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A novel serine protease inhibitor from the venom of Vespa bicolor Fabricius
Comparative Biochemistry and Physiology, Part B 153 (2009) 116–120
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part B j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b
A novel serine protease inhibitor from the venom of Vespa bicolor Fabricius Xinbo Yang a,b,1, Yakun Wang a,1, Zekuan Lu a, Lei Zhai a, Juguo Jiang c, Jingze Liu a,⁎, Haining Yu a,⁎ a b c
College of Life Sciences, Hebei Normal University, Shijiazhuang 050016, Hebei, China Biochemistry & Biology College, Yantai University, Yantai 264005, Shandong; China Shandong Shengli Co., Ltd., 2238 Tianchen Street, Jinan 250101, Shandong, China
a r t i c l e
i n f o
Article history: Received 20 December 2008 Received in revised form 6 February 2009 Accepted 16 February 2009 Available online 1 March 2009 Keywords: Bioactive peptides Serine protease inhibitor Vespa bicolor Wasp venom
a b s t r a c t Hornets possess highly toxic venoms, which are rich in toxin, enzymes and biologically active peptides. Many bioactive substances have been identified from wasp venoms but only a few serine protease inhibitors have been identified from two kinds of wasp venoms. In this work, a serine protease inhibitor named bicolin was purified and characterized from the venom of the wasp, Vespa bicolor Fabricius. The precursor encoding bicolin was cloned from the cDNA library of the venomous glands. It is a cysteine-rich small protein containing 54 amino acid residues including 6 half-cysteines. The peptide is homologous to serine protease inhibitors isolated from venoms of Anoplius samariensis and Pimpla hypochondriaca. Bicolin showed inhibitory ability against trypsin and thrombin. © 2009 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
The venoms of arthropods have attracted considerable interest as a potential source of pharmacological substances (Habermann, 1972; Hirai et al., 1979a,b; Nakajima, 1984). Wasp venom gland is biochemically, pharmacologically, and physiologically complex organ which fulfills a wide range of functions necessary for wasp survival, including predatoriness, defense, etc (Han et al., 2008; Yang et al., 2008). Over the past several decades, studies have focused on the bioactive compounds present in wasp venoms (Yasuhara et al., 1983; Argiolas and Pisano, 1984, 1985; Yu et al., 2007). These compounds include amines, small peptides, and proteins of high molecular mass such as enzymes, allergens and toxins (Konno et al., 2000; Mendes et al., 2004; Mendes et al., 2005; Xu et al., 2006a,b; Zhou et al., 2006). Only three serine protease inhibitors (peptide toxin As-fr-19, cysteinerich venom protein 2 (CVP2) and CVP4) are identified from two kinds of wasp venoms (Parkinson et al., 2004; Hisada et al., 2005). Vespa bicolor is one of the most dangerous species of vespine wasps, found in most provinces of China. It is a kind of social wasp. This hornet is aggressive and predatory. Stings by this hornet generally produce severe pain, local damage, cardiovascular system disorder and occasionally death in large vertebrates including man (Evans and Summers, 1986; Sakhuja et al., 1988; Korman et al., 1990; Watemberg et al., 1995; Chao and Lee, 1999; Chen et al., 2004). Little was reported on the chemical constituents of V. bicolor venom. Herein, we reported purification, characterization and biological activities of a serine protease inhibitor from V. bicolor venom.
2.1. Wasp venom
⁎ Corresponding authors. Tel.: +86 311 86268842; fax: +86 311 86268313. E-mail addresses: [email protected] (J. Liu), [email protected] (H. Yu). 1 These authors contributed equally to this paper. 1096-4959/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2009.02.010
The wasps of V. bicolor were collected in Hebei province of China. The collected wasps were stimulated by alternative current (6 V) lasting for 6–10 s. The wasp venom was secreted onto a clean glass plate (50 × 50 cm), immediately collected and stored at −20 °C. 2.2. Peptide purification The lyophilized wasp venom sample (0.1 g) was dissolved in 5 mL 0.1 M phosphate buffer solution, pH 6.0, and filtered through a 10-kDa cut-off Centriprep filter (Millipore, Bedford, CA). The yield of this filtration was about 30%. The filtrate was next lyophilized. Lyophilized filtrate was applied to a 5 × 250-mm Vydac C18 RP-HPLC (reversed-phase high-performance liquid chromatography) column (Sigma) equilibrated with 0.1% (v/v) trifluoroacetic acid/water. Elution (0.7 mL/ min) with collecting fraction of 3 mL was performed using 0.1% (v/v) TFA/water over 10 min, followed by a linear gradient of 0%–80% acetonitrile containing 0.1% (v/v) TFA in 0.1% (v/v) TFA /water over 100 min, and final elution with 80% acetonitrile containing 0.1% (v/v) TFA. The absorbance at 220 nm was monitored. UV-absorbing peaks were collected and lyophilized, and their effects on serine proteases were detected. 2.3. Structural analysis Peptide sample (1 mg) was first dissolved in 1 mL 0.1 M Tris–HCl buffer (pH 8.6) containing 6 M ultra-pure urea and 0.02 M 2-mercaptoethanol. Following flushing with nitrogen, 5 mg of iodoacetic acid was
X. Yang et al. / Comparative Biochemistry and Physiology, Part B 153 (2009) 116–120
added into the reaction mixture while maintaining the pH at 8.6 via the addition of 0.1 M NaOH and incubation for another 3 h. Finally, carboxymethylated (CM) peptide was desalted on a Vydac C8 RP-HPLC column (200 × 4.6 mm) and lyophilized. N-terminal amino acid sequence was directly determined by Edman degradation on an Applied Biosystems pulsed liquid-phase sequencer, model 491. Mass measurement was performed on a Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry instrument (Bruker Reflex), using α-cyano-4-hydroxycinnamic acid (HCCA) suspended in 100% acetonitrile as matrix. 2.4. SMART cDNA synthesis Total RNA was extracted using TRIzol (Life Technologies, Ltd.) from the 0.1 g venomous glands of V. bicolor. cDNA was synthesized by SMART™ techniques by using a SMART™ PCR cDNA synthesis kit (Clontech, Palo Alto, CA). The first strand was synthesized by using cDNA 3′ SMART CDS Primer II A, 5′-AAGCAGTGGTATCAACGCAGAGTACT (30) N-1 N-3′ (N = A, C, G or T; N-1 = A, G or C), and SMART II A oligonucleotide, 5′-AAGCAGTGGTATCAAC GCAGAGTACGCGGG-3′. The second strand was amplified using Advantage polymerase by 5′ PCR primer II A, 5′-AAGCAGTGGTATCAACGCAGAGT- 3′. The PCR conditions were: 2 min at 94 °C, followed by 30 cycles of 10 s at 92 °C, 30 s at 62 °C, 1 min at 72 °C. 2.5. Screening of cDNA encoding bicolin The cDNA synthesized by SMART™ techniques was used as template for PCR to screen the cDNAs encoding bicolin. Two pairs of oligonucleotide primers were used to screen the cDNA encoding biocolin. One pair of them is S1 (5′-gc(A/t/C/G)ca(t/C)cc(A/T/C/G)(C/T) T(A/T/C/G)TG(C/T)(C/T)T(A/T/C/G) (C/T)T(A/T/C/G)GA(C/T)-3′ based on the mature peptides of bicolin in the sense direction) and primer II A as mentioned in “SMART cDNA synthesis” in the antisense direction were used in PCR reactions. Another pair is S2 (5′ (G/A)TC(A/T/ C/G)A(A/G)(A/T/C/G)A(A/G)(A/G)CA(A/T/C/G)A(A/G)(A/T/C/G)GG (A/G)TG(A/t/C/G)CG-3′) and primer II A. The DNA polymerase was Advantage polymerase from Clontech (Palo Alto, CA) The PCR conditions were: 2 min at 94 °C, followed by 30 cycles of 10 s at 92 °C, 30 s at 50 °C, 40 s at 72 °C. Finally, the PCR products were cloned into pGEM®-T Easy vector (Promega, Madison, WI). DNA sequencing was performed on an Applied Biosystems DNA sequencer, model ABI PRISM 377.
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concentration under different chromogenic substrate concentrations (0–2 mM). The peptides were quantified by UV absorbance at 215 and 225 nm using the formula: concentration (mg/mL) = (A215 nm − A225 nm) × 0.144. 2.7. Recalcification time assay Platelet-poor plasma (PPP) was prepared by centrifuging the citrated whole blood twice at 2500 g for 15 min at 4 °C and used within 4 h. For the recalcification time assay (RT), certain amount of sample in 0.1 mL 0.9% NaCl was added to 0.1 mL PPP at 37 °C, and the mixture was incubated at 37 °C for 5 min. The time from the addition of 0.1 mL of 25 mM CaCl2 until the appearance of the first clot was recorded. The plasma aliquots incubated only with 0.1 mL of 0.9% NaCl were tested as controls (Xu et al., 2007). 3. Results 3.1. Purification of serine protease inhibitor peptide As illustrated in Fig. 1, the components from venoms of V. bicolor were considerably complex. About 40 peaks were eluted from venoms by C18 RP-HPLC (Fig. 1). The peak indicated as B (Bicolin) in Fig. 1 was found to exert significant trypsin-inhibitory activity. Ten batches of the purified peptide peaks were pooled and studied further. 3.2. Structural analysis Purified serine protease inhibitor indicated as B in Fig. 1 was named bicolin. It was subjected to amino acid sequence analysis by automated Edman degradation. Its amino acid sequence was `, and it molecular mass was 5749.4 Da analyzed by MALDI-TOF mass spectrometry (Fig. 2). 3.3. cDNA cloning A cDNA clone containing an insert around 360-base pairs was identified and isolated (GenBank accession number FJ749250). Both strands of the clone were sequenced (Fig. 3). It was found to have an open reading frame that encodes a polypeptide composed of 77 amino acids including the predicted signal peptide and the mature bicolin
2.6. Serine protease inhibitory assay The inhibition effects of the sample on the hydrolysis of synthetic chromogenic substrates by serine proteases (All the serine proteases are from Sigma) were assayed in 50 mM Tris–HCl, pH 7.8 at 37 °C according to previous method (Lai et al., 2004). The protease (final concentrations 10 nM for trypsin and thrombin, 21 nM and 23 nM for chymotrypsin, and 20 nM for elastase, respectively) and the inhibitor (final concentration 0.5 μM) were pre-incubated for 10 min at 37 °C (Lai et al., 2004). S-2238 (H-D-Phe-Pip-Arg-pNA, Kabi Vitrum, Stockholm, Sweden) and B-3133 (N-Benzoyl-Arg-4-Nitroanilidehydrochride-pNA, Sigma) were used as substrates for trypsin and thrombin, and elastase, respectively. The reaction was initiated by the addition of the substrate to a final concentration of 0.5 mM. The formation of p-nitroaniline was monitored continuously at 405 nm for 5 min. In the case of chymotrypsin, BTEE (N-benzoyl-tyrosine ethyl ester, Sigma) was used as the substrate and the reaction was monitored continuously at 253 nm for 5 min. The effect of the inhibitor was estimated by setting the initial velocity as 100%, obtained with enzyme alone (without inhibitor). The inhibition assay was carried out as described above and the Ki value was obtained by reciprocal plotting of the reaction velocity vs. inhibitor
Fig. 1. Isolation of serine protease inhibitor from the venoms of V. bicolor. The lyophilized venom sample (0.1 g) was dissolved in 5 mL 0.1 M phosphate buffer solution, pH 6.0, and filtered through a 10-kDa cut-off Centriprep filter (Millipore, Bedford, CA) and the filtrate was and lyophilized. Lyophilized filtrate was applied to a Hypersil BDS C18 RP-HPLC column (5 × 250-mm) equilibrated with 0.1% (v/v) trifluoroacetic acid/water. The elution was performed with the indicated gradient of acetonitrile in Fig. 1 at a flow rate of 0.7 mL/min, and purified serine protease inhibitor was indicated by a “B”. AM1 and AM2 are antimicrobial peptides (Chen et al., 2008).
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Fig. 2. MALDI-TOF mass spectrometry of bicolin.
Fig. 3. The nucleotide sequences encoding bicolin (A) and its sequence comparison (B) with As-fr-19 and CVP2. Mature peptides are boxed. ⁎Stop codon. The amino acids identical to the first line are shaded. Gaps (−) have been introduced to optimize the sequence homology.
sequence (Fig. 3A). The amino acid sequence deduced from the cDNA sequence matched well with the amino acid sequence determined by Edman degradation. The mature bicolin sequence deduced from the precursor has a theoretical molecular mass of 5747.6 Da that is 1.8 Da difference from the observed molecular mass (5749.4 Da). This difference is within the error tolerance, so it might be concluded that the deduced amino acid sequence was identical to the determined amino acid sequence by Edman degradation. The mature bicolin contains six half-cysteines. By Blast search, bicolin showed highly sequence similarity to As-fr-19 and CVP2 (Parkinson et al., 2004; Hisada et al., 2005) (Fig. 3B). They share six conserved halfcystemines in their sequences. The Blast search reveals that they belong to BPTI/Kunitz inhibitor family. 3.4. Serine protease inhibitory activity To examine the inhibitory specificity of sample to the serine proteases, the purified bicolin from the wasp venoms was assayed for inhibitory activity against four different proteases. Bicolin could inhibit trypsin and thrombin, but had no effect on the elastase and chymotrypsin (Table 1). Among those serine proteases, bicolin had the strongest inhibitory activity against trypsin with an inhibitory
constant (Ki) of 5.5 × 10− 7, but comparatively weaker inhibition toward thrombin with the inhibitory constant of 2.6 × 10− 5. 3.5. Anti-coagulant activity Considering bicolin's inhibitory activity against thrombin, its effect on plasma coagulation was assayed using platelet-poor plasma (ppp). As illustrated in Fig. 4, the addition of bicolin increased the
Table 1 Effect of bicolin on hydrolytic activity of different proteases. Proteases
Residual activitya
Inhibitory constant (Ki) (M)
Thrombin Trypsin Elastase Chymotrypsin
15 ± 2.7 5 ± 3.2 98 ± 4.9 99 ± 5.6
2.6 × 10− 5 5.5 × 10− 7 ND ND
The effect of the inhibitor was estimated by setting the initial velocity obtained in the presence of enzyme alone (without inhibitor) as 100%. The Ki value was obtained by reciprocal plotting of the reaction velocity vs. inhibitor concentration under different chromogenic substrate concentrations (0–2 mM). a Means ± Standard Deviation of three experiments at the inhibitor concentration of 10 µg/mL. ND: no detectable activity.
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work, bicolin was found to own thrombin-inhibitory activity and anticoagulation function, suggesting that bicolin might be responsible for the coagulopathy resulted from the wasp sting (Evans and Summers, 1986; Chao and Lee, 1999; Chen et al., 2004). Acknowledgements This work was supported by the Natural Science Foundation of Hebei province of China (08B029). References
Fig. 4. Dose dependent anticoagulant activity of bicolin. Ca2+-clotting time of control plasma is 7 min.
Ca2+-clotting time of ppp in vitro in a dose-dependent manner. At a bicolin concentration of 10 µg/mL, the ppp clotting time was longer than 2 h compared with the control. In addition, with an increase of preincubation time of bicolin with plasma, the clotting time was prolonged (data not shown). It is likely that bicolin exerts its anticoagulant activity by the way of inhibiting thrombin. 4. Discussion Vespid venoms contain mainly three groups of substances, i.e. high molecular mass proteins (enzymes, toxins and allergens), biological active amines (histamine, serotonin and catecholamines) and small peptides. Three kinds of bioactive peptides (kinins, mastoparans and chemotactic peptides) have been isolated and characterized from various species of vespid venoms (Ho et al., 1998). These peptides can exert variable functions. As an agonist of G proteins, mastoparans compete with G protein receptors (GPRs) for binding to the G proteins (GP) (Higashijima et al., 1988; Higashijima et al., 1990; Holler et al., 1999; dos Santos Cabrera et al., 2004). Moreover, such binding is often selective so as to make mastoparans a great interest in advanced medicine, such as improved peptidic drugs, to target GP (dos Santos Cabrera et al., 2004). In addition, wasp mastoparans and wasp chemotactic peptides have been found to exert antimicrobial (dos Santos Cabrera et al., 2004; Xu et al., 2006a,b; Yu et al., 2007). For example, chemotactic peptides from the wasp venom of V. magnifica could exert wide spectrum of antimicrobial activities against bacteria and fungi (Xu et al., 2006a,b). Recently, two serine protease inhibitor-like peptides (As-fr-19 and CVP2) containing 75-77 amino acid residues are identified from two kinds of wasp venoms of A. samariensis and P. hypochondriaca (Parkinson et al., 2004; Hisada et al., 2005). A pacifastin-like protease inhibitor cvp4 precursor containing 203 amino acid residues was screened by cDNA cloning from P. hypochondriaca (Parkinson et al., 2004). All these peptides or proteins were predicted to act as serine protease inhibitors just by similarity to other known serine protease inhibitor. Their serine protease inhibitory functions have not been investigated. So far no serine protease inhibitor has been reported from vespine wasps. In this work, a novel serine protease inhibitor peptide named bicolin was purified and characterized from the venom of the wasp, V. bicolor Fabricius. Its precursor was cloned from the cDNA library of the venomous glands. It showed similarity to the protease inhibitor precursors of CVP2 and As-fr-19 identified from P. hypochondriaca and A. samariensis, respectively. Bicolin could exert inhibitory activities against trypsin and thrombin. The biological significance of the presence of serine protease inhibitor in wasp venoms is still unknown. As-fr-19 has been suggested to serve as a potassium and/or calcium blocker and then participate in the longterm non-lethal paralysis on the prey (Hisada et al., 2005). In current
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Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part B j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b
A novel serine protease inhibitor from the venom of Vespa bicolor Fabricius Xinbo Yang a,b,1, Yakun Wang a,1, Zekuan Lu a, Lei Zhai a, Juguo Jiang c, Jingze Liu a,⁎, Haining Yu a,⁎ a b c
College of Life Sciences, Hebei Normal University, Shijiazhuang 050016, Hebei, China Biochemistry & Biology College, Yantai University, Yantai 264005, Shandong; China Shandong Shengli Co., Ltd., 2238 Tianchen Street, Jinan 250101, Shandong, China
a r t i c l e
i n f o
Article history: Received 20 December 2008 Received in revised form 6 February 2009 Accepted 16 February 2009 Available online 1 March 2009 Keywords: Bioactive peptides Serine protease inhibitor Vespa bicolor Wasp venom
a b s t r a c t Hornets possess highly toxic venoms, which are rich in toxin, enzymes and biologically active peptides. Many bioactive substances have been identified from wasp venoms but only a few serine protease inhibitors have been identified from two kinds of wasp venoms. In this work, a serine protease inhibitor named bicolin was purified and characterized from the venom of the wasp, Vespa bicolor Fabricius. The precursor encoding bicolin was cloned from the cDNA library of the venomous glands. It is a cysteine-rich small protein containing 54 amino acid residues including 6 half-cysteines. The peptide is homologous to serine protease inhibitors isolated from venoms of Anoplius samariensis and Pimpla hypochondriaca. Bicolin showed inhibitory ability against trypsin and thrombin. © 2009 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
The venoms of arthropods have attracted considerable interest as a potential source of pharmacological substances (Habermann, 1972; Hirai et al., 1979a,b; Nakajima, 1984). Wasp venom gland is biochemically, pharmacologically, and physiologically complex organ which fulfills a wide range of functions necessary for wasp survival, including predatoriness, defense, etc (Han et al., 2008; Yang et al., 2008). Over the past several decades, studies have focused on the bioactive compounds present in wasp venoms (Yasuhara et al., 1983; Argiolas and Pisano, 1984, 1985; Yu et al., 2007). These compounds include amines, small peptides, and proteins of high molecular mass such as enzymes, allergens and toxins (Konno et al., 2000; Mendes et al., 2004; Mendes et al., 2005; Xu et al., 2006a,b; Zhou et al., 2006). Only three serine protease inhibitors (peptide toxin As-fr-19, cysteinerich venom protein 2 (CVP2) and CVP4) are identified from two kinds of wasp venoms (Parkinson et al., 2004; Hisada et al., 2005). Vespa bicolor is one of the most dangerous species of vespine wasps, found in most provinces of China. It is a kind of social wasp. This hornet is aggressive and predatory. Stings by this hornet generally produce severe pain, local damage, cardiovascular system disorder and occasionally death in large vertebrates including man (Evans and Summers, 1986; Sakhuja et al., 1988; Korman et al., 1990; Watemberg et al., 1995; Chao and Lee, 1999; Chen et al., 2004). Little was reported on the chemical constituents of V. bicolor venom. Herein, we reported purification, characterization and biological activities of a serine protease inhibitor from V. bicolor venom.
2.1. Wasp venom
⁎ Corresponding authors. Tel.: +86 311 86268842; fax: +86 311 86268313. E-mail addresses: [email protected] (J. Liu), [email protected] (H. Yu). 1 These authors contributed equally to this paper. 1096-4959/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2009.02.010
The wasps of V. bicolor were collected in Hebei province of China. The collected wasps were stimulated by alternative current (6 V) lasting for 6–10 s. The wasp venom was secreted onto a clean glass plate (50 × 50 cm), immediately collected and stored at −20 °C. 2.2. Peptide purification The lyophilized wasp venom sample (0.1 g) was dissolved in 5 mL 0.1 M phosphate buffer solution, pH 6.0, and filtered through a 10-kDa cut-off Centriprep filter (Millipore, Bedford, CA). The yield of this filtration was about 30%. The filtrate was next lyophilized. Lyophilized filtrate was applied to a 5 × 250-mm Vydac C18 RP-HPLC (reversed-phase high-performance liquid chromatography) column (Sigma) equilibrated with 0.1% (v/v) trifluoroacetic acid/water. Elution (0.7 mL/ min) with collecting fraction of 3 mL was performed using 0.1% (v/v) TFA/water over 10 min, followed by a linear gradient of 0%–80% acetonitrile containing 0.1% (v/v) TFA in 0.1% (v/v) TFA /water over 100 min, and final elution with 80% acetonitrile containing 0.1% (v/v) TFA. The absorbance at 220 nm was monitored. UV-absorbing peaks were collected and lyophilized, and their effects on serine proteases were detected. 2.3. Structural analysis Peptide sample (1 mg) was first dissolved in 1 mL 0.1 M Tris–HCl buffer (pH 8.6) containing 6 M ultra-pure urea and 0.02 M 2-mercaptoethanol. Following flushing with nitrogen, 5 mg of iodoacetic acid was
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added into the reaction mixture while maintaining the pH at 8.6 via the addition of 0.1 M NaOH and incubation for another 3 h. Finally, carboxymethylated (CM) peptide was desalted on a Vydac C8 RP-HPLC column (200 × 4.6 mm) and lyophilized. N-terminal amino acid sequence was directly determined by Edman degradation on an Applied Biosystems pulsed liquid-phase sequencer, model 491. Mass measurement was performed on a Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry instrument (Bruker Reflex), using α-cyano-4-hydroxycinnamic acid (HCCA) suspended in 100% acetonitrile as matrix. 2.4. SMART cDNA synthesis Total RNA was extracted using TRIzol (Life Technologies, Ltd.) from the 0.1 g venomous glands of V. bicolor. cDNA was synthesized by SMART™ techniques by using a SMART™ PCR cDNA synthesis kit (Clontech, Palo Alto, CA). The first strand was synthesized by using cDNA 3′ SMART CDS Primer II A, 5′-AAGCAGTGGTATCAACGCAGAGTACT (30) N-1 N-3′ (N = A, C, G or T; N-1 = A, G or C), and SMART II A oligonucleotide, 5′-AAGCAGTGGTATCAAC GCAGAGTACGCGGG-3′. The second strand was amplified using Advantage polymerase by 5′ PCR primer II A, 5′-AAGCAGTGGTATCAACGCAGAGT- 3′. The PCR conditions were: 2 min at 94 °C, followed by 30 cycles of 10 s at 92 °C, 30 s at 62 °C, 1 min at 72 °C. 2.5. Screening of cDNA encoding bicolin The cDNA synthesized by SMART™ techniques was used as template for PCR to screen the cDNAs encoding bicolin. Two pairs of oligonucleotide primers were used to screen the cDNA encoding biocolin. One pair of them is S1 (5′-gc(A/t/C/G)ca(t/C)cc(A/T/C/G)(C/T) T(A/T/C/G)TG(C/T)(C/T)T(A/T/C/G) (C/T)T(A/T/C/G)GA(C/T)-3′ based on the mature peptides of bicolin in the sense direction) and primer II A as mentioned in “SMART cDNA synthesis” in the antisense direction were used in PCR reactions. Another pair is S2 (5′ (G/A)TC(A/T/ C/G)A(A/G)(A/T/C/G)A(A/G)(A/G)CA(A/T/C/G)A(A/G)(A/T/C/G)GG (A/G)TG(A/t/C/G)CG-3′) and primer II A. The DNA polymerase was Advantage polymerase from Clontech (Palo Alto, CA) The PCR conditions were: 2 min at 94 °C, followed by 30 cycles of 10 s at 92 °C, 30 s at 50 °C, 40 s at 72 °C. Finally, the PCR products were cloned into pGEM®-T Easy vector (Promega, Madison, WI). DNA sequencing was performed on an Applied Biosystems DNA sequencer, model ABI PRISM 377.
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concentration under different chromogenic substrate concentrations (0–2 mM). The peptides were quantified by UV absorbance at 215 and 225 nm using the formula: concentration (mg/mL) = (A215 nm − A225 nm) × 0.144. 2.7. Recalcification time assay Platelet-poor plasma (PPP) was prepared by centrifuging the citrated whole blood twice at 2500 g for 15 min at 4 °C and used within 4 h. For the recalcification time assay (RT), certain amount of sample in 0.1 mL 0.9% NaCl was added to 0.1 mL PPP at 37 °C, and the mixture was incubated at 37 °C for 5 min. The time from the addition of 0.1 mL of 25 mM CaCl2 until the appearance of the first clot was recorded. The plasma aliquots incubated only with 0.1 mL of 0.9% NaCl were tested as controls (Xu et al., 2007). 3. Results 3.1. Purification of serine protease inhibitor peptide As illustrated in Fig. 1, the components from venoms of V. bicolor were considerably complex. About 40 peaks were eluted from venoms by C18 RP-HPLC (Fig. 1). The peak indicated as B (Bicolin) in Fig. 1 was found to exert significant trypsin-inhibitory activity. Ten batches of the purified peptide peaks were pooled and studied further. 3.2. Structural analysis Purified serine protease inhibitor indicated as B in Fig. 1 was named bicolin. It was subjected to amino acid sequence analysis by automated Edman degradation. Its amino acid sequence was `, and it molecular mass was 5749.4 Da analyzed by MALDI-TOF mass spectrometry (Fig. 2). 3.3. cDNA cloning A cDNA clone containing an insert around 360-base pairs was identified and isolated (GenBank accession number FJ749250). Both strands of the clone were sequenced (Fig. 3). It was found to have an open reading frame that encodes a polypeptide composed of 77 amino acids including the predicted signal peptide and the mature bicolin
2.6. Serine protease inhibitory assay The inhibition effects of the sample on the hydrolysis of synthetic chromogenic substrates by serine proteases (All the serine proteases are from Sigma) were assayed in 50 mM Tris–HCl, pH 7.8 at 37 °C according to previous method (Lai et al., 2004). The protease (final concentrations 10 nM for trypsin and thrombin, 21 nM and 23 nM for chymotrypsin, and 20 nM for elastase, respectively) and the inhibitor (final concentration 0.5 μM) were pre-incubated for 10 min at 37 °C (Lai et al., 2004). S-2238 (H-D-Phe-Pip-Arg-pNA, Kabi Vitrum, Stockholm, Sweden) and B-3133 (N-Benzoyl-Arg-4-Nitroanilidehydrochride-pNA, Sigma) were used as substrates for trypsin and thrombin, and elastase, respectively. The reaction was initiated by the addition of the substrate to a final concentration of 0.5 mM. The formation of p-nitroaniline was monitored continuously at 405 nm for 5 min. In the case of chymotrypsin, BTEE (N-benzoyl-tyrosine ethyl ester, Sigma) was used as the substrate and the reaction was monitored continuously at 253 nm for 5 min. The effect of the inhibitor was estimated by setting the initial velocity as 100%, obtained with enzyme alone (without inhibitor). The inhibition assay was carried out as described above and the Ki value was obtained by reciprocal plotting of the reaction velocity vs. inhibitor
Fig. 1. Isolation of serine protease inhibitor from the venoms of V. bicolor. The lyophilized venom sample (0.1 g) was dissolved in 5 mL 0.1 M phosphate buffer solution, pH 6.0, and filtered through a 10-kDa cut-off Centriprep filter (Millipore, Bedford, CA) and the filtrate was and lyophilized. Lyophilized filtrate was applied to a Hypersil BDS C18 RP-HPLC column (5 × 250-mm) equilibrated with 0.1% (v/v) trifluoroacetic acid/water. The elution was performed with the indicated gradient of acetonitrile in Fig. 1 at a flow rate of 0.7 mL/min, and purified serine protease inhibitor was indicated by a “B”. AM1 and AM2 are antimicrobial peptides (Chen et al., 2008).
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Fig. 2. MALDI-TOF mass spectrometry of bicolin.
Fig. 3. The nucleotide sequences encoding bicolin (A) and its sequence comparison (B) with As-fr-19 and CVP2. Mature peptides are boxed. ⁎Stop codon. The amino acids identical to the first line are shaded. Gaps (−) have been introduced to optimize the sequence homology.
sequence (Fig. 3A). The amino acid sequence deduced from the cDNA sequence matched well with the amino acid sequence determined by Edman degradation. The mature bicolin sequence deduced from the precursor has a theoretical molecular mass of 5747.6 Da that is 1.8 Da difference from the observed molecular mass (5749.4 Da). This difference is within the error tolerance, so it might be concluded that the deduced amino acid sequence was identical to the determined amino acid sequence by Edman degradation. The mature bicolin contains six half-cysteines. By Blast search, bicolin showed highly sequence similarity to As-fr-19 and CVP2 (Parkinson et al., 2004; Hisada et al., 2005) (Fig. 3B). They share six conserved halfcystemines in their sequences. The Blast search reveals that they belong to BPTI/Kunitz inhibitor family. 3.4. Serine protease inhibitory activity To examine the inhibitory specificity of sample to the serine proteases, the purified bicolin from the wasp venoms was assayed for inhibitory activity against four different proteases. Bicolin could inhibit trypsin and thrombin, but had no effect on the elastase and chymotrypsin (Table 1). Among those serine proteases, bicolin had the strongest inhibitory activity against trypsin with an inhibitory
constant (Ki) of 5.5 × 10− 7, but comparatively weaker inhibition toward thrombin with the inhibitory constant of 2.6 × 10− 5. 3.5. Anti-coagulant activity Considering bicolin's inhibitory activity against thrombin, its effect on plasma coagulation was assayed using platelet-poor plasma (ppp). As illustrated in Fig. 4, the addition of bicolin increased the
Table 1 Effect of bicolin on hydrolytic activity of different proteases. Proteases
Residual activitya
Inhibitory constant (Ki) (M)
Thrombin Trypsin Elastase Chymotrypsin
15 ± 2.7 5 ± 3.2 98 ± 4.9 99 ± 5.6
2.6 × 10− 5 5.5 × 10− 7 ND ND
The effect of the inhibitor was estimated by setting the initial velocity obtained in the presence of enzyme alone (without inhibitor) as 100%. The Ki value was obtained by reciprocal plotting of the reaction velocity vs. inhibitor concentration under different chromogenic substrate concentrations (0–2 mM). a Means ± Standard Deviation of three experiments at the inhibitor concentration of 10 µg/mL. ND: no detectable activity.
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work, bicolin was found to own thrombin-inhibitory activity and anticoagulation function, suggesting that bicolin might be responsible for the coagulopathy resulted from the wasp sting (Evans and Summers, 1986; Chao and Lee, 1999; Chen et al., 2004). Acknowledgements This work was supported by the Natural Science Foundation of Hebei province of China (08B029). References
Fig. 4. Dose dependent anticoagulant activity of bicolin. Ca2+-clotting time of control plasma is 7 min.
Ca2+-clotting time of ppp in vitro in a dose-dependent manner. At a bicolin concentration of 10 µg/mL, the ppp clotting time was longer than 2 h compared with the control. In addition, with an increase of preincubation time of bicolin with plasma, the clotting time was prolonged (data not shown). It is likely that bicolin exerts its anticoagulant activity by the way of inhibiting thrombin. 4. Discussion Vespid venoms contain mainly three groups of substances, i.e. high molecular mass proteins (enzymes, toxins and allergens), biological active amines (histamine, serotonin and catecholamines) and small peptides. Three kinds of bioactive peptides (kinins, mastoparans and chemotactic peptides) have been isolated and characterized from various species of vespid venoms (Ho et al., 1998). These peptides can exert variable functions. As an agonist of G proteins, mastoparans compete with G protein receptors (GPRs) for binding to the G proteins (GP) (Higashijima et al., 1988; Higashijima et al., 1990; Holler et al., 1999; dos Santos Cabrera et al., 2004). Moreover, such binding is often selective so as to make mastoparans a great interest in advanced medicine, such as improved peptidic drugs, to target GP (dos Santos Cabrera et al., 2004). In addition, wasp mastoparans and wasp chemotactic peptides have been found to exert antimicrobial (dos Santos Cabrera et al., 2004; Xu et al., 2006a,b; Yu et al., 2007). For example, chemotactic peptides from the wasp venom of V. magnifica could exert wide spectrum of antimicrobial activities against bacteria and fungi (Xu et al., 2006a,b). Recently, two serine protease inhibitor-like peptides (As-fr-19 and CVP2) containing 75-77 amino acid residues are identified from two kinds of wasp venoms of A. samariensis and P. hypochondriaca (Parkinson et al., 2004; Hisada et al., 2005). A pacifastin-like protease inhibitor cvp4 precursor containing 203 amino acid residues was screened by cDNA cloning from P. hypochondriaca (Parkinson et al., 2004). All these peptides or proteins were predicted to act as serine protease inhibitors just by similarity to other known serine protease inhibitor. Their serine protease inhibitory functions have not been investigated. So far no serine protease inhibitor has been reported from vespine wasps. In this work, a novel serine protease inhibitor peptide named bicolin was purified and characterized from the venom of the wasp, V. bicolor Fabricius. Its precursor was cloned from the cDNA library of the venomous glands. It showed similarity to the protease inhibitor precursors of CVP2 and As-fr-19 identified from P. hypochondriaca and A. samariensis, respectively. Bicolin could exert inhibitory activities against trypsin and thrombin. The biological significance of the presence of serine protease inhibitor in wasp venoms is still unknown. As-fr-19 has been suggested to serve as a potassium and/or calcium blocker and then participate in the longterm non-lethal paralysis on the prey (Hisada et al., 2005). In current
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