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Molecular cloning and antifibrinolytic activity of a serine protease inhibitor from bumblebee (Bombus terrestris) venom
Toxicon 63 (2013) 1–6
Contents lists available at SciVerse ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Molecular cloning and antifibrinolytic activity of a serine protease inhibitor from bumblebee (Bombus terrestris) venom Yuling Qiu a, b, Kwang Sik Lee a, Young Moo Choo a, Dexin Kong b, c, Hyung Joo Yoon d, Byung Rae Jin a, * a
College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmaceutical Sciences, Tianjin Medical University, Tianjin 300070, China c Research Center of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China d Department of Agricultural Biology, National Academy of Agricultural Science, Suwon, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 July 2012 Received in revised form 8 September 2012 Accepted 6 November 2012 Available online 16 November 2012
Bumblebee (Bombus spp.) venom contains a variety of components, including bombolitin, phospholipase A2 (PLA2), serine proteases, and serine protease inhibitors. In this study, we identified a bumblebee (Bombus terrestris) venom serine protease inhibitor (Bt-KTI) that acts as a plasmin inhibitor. Bt-KTI consists of a 58-amino acid mature peptide that displays features consistent with snake venom Kunitz-type inhibitors, including six conserved cysteine residues and a P1 site. Recombinant Bt-KTI was expressed as a 6.5-kDa peptide in baculovirus-infected insect cells. The recombinant peptide demonstrated properties similar to Kunitz-type trypsin inhibitors. Bt-KTI showed no detectable inhibitory effects on factor Xa, thrombin, or tissue plasminogen activator; however, Bt-KTI strongly inhibited plasmin, indicating that it acts as an antifibrinolytic agent. These findings demonstrate the antifibrinolytic role of Bt-KTI as a plasmin inhibitor. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Antifibrinolytic agent Bumblebee Plasmin inhibitor Serine protease inhibitor Venom
1. Introduction The honeybee (Apis mellifera) and bumblebee (Bombus spp.) are the most abundant bee species worldwide. Bee venom, which serves as a weapon against intruders, contains various toxic components, including enzymes, peptides, and biogenic amines (Hoffman and Jacobson, 1996; Winningham et al., 2004; Hoffman, 2006; Son et al., 2007). Melittin and phospholipase A2 (PLA2) are the two major components of honeybee venom, whereas bumblebee venom contains three major components, i.e., bombolitin, PLA2, and serine proteases (Xin et al., 2009; Choo et al., 2010a,b; Qiu et al., 2011). PLA2 is the most
* Corresponding author. Tel./fax: þ82 51 200 7594. E-mail address: [email protected] (B.R. Jin). 0041-0101/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2012.11.004
commonly studied toxic enzyme of bee venom component (Six and Dennis, 2000). Bombolitin is structurally and biologically similar to melittin of honeybee venom and is the most abundant component in bumblebee venom (Gauldie et al., 1976; Argiolas and Pisano, 1985). Bumblebee venom also contains serine proteases and serine protease inhibitors similar to those demonstrated in snake venoms. In snake venom, certain serine proteases exhibit fibrin(ogen)olytic activity (Braud et al., 2000; Matsui et al., 2000; Kini, 2005; Swenson and Markland, 2005), whereas various serine protease inhibitors show antifibrinolytic activity (Masci et al., 2000; Flight et al., 2005, 2009; Millers et al., 2009). Our previous studies provided evidence for a fibrin(ogen)olytic role of bumblebee venom serine proteases, which act as prothrombin activators, thrombin-like proteases, and plasmin-like proteases (Choo et al., 2010b; Qiu et al., 2011), and an antifibrinolytic role
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of a bumblebee venom serine protease inhibitor, which acts as a plasmin inhibitor (Choo et al., 2012). These findings demonstrated a novel mechanism by which bumblebee venom affects the hemostatic system via venom serine protease inhibitor-mediated antifibrinolytic activity as well as venom serine protease-mediated fibrin(ogen)olytic activities (Choo et al., 2012). The antifibrinolytic role of a bumblebee venom Kunitztype serine protease inhibitor has been reported for Bombus ignitus, although the properties of venom serine protease inhibitors in other bee species have not been elucidated. Here, we report on the molecular cloning and characterization of a venom Kunitz-type serine protease inhibitor (Bt-KTI) from the bumblebee Bombus terrestris, a domesticated species that is widely used in greenhouses to pollinate crops (Velthuis and van Doorn, 2006). We expressed the recombinant Bt-KTI in baculovirus-infected insect cells. This present study demonstrates that Bt-KTI is a plasmin inhibitor that exhibits antifibrinolytic activity, thereby supporting the use of Bt-KTI as a potential antifibrinolytic agent. 2. Materials and methods 2.1. Bumblebees The B. terrestris (Hymenoptera: Apidae) bumblebees used in this study were supplied by the Department of Agricultural Biology, National Academy of Agricultural Science, Republic of Korea. The bumblebees were maintained at 28 C, 65% relative humidity, and in constant darkness, as described previously (Yoon et al., 2009).
of the Autographa californica nucleopolyhedrovirus (AcNPV) polyhedrin promoter. The recombinant baculoviruses were propagated in Sf9 cells cultured in TC100 medium (Gibco BRL, Gaithersburg, MD) at 27 C. The recombinant peptides were purified using the MagneHisÔ Protein Purification System (Promega). The protein concentrations were determined using a Bio-Rad Protein Assay Kit. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and western blot analysis were performed as described previously (Choo et al., 2012) using an Enhanced Chemiluminescence (ECL) Western Blotting Analysis System (Amersham Biosciences, Piscataway, NJ). 2.4. Measurement of protease and inhibitor activity Measurements of the protease and inhibitor activities were performed as previously described (Choo et al., 2012). Trypsin (200 ng, Sigma) or a-chymotrypsin (200 ng, Sigma) was incubated in 100 mM Tris–HCl (pH 8.0) containing 20 mM CaCl2 and 0.05% Triton X-100 with increasing amounts of Bt-KTI at 37 C for 30 min. The residual enzyme activity was determined at 405 nm or 410 nm using the following substrates: 0.2 mM BApNA (Sigma) for trypsin and 0.2 mM Suc-AAPF-pNA (Sigma) for a-chymotrypsin. Additionally, 200 ng of human plasmin (Sigma), thrombin (Sigma), tissue plasminogen activator (tPA; Sigma), or factor Xa (Novagen) was incubated with increasing amounts of BtKTI or aprotinin (Sigma) at 37 C for 30 min in 50 mM Tris– HCl buffer (pH 7.4), and the residual enzyme activity was determined at 405 nm using 0.2 mM of chromogenic substrate (Chromogenix, Mölndal, Sweden): S-2251 for plasmin, S-2238 for thrombin, S-2288 for tPA, and S-2222 for factor Xa.
2.2. Gene cloning and sequence analysis 2.5. Fibrinolytic cleavage assay A clone encoding Bt-KTI was selected from the expressed sequence tags (ESTs) generated from a cDNA library produced using the venom glands of B. terrestris worker bees (Qiu et al., 2011). The plasmid DNA was extracted using the Wizard Mini-Prep Kit (Promega, Madison, WI) and sequenced using an ABI 310 automated DNA sequencer (Perkin–Elmer Applied Biosystems, Foster City, CA). The sequences were compared using DNASIS and BLAST (http://www.ncbi.nlm.nih.gov/ BLAST). The genomic DNA was extracted from the fat body tissues of a single B. terrestris worker bee using the Wizard Genomic DNA Purification Kit (Promega); this DNA was then used as a template for PCR. The sequences of the oligonucleotide primers used for the amplification were forward (1– 24) 50 -ATGAATCATAAATTCATAGCATTA-30 , and reverse (249– 226) 50 -TTATACTGAGCATGATTGCTGACA-30 . The amplification primers were designed using the Bt-KTI cDNA sequence. All PCR products were verified by DNA sequence analysis.
Human fibrinogen (200 mg, Sigma) that had been clotted with 1 unit of thrombin in 50 mM Tris–HCl buffer (pH 7.4) containing 5 mM CaCl2 was incubated with plasmin (500 ng) or both plasmin and Bt-KTI (25 ng) at 37 C. The fibrinolytic cleavage was analyzed using 12% SDS–PAGE (Choo et al., 2012). 2.6. Fibrin plate assay The fibrin plate assay was performed with 5 ml of human fibrinogen (0.5%) clotted with three units of thrombin. Plasmin or a mixture of plasmin and Bt-KTI was dropped onto the fibrin plates, and the plates were incubated at 37 C for various periods of time. The fibrinolytic activity was determined by measuring the formation of a clear area (Choo et al., 2010b, 2012).
2.3. Protein expression and purification
2.7. Plasmin inhibitory assay
A baculovirus/Sf9 insect cell expression system (Je et al., 2001) was used to produce recombinant Bt-KTI. A Bt-KTI cDNA fragment containing the full-length open reading frame was inserted into the pBAC1 vector (Clontech, Palo Alto, CA) to generate an expression vector in which the expression of the recombinant protein was under the control
Human plasmin (30 nM, Sigma) was incubated with increasing amounts of Bt-KTI or aprotinin (Sigma) at 37 C for 30 min in 50 mM Tris–HCl buffer (pH 7.4), and the residual enzyme activity was determined at 405 nm using 200 mM S-2251. The initial reaction rate was determined by calculating the slope of the linear portion of the kinetic
Y. Qiu et al. / Toxicon 63 (2013) 1–6
curve. The inhibitory effect was expressed as the percent reduction in the initial hydrolysis rate; the reaction rate in the absence of inhibitor was taken to be 100%. The inhibitor concentration that decreased the rate of hydrolysis by 50% (IC50) was determined. The values of the inhibition constants (Ki) were calculated using the equation Ki ¼ IC50/ (1 þ S/Km) (Sinauridze et al., 2011).
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and then incubated with diluted (1:1000 v/v) antiserum against plasmin or the His-tag at room temperature for 1 h. After washing in TBST (10 mM Tris–HCl [pH 8.0], 100 mM NaCl, and 0.05% [w/v] Tween 20), the membrane was incubated with horseradish peroxidase-conjugated antimouse IgG diluted 1:5000 (v/v). After repeated washing, the membrane was incubated with ECL detection reagents (Amersham Biosciences) and exposed to X-ray film.
2.8. Electrophoretic mobility shift assay 3. Results and discussion The electrophoretic mobility shift assay was performed as previously described (Choo et al., 2012). Plasmin (1 mg) in 50 mM Tris–HCl buffer (pH 7.4) containing 5 mM CaCl2 was mixed with 0.5 mg of Bt-KTI and incubated at 37 C for 1 h. Samples were resolved on a 10% polyacrylamide gel at 4 C. Following electrophoresis, the proteins were blotted onto a sheet of nitrocellulose transfer membrane (Schleicher & Schuell, Dassel, Germany). The membrane was blocked by incubation in a 1% bovine serum albumin (BSA) solution
3.1. Gene cloning and the expression of Bt-KTI To characterize the serine protease inhibitor in bumblebee venom, we identified an EST for a gene encoding a venom serine protease inhibitor (Bt-KTI) in a B. terrestris cDNA library. The Bt-KTI gene consists of two exons encoding 82 amino acids (aa), including a predicted 24-aa signal peptide and a 58-aa mature peptide (Fig. 1A and B).
Fig. 1. The predicted amino acid sequence and structure of Bt-KTI. (A) The deduced amino acid sequence of Bt-KTI (GenBank accession no. JX273646). The cleavage site of the predicted signal sequence (solid arrowhead) is indicated. The start codon (ATG) is outlined by a box, and the termination codon is shown with an asterisk. The characteristic cysteine residues are indicated by squares. The P1 position is marked with a circle. (B) The genomic structure of the Bt-KTI gene (GenBank accession no. JX273645) inferred from an analysis of the Bt-KTI cDNA. The numbers indicate the position in the genomic sequence. (C) The alignment of the amino acid sequences for mature Bt-KTI and known Kunitz-type serine protease inhibitors. The characteristic cysteine residues are shown in bold. The P1 position is marked with an asterisk. The sources of the aligned sequences were B. terrestris (this study, GenBank accession no. JX273646), B. ignitus (GenBank accession no. JN381496), and P. textilis textilinin-1 (GenBank accession no. AF402324). The Bt-KTI sequence was used as a reference for the identity/ similarity (Id/Si) values.
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Database searches using the peptide sequence of mature BtKTI showed similarity to members of the Kunitz-type serine protease inhibitor family, such as those from B. ignitus (93% peptide sequence identity) and Pseudonaja textilis (54% peptide sequence identity) (Fig. 1C). In addition, the mature Bt-KTI peptide contains features consistent with bumblebee and snake venom Kunitz-type serine protease inhibitors (Shafqat et al.,1990; Siddigi et al.,1991; Masci et al., 2000; Lu et al., 2008; Choo et al., 2012), including six conserved cysteine residues and a P1 site (Fig.1C). These results suggest that Bt-KTI is structurally similar to other Kunitz-type serine protease inhibitors (Millers et al., 2009). The recombinant Bt-KTI, expressed in baculovirusinfected insect cells, was assessed by SDS–PAGE (Fig. 2A). The purified recombinant Bt-KTI, which contains additional 6 His residues, was present as a 6.5-kDa peptide. Using recombinant Bt-KTI, we investigated the inhibitory effects of the peptide. We found that Bt-KTI has a strong inhibitory activity against trypsin but no inhibitory effect against
Fig. 2. The expression and enzyme inhibition of Bt-KTI. (A) SDS–PAGE (left) and western blot analysis (right) of purified recombinant Bt-KTI expressed in baculovirus-infected Sf9 insect cells. Recombinant Bt-KTI was identified using a His-tag antibody. (B) Enzyme inhibition by Bt-KTI. Trypsin or chymotrypsin was incubated with increasing amounts of Bt-KTI, and the residual enzyme activity was then determined (n ¼ 3).
chymotrypsin (Fig. 2B), indicating that Bt-KTI is a Kunitztype trypsin inhibitor-like peptide. Together, these results demonstrate that Bt-KTI is a bee venom Kunitz-type serine protease inhibitor (Shafqat et al., 1990; Siddigi et al., 1991; Masci et al., 2000; Lu et al., 2008; Choo et al., 2012). 3.2. Antifibrinolytic activity of Bt-KTI Given that Bt-KTI is a Kunitz-type inhibitor, we investigated whether Bt-KTI functions in a manner similar to other venom serine protease inhibitors that exhibit antifibrinolytic activity (Masci et al., 2000; Flight et al., 2005, 2009; Choo et al., 2012). Based on an analysis of the degradation of human fibrin by plasmin over time, we observed that Bt-KTI significantly inhibited the transformation of fibrin to fibrin degradation products (FDPs) (Fig. 3A). Subsequently, we assayed the antifibrinolytic activity of Bt-KTI on a fibrin plate. This experiment showed that the addition of Bt-KTI inhibited the plasmin-mediated formation of a clear area in a dose- and time-dependent manner (Fig. 3B). Our results show that Bt-KTI inhibits the plasmin-mediated degradation of fibrin to FDPs, which is consistent with an antifibrinolytic activity of Bt-KTI. We then assessed whether Bt-KTI inhibits several other enzymes associated with the hemostatic system. The results indicate that Bt-KTI has no detectable inhibitory effect on factor Xa, thrombin, or tPA activities (Fig. 4A). In contrast, Bt-KTI strongly inhibited plasmin (Fig. 4B), indicating that Bt-KTI is an effective plasmin inhibitor; however, the inhibitory capacity of Bt-KTI was 1.5-fold less than that of aprotinin, which is widely used as a plasmin
Fig. 3. Plasmin inhibition by Bt-KTI. (A) Bt-KTI-mediated plasmin inhibition assay. The number indicates the time (in min) that fibrin was incubated with plasmin or both plasmin and Bt-KTI. The FDPs are shown. (B) The inhibitory activity of Bt-KTI against plasmin. Plasmin was dropped onto fibrin plates along with different amounts of Bt-KTI, and the plates were then incubated at 37 C for various periods of time.
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Fig. 5. Western blot analysis of plasmin–Bt-KTI complex formation via native gel electrophoresis. One microgram of plasmin was incubated with 0.5 mg of Bt-KTI, and the samples (plasmin, Bt-KTI, or plasmin–Bt-KTI mixture) were resolved on a 10% polyacrylamide gel. After electrophoresis, the protein samples were incubated with antiserum against plasmin (left) or His-tag (right). The plasmin, Bt-KTI, or plasmin–Bt-KTI complexes are indicated using arrows.
4. Conclusion
Fig. 4. Antifibrinolytic activity of Bt-KTI. (A) Inhibitory activity of Bt-KTI. Factor Xa, thrombin, or tPA was incubated with increasing amounts of BtKTI, and the residual enzyme activity was determined (n ¼ 3). (B) Comparison of the inhibitory ability of Bt-KTI with that of aprotinin. Plasmin was incubated with increasing amounts of Bt-KTI or aprotinin, and the residual enzyme activity was determined (n ¼ 3).
inhibitor (Davis and Whittington, 1995; Segal, 2000). In this experiment, the inhibitory constants (Ki) of Bt-KTI and aprotinin against plasmin were 2.01 nM and 1.33 nM, respectively. These results demonstrate the antifibrinolytic activity of Bt-KTI, thereby indicating that Bt-KTI is a plasmin inhibitor. Our findings suggest that Bt-KTI inhibits plasmin during fibrinolysis, indicating that Bt-KTI specifically targets plasmin, as has been demonstrated for the Kunitz-type venom serine protease inhibitor of B. ignitus (Choo et al., 2012) and textilinin-1, a Kunitz-type inhibitor from P. textilis venom (Masci et al., 2000; Flight et al., 2005, 2009; Millers et al., 2009). As shown in Fig. 5, we assessed the formation of plasmin–Bt-KTI complexes using native gel electrophoresis followed by western blotting. The electrophoretic mobility shift assay showed that Bt-KTI binds to plasmin, indicating the formation of a plasmin–Bt-KTI complex. These results are consistent with a mechanism for the antifibrinolytic activity of Bt-KTI involving the formation of a plasmin–BtKTI complex, as has been shown for the Kunitz-type venom serine protease inhibitor of B. ignitus (Choo et al., 2012). Thus, the ability of Bt-KTI to inhibit plasmin indicates that Bt-KTI is an antifibrinolytic agent.
Here, we demonstrated that Bt-KTI functions as an antifibrinolytic agent similar to B. ignitus (Choo et al., 2012) and snake (Masci et al., 2000; Flight et al., 2005, 2009; Millers et al., 2009) venom serine protease inhibitors. Given the similarity between the plasmin targeting of Bt-KTI and that of textilinin-1, an anti-bleeding agent (Masci et al., 2000; Flight et al., 2005, 2009), Bt-KTI likely functions as an antifibrinolytic agent that reduces bleeding at the sting site of victims. Our results identify Bt-KTI as a plasmin inhibitor, as has been demonstrated for the venom serine protease inhibitor of B. ignitus (Choo et al., 2012). The finding that BtKTI possesses antifibrinolytic activity similar to that of the B. ignitus venom serine protease inhibitor provides strong evidence that bumblebee venom serine protease inhibitors are plasmin inhibitors. Our identification of this antifibrinolytic serine protease inhibitor as a component of bumblebee venom will have significant implications for the use of the bee venom serine protease inhibitor as a potential clotting factor. Acknowledgments This work was supported by the Dong-A University Research Fund. Ethical statement This paper has no ethical problem. Conflict of interest statement The authors have no conflict of interest to declare.
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Contents lists available at SciVerse ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Molecular cloning and antifibrinolytic activity of a serine protease inhibitor from bumblebee (Bombus terrestris) venom Yuling Qiu a, b, Kwang Sik Lee a, Young Moo Choo a, Dexin Kong b, c, Hyung Joo Yoon d, Byung Rae Jin a, * a
College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmaceutical Sciences, Tianjin Medical University, Tianjin 300070, China c Research Center of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China d Department of Agricultural Biology, National Academy of Agricultural Science, Suwon, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 July 2012 Received in revised form 8 September 2012 Accepted 6 November 2012 Available online 16 November 2012
Bumblebee (Bombus spp.) venom contains a variety of components, including bombolitin, phospholipase A2 (PLA2), serine proteases, and serine protease inhibitors. In this study, we identified a bumblebee (Bombus terrestris) venom serine protease inhibitor (Bt-KTI) that acts as a plasmin inhibitor. Bt-KTI consists of a 58-amino acid mature peptide that displays features consistent with snake venom Kunitz-type inhibitors, including six conserved cysteine residues and a P1 site. Recombinant Bt-KTI was expressed as a 6.5-kDa peptide in baculovirus-infected insect cells. The recombinant peptide demonstrated properties similar to Kunitz-type trypsin inhibitors. Bt-KTI showed no detectable inhibitory effects on factor Xa, thrombin, or tissue plasminogen activator; however, Bt-KTI strongly inhibited plasmin, indicating that it acts as an antifibrinolytic agent. These findings demonstrate the antifibrinolytic role of Bt-KTI as a plasmin inhibitor. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Antifibrinolytic agent Bumblebee Plasmin inhibitor Serine protease inhibitor Venom
1. Introduction The honeybee (Apis mellifera) and bumblebee (Bombus spp.) are the most abundant bee species worldwide. Bee venom, which serves as a weapon against intruders, contains various toxic components, including enzymes, peptides, and biogenic amines (Hoffman and Jacobson, 1996; Winningham et al., 2004; Hoffman, 2006; Son et al., 2007). Melittin and phospholipase A2 (PLA2) are the two major components of honeybee venom, whereas bumblebee venom contains three major components, i.e., bombolitin, PLA2, and serine proteases (Xin et al., 2009; Choo et al., 2010a,b; Qiu et al., 2011). PLA2 is the most
* Corresponding author. Tel./fax: þ82 51 200 7594. E-mail address: [email protected] (B.R. Jin). 0041-0101/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2012.11.004
commonly studied toxic enzyme of bee venom component (Six and Dennis, 2000). Bombolitin is structurally and biologically similar to melittin of honeybee venom and is the most abundant component in bumblebee venom (Gauldie et al., 1976; Argiolas and Pisano, 1985). Bumblebee venom also contains serine proteases and serine protease inhibitors similar to those demonstrated in snake venoms. In snake venom, certain serine proteases exhibit fibrin(ogen)olytic activity (Braud et al., 2000; Matsui et al., 2000; Kini, 2005; Swenson and Markland, 2005), whereas various serine protease inhibitors show antifibrinolytic activity (Masci et al., 2000; Flight et al., 2005, 2009; Millers et al., 2009). Our previous studies provided evidence for a fibrin(ogen)olytic role of bumblebee venom serine proteases, which act as prothrombin activators, thrombin-like proteases, and plasmin-like proteases (Choo et al., 2010b; Qiu et al., 2011), and an antifibrinolytic role
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of a bumblebee venom serine protease inhibitor, which acts as a plasmin inhibitor (Choo et al., 2012). These findings demonstrated a novel mechanism by which bumblebee venom affects the hemostatic system via venom serine protease inhibitor-mediated antifibrinolytic activity as well as venom serine protease-mediated fibrin(ogen)olytic activities (Choo et al., 2012). The antifibrinolytic role of a bumblebee venom Kunitztype serine protease inhibitor has been reported for Bombus ignitus, although the properties of venom serine protease inhibitors in other bee species have not been elucidated. Here, we report on the molecular cloning and characterization of a venom Kunitz-type serine protease inhibitor (Bt-KTI) from the bumblebee Bombus terrestris, a domesticated species that is widely used in greenhouses to pollinate crops (Velthuis and van Doorn, 2006). We expressed the recombinant Bt-KTI in baculovirus-infected insect cells. This present study demonstrates that Bt-KTI is a plasmin inhibitor that exhibits antifibrinolytic activity, thereby supporting the use of Bt-KTI as a potential antifibrinolytic agent. 2. Materials and methods 2.1. Bumblebees The B. terrestris (Hymenoptera: Apidae) bumblebees used in this study were supplied by the Department of Agricultural Biology, National Academy of Agricultural Science, Republic of Korea. The bumblebees were maintained at 28 C, 65% relative humidity, and in constant darkness, as described previously (Yoon et al., 2009).
of the Autographa californica nucleopolyhedrovirus (AcNPV) polyhedrin promoter. The recombinant baculoviruses were propagated in Sf9 cells cultured in TC100 medium (Gibco BRL, Gaithersburg, MD) at 27 C. The recombinant peptides were purified using the MagneHisÔ Protein Purification System (Promega). The protein concentrations were determined using a Bio-Rad Protein Assay Kit. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and western blot analysis were performed as described previously (Choo et al., 2012) using an Enhanced Chemiluminescence (ECL) Western Blotting Analysis System (Amersham Biosciences, Piscataway, NJ). 2.4. Measurement of protease and inhibitor activity Measurements of the protease and inhibitor activities were performed as previously described (Choo et al., 2012). Trypsin (200 ng, Sigma) or a-chymotrypsin (200 ng, Sigma) was incubated in 100 mM Tris–HCl (pH 8.0) containing 20 mM CaCl2 and 0.05% Triton X-100 with increasing amounts of Bt-KTI at 37 C for 30 min. The residual enzyme activity was determined at 405 nm or 410 nm using the following substrates: 0.2 mM BApNA (Sigma) for trypsin and 0.2 mM Suc-AAPF-pNA (Sigma) for a-chymotrypsin. Additionally, 200 ng of human plasmin (Sigma), thrombin (Sigma), tissue plasminogen activator (tPA; Sigma), or factor Xa (Novagen) was incubated with increasing amounts of BtKTI or aprotinin (Sigma) at 37 C for 30 min in 50 mM Tris– HCl buffer (pH 7.4), and the residual enzyme activity was determined at 405 nm using 0.2 mM of chromogenic substrate (Chromogenix, Mölndal, Sweden): S-2251 for plasmin, S-2238 for thrombin, S-2288 for tPA, and S-2222 for factor Xa.
2.2. Gene cloning and sequence analysis 2.5. Fibrinolytic cleavage assay A clone encoding Bt-KTI was selected from the expressed sequence tags (ESTs) generated from a cDNA library produced using the venom glands of B. terrestris worker bees (Qiu et al., 2011). The plasmid DNA was extracted using the Wizard Mini-Prep Kit (Promega, Madison, WI) and sequenced using an ABI 310 automated DNA sequencer (Perkin–Elmer Applied Biosystems, Foster City, CA). The sequences were compared using DNASIS and BLAST (http://www.ncbi.nlm.nih.gov/ BLAST). The genomic DNA was extracted from the fat body tissues of a single B. terrestris worker bee using the Wizard Genomic DNA Purification Kit (Promega); this DNA was then used as a template for PCR. The sequences of the oligonucleotide primers used for the amplification were forward (1– 24) 50 -ATGAATCATAAATTCATAGCATTA-30 , and reverse (249– 226) 50 -TTATACTGAGCATGATTGCTGACA-30 . The amplification primers were designed using the Bt-KTI cDNA sequence. All PCR products were verified by DNA sequence analysis.
Human fibrinogen (200 mg, Sigma) that had been clotted with 1 unit of thrombin in 50 mM Tris–HCl buffer (pH 7.4) containing 5 mM CaCl2 was incubated with plasmin (500 ng) or both plasmin and Bt-KTI (25 ng) at 37 C. The fibrinolytic cleavage was analyzed using 12% SDS–PAGE (Choo et al., 2012). 2.6. Fibrin plate assay The fibrin plate assay was performed with 5 ml of human fibrinogen (0.5%) clotted with three units of thrombin. Plasmin or a mixture of plasmin and Bt-KTI was dropped onto the fibrin plates, and the plates were incubated at 37 C for various periods of time. The fibrinolytic activity was determined by measuring the formation of a clear area (Choo et al., 2010b, 2012).
2.3. Protein expression and purification
2.7. Plasmin inhibitory assay
A baculovirus/Sf9 insect cell expression system (Je et al., 2001) was used to produce recombinant Bt-KTI. A Bt-KTI cDNA fragment containing the full-length open reading frame was inserted into the pBAC1 vector (Clontech, Palo Alto, CA) to generate an expression vector in which the expression of the recombinant protein was under the control
Human plasmin (30 nM, Sigma) was incubated with increasing amounts of Bt-KTI or aprotinin (Sigma) at 37 C for 30 min in 50 mM Tris–HCl buffer (pH 7.4), and the residual enzyme activity was determined at 405 nm using 200 mM S-2251. The initial reaction rate was determined by calculating the slope of the linear portion of the kinetic
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curve. The inhibitory effect was expressed as the percent reduction in the initial hydrolysis rate; the reaction rate in the absence of inhibitor was taken to be 100%. The inhibitor concentration that decreased the rate of hydrolysis by 50% (IC50) was determined. The values of the inhibition constants (Ki) were calculated using the equation Ki ¼ IC50/ (1 þ S/Km) (Sinauridze et al., 2011).
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and then incubated with diluted (1:1000 v/v) antiserum against plasmin or the His-tag at room temperature for 1 h. After washing in TBST (10 mM Tris–HCl [pH 8.0], 100 mM NaCl, and 0.05% [w/v] Tween 20), the membrane was incubated with horseradish peroxidase-conjugated antimouse IgG diluted 1:5000 (v/v). After repeated washing, the membrane was incubated with ECL detection reagents (Amersham Biosciences) and exposed to X-ray film.
2.8. Electrophoretic mobility shift assay 3. Results and discussion The electrophoretic mobility shift assay was performed as previously described (Choo et al., 2012). Plasmin (1 mg) in 50 mM Tris–HCl buffer (pH 7.4) containing 5 mM CaCl2 was mixed with 0.5 mg of Bt-KTI and incubated at 37 C for 1 h. Samples were resolved on a 10% polyacrylamide gel at 4 C. Following electrophoresis, the proteins were blotted onto a sheet of nitrocellulose transfer membrane (Schleicher & Schuell, Dassel, Germany). The membrane was blocked by incubation in a 1% bovine serum albumin (BSA) solution
3.1. Gene cloning and the expression of Bt-KTI To characterize the serine protease inhibitor in bumblebee venom, we identified an EST for a gene encoding a venom serine protease inhibitor (Bt-KTI) in a B. terrestris cDNA library. The Bt-KTI gene consists of two exons encoding 82 amino acids (aa), including a predicted 24-aa signal peptide and a 58-aa mature peptide (Fig. 1A and B).
Fig. 1. The predicted amino acid sequence and structure of Bt-KTI. (A) The deduced amino acid sequence of Bt-KTI (GenBank accession no. JX273646). The cleavage site of the predicted signal sequence (solid arrowhead) is indicated. The start codon (ATG) is outlined by a box, and the termination codon is shown with an asterisk. The characteristic cysteine residues are indicated by squares. The P1 position is marked with a circle. (B) The genomic structure of the Bt-KTI gene (GenBank accession no. JX273645) inferred from an analysis of the Bt-KTI cDNA. The numbers indicate the position in the genomic sequence. (C) The alignment of the amino acid sequences for mature Bt-KTI and known Kunitz-type serine protease inhibitors. The characteristic cysteine residues are shown in bold. The P1 position is marked with an asterisk. The sources of the aligned sequences were B. terrestris (this study, GenBank accession no. JX273646), B. ignitus (GenBank accession no. JN381496), and P. textilis textilinin-1 (GenBank accession no. AF402324). The Bt-KTI sequence was used as a reference for the identity/ similarity (Id/Si) values.
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Database searches using the peptide sequence of mature BtKTI showed similarity to members of the Kunitz-type serine protease inhibitor family, such as those from B. ignitus (93% peptide sequence identity) and Pseudonaja textilis (54% peptide sequence identity) (Fig. 1C). In addition, the mature Bt-KTI peptide contains features consistent with bumblebee and snake venom Kunitz-type serine protease inhibitors (Shafqat et al.,1990; Siddigi et al.,1991; Masci et al., 2000; Lu et al., 2008; Choo et al., 2012), including six conserved cysteine residues and a P1 site (Fig.1C). These results suggest that Bt-KTI is structurally similar to other Kunitz-type serine protease inhibitors (Millers et al., 2009). The recombinant Bt-KTI, expressed in baculovirusinfected insect cells, was assessed by SDS–PAGE (Fig. 2A). The purified recombinant Bt-KTI, which contains additional 6 His residues, was present as a 6.5-kDa peptide. Using recombinant Bt-KTI, we investigated the inhibitory effects of the peptide. We found that Bt-KTI has a strong inhibitory activity against trypsin but no inhibitory effect against
Fig. 2. The expression and enzyme inhibition of Bt-KTI. (A) SDS–PAGE (left) and western blot analysis (right) of purified recombinant Bt-KTI expressed in baculovirus-infected Sf9 insect cells. Recombinant Bt-KTI was identified using a His-tag antibody. (B) Enzyme inhibition by Bt-KTI. Trypsin or chymotrypsin was incubated with increasing amounts of Bt-KTI, and the residual enzyme activity was then determined (n ¼ 3).
chymotrypsin (Fig. 2B), indicating that Bt-KTI is a Kunitztype trypsin inhibitor-like peptide. Together, these results demonstrate that Bt-KTI is a bee venom Kunitz-type serine protease inhibitor (Shafqat et al., 1990; Siddigi et al., 1991; Masci et al., 2000; Lu et al., 2008; Choo et al., 2012). 3.2. Antifibrinolytic activity of Bt-KTI Given that Bt-KTI is a Kunitz-type inhibitor, we investigated whether Bt-KTI functions in a manner similar to other venom serine protease inhibitors that exhibit antifibrinolytic activity (Masci et al., 2000; Flight et al., 2005, 2009; Choo et al., 2012). Based on an analysis of the degradation of human fibrin by plasmin over time, we observed that Bt-KTI significantly inhibited the transformation of fibrin to fibrin degradation products (FDPs) (Fig. 3A). Subsequently, we assayed the antifibrinolytic activity of Bt-KTI on a fibrin plate. This experiment showed that the addition of Bt-KTI inhibited the plasmin-mediated formation of a clear area in a dose- and time-dependent manner (Fig. 3B). Our results show that Bt-KTI inhibits the plasmin-mediated degradation of fibrin to FDPs, which is consistent with an antifibrinolytic activity of Bt-KTI. We then assessed whether Bt-KTI inhibits several other enzymes associated with the hemostatic system. The results indicate that Bt-KTI has no detectable inhibitory effect on factor Xa, thrombin, or tPA activities (Fig. 4A). In contrast, Bt-KTI strongly inhibited plasmin (Fig. 4B), indicating that Bt-KTI is an effective plasmin inhibitor; however, the inhibitory capacity of Bt-KTI was 1.5-fold less than that of aprotinin, which is widely used as a plasmin
Fig. 3. Plasmin inhibition by Bt-KTI. (A) Bt-KTI-mediated plasmin inhibition assay. The number indicates the time (in min) that fibrin was incubated with plasmin or both plasmin and Bt-KTI. The FDPs are shown. (B) The inhibitory activity of Bt-KTI against plasmin. Plasmin was dropped onto fibrin plates along with different amounts of Bt-KTI, and the plates were then incubated at 37 C for various periods of time.
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Fig. 5. Western blot analysis of plasmin–Bt-KTI complex formation via native gel electrophoresis. One microgram of plasmin was incubated with 0.5 mg of Bt-KTI, and the samples (plasmin, Bt-KTI, or plasmin–Bt-KTI mixture) were resolved on a 10% polyacrylamide gel. After electrophoresis, the protein samples were incubated with antiserum against plasmin (left) or His-tag (right). The plasmin, Bt-KTI, or plasmin–Bt-KTI complexes are indicated using arrows.
4. Conclusion
Fig. 4. Antifibrinolytic activity of Bt-KTI. (A) Inhibitory activity of Bt-KTI. Factor Xa, thrombin, or tPA was incubated with increasing amounts of BtKTI, and the residual enzyme activity was determined (n ¼ 3). (B) Comparison of the inhibitory ability of Bt-KTI with that of aprotinin. Plasmin was incubated with increasing amounts of Bt-KTI or aprotinin, and the residual enzyme activity was determined (n ¼ 3).
inhibitor (Davis and Whittington, 1995; Segal, 2000). In this experiment, the inhibitory constants (Ki) of Bt-KTI and aprotinin against plasmin were 2.01 nM and 1.33 nM, respectively. These results demonstrate the antifibrinolytic activity of Bt-KTI, thereby indicating that Bt-KTI is a plasmin inhibitor. Our findings suggest that Bt-KTI inhibits plasmin during fibrinolysis, indicating that Bt-KTI specifically targets plasmin, as has been demonstrated for the Kunitz-type venom serine protease inhibitor of B. ignitus (Choo et al., 2012) and textilinin-1, a Kunitz-type inhibitor from P. textilis venom (Masci et al., 2000; Flight et al., 2005, 2009; Millers et al., 2009). As shown in Fig. 5, we assessed the formation of plasmin–Bt-KTI complexes using native gel electrophoresis followed by western blotting. The electrophoretic mobility shift assay showed that Bt-KTI binds to plasmin, indicating the formation of a plasmin–Bt-KTI complex. These results are consistent with a mechanism for the antifibrinolytic activity of Bt-KTI involving the formation of a plasmin–BtKTI complex, as has been shown for the Kunitz-type venom serine protease inhibitor of B. ignitus (Choo et al., 2012). Thus, the ability of Bt-KTI to inhibit plasmin indicates that Bt-KTI is an antifibrinolytic agent.
Here, we demonstrated that Bt-KTI functions as an antifibrinolytic agent similar to B. ignitus (Choo et al., 2012) and snake (Masci et al., 2000; Flight et al., 2005, 2009; Millers et al., 2009) venom serine protease inhibitors. Given the similarity between the plasmin targeting of Bt-KTI and that of textilinin-1, an anti-bleeding agent (Masci et al., 2000; Flight et al., 2005, 2009), Bt-KTI likely functions as an antifibrinolytic agent that reduces bleeding at the sting site of victims. Our results identify Bt-KTI as a plasmin inhibitor, as has been demonstrated for the venom serine protease inhibitor of B. ignitus (Choo et al., 2012). The finding that BtKTI possesses antifibrinolytic activity similar to that of the B. ignitus venom serine protease inhibitor provides strong evidence that bumblebee venom serine protease inhibitors are plasmin inhibitors. Our identification of this antifibrinolytic serine protease inhibitor as a component of bumblebee venom will have significant implications for the use of the bee venom serine protease inhibitor as a potential clotting factor. Acknowledgments This work was supported by the Dong-A University Research Fund. Ethical statement This paper has no ethical problem. Conflict of interest statement The authors have no conflict of interest to declare.
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