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licking of hind legs in the hot plate test (55°C). .... to 55°C. Thereafter, the time till the first licking of the .... Gebhard, W, Tschesche, H, and Fritz, H, in Proteinase.
ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 6, pp. 711–719. © Pleiades Publishing, Ltd., 2009. Original Russian Text © S.A. Kozlov, Ya,A. Andreev, A.N. Murashev, D.I. Skobtsov, I.A. D’yachenko, E.V. Grishin 2009, published in Bioorganicheskaya Khimiya, 2009, Vol. 35, No. 6, pp. 789–498.

EXPERIMENTAL ARTICLE

New Polypeptide Components from the Heteractis crispa Sea Anemone with Analgesic Activity S. A. Kozlova,1 , Ya, A. Andreeva, A. N. Murashevb, D. I. Skobtsovb, I. A. D’yachenkob, and E. V. Grishina a

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia b Branch of the Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Pushchino Division Russian Academy of Sciences, Pushchino, Moscow oblast, 142292 Russia Received April 09, 2009; in final form, April 29, 2009

Abstract—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. Key words: the Heteractis crispa sea anemone; polypeptide inhibitors of the Kunitz type, structure, analgesic activity; functional expression DOI: 10.1134/S1068162009060065

INTRODUCTION Sea anemones are one of the most ancient predatory animals on the Earth.2 They predominantly occur in warm seas and lead a low-activity way of life with slow movement along the seabed. Anemones hunt for small fish, shellfish, and mollusca by paralyzing them by the thread cells on their tentacles around their oral fissure. These thread cells can cause itching and burning in humans, and tissue necrosis is also possible in the contact area. In general, it is associated with the cytotoxic effect of the basic components of venoms, cytolysins, on mammalian membranes [1]. A number of polypeptides with various biological activities were found in the anemone venoms along with membranolytic components. First of all, neurotoxins which are able to paralyze potential victims at low concentrations were identified. 1

Corresponding author; phone (495) 336-6540; fax: (495) 330-7301; e-mail: [email protected]. 2 Abbreviations: ASIC, proton-sensitive channel; BPTI, bovine pancreatic trypsin inhibitor; EDTA, ethylenediaminetetraacetic acid; IPTG, isopropyl-β-D-1-thiogalatopyranoside; TFA, trifluoroacetic acid; TRPV1, vanilloid receptor 1.

Many peptide toxins were isolated from anemone extracts and characterized. They selectively interacted with site 3 of the recognition of Na+ channels and retarded the kinetics of the channel inactivation. Three types of the blockers of Na+ channels are distinguished according to the special features of their primary structures [2, 3]. Toxins that consist of 40–50 amino acid residues are attributed to the first two types. Toxins of the third type are shorter (27–32 amino acid residues). Their physiological activity is more specific towards the Na+ channels of shellfishes and insects than towards those of mammals [4]. The anemone-produced inhibitors of K+ channels favor the paralytic effect. These polypeptide toxins are also divided into four structural groups similarly to the cytolytic components [2, 5]. The BgK and ShK toxins belong to the first type [6, 7]. They consist of 35–37 amino acid residues and contain three disulfide bonds. The second type involves polypeptide toxins, calycludines [8], the structural homologues of inhibitors of Kunitz-type proteases. The BDS-I and BDS-II inhibitors (40 amino acid residues) belong to the third type. They have a structural homology with the anemone toxins which affect Na+ channels [9]. Finally, the smallest SHTX I and SHTX II toxins

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(28 amino acid residues and two disulfide bonds) are attributed to the fourth type [10]. Along with the toxic components, anemones are able to produce small polypeptide molecules, which do not cause the death and paralysis of victims. For example, inhibitors of proteolytic enzymes were isolated from various species of anemones [11–13]. The same spatial structure is characteristic for toxins and peptide inhibitors. In addition, calycludines 1–3 [8] and the SHTX III protease inhibitor [10] inhibit both potentialactivated K+ channels and serine proteases. Anemones produce polypeptide components with an analgesic effect along with the neutral and toxic components. This is probably associated with the fact that their natural combinatorial library of polypeptides is directed to the maximum possible effect on any type of neuronal and muscular ionotropic receptors. Blocking or changing in the characteristics of a signal transduction (modulation of action) of a number of such receptors results in an analgesic effect. The analgesic effect after the administration of the anemone polypeptides was observed on animal models for the APETx1 selective inhibitor of the ASIC3 proton-sensitive channels [14] and for the APHC1 modulator of activity of the TRPV1 vanilloid receptor [15]. Data on the structure and biological activity of two new polypeptide components which were isolated from the alcohol extract of the Heteractis crispa sea anemone and exhibited the analgesic effect are given in this study. RESULTS AND DISCUSSION TRPV1 ionotropic receptors are known to be directly involved in mechanisms of the perception of transduction of a pain signal. For example, experiments with knockout mice demonstrated that the absence of this receptor resulted in a significant increase in the threshold of pain sensitivity towards heat and inflammatory stimuli [16]. The TRPV1 antagonists can probably ease pain in a number of pathologies. Investigations of the TRPV1 low-molecular inhibitors confirm the fact that the pharmacological block of this receptor could be used for the treatment of pain that arises from inflammation, cancer, and neuropathy [17, 18]. The search for new analgesic components from anemone extracts was first aimed at the isolation of inhibitors of the TRPV1 receptor. Polypeptide components with analgesic activity were isolated from the alcohol fraction of the H. crispa tropic anemone by the combination of methods of liquid chromatography. The preliminary stages of fractionation by hydrophobic chromatography on Polikhrom-1 and ion-exchange chromatography on Bio-Rex 70 and SP-Sephadex G-25 corresponded to the previously described stages [19] used for the isolation of the APHC1 modulator of the vanilloid receptor. In this study, a minor fraction was eluted after the main

active component, and its slight inhibiting activity towards the capsaicin-induced current trough the TRPV1 channels was observed in electrophysiological experiments. This fraction was obtained after ionexchange chromatography on SP-Sephadex G-25 and further fractionated by reverse-phase HPLC for the identification of its active components (Fig. 1). The first chromatographic fractionation in acidic buffer systems with 01% TFA (the data are not given) was ineffective. We used neutral buffer solutions and achieved a good resolution of separate chromatographic peaks. Two HPLC fractions exhibited the analgesic activity in the model experiments on animals. The first fraction contained the main APHC1 polypeptide component with a retention time of 34 min. The second fraction contained a new polypeptide component with a retention time of 43 min. The main APHC2 component of this fraction was isolated in the individual state by additional purification by reverse-phase chromatography on a Luna C18 column (2 × 150 mm). The molecular mass of this component was 6185 Da according to the MALDI mass spectrometry. This value is lower than that of the APHC1 analgesic peptide by 2 Da. Such a small difference in the molecular masses of two active components can be evidence of one or several substitutions of amino acid residues in the polypeptide structure or the possible amidation of the ë-terminal amino acid residue (taking into account the measurement error of a linear regime of the mass spectrometer +/–1 Da). Initially, the version of the peptide amidation seemed to be more convincing, because such a modification is widely occurring for components of natural venoms [20]. The determination of the first 23 amino acid residues of the N-terminal amino acid sequence of the 4vinylpyridine-alkylated APHC2 polypeptide demonstrated their complete coincidence with the corresponding N-terminal sequence of APHC1 (Fig. 2). The APHC2 fragment with possible substitutions was identified by the peptide mapping of the alkylated protein. An analysis of the molecular masses of the tryptic peptides revealed only one peptide from the central part of the molecule that differed from the corresponding fragments of APHC1 by 2 Da. This peptide was isolated from the hydrolysate by reverse-phase HPLC on a Luna C18 column (1 × 150 mm). Its amino acid sequence was determined, and a single substitution was found in position 3 (P V). This substitution provided the 2-Da difference of the peptide molecular masses. Finally, the complete amino acid sequence of the new APHC2 analgesic peptide was reconstructed (Fig. 2). A comparison of the APHC2 amino acid sequence according to the BLAST algorithm revealed a significant homology (in addition to the evident favorite, APHC1 that has 98% homology with APHC2, swiss prot B2G331) with three known protease inhibitors which were earlier isolated from anemones: inhibitor IV (87% homology, swiss prot P16344) [12], SHPI-1

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Fig. 1. Chromatographic fractionation of the fraction of inhibitors of the TRPV1 receptor on the Jupiter C5 column (Phenomenex, 4.6 × 150 mm). The fractions were eluted with a linear gradient of solution B in solution A (from 0 to 100% within 105 min) at a flow rate of 1.5 ml/min, where solution A was 10 mM ammonium acetate (pH 7.2) and solution B was 70% acetonitrile in solution A. The fraction containing the APCH2 polypeptide is cross hatched.

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APHC1 APHC2 APHC3 Inhibitor IV SHPI-1 SHPI-2 Fig. 2. Comparison of the amino acid sequences of the analgesic peptides isolated from the H. crispa sea anemone with the following protease inhibitors: inhibitor IV (P16344) from the H. crispa sea anemone and SHPI-1 (P31713) and SHPI-2 (P81129) from the Stichodactyla helianthus anemone. The amino acid residues which differ from those in APHC1 (B2G331) are in bold.

(83% homology, swiss prot P31713), and SHPI-2 (81% homology, swiss prot P81129) [21]. Such a high homology between the primary structures of the analgesic peptides and the protease inhibitors suggests some similarity of a number of physicochemical properties, including the retention time on chromatographic sorbents. In this connection, the analgesic activity of the chromatographic fraction after SPSephadex G-25 (the Inh VJ protease inhibitor was further obtained from this fraction [13]) was examined. This fraction was found to exhibit an analgesic effect in experiments on mice. An analysis of the component RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

composition by mass spectrometry indicated the presence of one main component and five minor polypeptides. The choice of the fractionation conditions resulted in the following scheme of separation of active molecules. The previously prepared [13] fraction of protease inhibitors was applied to a TSK gel CM-35W cationexchange column in a 10-mM ammonium acetate buffer. The main protein component (the InhVJ protease inhibitor) was not sorbed on this column. Further, two fractions were eluted with a step gradient (30 and 300 mM NaCl) and one of them (30 mM NaCl) exhib-

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ited the desired activity. The further fractionation was subsequently performed on the following reverse-phase sorbents: Delta-Pak C18-300 (Fig. 3a), RP-amide (Fig. 3b), and Luna C18 (Fig. 3c). As a result, the APHC3 single individual active component was isolated; its molecular mass was 6111 Da. The complexity of the fractionation scheme was explained by the presence of a large number of polypeptides with point substitutions (it was previously observed during the cloning of cDNA of APHC1) in the starting fraction of protease inhibitors [19]. The partial determination of the N-terminal amino acid sequence of APHC3 (32 amino acid residues) confirmed the high homology with APHC1 and the presence of only three substitutions of amino acid residues in positions 18, 23, and 31 (Fig. 2). We failed to identify all the peptide fragments after the tryptic cleavage of the polypeptide chain of the reduced and Cys-alkylated APHC3 polypeptide. Therefore, APHC3 was subjected to degradation by the Clu-C protease, and the structure of the missing peptide fragments was determined after their isolation from the peptide mixture by reverse-phase chromatography on the Luna C18 column (1 × 150 mm): 26–38 TGKCTPFIYGGCE, 39–45 GNGNNFE, 46–56 TLRACRGICRA. The molecular mass of the complete amino acid sequence reconstructed from the APHC3 peptides corresponded to that earlier determined by mass spectrometry. The sequence of the APHC3 peptide is given in Fig. 2 together with the most homologous proteins which were determined by the BLAST homology analysis3; APTH1, inhibitor IV, SHPI-1, and SHPI-2 proved to be the most homologous proteins (80–92% homology), similarly to APHC2. At first, only the preliminary data on the analgesic activity of the isolated peptides on mice were obtained due to the very limited content of the investigated natural proteins in natural sources. The scheme of the preparation of recombinant analogues of these polypeptides was elaborated for more detailed tests, and the products were obtained in sufficient amounts. For this purpose, synthetic nucleotide sequences which encoded the corresponding polypeptides and contained the EcoRI and XhoI restriction sites were obtained (Fig. 4) taking into account the frequency of the use of codons of the producing strain. They were further cloned with these restriction sites in the pET32b+ vector. Thioredoxin was used as a partner protein. It provided a higher yield and proper formation of the disulfide bonds in cysteine-rich proteins. The amino acid sequences of the analgesic peptides contained no Met residues, and the bromocyanogen cleavage of a peptide bond was used for the cleavage of the hybrid protein. Thus, the Met-encoding nucleotide sequence was inserted between the fragments that 3

BLAST means Basic Local Alignment Search Tool.

coded the polypeptides and thioredoxin. The BL21(DES) E. coli strain was transformed by the obtained constructions. The expression of the protein genes was induced by the addition of IPTG on the stage of exponential growth of the cells. The recombinant polypeptides were isolated according to the protocol described in the Experimental. The yield was approximately 8 mg/l of the culture. The accuracy of the synthesized sequence was confirmed by mass spectrometry of the recombinant products. A comparison of the chromatographic mobilities of the natural proteins with those of their recombinant analogues revealed no difference, suggesting adequate spatial arrangements of the chains of the recombinant polypeptides. The biological activity of the recombinant APHC2 and APHC3 in the model experiments on animals completely corresponded to that of the native polypeptides. The analgesic effect of APHC2 and APHC3 was determined on the models of heat stimulation in the heat plate test. This test of the determination of pain sensitivity is based on the activation of TRPV1 and, possibly, some other receptors. It is known that the reaction time of mice with the defect TRPV1 gene significantly differs from that for the wild-type mice precisely in these tests [16]. Intravenous administration of the APHC2 and APHC3 peptides (up to 1 mg/kg) caused no toxic effect and no behavioral disorders in mice. The analgesic activities of the APHC2 and APHC3 polypeptides were distinguished from each other and differed from those of APHC1 or BPTI (one of the most effective inhibitors of the Kunitz type) in the hot plate test, in spite of the high structural homology (Fig. 5). A peptide dose of 0.1 mg/kg was used for the analysis. This dose corresponded to the calculated dose that was previously used for the analysis of crude fractions and the purified natural polypeptides. APHC3 exhibited the highest analgesic potential. Its activity in this test was insignificantly higher than that of APHC1. APHC2 was weakly active, but its analgesic effect was statistically significant. The BPTI inhibitor of serine proteases was used as a control of the mediated pathways of the influence on the pain receptors and had no analgesic activity. Previously, the InhV1 protease inhibitor was shown to be predominantly specific to trypsin and α-chymotrypsin [13]. Therefore, the inhibition effect of the analgesic peptides was examined on these enzymes (Table 1). APHC2 and APHC3 demonstrated a weak inhibiting activity that was three orders lower than the activity of their nearest structural homologue, SHPI-1. Both peptides inhibited trypsin a little better than chymotrypsin. The function of the inhibitor of serine proteases may be considered as residual for APHC2 and APHC3, taking into account the fact that BPTI is inactive in the hot plate test. The basic biological target of these peptides is neuronal receptors.

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Fig. 3. Results of the step-by-step fractionation of the fraction of trypsin inhibitors from the H. crispa sea anemone by the following methods of reverse-phase chromatography: (a) chromatography on the Delta-Pak C18-300 column (Waters, 4.6 × 250 mm) in a linear gradient of acetonitrile in a 10-mM ammonium acetate buffer (pH 7.2) from 0 to 50% within 15 min at a flow rate of 2 ml/min, (b) chromatography on the RP-amide column (Supelco, 2 × 100 mm) in a linear gradient of acetonitrile in a 10-mM ammonium acetate buffer (pH 7.2) from 17.5 to 54% within 70 min at a flow rate of 0.3 ml/min, and (c) chromatography on the Luna C18 column (Phenomenex, 2 × 150 mm) in a linear gradient of acetonitrile in 0.1% TFA from 0 to 70% within 70 min at a flow rate of 0.15 ml/min. The fractions with the analgesic effect are crosshatched at every stage.

Electrophysiological studies of the APHC2 and APHC3 new polypeptide analgesics have still not been performed, but it is evident that they most probably are modulators of TRPV-1 receptors. Point substitutions of amino acid sequences in the Kunitz-type polypeptide structures of sea anemones can result in both a change in the activity (a decrease in the analgesic effect APCH3 > APCH1 > APCH2) and a complete change in the pharmacological properties of a molecule (the RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

SHPI-1 protease inhibitor and the APHC1–APHC3 analgesic peptides). According to provisional data, several dozens of genes of the polypeptide components possibly exist [19], and the investigation potential of the polypeptides of sea anemones is tremendous. We hope that our further studies of extracts from sea anemones will result in the discovery of modulators of new types and subtypes of ionic channels and other receptors.

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Fig. 4. Scheme of the assembly of the constructs for the preparation of the recombinant analogues on the basis of the pET32b+ plasmid vector. D1, D3, L1, R1, Rev1, and Rev2 are primers. TRX is thioredoxin.

EXPERIMENTAL Reagents and materials of the following companies were used in this study: Sigma (United States), ICN (United States), Fluka (Germany), Merck (Germany), Khimmed (Russia), and Kriokhrom (Russia). All of the solutions were prepared with the use of deionized water (resistance of 18.2 Mohm) prepared on a Millipore system (United State). Proteolytic enzymes, trypsin (EC 3.4.21.4), α-chymotrypsin (EC 3.4.21.1), and Glu-C endoproteinase (EC 3.4.21.19) were purchased from the Sigma company (United States). Experiments with the animals were carried out in a strict correspondence to the Legislation of the Russian Federation and European Convention of the Protection of Vertebrates Used for Experiments and Other Scientific Purposes. Table 1. Inhibition constants of trypsin and α-chymotrypsin calculated by the Dixon method Ki, M Polypeptide trypsin

Control

Fig. 5. Effect of the intravenous administration of the APHC1, APHC2, APHC3, and BPTI polypeptides at a dose of 0.1 mg/kg of the body weight on the delay time of the licking of hind legs in the hot plate test (55°C). The statistically significant differences (P < 0.05) are marked with asterisks.

Met

6His

0

chymotrypsin

APHC2

9 × 10–7

4.5 × 10–6

APHC3

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7 × 10–6

BPTI

6 × 10–14 [22]

1.8 × 10–13 [23]

SHPI-1

1 × 10–10 [21]

2.3 × 10–9 [21]

The final fractions after the fractionation of alkaline and aqueous extracts of the H. crispa sea anemone on SP-Sephadex G-25 (2.5 × 50 cm) (Pharmacia, Sweden) were kindly presented by the Pacific Institute of Bioorganic Chemistry (Far East Division, Russian Academy of Sciences). The procedure of the preparation of the anemone extracts and their fractionation were previously described in detail [13, 19]. HPLC of the polypeptides was performed on a KONTRON chromatograph (Italy). The APHC2 polypeptide was isolated from the starting fraction on a Jupiter C5 column (Phenomenex, United States, 4.6 × 150 mm) equilibrated with a starting buffer (10 mM ammonium acetate, pH 7.2). Fractions were eluted with a linear gradient of the acetonitrile concentration (from 0 to 70% within 105 min) at a flow rate of 1.5 ml/min. For the isolation of the APHC3 polypeptide, the starting fraction was first fractionated on an ion-exchange column with TSK-gel CM-35W (LKB, Sweden) in a step gradient (3% and 30%) of a 10-mM ammonium acetate buffer (pH 7.2) with 1 M NaCl at a flow rate of 2 ml/min. The fraction which was eluted with the first step of the gradient was further fractionated at the same pH (buffer A was 10 mM ammonium acetate and buffer B was 70% acetonitrile in buffer A) on a reverse-phase Delta-Pak C18-300 column (Waters, United States, 4.6 × 250 mm) at a flow rate of 2 ml/min in a linear gradient of buffer B (from 0 to 50% within 15 min). The single active fraction was further fractionated on a reverse-phase RP-amide column (Supelco, United States, 2 × 100 mm) in a linear gradient of the acetonitrile concentration in the same buffer systems (from 17.5 to 54% within 70 min) at a flow rate of 0.3 ml/min. The final purification of APHC3 was carried out on a reverse-phase Luna C18 column (Phenomenex, United States, 2 × 150 mm) in a linear concentration gradient of acetonitrile in 0.1% TFA (from 0 to 70% within 70 min) at a flow rate of 0.15 ml/min. Insignificant impurities of foreign proteins, which did not prevent

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Table 2. Structures of oligonucleotides that were used for the assembly of the genes encoding sequences of the mature polypeptides. The number of the polypeptide for which the gene was used is given in parentheses: (2) APHC2 and (3) APHC3 Oligonucleotide

Structure of the oligonucleotide (5'–3')

D1 (2)

CCTGGAACCGAAAGTTGTTGGTCCGTGCACCGCTTACTTCCGTCGTTTCT

D2 (2)

ACTTCGACTCTGAAACCGGTAAATGCACCCCTTTCATCTACGGTGGTTGC

D3 (2)

GAAGGTAACGGTAACAACTTCGAAACCCTGCGTGCTTGCCGTGCTATC

D1 (3)

CCTGGAACCGAAAGTTGTTGGTCCGTGCACCGCTTACTTCCCGCGTTTCT

D2 (3)

ACTTCAACTCTGAAACCGGTAAATGCACCCCTTTCATCTACGGTGGTTGC

D3 (3)

GAAGGTAACGGTAACAACTTCGAAACCCTGCGTGCTTGCCGTGGTATC

Rev1 (2)

CGGTTTCAGAGTCGAAGTAGAAACGACGGAAGTAAG

Rev1 (3)

CGGTTTCAGAGTTGAAGTAGAAACGCGGGAAGTAAG

Rev2 (2, 3)

GTTGTTACCGTTACCTTCGCAACCACCGTAGATGAAA

L1 (2, 3)

GGAATTCCATGGGTTCTATCTGCCTGGAACCGAAAGTTGTTG

R1 (2)

CTCTCGAGTCAAGCACGGCAGATAGCACGGCAAGCACGCAG

R1 (3)

CTCTCGAGTCAAGCACGGCAGATACCACGGCAAGCACGCAG

the determination of the biological activity, were removed by chromatography after the alkylation reaction. The protein concentration in the samples was determined according to the absorption spectrum recoded on a Hitachi U3210 spectrophotometer (Japan) in a 1-cm cell. Mass spectrometry was carried out on a time-offflight MALDI Ultraflex TOF-TOF mass spectrometer (Bruker Daltonics, Germany) or MALDI-LR (Micromass, Great Britain). A solution of 2,5-dihydroxybenzoic acid (10 mg/ml) or a solution of α-cyano-4hydroxycinnamic acid (10 mg/ml) in the mixture of 50% acetonitrile and 0.1% TFA were used as matrix solutions. The spectra were recorded in a regime of detection of positively charged ions in direct or reflector regimes. Alkylation of the cysteine residues of the polypeptides by 4-vinylpyridine was performed by the adapted procedure [24]. The polypeptides were dissolved in a 100-mM phosphate buffer (10 μl, pH 7.5) containing 6 M guanidine chloride and 3 mM EDTA, incubated for a night at 45°ë, cooled, degasified on an ultrasound bath, and treated with 5 μl of the reducing solution (300 mM phosphate buffer with pH 12.3, 200 mM RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

dithiotreitol, and 3 mM EDTA) for 1 h at 37°ë. The thiol groups of the cysteine residues were alkylated with a 10% solution of 4-vinylpyridine (3 μl) in methanol for 15 min at room temperature in the dark. The solution of the modified polypeptide was diluted with 0.1% TFA to a volume of 100–300 μl and demineralized on a reverse-phase Luna C18 column (Phenomenex, United States; 2 × 150 mm) in a linear or step gradient of the acetonitrile concentration. The N-terminal amino acid sequence was determined on a Procise 492 automatic peptide sequencer (Applied Biosystems, United States) by the Edman method using the producer’s program. The peptide mapping was carried out with a quantity of the substance of less than 1 nmol. The preliminary dried and alkylated samples were dissolved in a 100-mM ammonium bicarbonate buffer (10 μl, pH 8.5) containing 2 mM CaCl2, and the enzyme (trypsin or Glu-C endoprotease) solution (2–3 μl) was added in the same buffer solution to the final enzyme–substrate ratio of 1 : 20. The reaction mixture was kept for several hours at 37°ë with the mass spectrometry monitoring. The reaction products were fractionated by HPLC on a

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Luna C18 reverse-phase microcolumn (Phenomenex, United States; 1 × 150 mm) in the concentration gradient of acetonitrile in 0.1% TFA (from 0 to 60% within 60 min). The computer analysis of the measured and calculated masses of the peptides and proteins was performed using the GPMAW 4.0 program (Denmark). The expressing constructs were assembled from the synthetic oligonucleotide primers (Evrogen). The gene fragment that encoded the mature polypeptide was prepared by PCR from the primers which were chosen with consideration of the optimization of the use of codons for E. coli (Table 2). The corresponding D1, D2, D2, Rev1, and Rev2 internal primers (10 pmol of each) were added to the reaction mixture. The amplification was performed under the following conditions: DNA denaturing at 94°C for 30 s, the primer annealing at 55°C for 30 s, and DNA synthesis at 72°C for 30 s (20 cycles). The mixture was diluted with water by 1000 times and the amplification was performed under the same conditions with the use of R1 and L1 external primers. The amplification product (about 180 np) was extracted with the mixture of phenol and chloroform (1 : 1) and with chloroform, precipitated with ethanol in the presence of 0.3 M sodium acetate (pH 5.2), treated with the EcoR1/XhoI restrictases, purified by gel electrophoresis, and ligated with the pET32b plasmid (Novagen, United States) cleaved by the same restrictases. The ligates were transformed into cells of the XL1-Blue strain. The obtained clones were analyzed by PCR “on colonies” using a pair of universal primers (L1 and R1) and sequenced. Isolation of the recombinant analogues. Cells of the BL21(DE3) strain were transformed by the prepared constructs. The cells were grown in the LB medium (200 ml) containing ampicillin (100 μg/ml) at 37°C under intensive aeration to the optical absorption value (A600) of 0.6–0.8 (7–9 h). IPTG was added to the final concentration of 0.2–0.4 mM, and the reaction mixture was incubated for 12–16 h at 37°C. The bacterial cells were precipitated from the solution by centrifugation (5000 rev/min) at 4°C for 15 min. The cellular precipitate was resuspended in 20 ml of the starting buffer (20 mM Tris-HCl with pH 7.5 and 300 mM NaCl), placed in ice, and subjected to an ultrasound disintegration (4 × 3 min). The cellular lysate was centrifuged at 15000 rev/min for 20 min at 4°C, and the supernatant was applied onto an affinity column with Co2+sepharose (Clonetech, United States) equilibrated with the starting buffer. The column was washed with five total volumes of the same buffer, and the affinity-sorbed fraction was eluted with a Tris-HCl buffer (pH 7.5) containing 300 mM NaCl and 150 mM imidazole. Further, a 10-M solution of hydrochloric acid was added to the obtained fraction to the final concentration of 0.2 M, and the reaction mixture was treated with a 600-fold excess of BrCN (relative to the measured amount of the protein) for 14–16 h at room temperature in the dark,

evaporated to dryness, dissolved in 0.1% TFA, and applied to a Jupiter C4 reverse-phase column (Phenomenex, United States; 4.6 × 150 mm). The reaction products were fractionated in a linear gradient of the acetonitrile concentration in 0.1% TFA (from 10 to 70% within 40 min) at a flow rate of 1 ml/min. Detection was performed at 210 and 280 nm. The purity of the recombinant products was determined by mass spectrometry. Study of the analgesic activity of polypeptides was carried out on mice of the CD-1 line (NPP Pitomnik laboratonykh zhivotnykh FIBKh RAN) of the body mass 19–21 g. The animals were kept at temperature 23 ± 2°C. Each animal was used in an experiment one time. Peptides dissolved in isotonic solution up to the finel concentration of 0.1 mg/kg were intravenously introduced into the tail vein. Control animals were obtained pure isotonic solution into the tail vein. After 15 min, the mouse was placed onto a metal plate heated to 55°C. Thereafter, the time till the first licking of the hind foot was registered. A packet of programs ORIGIN (OriginLab., United States) was used for the statistical treatment of data. The reliability of differences between the control and experimental groups was determined by means of the variability analysis (ANOVA) and the Tukey post-hoc test. Inhibition of the proteolytic activity was determined spectrophotometrically in 96-well plates. An polypeptide under study was added at various concentrations to an aliquot of 0.5 μM trypsin or α-chymotrypsin in 50 mM Tris-HCl (pH 8.0); the volume of the reaction mixture was adjusted by buffer solution to 90 μl and incubated for 10 min at 37°C. The residual enzymatic activity was then determined by the addition of 10 μl of substrate. The trypsin activity was determined using 3 mM N-benzoyl-L-tyrosine p-nitroanilide and chymotrypsin activity, 3 mM N-benzoyl-L-tyrosine ethyl ester (BTEE) in 10% dimethylformamide and 100 mM Tris-HCl (pH 8.0) as substrates. Inhibition constants were determined by the Dixon method [25] after incubation for 10 min at 37°C measuring the quantity of the formed p-nitroanilin at 410 nm (trypsin) or tyrosine at 250 nm (α-chymotrypsin). ACKNOWLEDGMENTS We thank Ts. A. Egorov and A Kh. Musolyamova for the help in the determination of amino acid sequence of polypeptides, and I.N. Sokotun and E.P. Kozlovskaya for the gift of fractions of trypsin inhibitors. This work was suppoted by the Russian Foundadion for Basic Research, Program of fundamental studies Molecular and cellular Biology of the Presidium of Russian Academy of Sciences, anf the grants of the Federal Agency on Science and Innovations.

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NEW POLYPEPTIDE COMPONENTS FROM THE Heteractis crispa

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