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Up-regulation of the expressions of phospholipase A2 inhibitors in the liver of a venomous snake by its own venom phospholipase A2
Biochemical and Biophysical Research Communications 395 (2010) 377–381
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Up-regulation of the expressions of phospholipase A2 inhibitors in the liver of a venomous snake by its own venom phospholipase A2 Kohshi Kinkawa a, Ryoichi Shirai a, Shin Watanabe b, Michihisa Toriba b, Kyozo Hayashi a, Kiyoshi Ikeda a, Seiji Inoue a,* a b
Laboratory of Biochemistry, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan The Japan Snake Institute, Yabuzuka, Ohta, Gunma 379-2301, Japan
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Article history: Received 26 March 2010 Available online 9 April 2010 Keywords: Phospholipase A2 Phospholipase A2 inhibitor Viperidae Gene expression Snake venom Self-protection
a b s t r a c t Venomous snakes such as Gloydius brevicaudus have three distinct types of phospholipase A2 inhibitors (PLIa, PLIb, and PLIc) in their blood so as to protect themselves from their own venom phospholipases A2 (PLA2s). Expressions of these PLIs in G. brevicaudus liver were found to be enhanced by the intramuscular injection of its own venom. The enhancement of gene expressions of PLIa and PLIb in the liver was also found to be induced by acidic PLA2 contained in this venom. Furthermore, these effects of acidic PLA2 on gene expression of PLIs were shown to be unrelated to its enzymatic activity. These results suggest that these venomous snakes have developed the self-protective system against their own venom, by which the venom components up-regulate the expression of anti-venom proteins in their liver. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Snake venom is one of the most abundant sources of secretory phospholipases A2 (PLA2s), which exhibit a wide variety of pharmacological effects including neurotoxicity and myotoxicity [1]. Snake venom PLA2s are categorized into two groups (I and II), based on their amino acid sequences and disulfide bond patterns [2]. Elapidae venom contains group I PLA2s, whereas Viperidae venom contains group II ones. Venomous snakes have PLA2 inhibitory proteins (PLIs) in their blood so as to protect themselves from the leakage of their own venom PLA2s into the circulatory system [3–6]. Three types of PLIs (PLIa, PLIb, and PLIc) were purified from the blood plasma of the Chinese mamushi snake, Gloydius brevicaudus (renamed from Agkistrodon blomhoffii siniticus according to the present taxonomy) [7]. PLIa is a 75-kDa trimeric glycoprotein of 20-kDa subunits having a C-type lectin-like domain (CTLD), which is homologous to those of collectins, such as serum mannose-binding protein and lung surfactant-apoproteins [8,9]. It inhibits specifically the group II acidic PLA2 from its own venom [10]. Gloydius brevicaudus PLIb has nine tandem leucine-rich repeats in its sequence and shows a significant sequence homology to human leucine-rich a2-glycoprotein, a serum protein with unknown * Corresponding author. Address: Laboratory of Biochemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan. Fax: +81 72 690 1075. E-mail address: [email protected] (S. Inoue). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.04.024
function [11]. It selectively inhibits group II basic PLA2s from its own venom [7]. PLIc contains two homologous subunits, PLIc-A and PLIc-B, each of which has two tandem repeats of a cysteine pattern characteristic of the three-finger motifs found in Ly-6-related proteins [12,13]. It shows a broad spectrum of inhibition toward group-I, -II, and -III PLA2s [7,10]. These three PLIs are produced in liver and play an important role in the self-protective system of venomous snake against its own venom. However, there are no reports about the regulation of these self-protective proteins in venomous animals. In this study, we found that the injection of the venom into the venomous snake induced the enhancement of gene expressions of PLIs in liver. We also found that the acidic PLA2 in the venom was an actual factor enhancing the gene expressions of PLIa and PLIb. This is the first report showing that the venom component up-regulates the gene expressions of anti-venom proteins in liver.
2. Materials and methods 2.1. Materials Honey bee Cyt c was purchased from Sigma (St. Louis, MO, USA). Rabbit antisera were raised against G. brevicaudus PLIa, PLIb, and PLIc, which had been purified as described previously [7]. All other reagents and chemicals used were of the highest quality available.
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2.2. Animals
of 2.0 ml/min. PLA2 activities were measured fluorometrically using 1-palmitoyl-2-(10-pyrenyldecanoyl)-sn-glycero-3-phosphoryl -choline (10-Pyrene-PC; Cayman Chemical, Ann Arbor, MI, USA) as a substrate according to the method described previously [10]. Four fractions (fractions I–IV) were recovered and lyophilized.
Chinese mamushi snakes (G. brevicaudus) were bred at the Japan Snake Institute. The venom was collected, pooled, and lyophilized. Venom and protein samples in 50 ll PBS were intramuscularly injected into the snake’s back. Before injection and 0.5, 1, 2, 4, 8, 24 h after injection, approximately 50 ll of the blood was collected at a time from the caudal portion of the snake by capillary action into a hematocrit tube (Plasticrit plastic hematocrit tube, heparinized; Drummond Scientific Company, Broomall, PA, USA). The tube was centrifuged in a clinical hematocrit centrifuge for 15 min and the plasma was separated. Small pieces of liver were resected by a veterinarian from the individual snake before injection and 2, 4, 6 h after injection. The isolated pieces of liver were immediately treated with RNAlater solution (Qiagen, Venlo, The Netherlands). Animal care and treatment were conducted in conformity with institutional guidelines of The Japan Snake Institute.
2.5. Purification of venom PLA2s Gloydius brevicaudus acidic, neutral, and basic PLA2s were purified from the venom as described previously [9]. Fraction III obtained by Sephadex G-100 gel filtration was solubilized in 50 mM acetate buffer (pH 5.0) and applied onto an S-Sepharose column (10 100 mm; GE Healthcare) which had been equilibrated with the same buffer. The flow through fractions containing acidic PLA2 were further purified by a Q-Sepharose column equilibrated with 50 mM Tris–HCl buffer (pH 8.0) and the adsorbed proteins were eluted with the same buffer containing a linear concentration gradient of NaCl, from 0 to 2 M. The adsorbed proteins on the S-Sepharose column which had been equilibrated with 50 mM acetate buffer (pH 5.0) were eluted with the same buffer containing a linear concentration gradient of NaCl, from 0 to 2 M. The fractions exhibiting the activities of neutral and basic PLA2 were collected and dialyzed against 1% acetic acid and lyophilized. These preparations were further purified by reversed-phase HPLC on a Vydac 214TP C4 column (Grace, Deerfield, IL, USA) with 0.1% trifluoroacetic acid containing a linear concentration gradient of acetonitrile from 0% to 60%. The N-terminal amino acid sequences of the purified PLA2s were confirmed by use of a protein sequencer (Procise 491HT; Applied Biosystems, Foster City, CA, USA).
2.3. Western blotting Plasma samples diluted 10,000 fold with PBS were subjected to SDS–PAGE on a 12% gel, transferred electrophoretically onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked in Blocking One solution (Nacarai Tesque, Kyoto, Japan), and then probed separately with the rabbit antibodies against G. brevicaudus PLIa, PLIb, and PLIc. After washing, the proteins on the blot were detected by using the anti-rabbit IgG donkey antibody conjugated to horseradish peroxidase with ECL detection reagents (GE Healthcare, Buckinghamshire, England).
2.6. Preparation of p-bromophenacyl bromide (BPB) modified acidic PLA2
2.4. Gel filtration chromatography of G. brevicaudus venom One gram of the lyophilized G. brevicaudus venom was solubilized in 1% acetic acid and applied to a Sephadex G-100 column (40 900 mm; GE Healthcare), which had been equilibrated with 1% acetic acid. Fractions of 5 ml each were collected at a flow rate
Approximately 0.7 mg of the purified G. brevicaudus acidic PLA2 was dissolved in 10 ml of 50 mM Tris–HCl buffer (pH 7.5). A 100-ll aliquot of 10 mM BPB in ethanol was added to the PLA2 solution,
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Time (h) Fig. 1. Up-regulation of PLIa, PLIb, and PLIc by the intramuscular injection of the G. brevicaudus venom. (A) Western blot analyses of the blood plasma of G. brevicaudus after injection of the venom. The plasma was collected from G. brevicaudus 0, 0.5, 1, 2, 4, 8, 24 h after the intramuscular injection of 4 mg venom, subjected to SDS–PAGE, and transferred to PVDF membrane. PLIs on the blot were separately detected using rabbit antisera raised against G. brevicaudus PLIa, PLIb, and PLIc. (B) The expressions of PLIa, PLIb, and PLIc mRNAs in the liver of G. brevicaudus after the intramuscular injection of venom. A piece of liver was resected from the snake at 2 h intervals after the injection of 4 mg venom or PBS, and its total RNA was isolated, reverse transcribed, and quantified by real-time PCR. The mRNA levels of each PLIa (upper), PLIb (middle), and PLIc (lower) were quantified and normalized by those of G. brevicaudus b-actin, and expressed relatives to those before injection. Values are means ± SD of three (for venom) or two (for PBS) independent determinations.
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3. Results and discussion 3.1. Up-regulation of PLIs by the injection of venom
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Three types of PLA2 inhibitory proteins, PLIa, PLIb, and PLIc, are present in the blood plasma of Chinese mamushi G. brevicaudus [7]. It is accepted that these PLIs inhibit and neutralize its own venom PLA2s and thus function as self-protective proteins against its own venom. When 4 mg of G. brevicaudus venom was intramuscularly injected into this venomous snake, increased amounts of PLIs were found in the blood at 4–12 h after the injection (Fig. 1A). Since these plasma PLIs are synthesized in liver, the expressions of PLI genes in the liver were investigated by using quantitative PCR. As shown in Fig. 1B, gene expression of PLIa, PLIb, and PLIc was found to increase in a time-dependent manner to 3.7, 3.2, and 1.6-fold at 6 h after the venom injection. Gloydius brevicaudus venom was
II
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Total RNA was isolated from the piece of liver by use of RNeasy mini kit. cDNA was synthesized with SuperScript First Strand Synthesis System. Quantitative real-time PCR was performed using LightCycler Fast Start DNA Master SYBR Green I on a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) system. The primer set for b-actin was designed from the b-actin cDNA sequence determined above as follows; b-actin forward primer: 50 GAAAAGCTACGAACTGCCCG-30 , b-actin reverse primer: 50 -GTCAGC AATGCCAGGGTACA-30 . The primer sets for PLIa, PLIb, and PLIc-A were designed from their cDNA sequences [11,13,14] as follows; PLIa forward primer: 50 -GATCCTGATGGGCATGTGCT-30 , PLIa reverse primer: 50 -TCCCGACTTCCTTGTTGGTC-30 , PLIb forward primer: 50 -AGCAGCCTGACCAACATAAT-30 , PLIb reverse primer: 50 TCGTTGACCCAGGTTAGAAG-30 , PLIc-A forward primer: 50 -TCATCA GCATCGCTGTCAGT-30 , PLIc-A reverse primer: 50 -AGTCCCGGAAAT GGTTGGTC-30 . PCR was subjected to 600 s of 95 °C hot-start enzyme activation, and 40 cycles of 95 °C denaturation for 15 s, 55 °C annealing for 10 s and 72 °C extension for 9 s. Gene expression levels were normalized according to the level of b-actin expression.
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2.8. Real-time quantitative RT-PCR
To identify the inducing factors in G. brevicaudus venom for up-regulating PLIs in the liver, we separated the venom into four fractions by Sephadex G-100 column as shown in Fig. 2A. By the intramuscular injection of each fraction to the snake, the fraction III was found to contain the inducing factors for up-regulating PLIa and PLIb (Fig. 2B). Since the fraction III shows PLA2 activity and thus contains three types of venom PLA2s (acidic, neutral, and basic PLA2s), these PLA2s was supposed to be the inducing factors for upregulating PLA2 inhibitors in the liver. Therefore, the acidic, neutral, and basic PLA2s were purified from the venom and separately injected intramuscularly into snakes and mRNAs of PLIs in liver were quantified using real-time PCR. As shown in Fig. 3, acidic PLA2 was found to induce the enhancement of the expression of
mRNA Level (Fold increase)
Total RNA was isolated from G. brevicaudus liver by use of RNeasy mini kit (Qiagen). cDNA was synthesized with SuperScript First Strand Synthesis System (Invitrogen Corp., Carlsbad, CA, USA). The primer set was designed based on the cDNA sequence of green anole (Anolis carolinensis) b-actin (GenBank Accession No. AF199487) as follows; 50 -AGATCTGGCACCACA-30 and 50 -CCTGCT TGCTGATCC-30 , corresponding to sequences 188–202 and 1118– 1004 of A. carolinensis b-actin cDNA, respectively. DNA was amplified by Ready-to-go PCR beads (GE Healthcare, Buckinghamshire, England) using the primer set. The amplified 900 bp PCR product was purified and sequenced directly with a DYEnamic ET terminator cycle sequencing premix kit (GE Healthcare) on an ABI Prism 310 genetic analyzer (Applied Biosystems). The determined sequence of the cDNA fragment of G. brevicaudus b-actin has been deposited with the DDBJ/EMBL/GenBank nucleotide sequence databases under Accession No. AB550237. Analysis of DNA sequence data was performed by using the program GENETYX ver. 6 (Genetyx, Tokyo, Japan).
3.2. Acidic PLA2 from the venom is an inducing factor for up-regulation of PLIa and PLIb
Absorbance at 280 nm
2.7. cDNA sequence determination of G. brevicaudus b-actin
found to induce the enhancement of the expressions of PLIa, PLIb and PLIc in the liver, indicating that some inducing factors for up-regulating these PLIs in the liver are contained in the venom.
mRNA Level (Fold increase)
and reacted overnight at room temperature in the dark. The obtained preparation of BPB-modified PLA2 was purified by reversed-phase HPLC on a Vydac 214TP C4 column as described above.
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Fig. 2. The expressions of PLIa, PLIb, and PLIc mRNAs in the liver after the injection of the venom fractions separated by Sephadex G-100 column chromatography. (A) The separation of G. brevicaudus venom by Sephadex G-100 column. The venom was applied onto a Sephadex G-100 column, which had been equilibrated with 50 mM Tris–HCl buffer (pH 7.5) containing 1 mM EDTA. Each fraction contained 5 ml of the eluted solution. The eluate was separated into four fractions (I–IV). (B) The expressions of PLIa, PLIb, and PLIc mRNAs in the liver of G. brevicaudus after the injection of the fractions I–IV. The fractions injected were 0.42 mg of the fraction I, 0.14 mg of the fraction II, 0.28 mg of the fraction III, and 0.0012 mg of the fraction IV according to their contents in the venom. The mRNA levels of each PLIa (upper), PLIb (middle), and PLIc (lower) were measured by real-time quantitative RT-PCR and normalized by those of G. brevicaudus b-actin, and expressed relatives to those before injection. Values are means ± SD of three independent determinations.
K. Kinkawa et al. / Biochemical and Biophysical Research Communications 395 (2010) 377–381
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Fig. 3. The expressions of PLIa, PLIb, and PLIc mRNAs in the liver after the injection of the acidic, neutral, and basic PLA2s purified from the venom. The injected PLA2s were 34 lg of acidic PLA2 (A), 8.9 lg of neutral PLA2 (B), or 44 lg of basic PLA2 (C) according to their contents in the venom. The mRNA levels of each PLIa, PLIb, and PLIc were measured by real-time quantitative RT-PCR and normalized by those of G. brevicaudus b-actin, and expressed relatives to those before injection. Values are means ± SD of three independent determinations.
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3.3. PLA2 activity is not necessarily required for up-regulation of PLIs To test whether the enzymatic activity of acidic PLA2 is required for the induction of the up-regulation of PLIs in liver, the acidic PLA2 was modified and inactivated by BPB, which is known to alkylate specifically His-48 in the active site of PLA2 [16]. As shown in Fig. 4A, the injection of BPB-modified acidic PLA2 to the snake induced the enhancement of PLIa expression at 2 and 4 h after the injection, suggesting that the enzymatic activity of acidic PLA2 is not necessarily required for up-regulation of PLIa. Since PLIa could bind BPB-modified acidic PLA2 (S. Inoue et al., unpublished data), the BPB-modified acidic PLA2 might act through the formation of
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in liver was not enhanced by the injections of any fractions obtained from the venom by Sephadex G-100 chromatography and also by the injections of the purified PLA2s. The acidification of the venom in the fractionation procedures might inactivate the inducing factor for up-regulating PLIc in liver.
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PLIa and PLIb, suggesting that acidic PLA2 is an inducing factor for up-regulating PLIa and PLIb in liver. Since G. brevicaudus PLIa specifically binds and inhibits G. brevicaudus acidic PLA2 [10], the enhancement of PLIa expression by the acidic PLA2 seems favorable for the self-protection system in the venomous snake. In contrast to PLIa, the enhancement of PLIb expression in liver was up-regulated by acidic PLA2 but not by basic PLA2, although G. brevicaudus PLIb specifically binds and inhibits G. brevicaudus basic PLA2 [7]. PLIb has significant sequence homology with leucine-rich a2-glycoprotein (LRG), one of the mammalian serum glycoproteins with unknown function [11], and it might be a snake ortholog of mammalian LRG. We have recently reported that LRG was one of the acute-phase proteins and its expression was up-regulated by the mediator of acute-phase response [15]. Therefore, it is likely that the snake PLIb functions as an acute-phase protein and its expression is up-regulated by inflammatory mediators including lipid mediators produced by the enzymatic activity of the injected acidic PLA2. On the other hand, the expression of PLIc
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Fig. 4. The expressions of PLIa, PLIb, and PLIc mRNAs in the liver after the injection of BPB-modified acidic PLA2 and honey bee PLA2. The injected PLA2s were 34 lg of BPBmodified acidic PLA2 (A) or 6.2 lg of honey bee PLA2 (B). The mRNA levels of each PLIa, PLIb, and PLIc were measured by real-time quantitative RT-PCR and normalized by those of G. brevicaudus b-actin, and expressed relatives to those before injection. Values are means ± SD of two independent determinations.
K. Kinkawa et al. / Biochemical and Biophysical Research Communications 395 (2010) 377–381
PLIa–PLA2 complex. Alternatively, it is likely that the membrane receptor for the acidic PLA2 is present in the G. brevicaudus hepatocytes and regulates the gene expression of PLIa. BPB-modified PLA2 also induced the expression of PLIb in liver at 6 h after the injection. The immediate expression of PLIa and the delayed expression of PLIb seem to reflect the different modes of gene expression induced by BPB-modified acidic PLA2. Expressions of three types of PLIs might be regulated independently. Among three venom PLA2s, the acidic PLA2 has the highest enzymatic activity although it may depend on the substrate phospholipids. In order to investigate the effect of PLA2 activity on the expression of PLIs in liver, we have injected the snakes with honey bee PLA2, which has a higher enzymatic activity than the acidic PLA2. However, as shown in Fig. 4B, honey bee PLA2 was found to suppress the gene expressions of PLIs in liver. These results also indicate that gene expressions of PLIs in liver are unrelated to the enzymatic activity of acidic PLA2. In vitro studies using primary hepatocytes of G. brevicaudus snake will be required to elucidate the molecular mechanism of up-regulation by acidic PLA2. In conclusion, this study demonstrated that the gene expressions of PLIa and PLIb in G. brevicaudus liver are enhanced by the intramuscular injection of the acidic PLA2 derived from its own venom. The enzymatic activity of acidic PLA2 was not required for the up-regulations of PLIs, suggesting that a hypothetical acidic PLA2binding protein (it might be PLIa) would participate in the enhancement of gene expressions in the liver. Further investigations might help to elucidate the molecular mechanism of self-protection system in the venomous snake. References [1] R.M. Kini, Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes, Toxicon 42 (2003) 827–840. [2] R.L. Heinrikson, E.T. Krueger, P.S. Keim, Amino acid sequence of phospholipase A2-a from the venom of Crotalus adamanteus. A new classification of phospholipases A2 based upon structural determinants, J. Biol. Chem. 252 (1977) 4913–4921.
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[3] H. Kihara, Studies on phospholipase A in Trimeresurus flaoviridis venom. III. Purification and some properties of phospholipase A inhibitor in Habu serum, J. Biochem. 80 (1976) 341–349. [4] G. Lambeau, M. Lazdunski, Receptors for a growing family of secreted phospholipases A2, Trends Pharmacol. Sci. 20 (1999) 162–170. [5] R.D. Dunn, K.W. Broady, Snake inhibitors of phospholipase A2 enzymes, Biochim. Biophys. Acta 1533 (2001) 29–37. [6] C.L. Fortes-Dias, Endogenous inhibitors of snake venom phospholipases A2 in the blood plasma of snakes, Toxicon 40 (2002) 481–484. [7] N. Ohkura, H. Okuhara, S. Inoue, K. Ikeda, K. Hayashi, Purification and characterization of three distinct types of phospholipase A2 inhibitors from the blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus, Biochem. J. 325 (1997) 527–531. [8] S. Inoue, H. Kogaki, K. Ikeda, Y. Samejima, T. Omori-Satoh, Amino acid sequences of the two subunits of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis: sequence homologies with pulmonary surfactant apoprotein and animal lectins, J. Biol. Chem. 266 (1991) 1001–1007. [9] N. Ohkura, S. Inoue, K. Ikeda, K. Hayashi, Isolation and amino acid sequence of a phospholipase A2 inhibitor from the blood plasma of Agkistrodon blomhoffii siniticus, J. Biochem. 113 (1993) 413–419. [10] S. Inoue, A. Shimada, N. Ohkura, K. Ikeda, Y. Samejima, T. Omori-Satoh, K. Hayashi, Specificity of two types of phospholipase A2 inhibitors from the plasma of venomous snakes, Biochem. Mol. Biol. Int. 41 (1997) 529–537. [11] K. Okumura, N. Ohkura, S. Inoue, K. Ikeda, K. Hayashi, A novel phospholipase A2 inhibitor with leucine-rich repeats from the blood plasma of Agkistrodon blomhoffii siniticus. Sequence homologies with human leucine-rich a2glycoprotein, J. Biol. Chem. 273 (1998) 19469–19475. [12] N. Ohkura, S. Inoue, K. Ikeda, K. Hayashi, The two subunits of a phospholipase A2 inhibitor from the plasma of Thailand cobra having structural similarity to urokinase-type plasminogen activator receptor and Ly-6 related proteins, Biochem. Biophys. Res. Commun. 204 (1994) 1212–1218. [13] K. Okumura, S. Inoue, N. Ohkura, K. Ikeda, K. Hayashi, CDNA cloning of the two subunits of phospholipase A2 inhibitor PLIc from blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus, IUBMB Life 48 (1999) 99–104. [14] K. Okumura, S. Inoue, K. Ikeda, K. Hayashi, CDNA cloning and bacterial expression of phospholipase A2 inhibitor PLIa from the serum of the Chinese mamushi, Agkistrodon blomhoffii siniticus, Biochim. Biophys. Acta 1441 (1999) 51–60. [15] R. Shirai, F. Hirano, N. Ohkura, K. Ikeda, S. Inoue, Up-regulation of the expression of leucine-rich a2-glycoprotein in hepatocytes by the mediators of acute-phase response, Biochem. Biophys. Res. Commun. 382 (2009) 776– 779. [16] M.F. Roberts, R.A. Deems, T.C. Mincey, E.A. Dennis, Chemical modification of the histidine residue in phospholipase A2 (Naja naja naja). A case of half-site reactivity, J. Biol. Chem. 252 (1977) 2405–2411.
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Up-regulation of the expressions of phospholipase A2 inhibitors in the liver of a venomous snake by its own venom phospholipase A2 Kohshi Kinkawa a, Ryoichi Shirai a, Shin Watanabe b, Michihisa Toriba b, Kyozo Hayashi a, Kiyoshi Ikeda a, Seiji Inoue a,* a b
Laboratory of Biochemistry, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan The Japan Snake Institute, Yabuzuka, Ohta, Gunma 379-2301, Japan
a r t i c l e
i n f o
Article history: Received 26 March 2010 Available online 9 April 2010 Keywords: Phospholipase A2 Phospholipase A2 inhibitor Viperidae Gene expression Snake venom Self-protection
a b s t r a c t Venomous snakes such as Gloydius brevicaudus have three distinct types of phospholipase A2 inhibitors (PLIa, PLIb, and PLIc) in their blood so as to protect themselves from their own venom phospholipases A2 (PLA2s). Expressions of these PLIs in G. brevicaudus liver were found to be enhanced by the intramuscular injection of its own venom. The enhancement of gene expressions of PLIa and PLIb in the liver was also found to be induced by acidic PLA2 contained in this venom. Furthermore, these effects of acidic PLA2 on gene expression of PLIs were shown to be unrelated to its enzymatic activity. These results suggest that these venomous snakes have developed the self-protective system against their own venom, by which the venom components up-regulate the expression of anti-venom proteins in their liver. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Snake venom is one of the most abundant sources of secretory phospholipases A2 (PLA2s), which exhibit a wide variety of pharmacological effects including neurotoxicity and myotoxicity [1]. Snake venom PLA2s are categorized into two groups (I and II), based on their amino acid sequences and disulfide bond patterns [2]. Elapidae venom contains group I PLA2s, whereas Viperidae venom contains group II ones. Venomous snakes have PLA2 inhibitory proteins (PLIs) in their blood so as to protect themselves from the leakage of their own venom PLA2s into the circulatory system [3–6]. Three types of PLIs (PLIa, PLIb, and PLIc) were purified from the blood plasma of the Chinese mamushi snake, Gloydius brevicaudus (renamed from Agkistrodon blomhoffii siniticus according to the present taxonomy) [7]. PLIa is a 75-kDa trimeric glycoprotein of 20-kDa subunits having a C-type lectin-like domain (CTLD), which is homologous to those of collectins, such as serum mannose-binding protein and lung surfactant-apoproteins [8,9]. It inhibits specifically the group II acidic PLA2 from its own venom [10]. Gloydius brevicaudus PLIb has nine tandem leucine-rich repeats in its sequence and shows a significant sequence homology to human leucine-rich a2-glycoprotein, a serum protein with unknown * Corresponding author. Address: Laboratory of Biochemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan. Fax: +81 72 690 1075. E-mail address: [email protected] (S. Inoue). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.04.024
function [11]. It selectively inhibits group II basic PLA2s from its own venom [7]. PLIc contains two homologous subunits, PLIc-A and PLIc-B, each of which has two tandem repeats of a cysteine pattern characteristic of the three-finger motifs found in Ly-6-related proteins [12,13]. It shows a broad spectrum of inhibition toward group-I, -II, and -III PLA2s [7,10]. These three PLIs are produced in liver and play an important role in the self-protective system of venomous snake against its own venom. However, there are no reports about the regulation of these self-protective proteins in venomous animals. In this study, we found that the injection of the venom into the venomous snake induced the enhancement of gene expressions of PLIs in liver. We also found that the acidic PLA2 in the venom was an actual factor enhancing the gene expressions of PLIa and PLIb. This is the first report showing that the venom component up-regulates the gene expressions of anti-venom proteins in liver.
2. Materials and methods 2.1. Materials Honey bee Cyt c was purchased from Sigma (St. Louis, MO, USA). Rabbit antisera were raised against G. brevicaudus PLIa, PLIb, and PLIc, which had been purified as described previously [7]. All other reagents and chemicals used were of the highest quality available.
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2.2. Animals
of 2.0 ml/min. PLA2 activities were measured fluorometrically using 1-palmitoyl-2-(10-pyrenyldecanoyl)-sn-glycero-3-phosphoryl -choline (10-Pyrene-PC; Cayman Chemical, Ann Arbor, MI, USA) as a substrate according to the method described previously [10]. Four fractions (fractions I–IV) were recovered and lyophilized.
Chinese mamushi snakes (G. brevicaudus) were bred at the Japan Snake Institute. The venom was collected, pooled, and lyophilized. Venom and protein samples in 50 ll PBS were intramuscularly injected into the snake’s back. Before injection and 0.5, 1, 2, 4, 8, 24 h after injection, approximately 50 ll of the blood was collected at a time from the caudal portion of the snake by capillary action into a hematocrit tube (Plasticrit plastic hematocrit tube, heparinized; Drummond Scientific Company, Broomall, PA, USA). The tube was centrifuged in a clinical hematocrit centrifuge for 15 min and the plasma was separated. Small pieces of liver were resected by a veterinarian from the individual snake before injection and 2, 4, 6 h after injection. The isolated pieces of liver were immediately treated with RNAlater solution (Qiagen, Venlo, The Netherlands). Animal care and treatment were conducted in conformity with institutional guidelines of The Japan Snake Institute.
2.5. Purification of venom PLA2s Gloydius brevicaudus acidic, neutral, and basic PLA2s were purified from the venom as described previously [9]. Fraction III obtained by Sephadex G-100 gel filtration was solubilized in 50 mM acetate buffer (pH 5.0) and applied onto an S-Sepharose column (10 100 mm; GE Healthcare) which had been equilibrated with the same buffer. The flow through fractions containing acidic PLA2 were further purified by a Q-Sepharose column equilibrated with 50 mM Tris–HCl buffer (pH 8.0) and the adsorbed proteins were eluted with the same buffer containing a linear concentration gradient of NaCl, from 0 to 2 M. The adsorbed proteins on the S-Sepharose column which had been equilibrated with 50 mM acetate buffer (pH 5.0) were eluted with the same buffer containing a linear concentration gradient of NaCl, from 0 to 2 M. The fractions exhibiting the activities of neutral and basic PLA2 were collected and dialyzed against 1% acetic acid and lyophilized. These preparations were further purified by reversed-phase HPLC on a Vydac 214TP C4 column (Grace, Deerfield, IL, USA) with 0.1% trifluoroacetic acid containing a linear concentration gradient of acetonitrile from 0% to 60%. The N-terminal amino acid sequences of the purified PLA2s were confirmed by use of a protein sequencer (Procise 491HT; Applied Biosystems, Foster City, CA, USA).
2.3. Western blotting Plasma samples diluted 10,000 fold with PBS were subjected to SDS–PAGE on a 12% gel, transferred electrophoretically onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked in Blocking One solution (Nacarai Tesque, Kyoto, Japan), and then probed separately with the rabbit antibodies against G. brevicaudus PLIa, PLIb, and PLIc. After washing, the proteins on the blot were detected by using the anti-rabbit IgG donkey antibody conjugated to horseradish peroxidase with ECL detection reagents (GE Healthcare, Buckinghamshire, England).
2.6. Preparation of p-bromophenacyl bromide (BPB) modified acidic PLA2
2.4. Gel filtration chromatography of G. brevicaudus venom One gram of the lyophilized G. brevicaudus venom was solubilized in 1% acetic acid and applied to a Sephadex G-100 column (40 900 mm; GE Healthcare), which had been equilibrated with 1% acetic acid. Fractions of 5 ml each were collected at a flow rate
Approximately 0.7 mg of the purified G. brevicaudus acidic PLA2 was dissolved in 10 ml of 50 mM Tris–HCl buffer (pH 7.5). A 100-ll aliquot of 10 mM BPB in ethanol was added to the PLA2 solution,
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Time (h) Fig. 1. Up-regulation of PLIa, PLIb, and PLIc by the intramuscular injection of the G. brevicaudus venom. (A) Western blot analyses of the blood plasma of G. brevicaudus after injection of the venom. The plasma was collected from G. brevicaudus 0, 0.5, 1, 2, 4, 8, 24 h after the intramuscular injection of 4 mg venom, subjected to SDS–PAGE, and transferred to PVDF membrane. PLIs on the blot were separately detected using rabbit antisera raised against G. brevicaudus PLIa, PLIb, and PLIc. (B) The expressions of PLIa, PLIb, and PLIc mRNAs in the liver of G. brevicaudus after the intramuscular injection of venom. A piece of liver was resected from the snake at 2 h intervals after the injection of 4 mg venom or PBS, and its total RNA was isolated, reverse transcribed, and quantified by real-time PCR. The mRNA levels of each PLIa (upper), PLIb (middle), and PLIc (lower) were quantified and normalized by those of G. brevicaudus b-actin, and expressed relatives to those before injection. Values are means ± SD of three (for venom) or two (for PBS) independent determinations.
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3. Results and discussion 3.1. Up-regulation of PLIs by the injection of venom
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Three types of PLA2 inhibitory proteins, PLIa, PLIb, and PLIc, are present in the blood plasma of Chinese mamushi G. brevicaudus [7]. It is accepted that these PLIs inhibit and neutralize its own venom PLA2s and thus function as self-protective proteins against its own venom. When 4 mg of G. brevicaudus venom was intramuscularly injected into this venomous snake, increased amounts of PLIs were found in the blood at 4–12 h after the injection (Fig. 1A). Since these plasma PLIs are synthesized in liver, the expressions of PLI genes in the liver were investigated by using quantitative PCR. As shown in Fig. 1B, gene expression of PLIa, PLIb, and PLIc was found to increase in a time-dependent manner to 3.7, 3.2, and 1.6-fold at 6 h after the venom injection. Gloydius brevicaudus venom was
II
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Total RNA was isolated from the piece of liver by use of RNeasy mini kit. cDNA was synthesized with SuperScript First Strand Synthesis System. Quantitative real-time PCR was performed using LightCycler Fast Start DNA Master SYBR Green I on a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) system. The primer set for b-actin was designed from the b-actin cDNA sequence determined above as follows; b-actin forward primer: 50 GAAAAGCTACGAACTGCCCG-30 , b-actin reverse primer: 50 -GTCAGC AATGCCAGGGTACA-30 . The primer sets for PLIa, PLIb, and PLIc-A were designed from their cDNA sequences [11,13,14] as follows; PLIa forward primer: 50 -GATCCTGATGGGCATGTGCT-30 , PLIa reverse primer: 50 -TCCCGACTTCCTTGTTGGTC-30 , PLIb forward primer: 50 -AGCAGCCTGACCAACATAAT-30 , PLIb reverse primer: 50 TCGTTGACCCAGGTTAGAAG-30 , PLIc-A forward primer: 50 -TCATCA GCATCGCTGTCAGT-30 , PLIc-A reverse primer: 50 -AGTCCCGGAAAT GGTTGGTC-30 . PCR was subjected to 600 s of 95 °C hot-start enzyme activation, and 40 cycles of 95 °C denaturation for 15 s, 55 °C annealing for 10 s and 72 °C extension for 9 s. Gene expression levels were normalized according to the level of b-actin expression.
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2.8. Real-time quantitative RT-PCR
To identify the inducing factors in G. brevicaudus venom for up-regulating PLIs in the liver, we separated the venom into four fractions by Sephadex G-100 column as shown in Fig. 2A. By the intramuscular injection of each fraction to the snake, the fraction III was found to contain the inducing factors for up-regulating PLIa and PLIb (Fig. 2B). Since the fraction III shows PLA2 activity and thus contains three types of venom PLA2s (acidic, neutral, and basic PLA2s), these PLA2s was supposed to be the inducing factors for upregulating PLA2 inhibitors in the liver. Therefore, the acidic, neutral, and basic PLA2s were purified from the venom and separately injected intramuscularly into snakes and mRNAs of PLIs in liver were quantified using real-time PCR. As shown in Fig. 3, acidic PLA2 was found to induce the enhancement of the expression of
mRNA Level (Fold increase)
Total RNA was isolated from G. brevicaudus liver by use of RNeasy mini kit (Qiagen). cDNA was synthesized with SuperScript First Strand Synthesis System (Invitrogen Corp., Carlsbad, CA, USA). The primer set was designed based on the cDNA sequence of green anole (Anolis carolinensis) b-actin (GenBank Accession No. AF199487) as follows; 50 -AGATCTGGCACCACA-30 and 50 -CCTGCT TGCTGATCC-30 , corresponding to sequences 188–202 and 1118– 1004 of A. carolinensis b-actin cDNA, respectively. DNA was amplified by Ready-to-go PCR beads (GE Healthcare, Buckinghamshire, England) using the primer set. The amplified 900 bp PCR product was purified and sequenced directly with a DYEnamic ET terminator cycle sequencing premix kit (GE Healthcare) on an ABI Prism 310 genetic analyzer (Applied Biosystems). The determined sequence of the cDNA fragment of G. brevicaudus b-actin has been deposited with the DDBJ/EMBL/GenBank nucleotide sequence databases under Accession No. AB550237. Analysis of DNA sequence data was performed by using the program GENETYX ver. 6 (Genetyx, Tokyo, Japan).
3.2. Acidic PLA2 from the venom is an inducing factor for up-regulation of PLIa and PLIb
Absorbance at 280 nm
2.7. cDNA sequence determination of G. brevicaudus b-actin
found to induce the enhancement of the expressions of PLIa, PLIb and PLIc in the liver, indicating that some inducing factors for up-regulating these PLIs in the liver are contained in the venom.
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and reacted overnight at room temperature in the dark. The obtained preparation of BPB-modified PLA2 was purified by reversed-phase HPLC on a Vydac 214TP C4 column as described above.
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Fig. 2. The expressions of PLIa, PLIb, and PLIc mRNAs in the liver after the injection of the venom fractions separated by Sephadex G-100 column chromatography. (A) The separation of G. brevicaudus venom by Sephadex G-100 column. The venom was applied onto a Sephadex G-100 column, which had been equilibrated with 50 mM Tris–HCl buffer (pH 7.5) containing 1 mM EDTA. Each fraction contained 5 ml of the eluted solution. The eluate was separated into four fractions (I–IV). (B) The expressions of PLIa, PLIb, and PLIc mRNAs in the liver of G. brevicaudus after the injection of the fractions I–IV. The fractions injected were 0.42 mg of the fraction I, 0.14 mg of the fraction II, 0.28 mg of the fraction III, and 0.0012 mg of the fraction IV according to their contents in the venom. The mRNA levels of each PLIa (upper), PLIb (middle), and PLIc (lower) were measured by real-time quantitative RT-PCR and normalized by those of G. brevicaudus b-actin, and expressed relatives to those before injection. Values are means ± SD of three independent determinations.
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Fig. 3. The expressions of PLIa, PLIb, and PLIc mRNAs in the liver after the injection of the acidic, neutral, and basic PLA2s purified from the venom. The injected PLA2s were 34 lg of acidic PLA2 (A), 8.9 lg of neutral PLA2 (B), or 44 lg of basic PLA2 (C) according to their contents in the venom. The mRNA levels of each PLIa, PLIb, and PLIc were measured by real-time quantitative RT-PCR and normalized by those of G. brevicaudus b-actin, and expressed relatives to those before injection. Values are means ± SD of three independent determinations.
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3.3. PLA2 activity is not necessarily required for up-regulation of PLIs To test whether the enzymatic activity of acidic PLA2 is required for the induction of the up-regulation of PLIs in liver, the acidic PLA2 was modified and inactivated by BPB, which is known to alkylate specifically His-48 in the active site of PLA2 [16]. As shown in Fig. 4A, the injection of BPB-modified acidic PLA2 to the snake induced the enhancement of PLIa expression at 2 and 4 h after the injection, suggesting that the enzymatic activity of acidic PLA2 is not necessarily required for up-regulation of PLIa. Since PLIa could bind BPB-modified acidic PLA2 (S. Inoue et al., unpublished data), the BPB-modified acidic PLA2 might act through the formation of
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in liver was not enhanced by the injections of any fractions obtained from the venom by Sephadex G-100 chromatography and also by the injections of the purified PLA2s. The acidification of the venom in the fractionation procedures might inactivate the inducing factor for up-regulating PLIc in liver.
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PLIa and PLIb, suggesting that acidic PLA2 is an inducing factor for up-regulating PLIa and PLIb in liver. Since G. brevicaudus PLIa specifically binds and inhibits G. brevicaudus acidic PLA2 [10], the enhancement of PLIa expression by the acidic PLA2 seems favorable for the self-protection system in the venomous snake. In contrast to PLIa, the enhancement of PLIb expression in liver was up-regulated by acidic PLA2 but not by basic PLA2, although G. brevicaudus PLIb specifically binds and inhibits G. brevicaudus basic PLA2 [7]. PLIb has significant sequence homology with leucine-rich a2-glycoprotein (LRG), one of the mammalian serum glycoproteins with unknown function [11], and it might be a snake ortholog of mammalian LRG. We have recently reported that LRG was one of the acute-phase proteins and its expression was up-regulated by the mediator of acute-phase response [15]. Therefore, it is likely that the snake PLIb functions as an acute-phase protein and its expression is up-regulated by inflammatory mediators including lipid mediators produced by the enzymatic activity of the injected acidic PLA2. On the other hand, the expression of PLIc
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Fig. 4. The expressions of PLIa, PLIb, and PLIc mRNAs in the liver after the injection of BPB-modified acidic PLA2 and honey bee PLA2. The injected PLA2s were 34 lg of BPBmodified acidic PLA2 (A) or 6.2 lg of honey bee PLA2 (B). The mRNA levels of each PLIa, PLIb, and PLIc were measured by real-time quantitative RT-PCR and normalized by those of G. brevicaudus b-actin, and expressed relatives to those before injection. Values are means ± SD of two independent determinations.
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PLIa–PLA2 complex. Alternatively, it is likely that the membrane receptor for the acidic PLA2 is present in the G. brevicaudus hepatocytes and regulates the gene expression of PLIa. BPB-modified PLA2 also induced the expression of PLIb in liver at 6 h after the injection. The immediate expression of PLIa and the delayed expression of PLIb seem to reflect the different modes of gene expression induced by BPB-modified acidic PLA2. Expressions of three types of PLIs might be regulated independently. Among three venom PLA2s, the acidic PLA2 has the highest enzymatic activity although it may depend on the substrate phospholipids. In order to investigate the effect of PLA2 activity on the expression of PLIs in liver, we have injected the snakes with honey bee PLA2, which has a higher enzymatic activity than the acidic PLA2. However, as shown in Fig. 4B, honey bee PLA2 was found to suppress the gene expressions of PLIs in liver. These results also indicate that gene expressions of PLIs in liver are unrelated to the enzymatic activity of acidic PLA2. In vitro studies using primary hepatocytes of G. brevicaudus snake will be required to elucidate the molecular mechanism of up-regulation by acidic PLA2. In conclusion, this study demonstrated that the gene expressions of PLIa and PLIb in G. brevicaudus liver are enhanced by the intramuscular injection of the acidic PLA2 derived from its own venom. The enzymatic activity of acidic PLA2 was not required for the up-regulations of PLIs, suggesting that a hypothetical acidic PLA2binding protein (it might be PLIa) would participate in the enhancement of gene expressions in the liver. Further investigations might help to elucidate the molecular mechanism of self-protection system in the venomous snake. References [1] R.M. Kini, Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes, Toxicon 42 (2003) 827–840. [2] R.L. Heinrikson, E.T. Krueger, P.S. Keim, Amino acid sequence of phospholipase A2-a from the venom of Crotalus adamanteus. A new classification of phospholipases A2 based upon structural determinants, J. Biol. Chem. 252 (1977) 4913–4921.
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