Козлов Сергей Александрович

Доктор химических наук

Руководитель подразделения (Лаборатория нейрорецепторов и нейрорегуляторов)

Тел.: +7 (495) 336 4022

Эл. почта: serg@ibch.ru

Избранные публикации

  1. Osmakov D.I., Koshelev S.G., Andreev Y.A., Dyachenko I.A., Bondarenko D.A., Murashev A.N., Grishin E.V., Kozlov S.A. (2015). Conversed mutagenesis of an inactive peptide to ASIC3 inhibitor for active sites determination. Toxicon , [+]

    Peptide Ugr9-1 from the venom of sea anemone Urticina grebelnyi selectively inhibits the ASIC3 channel and significantly reverses inflammatory and acid-induced pain in vivo. A close homolog peptide Ugr 9-2 does not have these features. To find the pharmacophore residues and explore structure-activity relationships of Ugr 9-1, we performed site-directed mutagenesis of Ugr 9-2 and replaced several positions by the corresponding residues from Ugr 9-1. Mutant peptides Ugr 9-2 T9F and Ugr 9-2 Y12H were able to inhibit currents of the ASIC3 channels 2.2 times and 1.3 times weaker than Ugr 9-1, respectively. Detailed analysis of the spatial models of Ugr 9-1, Ugr 9-2 and both mutant peptides revealed the presence of the basic-aromatic clusters on opposite sides of the molecule, each of which is responsible for the activity. Additionally, Ugr9-1 mutant with truncated N- and C-termini retained similar with the Ugr9-1 action in vitro and was equally potent in vivo model of thermal hypersensitivity. All together, these results are important for studying the structure-activity relationships of ligand-receptor interaction and for the future development of peptide drugs from animal toxins.

  2. Dubovskii P.V., Vassilevski A.A., Kozlov S.A., Feofanov A.V., Grishin E.V., Efremov R.G. (2015). Latarcins: versatile spider venom peptides. Cell. Mol. Life Sci. 72 (23), 4501–22 [+]

    Arthropod venoms feature the presence of cytolytic peptides believed to act synergetically with neurotoxins to paralyze prey or deter aggressors. Many of them are linear, i.e., lack disulfide bonds. When isolated from the venom, or obtained by other means, these peptides exhibit common properties. They are cationic; being mostly disordered in aqueous solution, assume amphiphilic α-helical structure in contact with lipid membranes; and exhibit general cytotoxicity, including antifungal, antimicrobial, hemolytic, and anticancer activities. To suit the pharmacological needs, the activity spectrum of these peptides should be modified by rational engineering. As an example, we provide a detailed review on latarcins (Ltc), linear cytolytic peptides from Lachesana tarabaevi spider venom. Diverse experimental and computational techniques were used to investigate the spatial structure of Ltc in membrane-mimicking environments and their effects on model lipid bilayers. The antibacterial activity of Ltc was studied against a panel of Gram-negative and Gram-positive bacteria. In addition, the action of Ltc on erythrocytes and cancer cells was investigated in detail with confocal laser scanning microscopy. In the present review, we give a critical account of the progress in the research of Ltc. We explore the relationship between Ltc structure and their biological activity and derive molecular characteristics, which can be used for optimization of other linear peptides. Current applications of Ltc and prospective use of similar membrane-active peptides are outlined.

  3. Astafieva A.A., Enyenihi A.A., Rogozhin E.A., Kozlov S.A., Grishin E.V., Odintsova T.I., Zubarev R.A., Egorov T.A. (2015). Novel proline-hydroxyproline glycopeptides from the dandelion (Taraxacum officinale Wigg.) flowers: de novo sequencing and biological activity. Plant Sci. 238, 323–9 [+]

    Two novel homologous peptides named ToHyp1 and ToHyp2 that show no similarity to any known proteins were isolated from Taraxacum officinale Wigg. flowers by multidimensional liquid chromatography. Amino acid and mass spectrometry analyses demonstrated that the peptides have unusual structure: they are cysteine-free, proline-hydroxyproline-rich and post-translationally glycosylated by pentoses, with 5 carbohydrates in ToHyp2 and 10 in ToHyp1. The ToHyp2 peptide with a monoisotopic molecular mass of 4350.3Da was completely sequenced by a combination of Edman degradation and de novo sequencing via top down multistage collision induced dissociation (CID) and higher energy dissociation (HCD) tandem mass spectrometry (MS(n)). ToHyp2 consists of 35 amino acids, contains eighteen proline residues, of which 8 prolines are hydroxylated. The peptide displays antifungal activity and inhibits growth of Gram-positive and Gram-negative bacteria. We further showed that carbohydrate moieties have no significant impact on the peptide structure, but are important for antifungal activity although not absolutely necessary. The deglycosylated ToHyp2 peptide was less active against the susceptible fungus Bipolaris sorokiniana than the native peptide. Unique structural features of the ToHyp2 peptide place it into a new family of plant defense peptides. The discovery of ToHyp peptides in T. officinale flowers expands the repertoire of molecules of plant origin with practical applications.

  4. Миков А.Н., Козлов С.А. (2015). Структурные особенности цистеин-богатых полипептидов из ядов морских анемон. Биоорг. хим. 41 (5), 511–523 [+]
    В обзоре рассмотрены известные полипептидные токсины из ядов морских анемон, в первую очередь, с учетом их классификации по структурным признакам. Внутри
    каждого класса проанализированы наиболее яркие функциональные особенности этих полипептидов.
  5. Mikov A.N., Fedorova I.M., Potapieva N.N., Maleeva E.E., Andreev Y.A., Zaitsev A.V., Kim K.K., Bocharov E.V., Bozin T.N., Altukhov D.A., Lipkin A.V., Kozlov S.A., Tikhonov D.B., Grishin E.V. (2015). ω-Tbo-IT1-New Inhibitor of Insect Calcium Channels Isolated from Spider Venom. Sci Rep 5, 17232 [+]

    Novel disulfide-containing polypeptide toxin was discovered in the venom of the Tibellus oblongus spider. We report on isolation, spatial structure determination and electrophysiological characterization of this 41-residue toxin, called ω-Tbo-IT1. It has an insect-toxic effect with LD50 19 μg/g in experiments on house fly Musca domestica larvae and with LD50 20 μg/g on juvenile Gromphadorhina portentosa cockroaches. Electrophysiological experiments revealed a reversible inhibition of evoked excitatory postsynaptic currents in blow fly Calliphora vicina neuromuscular junctions, while parameters of spontaneous ones were not affected. The inhibition was concentration dependent, with IC50 value 40 ± 10 nM and Hill coefficient 3.4 ± 0.3. The toxin did not affect frog neuromuscular junctions or glutamatergic and GABAergic transmission in rat brains. Ca(2+) currents in Calliphora vicina muscle were not inhibited, whereas in Periplaneta americana cockroach neurons at least one type of voltage gated Ca(2+) current was inhibited by ω-Tbo-IT1. Thus, the toxin apparently acts as an inhibitor of presynaptic insect Ca(2+) channels. Spatial structure analysis of the recombinant ω-Tbo-IT1 by NMR spectroscopy in aqueous solution revealed that the toxin comprises the conventional ICK fold containing an extended β-hairpin loop and short β-hairpin loop which are capable of making "scissors-like mutual motions".

  6. Pluzhnikov K.A., Kozlov S.A., Vassilevski A.A., Vorontsova O.V., Feofanov A.V., Grishin E.V. (2014). Linear antimicrobial peptides from Ectatomma quadridens ant venom. Biochimie 107 Pt B, 211–5 [+]

    Venoms from three poneromorph ant species (Paraponera clavata, Ectatomma quadridens and Ectatomma tuberculatum) were investigated for the growth inhibition of Gram-positive and Gram-negative bacteria. It was shown that the venom of E. quadridens and its peptide fraction in particular possess marked antibacterial action. Three linear antimicrobial peptides sharing low similarity to the well-known ponericin peptides were isolated from this ant venom by means of size-exclusion and reversed-phase chromatography. The peptides showed antimicrobial activity at low micromolar concentrations. Their primary structure was established by direct Edman sequencing in combination with mass spectrometry. The most active peptide designated ponericin-Q42 was chemically synthesized. Its secondary structure was investigated in aqueous and membrane-mimicking environment, and the peptide was shown to be partially helical already in water, which is unusual for short linear peptides. Analysis of its activity on different bacterial strains, human erythrocytes and chronic myelogenous leukemia K562 cells revealed that the peptide shows broad spectrum cytolytic activity at micromolar and submicromolar concentrations. Ponericin-Q42 also possesses weak toxic activity on flesh fly larvae with LD50 of ∼105 μg/g.

  7. Vassilevski A.A., Sachkova M.Y., Ignatova A.A., Kozlov S.A., Feofanov A.V., Grishin E.V. (2013). Spider toxins comprising disulfide-rich and linear amphipathic domains: a new class of molecules identified in the lynx spider Oxyopes takobius. FEBS J. 280 (23), 6247–61 [+]

    In addition to the conventional neurotoxins and cytotoxins, venom of the lynx spider Oxyopes takobius was found to contain two-domain modular toxins named spiderines: OtTx1a, 1b, 2a and 2b. These toxins show both insecticidal activity (a median lethal dose against flesh fly larvae of 75 μg·g(-1) ) and potent antimicrobial effects (minimal inhibitory concentrations in the range 0.1-10 μm). Full sequences of the purified spiderines were established by a combination of Edman degradation, mass spectrometry and cDNA cloning. They are relatively large molecules (~ 110 residues, 12.0-12.5 kDa) and consist of two distinct modules separated by a short linker. The N-terminal part (~ 40 residues) contains no cysteine residues, is highly cationic, forms amphipathic α-helical structures in a membrane-mimicking environment, and shows potent cytolytic effects on cells of various origins. The C-terminal part (~ 60 residues) is disulfide-rich (five S-S bonds), and contains the inhibitor cystine knot (ICK/knottin) signature. The N-terminal part of spiderines is very similar to linear cytotoxic peptides found in various organisms, whereas the C-terminal part corresponds to the usual spider neurotoxins. We synthesized the modules of OtTx1a and compared their activity to that of full-length mature toxin produced recombinantly, highlighting the importance of the N-terminal part, which retained full-length toxin activity in both insecticidal and antimicrobial assays. The unique structure of spiderines completes the range of two-domain spider toxins.

  8. Osmakov D.I., Kozlov S.A., Andreev Y.A., Koshelev S.G., Sanamyan N.P., Sanamyan K.E., Dyachenko I.A., Bondarenko D.A., Murashev A.N., Mineev K.S., Arseniev A.S., Grishin E.V. (2013). Sea Anemone Peptide with Uncommon β-Hairpin Structure Inhibits Acid-sensing Ion Channel 3 (ASIC3) and Reveals Analgesic Activity. J. Biol. Chem. 288 (32), 23116–27 [+]

    Three novel peptides were isolated from the venom of the sea anemone Urticina grebelnyi. All of them are 29 amino acid peptides cross-linked by two disulfide bridges, with a primary structure similar to other sea anemone peptides belonging to structural group 9a. The structure of the gene encoding the shared precursor protein of the identified peptides was determined. One peptide, π-AnmTX Ugr 9a-1 (short name Ugr 9-1), produced a reversible inhibition effect on both the transient and the sustained current of human ASIC3 channels expressed in Xenopus laevis oocytes. It completely blocked the transient component (IC50 10 ± 0.6 μm) and partially (48 ± 2%) inhibited the amplitude of the sustained component (IC50 1.44 ± 0.19 μm). Using in vivo tests in mice, Ugr 9-1 significantly reversed inflammatory and acid-induced pain. The other two novel peptides, AnmTX Ugr 9a-2 (Ugr 9-2) and AnmTX Ugr 9a-3 (Ugr 9-3), did not inhibit the ASIC3 current. NMR spectroscopy revealed that Ugr 9-1 has an uncommon spatial structure, stabilized by two S-S bridges, with three classical β-turns and twisted β-hairpin without interstrand disulfide bonds. This is a novel peptide spatial structure that we propose to name boundless β-hairpin.

  9. Andreev Y.A., Kozlov S.A., Korolkova Y.V., Dyachenko I.A., Bondarenko D.A., Skobtsov D.I., Murashev A.N., Kotova P.D., Rogachevskaja O.A., Kabanova N.V., Kolesnikov S.S., Grishin E.V. (2013). Polypeptide Modulators of TRPV1 Produce Analgesia without Hyperthermia. Mar Drugs 11 (12), 5100–15 [+]Transient receptor potential vanilloid 1 receptors (TRPV1) play a significant physiological role. The study of novel TRPV1 agonists and antagonists is essential. Here, we report on the characterization of polypeptide antagonists of TRPV1 based on in vitro and in vivo experiments. We evaluated the ability of APHC1 and APHC3 to inhibit TRPV1 using the whole-cell patch clamp approach and single cell Ca2+ imaging. In vivo tests were performed to assess the biological effects of APHC1 and APHC3 on temperature sensation, inflammation and core body temperature. In the electrophysiological study, both polypeptides partially blocked the capsaicin-induced response of TRPV1, but only APHC3 inhibited acid-induced (pH 5.5) activation of the receptor. APHC1 and APHC3 showed significant antinociceptive and analgesic activity in vivo at reasonable doses (0.01-0.1 mg/kg) and did not cause hyperthermia. Intravenous administration of these polypeptides prolonged hot-plate latency, blocked capsaicin- and formalin-induced behavior, reversed CFA-induced hyperalgesia and produced hypothermia. Notably, APHC3's ability to inhibit the low pH-induced activation of TRPV1 resulted in a reduced behavioural response in the acetic acid-induced writhing test, whereas APHC1 was much less effective. The polypeptides APHC1 and APHC3 could be referred to as a new class of TRPV1 modulators that produce a significant analgesic effect without hyperthermia. ID:994
  10. Dubinnyi M.A., Osmakov D.I., Koshelev S.G., Kozlov S.A., Andreev Y.A., Zakaryan N.A., Dyachenko I.A., Bondarenko D.A., Arseniev A.S., Grishin E.V. (2012). Lignan from thyme possessing inhibitory effect on ASIC3 current. J. Biol. Chem. , [+]

    Novel compound was identified in acidic extract of Thymus armeniacus collected in the Lake Sevan region of Armenia. This compound, named sevanol, to our knowledge is the first low molecular weight natural molecule that has a reversible inhibition effect both on transient and sustained current of human ASIC3 channels expressed in Xenopus laevis oocytes. Sevanol completely blocked the transient component (IC50 353+/-23 μM) and partially (~45%) inhibited the amplitude of sustained component (IC50 of 234+/-53 μM). Other types of ASICs channels were intact to sevanol application except ASIC1a that showed more than 6 times less affinity to it as compared with inhibitory action on ASIC3 channel. To elucidate sevanol structure the set of NMR spectra in two solvents: d6-DMSO and D2O was collected and the complete chemical structure was confirmed by LC-ESI+-MS fragmentation. This compound is a new lignan built up of epiphyllic acid and two isocytril esters in positions 9, 10. In vivo administration of sevanol (1-10 mg/kg) significantly reversed of thermal hyperalgesia induced by complete Freund adjuvant (CFA) injection and reduced response to acid in writhing test. Thus we assume the probable considerable role of sevanol in known analgesic and anti-inflammatory properties of thyme.

  11. Andreev Y.A., Vassilevski A.A., Kozlov S.A. (2012). Molecules to selectively target receptors for treatment of pain and neurogenic inflammation. Recent Pat Inflamm Allergy Drug Discov 6 (1), 35–45 [+]

    Receptors that are involved in generation and transduction of pain signals attract much interest from the scientific and corporate communities. Good commercial prospects for successful development of effective analgesic drugs stimulate significantly the research. This article provides a brief overview of the key molecular targets, i.e. cell receptors, inhibition of which can lead to analgesia. Today transient receptor potential (TRP), purinergic (P2X) receptors and acidsensing ion channels (ASIC) are considered to be the most important proteins for perception of pain stimuli. These ionotropic receptors also participate in the development of inflammation; their hyperactivity leads to many pathological conditions and is closely associated with acute and inflammatory pain. Development of molecules capable to selectively modulate these receptors, their in vitro and in vivo effects, as well as perspectives for practical application described in patents and research articles are reviewed in this paper.

  12. Lazarev V.N., Polina N.F., Shkarupeta M.M., Kostrjukova E.S., Vassilevski A.A., Kozlov S.A., Grishin E.V., Govorun V.M. (2011). Spider venom peptides for gene therapy of Chlamydia infection. Antimicrobial agents and chemotherapy 55 (11), 5367–9 [+]

    Spider venoms are vast natural pharmacopoeias selected by evolution. The venom of the ant spider Lachesana tarabaevi contains a wide variety of antimicrobial peptides. We tested six of them (latarcins 1, 2a, 3a, 4b, 5, and cytoinsectotoxin 1a) for their ability to suppress Chlamydia trachomatis infection. HEK293 cells were transfected with plasmid vectors harboring the genes of the selected peptides. Controlled expression of the transgenes led to a significant decrease of C. trachomatis viability inside the infected cells.

  13. Dubovskii P.V., Vassilevski A.A., Samsonova O.V., Egorova N.S., Kozlov S.A., Feofanov A.V., Arseniev A.S., Grishin E.V. (2011). Novel lynx spider toxin shares common molecular architecture with defense peptides from frog skin. FEBS J. 278 (22), 4382–93 [+]

    A unique 30-residue cationic peptide oxyopinin 4a (Oxt 4a) was identified in the venom of the lynx spider Oxyopes takobius (Oxyopidae). Oxt 4a contains a single N-terminally located disulfide bond, Cys4-Cys10, and is structurally different from any spider toxin studied so far. According to NMR findings, the peptide is disordered in water, but assumes a peculiar torpedo-like structure in detergent micelles. It features a C-terminal amphipathic α-helical segment (body; residues 12-25) and an N-terminal disulfide-stabilized loop (head; residues 1-11), and has an unusually high density of positive charge in the head region. Synthetic Oxt 4a was produced and shown to possess strong and broad-spectrum cytolytic and antimicrobial activity. cDNA cloning showed that the peptide is synthesized in the form of a conventional prepropeptide with an acidic prosequence. Unlike other arachnid toxins, Oxt 4a exhibits striking similarity with defense peptides from the skin of ranid frogs that contain the so-called Rana-box motif (a C-terminal disulfide-enclosed loop). Parallelism or convergence is apparent on several levels: the structure, function and biosynthesis of a lynx spider toxin are mirrored by those of Rana-box peptides from frogs. DATABASE: The protein sequence of oxyopinin 4a (Oxt 4a) has been submitted to the UniProt Knowledgebase (UniProtKB) under the accession number P86350. The coordinates and chemical shifts of Oxt 4a in complex with dodecylphosphocholine micelles have been deposited in the Protein Data Bank and Biological Magnetic Resonance Bank under the accession codes 2L3I and 17194, respectively. The nucleotide sequence encoding Oxt 4a has been submitted to the EMBL Nucleotide Sequence Database under the accession number FN997582.

  14. Kozlov S., Grishin E. (2011). The mining of toxin-like polypeptides from EST database by single residue distribution analysis. BMC Genomics 12, 88 [+]

    Novel high throughput sequencing technologies require permanent development of bioinformatics data processing methods. Among them, rapid and reliable identification of encoded proteins plays a pivotal role. To search for particular protein families, the amino acid sequence motifs suitable for selective screening of nucleotide sequence databases may be used. In this work, we suggest a novel method for simplified representation of protein amino acid sequences named Single Residue Distribution Analysis, which is applicable both for homology search and database screening.

  15. Andreev Y.A., Kozlov S.A., Vassilevski A.A., Grishin E.V. (2010). Cyanogen bromide cleavage of proteins in salt and buffer solutions. Anal. Biochem. 407 (1), 144–6 [+]

    Protocols for recombinant polypeptide production should provide high yields and be efficient, user friendly, and time saving. To perform cyanogen bromide (CNBr) cleavage of fusion proteins, the majority of researchers first desalted and vacuum-dried samples and then dissolved them in aqueous formic or trifluoroacetic acid. We propose to exclude the desalting step and run CNBr cleavage directly. We show that the commonly used Tris-HCl, sodium phosphate, NaCl, imidazole, and guanidine-HCl do not interfere with the reaction under acidic conditions. Omitting the desalting step does not decrease the final yields of target products, as demonstrated for fusion proteins of different origin and composition.

  16. Vassilevski A.A., Kozlov S.A., Egorov T.A., Grishin E.V. (2010). Purification and characterization of biologically active peptides from spider venoms. Methods Mol. Biol. 615, 87–100 [+]

    Spider venoms represent invaluable sources of biologically active compounds suitable for use in life science research and also having a significant potential for biotechnology and therapeutic applications. The methods reported herewith are based on our long experience of spider venom fractionation and peptides purification. We routinely screen new peptides for antimicrobial and insecticidal activities and our detailed protocols are also reported here. So far these have been tested on species of Central Asian and European spiders from the families Agelenidae, Eresidae, Gnaphosidae, Lycosidae, Miturgidae, Oxyopidae, Philodromidae, Pisauridae, Segestriidae, Theridiidae, Thomisidae, and Zodariidae. The reported protocols should be easily adaptable for use with other arthropod species.

  17. Vassilevski A.A., Kozlov S.A., Grishin E.V. (2009). Molecular diversity of spider venom. Biochemistry Mosc. 74 (13), 1505–34 [+]

    Spider venom, a factor that has played a decisive role in the evolution of one of the most successful groups of living organisms, is reviewed. Unique molecular diversity of venom components including substances of variable structure (from simple low molecular weight compounds to large multidomain proteins) with different functions is considered. Special attention is given to the structure, properties, and biosynthesis of toxins of polypeptide nature.

  18. Andreev Y.A., Kozlov S.A., Kozlovskaya E.P., Grishin E.V. (2009). Analgesic effect of a polypeptide inhibitor of the TRPV1 receptor in noxious heat pain models. Dokl. Biochem. Biophys. 424, 46–8 ID:635
  19. Kozlov S.A., Andreev Ia.A., Murashev A.N., Skobtsov D.I., Diachenko I.A., Grishin E.V. (2009). [New polypeptide components from the Heteractis crispa sea anemone with analgesic activity]. Bioorg. Khim. 35 (6), 789–98 [+]

    Two new polypeptide components which exhibited an analgesic effect in experiments on mice were isolated from the Heteractis crispa sea tropical anemone by the combination of chromatographic methods. The APHC2 and APHC3 new polypeptides consisted of 56 amino acid residues and contained six cysteine residues. Their complete amino acid sequence was determined by the methods of Edman sequencing, mass spectrometry, and peptide mapping. An analysis of the primary structure of the new peptides allowed for their attribution to a large group of trypsin inhibitors of the Kunitz type. An interesting biological function of the new polypeptides was their analgesic effect on mammals, which is possibly realized via the modulation of the activity of the TRPV1 receptor and was not associated with the residual inhibiting activity towards trypsin and chymotrypsin. The analgesic activity of the APHC3 polypeptide was measured on the hot plate model of acute pain and was significantly higher than that, of APHC2. Methods of preparation of the recombinant analogues were created for both polypeptides.

  20. Andreev Y.A., Kozlov S.A., Koshelev S.G., Ivanova E.A., Monastyrnaya M.M., Kozlovskaya E.P., Grishin E.V. (2008). Analgesic compound from sea anemone Heteractis crispa is the first polypeptide inhibitor of vanilloid receptor 1 (TRPV1). J. Biol. Chem. 283 (35), 23914–21 [+]

    Первый ингибитор термочувствительных рецепторов TRPV1, активно принимающих участие в механизмах воспаления и передачи болевых ощущений, был выделен из экстракта нематоцистов морской анемоны Heteractis crispa. В экспериментах in vivo на мышах рекомбинантный аналог природного полипептида, названного АРНС1, проявил значимую анальгетическую активность, сопоставимую с действием опиоидов. В отличие от опиоидов, действие АРНС1 не приводит к наркотическому эффекту; поэтому обнаруженный полипептид имеет огромное практическое значение для разработки новых обезболивающих и противовоспалительных средств.

  21. Shlyapnikov Y.M., Andreev Y.A., Kozlov S.A., Vassilevski A.A., Grishin E.V. (2008). Bacterial production of latarcin 2a, a potent antimicrobial peptide from spider venom. Protein Expr. Purif. 60 (1), 89–95 [+]

    Natural venoms are promising sources of candidate therapeutics including antibiotics. A recently described potent antimicrobial peptide latarcin 2a (Ltc 2a) from Lachesana tarabaevi spider venom shows a broad-spectrum antibacterial activity. This peptide consists of 26 amino acid residues and therefore its production using chemical synthesis, although trivial, is costly. We describe an easy approach to Ltc 2a production in Escherichia coli using the conventional fusion partner thioredoxin. Latarcin 2a synthetic gene was cloned into the expression vector pET-32b, which was then used to transform E. coli BL21(DE3) strain. His-tagged fusion purification was achieved using metal-chelate affinity chromatography. Since no methionine residues are present in the latarcin 2a sequence, cyanogen bromide could be effectively utilized to separate the target product from the carrier protein. Reverse-phase HPLC was used as the final step of purification; the final yield was approximately 3 mg/L of bacterial culture. To increase the yields, we attempted incorporation of Ltc 2a tandem repeats into the fusion protein; however, production rates greatly decreased due to enhanced fusion toxicity. Moreover, we probed constructs to produce an Ltc 2a dimer and the Ltc 2a propeptide to study their functional properties. Recombinant peptides were produced at appreciable yields and biological tests to determine their activities were performed. Latarcin 2a is the first linear peptide from spider venom and one of the first membrane-active peptides from venomous animals to be biosynthetically produced.

  22. Vassilevski A.A., Kozlov S.A., Samsonova O.V., Egorova N.S., Karpunin D.V., Pluzhnikov K.A., Feofanov A.V., Grishin E.V. (2008). Cyto-insectotoxins, a novel class of cytolytic and insecticidal peptides from spider venom. Biochem. J. 411 (3), 687–96 [+]

    Eight linear cationic peptides with cytolytic and insecticidal activity, designated cyto-insectotoxins (CITs), were identified in Lachesana tarabaevi spider venom. The peptides showed antibiotic activity towards Gram-positive and Gram-negative bacteria at micromolar concentrations as well as toxicity to insects. The primary structures of the toxins were established by direct Edman sequencing in combination with enzymatic and chemical polypeptide degradation and MS. CITs represent a novel class of cytolytic molecules and spider venom toxins. They are the first example of molecules showing equally potent antimicrobial and insecticidal effects. Analysis of L. tarabaevi venom gland expressed sequence tag database revealed the primary structures of the protein precursors; eight peptides homologous with the purified toxins were additionally predicted. CIT precursors share a conventional prepropeptide structure with an acidic prosequence and a processing motif common to most spider toxin precursors. The most abundant peptide, CIT 1a, was chemically synthesized, and its lytic activity on different bacterial strains, human erythrocytes and lymphocytes, insect cells, planar lipid bilayers and lipid vesicles was characterized. The spider L. tarabaevi is suggested to have evolved to rely on a unique set of linear cytolytic toxins, as opposed to the more common disulfide-containing spider neurotoxins.

  23. Kozlov S.A., Vassilevski A.A., Feofanov A.V., Surovoy A.Y., Karpunin D.V., Grishin E.V. (2006). Latarcins, antimicrobial and cytolytic peptides from the venom of the spider Lachesana tarabaevi (Zodariidae) that exemplify biomolecular diversity. J. Biol. Chem. 281 (30), 20983–92 [+]

    Семейство коротких линейных полипептидных молекул — латарцинов — было найдено в яде паука Lachesana tarabaevi, часть из них была получена химическим синтезом и исследована на антибактериальное действие. Бактерицидная активность латарцинов соответствовала лучшим антимикробным пептидам, обнаруженным у различных видов животных. Большой терапевтический потенциал этих простых по структуре полипептидных молекул будет реализован, с большой долей вероятности, в самое ближайшее время.

  24. Kozlov S., Malyavka A., McCutchen B., Lu A., Schepers E., Herrmann R., Grishin E. (2005). A novel strategy for the identification of toxinlike structures in spider venom. Proteins 59 (1), 131–40 [+]

    We compared two different approaches to sequence information analysis from the expressed sequence tag (EST) library constructed for the venom glands of the spider Agelena orientalis. Some results were more illustrative and reliable by the contig analysis technique, whereas our novel method, with specific structural markers introduced for protein structure detection, allowed us to overcome some limitations of the contig analysis. A novel technique was suggested for the identification in data banks of the spider's ion channel inhibitor toxins using primary structure features common to all spiders. Analysis of about 150 polypeptides made it possible to introduce 3 primary structure motifs for spider toxins: the Principal Structural Motif (PSM), which postulates the existence of 6 amino acid residues between the first and second cysteine residue and the Cys-Cys sequence at a distance of 5-10 amino acid residues from the second cysteine; the Extra Structural Motif (ESM), which postulates the existence of a pair of CXC fragments in the C-region; and the Processing Quadruplet Motif (PQM), which specifies the Arg residue at position -1 and Glu residues at positions -2, -3, and/or -4 in the precursor sequences just before the postprocessing site. In the processed data bank we found 48 toxinlike structures with ion channel inhibitor motifs. These include agelenin earlier isolated from Agelena opulenta and 25 more homologous sequences, 15 homologs of mu-agatoxin 2 from the spider Agelenopsis aperta, 3 structures with low homology to omega-agatoxin-IIIA, and 4 new structures. Also we showed that toxinlike structures exceed two thirds of the overall database sequences.

  25. Korolkova Y.V., Kozlov S.A., Lipkin A.V., Pluzhnikov K.A., Hadley J.K., Filippov A.K., Brown D.A., Angelo K., Strøbaek D., Jespersen T., Olesen S.P., Jensen B.S., Grishin E.V. (2001). An ERG channel inhibitor from the scorpion Buthus eupeus. J. Biol. Chem. 276 (13), 9868–76 [+]

    Первый селективный блокатор K+ каналов ERG-типа BeKm, выделенный из яда скорпиона Buthus eupeus, был экспрессирован в гетерологичной системе и его биологические свойства, а также свойства нескольких мутантных аналогов, были изучены на M-токах культуры клеток NG108-15. Анализ активности мутантных форм позволили установить наиболее значимые аминокислотные остатки в структуре токсина, а сам токсин стал первым опубликованным селективным ингибитором hERG каналов, который в настоящий момент продается на рынке биологических препаратов.