage to transport and synthetic functions in fibroblasts was reversible upon removal of the toxin after prolonged exposure. It is proposed that adrenal cells may carry a cell-surface receptor to which α-toxin binds specifically, thereby allowing the toxin to exert its cell damaging effect.     

Scorpion toxins from Buthus martensii Karsch all possess a predicted α-tight-turn

We have purified a new toxin (BmK 17[4]) from Asian scorption (Buthus martensii Karsch) venom that possesses a distinctive structural motif in its N-terminal (positions 8–12) that is similarly found in two other previously described α-like toxins. BmK 17[4] prolongs action potentials (APs) in frog nerve and was purified using gel filtration, ion exchange, fast protein liquid chromatography (FPLC), and high-performance liquid chromatography (HPLC). BmK 17[4] significantly prolonged frog APs but it did not alter APs from an insect ventral nerve cord at similar doses. When applied to voltage-clamped frog muscle single fibers, BmK 17[4] prolonged fast inactivation. Because the polypeptide prolongs APs when both K⁺ and Ca²⁺ channels were blocked, BMK 17[4] acts to selectively alter Na⁺ channel inactivation. The N-terminal sequence of BmK 17[4] was found to be VRDAYIAKPENCVYXC—. The molar mass of BmK 17[4] was determined by LC/MS/MS to be 7097 Daltons. The N-terminal motif (KPENC), which introduces a reverse turn in residues 8–12, does not appear in previously characterized BmK α-toxins and may be characteristic of α-like toxins. Sequence similarity database searches were used to test whether the N-terminal sequences of α-like polypeptide toxins from B. martensii Karsch possess a distinctive structural motif in its 5-residue reverse turn (α-turn) that is conserved. Sequence similarities with putative polypetides encoded by cDNAs obtained from a cDNA library [Zhu, S. Y., Li, W. X., Zenq, X. C., et al. (2000) Nine novel precursors of Buthus martensii scorpiox alpha-toxin homologues. Toxicon 38, 1653–1661] from BmK venom glands showed that an active polypeptide toxin cleaved from the putative propolypeptide toxin BmK M9 is likely identical to BmK 17[4]. Sequence comparisons with toxins and putative toxins from B. martensii Karsch and other species revealed that a group of these toxins possess a common structural motif in their α-turn. A neighbor-joining phylogenetic analysis suggests that there are two phylogenetic sister groups of related BmK polypeptides; one possesses the KPENC motif and the other possesses a modifed version (KPHNC) of it.     

The giant panda is closer to a bear,judged by α- and β-hemoglobin sequences

Summary Using - and -hemoglobin sequences, we made a maximum likelihood inference as to the phylogenetic relationship among carnivores, including the two pandas, giant and lesser. Molecular phylogenetic studies up to 1985 had consistently indicated that the giant panda is more closely related to the bear than to the lesser panda. In 1986, however, a contradictory tree was constructed, using hemoglobins and so on, in which the two pandas link together (Tagle et al. 1986). In contrast to that tree, our conclusion supports the close relationship between bear and giant panda. The surface impression of a close relationship between the two pandas drawn from pairwise amino acid differences is explained by a rapid rate of hemoglobin evolution in the bear compared to that in the two pandas.Offprint requests to: T. Hashimoto     

Molecular characterization of a novel β-1,3-exoglucanase related to mycoparasitism of Trichoderma harzianum

Trichoderma harzianum, a soil-borne filamentous fungus, is capable of parasitizing several plant pathogenic fungi. Secretion of lytic enzymes, mainly glucanases and chitinases, is considered the most crucial step of the mycoparasitic process. The lytic enzymes degrade the cell walls of the pathogenic fungi, enabling Trichoderma to utilize both their cell walls and cellular contents for nutrition. We have purified a 110 kDa novel extracellular β-1,3-exoglucanase from T. harzianum, grown with laminarin or in dual cultures with host fungi. The corresponding gene, lam1.3, and its cDNA were isolated and their nucleotide sequences determined. The deduced amino-acid sequence predicted a molecular mass of 110.7 kDa of a mature protein excluding a signal peptide. LAM1.3 showed high homology to EXG1, a β-1,3-exoglucanase of the phytopathogenic fungus Cochliobolus carbonum, and a lower homology to BGN13.1, a β-1,3-endoglucanase isolated from T. harzianum. However, it contains a unique C-terminal embodying cysteine motifs. The expression of lam1.3 in growth with laminarin, but not with glucose, was found to be a result of differential accumulation of the corresponding mRNA.     

Klotho is a substrate for α-, β- and γ-secretase

Klotho is an anti-aging protein with different functions of the full-length membrane protein and the secreted hormone-like form. Using overexpression and knock-down approaches as well as embryonic fibroblasts of knock-out mice we present evidence that Klotho is shedded by the α-secretases ADAM10 and 17 as well as by the β-secretase β-APP cleaving enzyme 1. The remaining membrane-bound fragment is a substrate for regulated intramembrane proteolysis by γ-secretase. Our data suggest that therapeutic approaches targeting these proteases should be carefully analyzed for potential side effects on Klotho-mediated physiological processes.     

Cardiovascular effects of peptides related to the enkephalins and β-casomorphin

In the urethane-anesthetized rat, intravenous injections of morphine produced a short-lasting fall in heart rate and blood pressure. The fall in heart rate (which is vagal in origin) was used here to bioassay peptides related to the enkephalins [-enk] and β-casomorphin [β-C, H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH]. A > 10% decrease in heart rate was used as a quantal index of response and the intravenous ED50 [μmol/kg] were estimated as: methionine-enk, 1.3 ± .24; leucine-enk, 1.5 ± .20; [D-Ala², D-Leu⁵]-enk, 0.45 ± .05; [D-Ala², NMePhe⁴, Met(0)⁵-ol]-enk, 0.0011 ± .0002; H-Tyr-Arg-OH, > 2.0; β-C (1–7), > 2.0; β-C(1–5), > 2.0; β-C(1–4), > 4.0; β-C(1–3), > 2.0; morphine sulfate, 0.11 ± .03; and human β-endorphin,, 0.07 ± .01. One β-C derivative, H-Tyr-Pro-Phe-Pro-NH₂ [β-C(1–4)-NH₂], was active at 0.32 ± .08. Naloxone pretreatment blocked the bradycardia produced by the enkephalins and β-C(1–4)-NH₂. The bioassay described here, based on heart rate, may prove to be useful for the rapid detection and estimation of the $\text{in vivo}$ pharmacological activities of new opioid peptides.     

Scorpion β-toxin interference with NaV channel voltage sensor gives rise to excitatory and depressant modes

Scorpion β toxins, peptides of ～70 residues, specifically target voltage-gated sodium (Na(V)) channels to cause use-dependent subthreshold channel openings via a voltage-sensor trapping mechanism. This excitatory action is often overlaid by a not yet understood depressant mode in which Na(V) channel activity is inhibited. Here, we analyzed these two modes of gating modification by β-toxin Tz1 from Tityus zulianus on heterologously expressed Na(V)1.4 and Na(V)1.5 channels using the whole cell patch-clamp method. Tz1 facilitated the opening of Na(V)1.4 in a use-dependent manner and inhibited channel opening with a reversed use dependence. In contrast, the opening of Na(V)1.5 was exclusively inhibited without noticeable use dependence. Using chimeras of Na(V)1.4 and Na(V)1.5 channels, we demonstrated that gating modification by Tz1 depends on the specific structure of the voltage sensor in domain 2. Although residue G658 in Na(V)1.4 promotes the use-dependent transitions between Tz1 modification phenotypes, the equivalent residue in Na(V)1.5, N803, abolishes them. Gating charge neutralizations in the Na(V)1.4 domain 2 voltage sensor identified arginine residues at positions 663 and 669 as crucial for the outward and inward movement of this sensor, respectively. Our data support a model in which Tz1 can stabilize two conformations of the domain 2 voltage sensor: a preactivated outward position leading to Na(V) channels that open at subthreshold potentials, and a deactivated inward position preventing channels from opening. The results are best explained by a two-state voltage-sensor trapping model in that bound scorpion β toxin slows the activation as well as the deactivation kinetics of the voltage sensor in domain 2.     

Transformation of a Fragment of β-Structural Bacteriophage T4 Adhesin to Stable α-Helical Trimer

Gene product 12 of bacteriophage T4, adhesin, serves to adhere the virus to host cells. Adhesin is a fibrous homotrimer, and a novel tertiary structure element, a -helix, is supposed to be a major structural feature of this protein. We have constructed two truncated gp12 mutants, 12N1 and 12N2, containing 221 and 135 N-terminal residues, respectively. When expressed in E. coli cells, these gp12 fragments formed labile -structural trimers. Another hybrid protein, 12FN, containing 179 N-terminal amino acid residues of gp12 fused to the C-terminal domain (31 amino acids) of T4 fibritin, was shown to have a trimeric proteolytically resistant a-helical structure. This structure is probably similar to that of fibritin, which has a triple -helical coiled-coil structure. Hence, we have demonstrated the possibility of global transformation of fibrous protein structure using fusion with a C-terminal domain that initiates trimerization.     

Phospholipid hydrolysis caused by Clostridium perfringens α-toxin facilitates the targeting of perfringolysin O to membrane bilayers

Clostridium perfringens causes gas gangrene and gastrointestinal disease in humans. These pathologies are mediated by potent extracellular protein toxins, particularly α-toxin and perfringolysin O (PFO). While α-toxin hydrolyzes phosphatidylcholine and sphingomyelin, PFO forms large transmembrane pores on cholesterol-containing membranes. It has been suggested that the ability of PFO to perforate the membrane of target cells is dictated by how much free cholesterol molecules are present. Given that C. perfringens α-toxin cleaves the phosphocholine headgroup of phosphatidylcholine, we reasoned that α-toxin may increase the number of free cholesterol molecules in the membrane. Our present studies reveal that α-toxin action on membrane bilayers facilitates the PFO?cholesterol interaction as evidenced by a reduction in the amount of cholesterol required in the membrane for PFO binding and pore formation. These studies suggest a mechanism for the concerted action of α-toxin and PFO during C. perfringens pathogenesis.     

How much can a T-cell antigen receptor adapt to structurally distinct antigenic peptides?

Binding degeneracy is thought to constitute a fundamental property of the T-cell antigen receptor (TCR), yet its structural basis is poorly understood. We determined the crystal structure of a complex involving the BM3.3 TCR and a peptide (pBM8) bound to the H-2K(bm8) major histocompatibility complex (MHC) molecule, and compared it with the structures of the BM3.3 TCR bound to H-2K(b) molecules loaded with two peptides that had a minimal level of primary sequence identity with pBM8. Our findings provide a refined structural view of the basis of BM3.3 TCR cross-reactivity and a structural explanation for the long-standing paradox that a TCR antigen-binding site can be both specific and degenerate. We also measured the thermodynamic features and biological penalties that incurred during cross-recognition. Our data illustrate the difficulty for a given TCR in adapting to distinct peptide-MHC surfaces while still maintaining affinities that result in functional in vivo responses. Therefore, when induction of protective effector T cells is used as the ultimate criteria for adaptive immunity, TCRs are probably much less degenerate than initially assumed.     

Characterization of a cDNA encoding a protein with limited similarity to β1, 3-N-acetylglucosaminyltransferase

Glycosyltransferases constitute a large group of enzymes that are involved in a wide range of functions in all living organisms. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human putative glycosyltransferase gene named beta3GnTL1. Its cDNA is 1372 base pair in length, encoding a predicted protein with 361 amino acid residues. The human beta3GnTL1 is located to chromosome 17q25.3 by comparison of its cDNA with human gemome database. RT-PCR result shows the beta3GnTL1 is expressed at various levels in most of tissues examined.     

Binding of neuronal α-synuclein to β-III tubulin and accumulation in a model of multiple system atrophy

Multiple system atrophy (MSA) is a neurodegenerative disease caused by α-synuclein (α-syn) accumulation in oligodendrocytes and neurons. We generated a transgenic (Tg) mouse model in which human α-syn was overexpressed in oligodendrocytes. Our previous studies have revealed that oligodendrocytic α-syn inclusions induced neuronal α-syn accumulation, thereby resulting in progressive neuronal degeneration in mice. We also demonstrated that an insoluble complex of α-syn and β-III tubulin in microtubules progressively accumulated in neurons, thereby leading to neuronal degeneration. In the present study, we demonstrated that neuronal accumulation of the insoluble complex was derived from binding of α-syn to β-III tubulin and not from α-syn self-aggregation. Thus, interaction between α-syn and β-III tubulin plays an important role in neuronal α-syn accumulation in an MSA mouse model.