- Email: [email protected]
Effects of the bumblebee (Bombus ignitus) venom serine protease inhibitor on serine protease and phospholipase A2 of B. ignitus venom
Journal of Asia-Pacific Entomology 15 (2012) 543–545
Contents lists available at SciVerse ScienceDirect
Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape
Short Communication
Effects of the bumblebee (Bombus ignitus) venom serine protease inhibitor on serine protease and phospholipase A2 of B. ignitus venom Young Moo Choo a, Hyung Joo Yoon b, Byung Rae Jin a,⁎ a b
College of Natural Resources and Life Science, Dong-A University, Busan 604–714, Republic of Korea Department of Agricultural Biology, National Academy of Agricultural Science, RDA, Suwon 441–100, Republic of Korea
a r t i c l e
i n f o
Article history: Received 9 April 2012 Revised 14 May 2012 Accepted 14 May 2012 Keywords: Bumblebee Bombus ignitus Serine protease Serine protease inhibitor Venom
a b s t r a c t Bumblebee venom contains serine proteases and serine protease inhibitors. In this study, we characterized whether the bumblebee (Bombus ignitus) venom serine protease inhibitor (Bi-KTI) inhibits B. ignitus venom serine protease (Bi-VSP) or phospholipase A2 (Bi-PLA2). Bi-KTI did not inhibit Bi-VSP activity at pH 5.4 or 7.4, whereas Bi-KTI slightly inhibited Bi-VSP activity at pH 7.4 after a 30 min preincubation. The Bi-VSP activity that converts prothrombin into thrombin and fibrin into fibrin degradation products was not significantly affected by Bi-KTI. Additionally, Bi-KTI or Bi-VSP did not inhibit Bi-PLA2 activity. These findings indicate that each bee venom component appears to a play a toxic role via a unique function. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2012. Published by Elsevier B.V. All rights reserved.
Introduction
Materials and methods
Bumblebee (Bombus spp.) venom contains a variety of components, including bombolitin, phospholipase A2 (PLA2), serine proteases, and serine protease inhibitors (Xin et al., 2009; Choo et al., 2010a, b, 2012; Qiu et al., 2011, 2012a, b). Serine proteases and serine protease inhibitors have also been found in snake venoms, and a number of serine proteases exhibit fibrin(ogen)olytic activity (Braud et al., 2000; Matsui et al., 2000; Kini, 2005), whereas serine protease inhibitors show antifibrinolytic activity (Masci et al., 2000; Flight et al., 2005, 2009; Miller et al., 2009). Our previous studies focused on the fibrin(ogen)olytic activity 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, 2012b). Additionally, our previous study focused on the antifibrinolytic role of a bee venom serine protease inhibitor that acts as a plasmin inhibitor (Choo et al., 2012). Although bumblebee venom is involved in the hemostatic system through the antifibrinolytic activity of the venom serine protease inhibitor (Choo et al., 2012) and through venom serine protease-mediated fibrin(ogen)olytic activity (Choo et al., 2010b; Qiu et al., 2011, 2012b), the mechanism by which venom serine protease inhibitors affect venom components, including serine proteases and PLA2, remains poorly understood. In this study, we investigated whether bumblebee (Bombus ignitus) venom serine protease inhibitor (Bi-KTI) inhibited the activity of B. ignitus venom serine protease (Bi-VSP) and PLA2 (Bi-PLA2).
Inhibitory activity of Bi-KTI against Bi-VSP The inhibitory activity assay of Bi-KTI against Bi-VSP was performed using a 96-well plate with a 200 μl final volume of buffer per well. Bi-VSP (400 ng) or a mixture of Bi-VSP (400 ng) and Bi-KTI (13.3 ng), with or without a 30 min preincubation at 37 °C, was incubated with a thrombin fluorogenic substrate (50 μM Boc-Val-Pro-Arg-7-amido-4methylcoumarin hydrochloride; Sigma, St. Louis, MO, USA) in 50 mM citrate-NaOH (pH 5.4) or 50 mM Tris–HCl (pH 7.4) at 25 °C. Fluorescence was recorded at an emission wavelength of 380 and an excitation wavelength of 440 nm every 50 s for 700 s using a dual scanning microplate spectrofluorometer (Gemini XPS, Molecular Devices, Sunnyvale, CA, USA). Hydrolysis of the substrate by Bi-VSP was expressed as relative fluorescence units (RFU). Prothrombin activation assay Human prothrombin (200 μg, Sigma) was incubated with Bi-VSP (20 ng) or a mixture of Bi-VSP (20 ng) and Bi-KTI (0.66 ng) in 50 mM Tris–HCl buffer (pH 7.4) containing 5 mM CaCl2 at 37 °C. Aliquots were removed at various time points and analyzed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fibrin degradation assay
⁎ Corresponding author: Tel./fax: + 82 51 200 7594. E-mail address: [email protected] (B.R. Jin).
Human fibrinogen (200 μg, Sigma), clotted with one unit of thrombin in 50 mM Tris–HCl buffer (pH 7.4) containing 5 mM CaCl2, was incubated
1226-8615/$ – see front matter © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2012. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aspen.2012.05.011
544
Y.M. Choo et al. / Journal of Asia-Pacific Entomology 15 (2012) 543–545
with Bi-VSP (20 ng) or a mixture of Bi-VSP (20 ng) and Bi-KTI (0.66 ng) at 37 °C. Aliquots were removed at various time points and analyzed using 12% SDS-PAGE. Inhibitory activity of Bi-KTI and Bi-VSP against Bi-PLA2 Hydrolysis of 1-O-(6-dabcyl-aminohexanoyl)-2-O-(6-[12-BODIPYdodecanoyl]amino-hexanoyl)-sn-3-glycerylphophatidylcholine (DBPC) by Bi-PLA2 was performed as described previously (Xin et al., 2009). The inhibitory activity assay for Bi-KTI and Bi-VSP against Bi-PLA2 was performed using a 96-well plate with a 200 μl final volume of buffer per well. Each of the following compounds were pre-incubated in 100 mM Tris–HCl buffer (pH 7.4) at room temperature for 24 h: BiPLA2 (800 ng); Bi-VSP (400 ng); a mixture of Bi-PLA2 (800 ng) and BiVSP (400 ng); and a mixture of Bi-PLA2 (800 ng), Bi-VSP (400 ng), and Bi-KTI (13.3 ng). An aliquot of 1.78 μM DBPC was added to each well, and the reaction was incubated at 37 °C for 30 min. The fluorescence end points were measured at an emission wavelength of 500 nm and an excitation wavelength of 530 nm using a dual scanning microplate spectrofluorometer (Gemini XPS). The hydrolysis of DBPC by Bi-PLA2 was expressed as RFU. Results and discussion Bee venom contains both serine proteases and serine protease inhibitors (Choo et al., 2010b, 2012; Qiu et al., 2011, 2012b). To investigate whether Bi-KTI inhibited Bi-VSP, we assessed the inhibition of Bi-VSP at pH 5.4 or 7.4 with or without Bi-KTI. As a result, when
Bi-VSP was incubated with Bi-KTI without a preincubation, Bi-KTI did not inhibit Bi-VSP activity (Fig. 1A), whereas Bi-KTI slightly inhibited Bi-VSP activity at pH 7.4 following a 30 min preincubation (Fig. 1B). We also found that Bi-VSP was active at pH 7.4 (human blood condition) but not at pH 5.4 (bee venom condition). This result is consistent with a previous result that Bi-VSP activity is almost completely inhibited at the acidic pH of 5.4 of B. ignitus venom (Choo et al., 2011). Bi-VSP acts as both a prothrombin activator and fibrin(ogen)olytic enzyme (Choo et al., 2010b). Thus, we also assessed whether Bi-KTI inhibited Bi-VSP by determining the time course of prothrombin activation and fibrin degradation. Prothrombin was cleaved by Bi-VSP to produce thrombin when Bi-VSP was treated without Bi-KTI (Fig. 1C), and fibrin was degraded into fibrin degradation products (FDPs) by Bi-VSP (Fig. 1D). Moreover, Bi-VSP activity in the presence of Bi-KTI was similar to that of Bi-VSP alone (Choo et al., 2012). These results showed that Bi-KTI did not inhibit the activation of prothrombin or the degradation of fibrin into fibrin degradation products by Bi-VSP. Bee venom also contains a PLA2 that acts as a major toxic component (Xin et al., 2009; Choo et al., 2010b). Thus, we investigated whether Bi-VSP or Bi-KTI affected Bi-PLA2. We pre-incubated Bi-PLA2 with BiVSP or a mixture of Bi-VSP and Bi-KTI for 24 h and found that Bi-VSP and Bi-KTI did not inhibit Bi-PLA2 activity (Fig. 2). In conclusion, these data reveal that Bi-KTI was not significantly affected by the activity of B. ignitus venom components. The observation that bee venom contains Bi-VSP, Bi-KTI, and Bi-PLA2 suggests that each venom component must have its own function (Choo et al., 2010b, 2012). Considering the specific role of Bi-KTI as a plasmin
Fig. 1. Inhibitory activity of Bombus ignitus venom serine protease inhibitor (Bi-KTI) on B. ignitus venom serine protease (Bi-VSP). Bi-VSP or a mixture of Bi-VSP and Bi-KTI was incubated at pH 5.4 or 7.4 with (A) or without (B) a 30 min preincubation. The kinetic curves for digestion of the Bi-VSP substrate are expressed as relative fluorescence units (RFU). (C) Prothrombin activation of Bi-VSP in the presence of Bi-KTI. Human prothrombin activation of Bi-VSP was analyzed by 12% SDS-PAGE. Number indicates the time (min) that prothrombin was incubated with Bi-VSP. Prothrombin and thrombin are shown. (D) Fibrin(ogen)olytic activity of Bi-VSP in the presence of Bi-KTI. Bi-VSP fibrinolytic activity was analyzed by 12% SDS-PAGE. Number indicates the time (min) that fibrin was incubated with Bi-VSP. Human fibrin chains and fibrin degradation products (FDPs) are shown.
Y.M. Choo et al. / Journal of Asia-Pacific Entomology 15 (2012) 543–545
545
References
Fig. 2. Inhibitory activity of Bombus ignitus venom serine protease inhibitor (Bi-KTI) or B. ignitus venom serine protease (Bi-VSP) on phospholipase A2 (Bi-PLA2). Bi-PLA2, Bi-VSP, a mixture of Bi-PLA2 and Bi-VSP, or a mixture of Bi-PLA2, Bi-VSP, and Bi-KTI was pre-incubated for 24 h. The hydrolysis of 1-O-(6-dabcyl-aminohexanoyl)-2-O-(6-[12BODIPY-dodecanoyl]amino-hexanoyl)-sn-3-glycerylphophatidylcholine (DBPC) by PLA2 is expressed as relative fluorescence units (RFU).
inhibitor (Choo et al., 2012), our findings show that Bi-KTI is not a target of Bi-VSP. Taken together, these results indicate that each component of bee venom appears to play a toxic role with a unique function.
Acknowledgments This study was supported by the Dong-A University Research Fund.
Braud, S., Bon, C., Wisner, A., 2000. Snake venom proteins acting on hemostasis. Biochimie 82, 851–859. Choo, Y.M., Lee, K.S., Yoon, H.J., Je, Y.H., Lee, S.W., Sohn, H.D., Jin, B.R., 2010a. Molecular cloning and antimicrobial activity of bombolitin, a component of bumblebee Bombus ignitus venom. Comp. Biochem. Physiol. B 156, 168–173. Choo, Y.M., Lee, K.S., Yoon, H.J., Kim, B.Y., Sohn, M.R., Roh, J.Y., Je, Y.H., Kim, N.J., Kim, I., Woo, S.D., Sohn, H.D., Jin, B.R., 2010b. Dual strategy of bee venom serine protease: prophenoloxidase-activating factor in arthropods and fibrin(ogen)olytic enzyme in mammals. PLoS One 5, e10393. Choo, Y.M., Qiu, Y., Yoon, H.J., Lee, K.Y., Sohn, H.D., Jin, B.R., 2011. Enzymatic properties of a bee venom serine protease from the bumblebee Bombus ignitus. J. Asia Pac. Entomol. 14, 249–251. Choo, Y.M., Lee, K.S., Yoon, H.J., Qiu, Y., Wan, H., Sohn, M.R., Sohn, H.D., Jin, B.R., 2012. Antifibrinolytic role of a bee venom serine protease inhibitor that acts as a plasmin inhibitor. PLoS One 7, e32269. Flight, S., Johnson, L., Trabi, M., Gaffney, P., Lavin, M., de Jersey, J., Masci, P., 2005. Comparison of textinin-1 with aprotinin as serine protease inhibitors and as antifibrinolytic agents. Pathophysiol. Haemost. Thromb. 34, 188–193. Flight, S.M., Johnson, L.A., Du, Q.S., Warner, R.L., Trabi, M., Gaffney, P.J., Lavin, M.F., de Jersey, J., Masci, P.P., 2009. Textilinin-1, an alternative anti-bleeding agent to aprotinin: Importance of plasmin inhibition in controlling blood loss. Br. J. Haematol. 145, 207–211. Kini, R.M., 2005. Serine proteases affecting blood coagulation and fibrinolysis from snake venoms. Pathophysiol. Haemost. Thromb. 34, 200–204. Masci, P.P., Whitaker, A.N., Sparrow, L.G., de Jersey, J., Winzor, D.J., Watters, D.J., Lavin, M.F., Gaffney, P.J., 2000. Textilinins from Pseudonaja textilis textilis. Characterization of two plasmin inhibitors that reduce bleeding in an animal model. Blood Coagul. Fibrinolysis 11, 385–393. Matsui, T., Fujimura, Y., Titani, K., 2000. Snake venom proteases affecting hemostasis and thrombisis. Biochim. Biophys. Acta 1477, 146–156. Miller, E.K.I., Trabi, M., Masci, P.P., Lavin, M.F., de Jersey, J., Guddat, L.W., 2009. Crystal structure of textilinin-1, a Kunitz-type serine protease inhibitor from the venom of the Australian common brown snake (Pseudonaja textiles). FEBS J. 276, 3163–3175. Qiu, Y., Choo, Y.M., Yoon, H.J., Jia, J., Cui, Z., Wang, D., Kim, D.H., Sohn, H.D., Jin, B.R., 2011. Fibrin(ogen)olytic activity of bumblebee venom serine protease. Toxicol. Appl. Pharmacol. 255, 207–213. Qiu, Y., Choo, Y.M., Yoon, H.J., Jin, B.R., 2012a. Molecular cloning and antibacterial activity of bombolitin isolated from the venom of a bumblebee, Bombus terrestris. J. Asia-Pac. Entomol. 15, 21–25. Qiu, Y., Choo, Y.M., Yoon, H.J., Jin, B.R., 2012b. Molecular cloning and fibrin(ogen)olytic activity of a bumblebee (Bombus hypocrite sapporoensis) venom serine protease. J. Asia-Pac. Entomol. 15, 79–82. Xin, Y., Choo, Y.M., Hu, Z., Lee, K.S., Yoon, H.J., Cui, Z., Sohn, H.D., Jin, B.R., 2009. Molecular cloning and characterization of a venom phospholipase A2 from the bumblebee Bombus ignitus. Comp. Biochem. Physiol. B 154, 195–202.
Contents lists available at SciVerse ScienceDirect
Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape
Short Communication
Effects of the bumblebee (Bombus ignitus) venom serine protease inhibitor on serine protease and phospholipase A2 of B. ignitus venom Young Moo Choo a, Hyung Joo Yoon b, Byung Rae Jin a,⁎ a b
College of Natural Resources and Life Science, Dong-A University, Busan 604–714, Republic of Korea Department of Agricultural Biology, National Academy of Agricultural Science, RDA, Suwon 441–100, Republic of Korea
a r t i c l e
i n f o
Article history: Received 9 April 2012 Revised 14 May 2012 Accepted 14 May 2012 Keywords: Bumblebee Bombus ignitus Serine protease Serine protease inhibitor Venom
a b s t r a c t Bumblebee venom contains serine proteases and serine protease inhibitors. In this study, we characterized whether the bumblebee (Bombus ignitus) venom serine protease inhibitor (Bi-KTI) inhibits B. ignitus venom serine protease (Bi-VSP) or phospholipase A2 (Bi-PLA2). Bi-KTI did not inhibit Bi-VSP activity at pH 5.4 or 7.4, whereas Bi-KTI slightly inhibited Bi-VSP activity at pH 7.4 after a 30 min preincubation. The Bi-VSP activity that converts prothrombin into thrombin and fibrin into fibrin degradation products was not significantly affected by Bi-KTI. Additionally, Bi-KTI or Bi-VSP did not inhibit Bi-PLA2 activity. These findings indicate that each bee venom component appears to a play a toxic role via a unique function. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2012. Published by Elsevier B.V. All rights reserved.
Introduction
Materials and methods
Bumblebee (Bombus spp.) venom contains a variety of components, including bombolitin, phospholipase A2 (PLA2), serine proteases, and serine protease inhibitors (Xin et al., 2009; Choo et al., 2010a, b, 2012; Qiu et al., 2011, 2012a, b). Serine proteases and serine protease inhibitors have also been found in snake venoms, and a number of serine proteases exhibit fibrin(ogen)olytic activity (Braud et al., 2000; Matsui et al., 2000; Kini, 2005), whereas serine protease inhibitors show antifibrinolytic activity (Masci et al., 2000; Flight et al., 2005, 2009; Miller et al., 2009). Our previous studies focused on the fibrin(ogen)olytic activity 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, 2012b). Additionally, our previous study focused on the antifibrinolytic role of a bee venom serine protease inhibitor that acts as a plasmin inhibitor (Choo et al., 2012). Although bumblebee venom is involved in the hemostatic system through the antifibrinolytic activity of the venom serine protease inhibitor (Choo et al., 2012) and through venom serine protease-mediated fibrin(ogen)olytic activity (Choo et al., 2010b; Qiu et al., 2011, 2012b), the mechanism by which venom serine protease inhibitors affect venom components, including serine proteases and PLA2, remains poorly understood. In this study, we investigated whether bumblebee (Bombus ignitus) venom serine protease inhibitor (Bi-KTI) inhibited the activity of B. ignitus venom serine protease (Bi-VSP) and PLA2 (Bi-PLA2).
Inhibitory activity of Bi-KTI against Bi-VSP The inhibitory activity assay of Bi-KTI against Bi-VSP was performed using a 96-well plate with a 200 μl final volume of buffer per well. Bi-VSP (400 ng) or a mixture of Bi-VSP (400 ng) and Bi-KTI (13.3 ng), with or without a 30 min preincubation at 37 °C, was incubated with a thrombin fluorogenic substrate (50 μM Boc-Val-Pro-Arg-7-amido-4methylcoumarin hydrochloride; Sigma, St. Louis, MO, USA) in 50 mM citrate-NaOH (pH 5.4) or 50 mM Tris–HCl (pH 7.4) at 25 °C. Fluorescence was recorded at an emission wavelength of 380 and an excitation wavelength of 440 nm every 50 s for 700 s using a dual scanning microplate spectrofluorometer (Gemini XPS, Molecular Devices, Sunnyvale, CA, USA). Hydrolysis of the substrate by Bi-VSP was expressed as relative fluorescence units (RFU). Prothrombin activation assay Human prothrombin (200 μg, Sigma) was incubated with Bi-VSP (20 ng) or a mixture of Bi-VSP (20 ng) and Bi-KTI (0.66 ng) in 50 mM Tris–HCl buffer (pH 7.4) containing 5 mM CaCl2 at 37 °C. Aliquots were removed at various time points and analyzed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fibrin degradation assay
⁎ Corresponding author: Tel./fax: + 82 51 200 7594. E-mail address: [email protected] (B.R. Jin).
Human fibrinogen (200 μg, Sigma), clotted with one unit of thrombin in 50 mM Tris–HCl buffer (pH 7.4) containing 5 mM CaCl2, was incubated
1226-8615/$ – see front matter © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2012. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aspen.2012.05.011
544
Y.M. Choo et al. / Journal of Asia-Pacific Entomology 15 (2012) 543–545
with Bi-VSP (20 ng) or a mixture of Bi-VSP (20 ng) and Bi-KTI (0.66 ng) at 37 °C. Aliquots were removed at various time points and analyzed using 12% SDS-PAGE. Inhibitory activity of Bi-KTI and Bi-VSP against Bi-PLA2 Hydrolysis of 1-O-(6-dabcyl-aminohexanoyl)-2-O-(6-[12-BODIPYdodecanoyl]amino-hexanoyl)-sn-3-glycerylphophatidylcholine (DBPC) by Bi-PLA2 was performed as described previously (Xin et al., 2009). The inhibitory activity assay for Bi-KTI and Bi-VSP against Bi-PLA2 was performed using a 96-well plate with a 200 μl final volume of buffer per well. Each of the following compounds were pre-incubated in 100 mM Tris–HCl buffer (pH 7.4) at room temperature for 24 h: BiPLA2 (800 ng); Bi-VSP (400 ng); a mixture of Bi-PLA2 (800 ng) and BiVSP (400 ng); and a mixture of Bi-PLA2 (800 ng), Bi-VSP (400 ng), and Bi-KTI (13.3 ng). An aliquot of 1.78 μM DBPC was added to each well, and the reaction was incubated at 37 °C for 30 min. The fluorescence end points were measured at an emission wavelength of 500 nm and an excitation wavelength of 530 nm using a dual scanning microplate spectrofluorometer (Gemini XPS). The hydrolysis of DBPC by Bi-PLA2 was expressed as RFU. Results and discussion Bee venom contains both serine proteases and serine protease inhibitors (Choo et al., 2010b, 2012; Qiu et al., 2011, 2012b). To investigate whether Bi-KTI inhibited Bi-VSP, we assessed the inhibition of Bi-VSP at pH 5.4 or 7.4 with or without Bi-KTI. As a result, when
Bi-VSP was incubated with Bi-KTI without a preincubation, Bi-KTI did not inhibit Bi-VSP activity (Fig. 1A), whereas Bi-KTI slightly inhibited Bi-VSP activity at pH 7.4 following a 30 min preincubation (Fig. 1B). We also found that Bi-VSP was active at pH 7.4 (human blood condition) but not at pH 5.4 (bee venom condition). This result is consistent with a previous result that Bi-VSP activity is almost completely inhibited at the acidic pH of 5.4 of B. ignitus venom (Choo et al., 2011). Bi-VSP acts as both a prothrombin activator and fibrin(ogen)olytic enzyme (Choo et al., 2010b). Thus, we also assessed whether Bi-KTI inhibited Bi-VSP by determining the time course of prothrombin activation and fibrin degradation. Prothrombin was cleaved by Bi-VSP to produce thrombin when Bi-VSP was treated without Bi-KTI (Fig. 1C), and fibrin was degraded into fibrin degradation products (FDPs) by Bi-VSP (Fig. 1D). Moreover, Bi-VSP activity in the presence of Bi-KTI was similar to that of Bi-VSP alone (Choo et al., 2012). These results showed that Bi-KTI did not inhibit the activation of prothrombin or the degradation of fibrin into fibrin degradation products by Bi-VSP. Bee venom also contains a PLA2 that acts as a major toxic component (Xin et al., 2009; Choo et al., 2010b). Thus, we investigated whether Bi-VSP or Bi-KTI affected Bi-PLA2. We pre-incubated Bi-PLA2 with BiVSP or a mixture of Bi-VSP and Bi-KTI for 24 h and found that Bi-VSP and Bi-KTI did not inhibit Bi-PLA2 activity (Fig. 2). In conclusion, these data reveal that Bi-KTI was not significantly affected by the activity of B. ignitus venom components. The observation that bee venom contains Bi-VSP, Bi-KTI, and Bi-PLA2 suggests that each venom component must have its own function (Choo et al., 2010b, 2012). Considering the specific role of Bi-KTI as a plasmin
Fig. 1. Inhibitory activity of Bombus ignitus venom serine protease inhibitor (Bi-KTI) on B. ignitus venom serine protease (Bi-VSP). Bi-VSP or a mixture of Bi-VSP and Bi-KTI was incubated at pH 5.4 or 7.4 with (A) or without (B) a 30 min preincubation. The kinetic curves for digestion of the Bi-VSP substrate are expressed as relative fluorescence units (RFU). (C) Prothrombin activation of Bi-VSP in the presence of Bi-KTI. Human prothrombin activation of Bi-VSP was analyzed by 12% SDS-PAGE. Number indicates the time (min) that prothrombin was incubated with Bi-VSP. Prothrombin and thrombin are shown. (D) Fibrin(ogen)olytic activity of Bi-VSP in the presence of Bi-KTI. Bi-VSP fibrinolytic activity was analyzed by 12% SDS-PAGE. Number indicates the time (min) that fibrin was incubated with Bi-VSP. Human fibrin chains and fibrin degradation products (FDPs) are shown.
Y.M. Choo et al. / Journal of Asia-Pacific Entomology 15 (2012) 543–545
545
References
Fig. 2. Inhibitory activity of Bombus ignitus venom serine protease inhibitor (Bi-KTI) or B. ignitus venom serine protease (Bi-VSP) on phospholipase A2 (Bi-PLA2). Bi-PLA2, Bi-VSP, a mixture of Bi-PLA2 and Bi-VSP, or a mixture of Bi-PLA2, Bi-VSP, and Bi-KTI was pre-incubated for 24 h. The hydrolysis of 1-O-(6-dabcyl-aminohexanoyl)-2-O-(6-[12BODIPY-dodecanoyl]amino-hexanoyl)-sn-3-glycerylphophatidylcholine (DBPC) by PLA2 is expressed as relative fluorescence units (RFU).
inhibitor (Choo et al., 2012), our findings show that Bi-KTI is not a target of Bi-VSP. Taken together, these results indicate that each component of bee venom appears to play a toxic role with a unique function.
Acknowledgments This study was supported by the Dong-A University Research Fund.
Braud, S., Bon, C., Wisner, A., 2000. Snake venom proteins acting on hemostasis. Biochimie 82, 851–859. Choo, Y.M., Lee, K.S., Yoon, H.J., Je, Y.H., Lee, S.W., Sohn, H.D., Jin, B.R., 2010a. Molecular cloning and antimicrobial activity of bombolitin, a component of bumblebee Bombus ignitus venom. Comp. Biochem. Physiol. B 156, 168–173. Choo, Y.M., Lee, K.S., Yoon, H.J., Kim, B.Y., Sohn, M.R., Roh, J.Y., Je, Y.H., Kim, N.J., Kim, I., Woo, S.D., Sohn, H.D., Jin, B.R., 2010b. Dual strategy of bee venom serine protease: prophenoloxidase-activating factor in arthropods and fibrin(ogen)olytic enzyme in mammals. PLoS One 5, e10393. Choo, Y.M., Qiu, Y., Yoon, H.J., Lee, K.Y., Sohn, H.D., Jin, B.R., 2011. Enzymatic properties of a bee venom serine protease from the bumblebee Bombus ignitus. J. Asia Pac. Entomol. 14, 249–251. Choo, Y.M., Lee, K.S., Yoon, H.J., Qiu, Y., Wan, H., Sohn, M.R., Sohn, H.D., Jin, B.R., 2012. Antifibrinolytic role of a bee venom serine protease inhibitor that acts as a plasmin inhibitor. PLoS One 7, e32269. Flight, S., Johnson, L., Trabi, M., Gaffney, P., Lavin, M., de Jersey, J., Masci, P., 2005. Comparison of textinin-1 with aprotinin as serine protease inhibitors and as antifibrinolytic agents. Pathophysiol. Haemost. Thromb. 34, 188–193. Flight, S.M., Johnson, L.A., Du, Q.S., Warner, R.L., Trabi, M., Gaffney, P.J., Lavin, M.F., de Jersey, J., Masci, P.P., 2009. Textilinin-1, an alternative anti-bleeding agent to aprotinin: Importance of plasmin inhibition in controlling blood loss. Br. J. Haematol. 145, 207–211. Kini, R.M., 2005. Serine proteases affecting blood coagulation and fibrinolysis from snake venoms. Pathophysiol. Haemost. Thromb. 34, 200–204. Masci, P.P., Whitaker, A.N., Sparrow, L.G., de Jersey, J., Winzor, D.J., Watters, D.J., Lavin, M.F., Gaffney, P.J., 2000. Textilinins from Pseudonaja textilis textilis. Characterization of two plasmin inhibitors that reduce bleeding in an animal model. Blood Coagul. Fibrinolysis 11, 385–393. Matsui, T., Fujimura, Y., Titani, K., 2000. Snake venom proteases affecting hemostasis and thrombisis. Biochim. Biophys. Acta 1477, 146–156. Miller, E.K.I., Trabi, M., Masci, P.P., Lavin, M.F., de Jersey, J., Guddat, L.W., 2009. Crystal structure of textilinin-1, a Kunitz-type serine protease inhibitor from the venom of the Australian common brown snake (Pseudonaja textiles). FEBS J. 276, 3163–3175. Qiu, Y., Choo, Y.M., Yoon, H.J., Jia, J., Cui, Z., Wang, D., Kim, D.H., Sohn, H.D., Jin, B.R., 2011. Fibrin(ogen)olytic activity of bumblebee venom serine protease. Toxicol. Appl. Pharmacol. 255, 207–213. Qiu, Y., Choo, Y.M., Yoon, H.J., Jin, B.R., 2012a. Molecular cloning and antibacterial activity of bombolitin isolated from the venom of a bumblebee, Bombus terrestris. J. Asia-Pac. Entomol. 15, 21–25. Qiu, Y., Choo, Y.M., Yoon, H.J., Jin, B.R., 2012b. Molecular cloning and fibrin(ogen)olytic activity of a bumblebee (Bombus hypocrite sapporoensis) venom serine protease. J. Asia-Pac. Entomol. 15, 79–82. Xin, Y., Choo, Y.M., Hu, Z., Lee, K.S., Yoon, H.J., Cui, Z., Sohn, H.D., Jin, B.R., 2009. Molecular cloning and characterization of a venom phospholipase A2 from the bumblebee Bombus ignitus. Comp. Biochem. Physiol. B 154, 195–202.