Functional analysis of the α-neurotoxin, BmαTX14, derived from the Chinese scorpion, Buthus martensii Karsch

The gene encoding the BmαTX14 (α-neurotoxin TX14) protein, derived from the cDNA library of the Chinese scorpion Buthus martensii Karsch, was expressed in Pichia pastoris. The recombinant protein was purified by metal chelate affinity chromatography and gel filtration chromatography. Using patch-clamp technique, electrophysiological activity of rBmαTX14 was identified. In the neurons isolated from mice trigeminal root ganglion, the Na⁺ current amplitude was reduced by 80% under whole cell patch-clamp recording. There were no apparent modifications to the gating mechanism in the presence of rBmαTX14. Although BmαTX14 shared a high amino acid sequence similarity with other typical α-toxins, it has different effects on neurons. Further electrophysiological analysis suggested that rBmαTX14 selectively blocked Na⁺ channels and is a member of a new group of scorpion toxins.     

我国不同地区东亚钳蝎（Buthus martensii Karsch）毒在三种…

用ＳＤＳ－不连续聚丙烯酰胺凝胶板电泳、ｐＨ４．５－不连续聚丙烯酰胺凝胶板电泳和聚丙烯酰胺凝胶圆盘等电聚焦电泳,分离我国不同地区东亚钳蝎毒。其图谱表明：不同产地的东亚钳蝎毒蛋白的种类及含量存在一定的差异,地区相近,差异较小,反之亦然。     

我国不同地区东亚钳蝎(Buthus martensii Karsch)毒在三种类型电泳中的比较研究

用SDS—不连续聚丙烯酰胺凝胶板电泳、pH4.5—不连续聚丙烯酰胺凝胶板电泳和聚丙烯酰胺凝胶圆盘等电聚焦电泳,分离我国不同地区东亚钳蝎毒。其图谱表明:不同产地的东亚钳蝎毒蛋白的种类及含量存在一定的差异,地区相近,差异较小,反之亦然。     

Kbot55, purified from Buthus occitanus tunetanus venom,represents the first member of a novel α-KTx subfamily

Kbot55 is a 39 amino acid peptide isolated from the venom of the Tunisian scorpion Buthus occitanus tunetanus. This peptide is cross-linked by 3 disulfide bridges and has a molecular mass of 4128.65 Da. Kbot55 is very low represented in the venom and thus represents a challenge for biochemical characterization. In this study, Kbot55 has been subjected to a screening on ion channels expressed in Xenopus laevis oocytes. It was found that Kbot55 targets voltage-gated potassium channels with high affinity. Kbot55 shows very low amino acid identity with other scorpion potassium toxins and therefore was considered a bona fide novel type of scorpion toxin. Sequence alignment analysis indicated that Kbot55 is the first representative of the new α-Ktx31 subfamily and therefore was classified as α-Ktx31.1.     

Ts17, a Tityus serrulatus β-toxin structurally related to α-scorpion toxins

Ts17 was purified from the venom of the scorpion Tityus serrulatus, the most dangerous scorpion species in Brazil. The activity on Na_v1.1-Na_v1.7 channels was electrophysiologically characterized by patch-clamp technique. Ts17 amino acid sequence indicated high similarity to alpha-scorpion toxins; however, it presented beta-toxin activity, altering the kinetics of the Na⁺-channels. The most affected subtypes during activation (with and without prepulse) and inactivation phases were Na_v1.2 and Na_v1.5, respectively. For recovery from inactivation, the most affected voltage-gated sodium channel was Na_v1.5. Circular dichroism spectra showed that Ts17 presents mainly β-sheet and unordered structures at all analyzed pHs, and the maximum value of α-helix was found at pH 4.0 (13.3 %). Based on the results, Ts17 might be used as a template to develop a new cardiac drug.Key contributionPurification of Ts17 from Tityus serrulatus, electrophysiological characterization of Ts17 on voltage-gated sodium channel subtypes, β-toxin classification.     

Modular Organization of α-Toxins from Scorpion Venom Mirrors Domain Structure of Their Targets,Sodium Channels

To gain success in the evolutionary “arms race,” venomous animals such as scorpions produce diverse neurotoxins selected to hit targets in the nervous system of prey. Scorpion α-toxins affect insect and/or mammalian voltage-gated sodium channels (Na_vs) and thereby modify the excitability of muscle and nerve cells. Although more than 100 α-toxins are known and a number of them have been studied into detail, the molecular mechanism of their interaction with Na_vs is still poorly understood. Here, we employ extensive molecular dynamics simulations and spatial mapping of hydrophobic/hydrophilic properties distributed over the molecular surface of α-toxins. It is revealed that despite the small size and relatively rigid structure, these toxins possess modular organization from structural, functional, and evolutionary perspectives. The more conserved and rigid “core module” is supplemented with the “specificity module” (SM) that is comparatively flexible and variable and determines the taxon (mammal versus insect) specificity of α-toxin activity. We further show that SMs in mammal toxins are more flexible and hydrophilic than in insect toxins. Concomitant sequence-based analysis of the extracellular loops of Na_vs suggests that α-toxins recognize the channels using both modules. We propose that the core module binds to the voltage-sensing domain IV, whereas the more versatile SM interacts with the pore domain in repeat I of Na_vs. These findings corroborate and expand the hypothesis on different functional epitopes of toxins that has been reported previously. In effect, we propose that the modular structure in toxins evolved to match the domain architecture of Na_vs.     

Synthesis of α-galacto-oligosaccharides by a cloned α-galactosidase from Bifidobacterium adolescentis

A genomic library of Bifidobacterium adolescentis was constructed in Escherichia coli and a gene encoding an -galactosidase was isolated. The identified open reading frame showed high similarity and identity with bacterial -galactosidases, which belong to Family 36 of the glycosyl hydrolases. For the purification of the enzyme from the medium a single chromatography step was sufficient. The yield of the recombinant enzyme was 100 times higher than from B. adolescentis itself. In addition to hydrolytic activity the -galactosidase showed transglycosylation activity and can be used for the production of -galacto-oligosaccharides.     

Purification of a non-chloroplastic α-glucan phosphorylase from spinach leaves

The non-chloroplastic -glucan phosphorylase (EC 2.4.1.1) from spinach leaves has been purified to homogeneity as revealed by dodecylsulfate gel electrophoresis. Both purification and separation from the chloroplastic phosphorylase were achieved by chromatography on Sepharose-bound dextrin. The chloroplastic phosphorylase did not bind to Sepharose-dextrin and was removed from the column by washing with buffer, as verified by polyacrylamide gel electrophoresis of the buffer eluate and by chromatography of a preparation from isolated intact chloroplasts. The non-chloroplastic phosphorylase did bind to a high extent to Sepharose-dextrin and could be eluted by a dextrin gradient. Based on dodecylsulfate gel electrophoresis and pyridoxal phosphate determination, a molecular weight of about 90,000 was found for the monomer. Molecular-weight determination by porosity density gradient electrophoresis and gel filtration on Sephadex G-200 suggested that the native enzyme is a dimer, as are other phosphorylases.Abbreviations DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetic acid - G1P glucose 1-phosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - PMSF phenylmethyl sulphonyl fluoride - SDS sodium dodecylsulfate - Tris Tris (hydroxymethyl)aminomethane Dedicated to Professor Dr. A. Pirson on the occasion of his 70th birthday     

Isolation of a Raw Starch-Binding Fragment from Barley α-Amylase

Barley α-amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with β-cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel β-sheets in domain C and the α₇-and α₈-helices of the (α/β)₈ domain.     

A new thermostable α-l-arabinofuranosidase from a novel thermophilic bacterium

An alpha-L-arabinofuranosidase gene was identified in a sequenced genome of a novel thermophilic bacterium, which belongs to the recently described phylum of Thermomicrobia. Amino acid sequence comparison of the enzyme (designated AraF) revealed similarity to glycoside hydrolases of family 51. The gene was cloned into Escherichia coli and its recombinant product expressed and purified. The enzyme appeared to be a hexamer. AraF was optimally active at 70 degrees C (over 10 min) and pH 6 having 92% residual activity after 1 h at 70 degrees C. AraF had a Km) value of 0.6 mM and V(max) value of 122 U mg(-1) on p-nitrophenyl-alpha-L-arabinofuranoside. AraF was almost equally active on branched arabinan and debranched arabinan, properties not previously found in alpha-L-arabinofuranosidases in GH family 51.     

Purification and Characterization of a Novel α-Agarase from a Thalassomonas sp.

An agar-degrading Thalassomonas bacterium, strain JAMB-A33, was isolated from the sediment off Noma Point, Japan, at a depth of 230 m. A novel -agarase from the isolate was purified to homogeneity from cultures containing agar as a carbon source. The molecular mass of the purified enzyme, designated as agaraseA33, was 85 kDa on both SDS-PAGE and gel-filtration chromatography, suggesting that it is a monomer. The optimal pH and temperature for activity were about 8.5 and 45°C, respectively. The enzyme had a specific activity of 40.7 U/mg protein. The pattern of agarose hydrolysis showed that the enzyme is an endo-type -agarase, and the final main product was agarotetraose. The enzyme degraded not only agarose but also agarohexaose, neoagarohexaose, and porphyran.     

Thermostable α-1,4- and α-1,6-glucosidase enzymes from Bacillus sp. Isolated from a marine environment

Penaeus vannamei (the shrimp) is an omnivorous species and it can be assumed that a high level of carbohydrates is necessary for its growth. -1,4- and 1,6-glucosidases are important enzymes necessary for the ultimate liberation of glucose residues from various carbohydrates, principally starch. However, the shrimp's hepatopancreas produces only -1,4-glucosidases, which limits the growth rate in different sources of starch. In order to identify strains with -1,4- and 1,6-glucosidase enzymes with potential uses in shrimp feed production, Bacillus strains were isolated from marine environments. One strain produced large amounts of an extracellular thermostable -glucosidase that permitted good growth on starch. The organism was identified by polymorphism (restriction-fragment-length polymorphism, RFLP), sequenced, and named B. subtilis LMM-12.     

Isolation and characterization of a novel GH67 α-glucuronidase from a mixed culture

Hemicelluloses represent a large reservoir of carbohydrates that can be utilized for renewable products. Hydrolysis of hemicellulose into simple sugars is inhibited by its various chemical substituents. The glucuronic acid substituent is removed by the enzyme α-glucuronidase. A gene (deg75-AG) encoding a putative α-glucuronidase enzyme was isolated from a culture of mixed compost microorganisms. The gene was subcloned into a prokaryotic vector, and the enzyme was overexpressed and biochemically characterized. The DEG75-AG enzyme had optimum activity at 45?°C. Unlike other α-glucuronidases, the DEG75-AG had a more basic pH optimum of 7-8. When birchwood xylan was used as substrate, the addition of DEG75-AG increased hydrolysis twofold relative to xylanase alone.     

Two recombinant α-like scorpion toxins from Mesobuthus eupeus with differential affinity toward insect and mammalian Na channels

α-Scorpion toxins are modulators of voltage-gated Na⁺ channels (Na_vs), which bind to the receptor site 3 to inhibit the fast inactivation of the channels. MeuNaTxα-12 and MeuNaTxα-13 are two new α-scorpion toxin-like peptides identified by cDNA cloning from the scorpion Mesobuthus eupeus with unknown functions. Here, we report their recombinant production, oxidative refolding, structural and functional features. By in vitro renaturation from bacterial inclusion bodies and further purification through reverse phase high-performance liquid chromatography, we obtained high purity recombinant products with a native-like conformation identified by circular dichroism analysis. Two-electrode voltage clamp recordings on five cloned mammalian Na_v subtypes (rNa_v1.1, rNa_v1.2, rNa_v1.4, rNa_v1.5, and mNa_v1.6) and the insect counterpart DmNa_v1, all expressed in Xenopus laevis oocytes, showed that these two peptides inhibited rapid inactivation of the sensitive Na⁺ channels with significant preference for DmNa_v1. The half maximal effective concentrations (EC₅₀) of MeuNaTxα-12 and MeuNaTxα-13 for this channel are 19.95 ± 2.99 nM and 65.50 ± 7.28 nM, respectively, showing 45 and 38 folds higher affinities than for rNa_v1.1, the most sensitive mammalian channel among the five isoforms. Our functional data confirms that these two peptides belong to the α-like scorpion toxin group. A combined analysis of the site 3 sequences and the pharmacological data illuminates the importance of the loop L_D4:S5–S6 of the channel in interacting with the toxins whereas affinity variations between MeuNaTxα-12 and MeuNaTxα-13 highlight a key functional role of a cationic side chain at position 28 of MeuNaTxα-12. Successful expression together with structural and functional characterization of these two new α-like scorpion toxins lays basis for further studies of their structure–function relationship.     

Grafting a short chameleon sequence from αB crystallin into a β-sheet scaffold protein

Many protein and peptide sequences are self-assembled into β-sheet-rich fibrous structures called amyloids. Their atomic details provide insights into fundamental knowledge related to amyloid diseases. To study the detailed structure of the amyloid, we have developed a model system that mimics the self-assembling process of the amyloid within a water-soluble protein, termed peptide self-assembly mimic (PSAM). PSAM enables capturing of a peptide sequence within a water-soluble protein, thus making structural and energetics-related studies possible. In this work, we extend our PSAM approach to a naturally occurring chameleon sequence from αB crystallin. We chose “Val–Leu–Gly–Asp–Val (VLGDV)”, a five amino-acid sequence, which forms a β-turn in the native structure and a β-barrel in the amyloid oligomer cylindrin, as a grafting sequence to the PSAM scaffold. The crystal structure revealed that the sequence grafting induced β-sheet bending at the grafted site. We further investigated the role of the central glycine residue and found that its role in the β-sheet bending is dependent on the neighboring residues. The ability of PSAM to observe the structural alterations induced by the grafted sequence provides an opportunity to evaluate the structural impact of a sequence from the peptide self-assembly.     

Structure-Function Elucidation of a New α-Conotoxin,Lo1a,from Conus longurionis

α-Conotoxins are peptide toxins found in the venom of marine cone snails and potent antagonists of various subtypes of nicotinic acetylcholine receptors (nAChRs). nAChRs are cholinergic receptors forming ligand-gated ion channels in the plasma membranes of certain neurons and the neuromuscular junction. Because nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, nAChR dysfunctions have been implicated in a variety of severe pathologies such as epilepsy, myasthenic syndromes, schizophrenia, Parkinson disease, and Alzheimer disease. To expand the knowledge concerning cone snail toxins, we examined the venom of Conus longurionis. We isolated an 18-amino acid peptide named α-conotoxin Lo1a, which is active on nAChRs. To the best of our knowledge, this is the first characterization of a conotoxin from this species. The peptide was characterized by electrophysiological screening against several types of cloned nAChRs expressed in Xenopus laevis oocytes. The three-dimensional solution structure of the α-conotoxin Lo1a was determined by NMR spectroscopy. Lo1a, a member of the α4/7 family, blocks the response to acetylcholine in oocytes expressing α₇ nAChRs with an IC₅₀ of 3.24 ± 0.7 μm. Furthermore, Lo1a shows a high selectivity for neuronal versus muscle subtype nAChRs. Because Lo1a has an unusual C terminus, we designed two mutants, Lo1a-ΔD and Lo1a-RRR, to investigate the influence of the C-terminal residue. Lo1a-ΔD has a C-terminal Asp deletion, whereas in Lo1a-RRR, a triple-Arg tail replaces the Asp. They blocked the neuronal nAChR α₇ with a lower IC₅₀ value, but remarkably, both adopted affinity for the muscle subtype α₁β₁δϵ.     

Purification and characterization of a maltooligosaccharide-forming α-amylase from a new Bacillus subtilis KCC103

A maltooligosaccharide-forming α-amylase was produced by a new soil isolate Bacillus subtilis KCC103. In contrast to other Bacillus species, the synthesis of α-amylase in KCC103 was not catabolite-repressed. The α-amylase was purified in one step using anion exchange chromatography after concentration of crude enzyme by acetone precipitation. The purified α-amylase had a molecular mass of 53 kDa. It was highly active over a broad pH range from 5 to 7 and stable in a wide pH range between 4 and 9. Though optimum temperature was 65–70 °C, it was rapidly deactivated at 70 °C with a half-life of 7 min and at 50 °C, the half-life was 94 min. The K _m and V _max for starch hydrolysis were 2.6 mg ml⁻¹ and 909 U mg⁻¹, respectively. Ca²⁺ did not enhance the activity and stability of the enzyme; however, EDTA (50 mM) abolished 50% of the activity. Hg²⁺, Ag²⁺, and p-hydroxymercurybenzoate severely inhibited the activity indicating the role of sulfydryl group in catalysis. The α-amylase displayed endolytic activity and formed maltooligosaccharides on hydrolysis of soluble starch at pH 4 and 7. Small maltooligosaccharides (D2–D4) were formed more predominantly than larger maltooligosaccharides (D5–D7). This maltooligosaccharide forming endo-α-amylase is useful in bread making as an antistaling agent and it can be produced economically using low-cost sugarcane bagasse.