- Email: [email protected]
Molecular cloning of serine proteases from elapid snake venoms
ARTICLE IN PRESS
Toxicon 49 (2007) 1200–1207 www.elsevier.com/locate/toxicon
Molecular cloning of serine proteases from elapid snake venoms Yang Jin1, Wen-Hui Lee1, Yun Zhang Biotoxin Units, Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China Received 27 October 2006; received in revised form 16 February 2007; accepted 19 February 2007 Available online 27 February 2007
Abstract Serine proteases are widely distributed in viperid snake venoms, but rare in elapid snake venoms. Previously, we have identified a fibrinogenolytic enzyme termed OhS1 from the venom of Ophiophagus hannah. The results indicated that OhS1 might be a serine protease, but there was no structural evidence previously. In the present study, the primary structure of OhS1 was determined by protein sequencing, in combination with RT-PCR and 50 -RACE methods. OhS1 precursor is composed of an 18-amino acid signal peptide, a 6-amino acid putative activation peptide and 236-amino acid mature protein. OhS1 homologues from Naja atra and Bungarus multicinctus were also cloned and reported. These elapid venom serine proteases exhibited 60% sequence identity with serine proteases from the snake venoms of the Viperidae and Colubridae family. Phylogenetic analysis indicated that snake venom serine protease might have a common ancestor. r 2007 Elsevier Ltd. All rights reserved. Keywords: Serine protease; Elapidae; Cloning
1. Introduction Snake venoms are complex mixtures of various molecules, most of which are peptide toxins, hydrolytic enzymes and non-lethal proteins possessing various biological functions. Serine proteases are among the best-characterized components of living organisms. Snake venom serine proteases (SVSPs) are classified into the trypsin family S1 of Clan SA. SVSPs possess an identical trypsin fold consisting of two b-barrels and the conserved catalytic triad, Ser195, His57 and Asp102 (Perona and Craik, 1997). These enzymes normally contain Corresponding author. Tel.: +86 871 5198515;
fax: +86 871 5191823. E-mail address: [email protected] (Y. Zhang). 1 These authors contributed equally to this work. 0041-0101/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2007.02.013
12 cysteine residues and a C-terminal extension which is the characteristic of SVSPs (Parry et al., 1998). SVSPs undergo fast adaptive evolution to generate variants of diverse functions (Deshimaru et al., 1996; Fry et al., 2006), including specifically degrading or activating blood components involved in coagulation and fibrinolysis (Markland, 1998; Serrano and Maroun, 2005; Zhang et al. 1995, 1997), activating kallikrein/kinin system (Hung and Chiou, 2001; Matsui et al., 1998) or affecting platelet aggregation (Laing et al., 2005; Dekhil et al., 2003). The king cobra (Ophiophagus hannah), which belongs to the Elapidae family, is a large poisonous snake inhabiting in south Asia and injects a large amount of venom once. Although the majority of the venom components are neurotoxins and phospholipase A2, it also contains high-molecular-weight
ARTICLE IN PRESS Y. Jin et al. / Toxicon 49 (2007) 1200–1207
proteins targeting at blood coagulation system, such as factor X activator, L-amino acid oxidase, metalloproteinase and platelet GPVI agonist (Lee et al., 1995; Li et al., 1994; Tan and Saifuddin, 1990; Du et al., 2002). However, there are few reports on the serine proteases from elapid snake venoms. Previously, we have isolated an arginine/lysine amidase termed OhS1 from the venom of king cobra. OhS1 is a single-chain protein with an apparent molecular weight of 43 kDa and possesses potent fibrinogenase and amidase activity. Its enzymatic activity could be completely inhibited by serine protease inhibitors, e.g., PMSF, DFP and soybean trypsin inhibitor, but not affected by EDTA (Zhang et al., 1994). Though these characteristics suggested that OhS1 is a serine protease, there was no structural information to confirm its identity. In this work, the cDNA of OhS1, as well as its homologues from two other elapid snakes were cloned and analyzed. 2. Materials and methods 2.1. Materials O. hannah crude venom was the stock of Kunming Institute of Zoology. Sephadex G-100 (superfine) and Resource Q column (1 ml) were products of Amersham Biosciences (Uppsala, Sweden). Human fibrinogen was purchased from Sigma Chemical (St. Louis, MO, USA). The RNeasy RNA isolation kit was a product of Qiagen (Valencia, CA, USA). Superscript II reverse transcription polymerase and 50 -RACE kit were products of Invitrogen. Agarose gel extraction kit, rTaq DNA polymerase, synthesized oligonucleotides and Escherichia coli JM109 competent cells were purchased from TaKaRa (Dalian, China). pGEM-T vector system was product of Promega Corporation Ltd. (Madison, WI, USA). All other chemicals were of analytic grade. 2.2. Isolation, purity examination and biological assay OhS1 was isolated according to our previous work (Zhang et al., 1994). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method described (Laemmli, 1970). In a separate experiment, the purity of final preparation was determined by high-performance liquid chromatography (HPLC) on a C4 reversed-
1201
phase (RP) column (ZORBAX, Agilent Technolgies, USA) using a linear gradient of 10–40% acetonitrile in 0.1% trifluoroacetic acid (TFA) run for 30 min. The preparation eluted from C4 column chromatography was subjected to ABI model 476A protein sequencer to determine the N-terminal amino acid sequence. Fibrinogenlytic and amidolytic activities were conducted as described previously (Zhang et al., 1994).
2.3. Venom gland RNA isolation and cloning of OhS1 Living O. hannah snake was collected in Guangxi province, China. Total RNA was isolated from the venom gland of O. hannah using a homogenizer and RNeasy RNA isolation kit according to the manufacture’s instruction. RNA purity and concentration were estimated by checking the optical density at 280 and 260 nm. RT-PCR and 50 -RACE methods were used to obtain the full-length cDNA of OhS1. Briefly, firststrand cDNAs were generated from total RNA using an adaptor-oligo(dT) primer (50 -GGC CAC GCG TCG ACT AGT AC(T)17-30 ). A forward degenerate primer (termed P1: 50 -ATH GGN GGN TTY GAR TGY AAY GA) was designed based on the determined amino-terminal sequence. A reverse primer (P2: 50 -GGC CAC GCG TCG ACT AGT AC-30 ) was designed according to the adaptoroligo(dT) primer. The PCR was carried out in a final volume of 50 ml containing 200 pmol of each primer (P1/P2), using 0.5 ml RT reaction mixture as DNA template. The cycling condition is as followed: 5 min at 94 1C, followed by 33 cycles of 15 s at 94 1C, 30 min at 52 1C and 2 min at 72 1C. The last cycle was followed by an extension step at 72 1C for 10 min. The PCR products were separated in a 1% agarose gel electrophoresis, and the target fragment was extracted and subcloned into pGEM-T vector. The ligation products were transformed into E. coli strain JM109. Five positive clones were sequenced and all the cDNAs were the same. Finally, two primers (P3: 50 -ACT GTT CAG CCT GAT CAA CAT GA-30 ; P4: 50 -AGA GGT GCG ATG TGT TCA CTA TA-30 ) were designed for 50 -RACE reactions based on the nucleotide sequence determined in the previous experiments. The reactions were performed according to the manufacture’s protocol. The longest DNA fragment was ligated into pGEM-T vector and applied to sequencing.
ARTICLE IN PRESS 1202
Y. Jin et al. / Toxicon 49 (2007) 1200–1207
2.4. Cloning of OhS1 homologues from Naja atra and Bungarus multicinctus Living N. atra and B. multicinctus snakes were collected in Hunan province, China. Total RNA extraction and first-strand cDNA synthesis were performed as described above. Forward primer (P5: 50 -GAG TGC AGA GCT GAA ACT ATG-30 ) was designed according to the 50 -untranslated region of OhS1 cDNA. Reverse primer (P6: 50 -ATT TCC TGC AAT AAT ACC CT-30 ) was designed according to coding region approaching to the termination code of OhS1. PCR reaction was conducted as follows. The reaction mixture was subjected to an initial incubation at 94 1C for 5 min before adding rTaq DNA polymerase. It was then subjected to 30 cycles of 15 s at 94 1C, 30 min at 52 1C and 2 min at 72 1C. The last cycle was followed by an extension step at 72 1C for 10 min. 2.5. Sequence analysis and phylogenetic tree construction Sequence similarity between OhS1 and other venom serine protease sequences was assessed using BLASTP. Multi-sequence alignment was conducted by CLUSTX (Higgins et al., 1994) and phylogenetic tree was constructed by neighbor-joining method with MEGA 3 (Kumar et al., 2004).
3. Results and discussion OhS1 was purified according to the method described previously (Zhang et al., 1994). The final preparation was found to be homogeneous estimated by both SDS-PAGE and HPLC RP C4 column (Fig. 1). It was revealed to be a single band with an apparent molecular weight of 43 kDa on the SDS-PAGE analysis under reducing conditions. The result is in accordance with that reported previously (Zhang et al., 1994). To further confirm the identity of OhS1 isolated this time. Fibrinogenolytic activity and amidolytic activity were examined. The enzyme gave a similar fibrinogendegrading profile and substrate specificity similar to that previously reported (Zhang et al., 1994). The OhS1 preparation eluted from C4 column was subjected to protein sequencing and the N-terminal sequence was determined to be IIGGFECNEYEHRSLVHLYNSSG. It shares great similarity with other snake venom thrombin-like enzymes. Based on the N-terminal sequence, a degenerate primer P1 was designed according to the fragment IGGFECNE. Using the RT-PCR and 50 -RACE methods, a full-length cDNA coding for OhS1 was obtained (Fig. 2). The obtained OhS1 cDNA contains a short (15-bp) 50 -untranslation region (50 -UTR), an open reading frame of 783bp and a 110-bp 30 -untranslation region (30 -UTR) containing
Fig. 1. Purity and molecular weight of OhS1. Purity of final preparation was determined by high-performance liquid chromatography (HPLC) on a C4 reversed-phase column using a linear gradient of 10–40% acetonitrile in 0.1% trifluoroacetic acid (TFA) run for 30 min. Insert: SDS-PAGE profile of purified OhS1 under reduced condition (R) and non-reduced condition (N). Molecular weight standards (M) were b-galactosidase (116.0 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45.0 kDa), lactate dehydrgenase (35.0 kDa), restriction endonuclease Bsp981 (25.0 kDa) from top to bottom, respectively.
ARTICLE IN PRESS Y. Jin et al. / Toxicon 49 (2007) 1200–1207
1203
Fig. 2. Nucleotide acid sequence and deduced amino acid sequence of OhS1. Putative signal peptide is in bold. Putative activation peptide is in bold and italic. The N-terminal sequence determined experimentally is boxed. Predicted N-glycosylation positions are indicated in dark.
a poly-adenylation signal, AATAAA (Fig. 2). The cDNA encodes a 260-amino acid precursor, comprising of a signal peptide, activation peptide and mature protein. The fragment containing the first 18 amino acid residues was predicted to be signal peptide by SignalP3.0 (Bendtsen et al., 2004). The six-residue fragment between the signal peptide and the mature enzyme is putative activation peptide. Among the serine proteases from viperid and crotalid snake venoms, they share a highly conserved activation peptide sequence: QR/KSSDR. Interestingly, the activation peptide sequence of OhS1 is VTPFDR. Only the last two residues are conserved comparing with other SVSPs. It is also different from that of colubrid SVSPs, though they share significant sequence identity with OhS1 (Fig. 3). The difference indicates that these homologues might have different activation mechanisms. The experimentally determined N-terminal sequence matched perfectly with the N-terminal
sequence of the predicted mature form of OhS1 (Fig. 2). The mature form of OhS1 is composed of 236 amino acid residues with a predicted molecular mass of 25968 Da, which is less than the apparent molecular weight of 43 kDa as judged by SDSPAGE. The apparent molecular weight of SVSPs varies from 25 to 70 kDa, though generally they are composed of around 230 amino acid residues. Many SVSPs are characterized as glycoproteins, and the carbohydrate content in SVSPs is responsible for the considerable difference in molecular mass. Five potential N-glycosylation sites were found in the sequence of OhS1 (Fig. 2), indicating that the carbohydrate moieties are responsible for its high molecular mass. The function of glycosylation in SVSPs is not well known. The carbohydrates in TSV-PA have been documented to have little influence on its amidolytic activity and plasminogen activation property (Parry et al., 1998); however, other work indicated that glycosylation of SVSPs
ARTICLE IN PRESS 1204
Y. Jin et al. / Toxicon 49 (2007) 1200–1207
Fig. 3. Sequence alignment of snake venom serine proteases. The sequences used in the alignment are OhS1 (GenBank accession number: EF080837), NaSP (GenBank accession number: EF080834), BmSP (GenBank accession number: EF080838), bothrombin from Bothrops jararaca (GenBank accession number: P81661), flavoxobin from Trimeresurus flavoviridis (GenBank accession number: P05620), TSV-PA from T. stejnegeri (GenBank accession number: Q91516), ancrod from Calloselasma rhodostoma (GenBank accession number: P47797), rCCPPP from Cerastes cerastes (GenBank accession number: CAD86932), KN-BJ2 from B. jararaca (GenBank accession number: O13069), Factor V activator from Macrovipera lebetina (Vr-FVA, GenBank accession number: Q9PT41), LhSP from Lapemis hardwickii (GenBank accession number: AAV98367), human thrombin (hThrombin, GenBank accession number: CAJ01369), bovine trypsin (bTrypsin, GenBank accession number: Q29463), serine protease from Philodryas olfersii (Po-Klk, GenBank accession number: AAZ75628) and serine protease from Varanus mitchelli (Vm-klk, GenBank accession number: AAZ75621). Cysteine residues are indicated by asterisks.
ARTICLE IN PRESS Y. Jin et al. / Toxicon 49 (2007) 1200–1207
has a stabilizing effect (Murayama et al., 2003) or contributes to resistance against protease inhibitors (Zhu et al., 2005).
1205
Partial cDNAs encoding serine proteases were cloned from the venom gland of N. atra and B. multicinctus by RT-PCR, with a pair of primers
Fig. 4. Phylogenetic tree of snake venom serine proteases. Venom serine proteases from snakes were aligned by CLUSTALX and phylogenetic tree was constructed by MEGA 3. The amino acid sequences were randomly picked from the GenBank database. The species name and the GenBank accession number are indicated in the tree. Bos taurus trypsin was used as out-group (Q29463).
ARTICLE IN PRESS 1206
Y. Jin et al. / Toxicon 49 (2007) 1200–1207
designed based on the nucleotide sequence of OhS1. The two predicted proteins, designed as NaSP and BmSP, shared a considerable high degree (99.6%) of sequence identity (Fig. 3), suggesting that this gene is highly conserved in both species. Mature OhS1 shared 80% amino acid sequence identity with predicted mature NaSP and BmSP, respectively. A significant difference between OhS1 and NaSP or BmSP is at the potential activation peptide domain. Unlike most SVSPs, NaSP and BmSP possess a considerable long activation peptide (Fig. 3). When comparing the whole amino acid sequence of OhS1 with that of NaSP and BmSP, lower identity degree of 62% is observed. Like trypsin, OhS1 has the conserved catalytic triad at the conserved position (Fig. 3). Generally, SVSPs contain 12 cysteine residues, 10 of which form disulfide bonds, homologue to trypsin (Itoh et al., 1987). Besides the 12 conserved cysteine residues, OhS1 has two additional cysteine residues (Fig. 3). The additional cysteine residues are also found in NaSP, BmSP and kallikrein-like proteases from a Colubridae snake, Philodryas olfersii. OhS1 also contains a C-terminal extension that is a typical motif of SVSPs. Sequence comparison of OhS1 with SVSPs revealed that OhS1 shared a moderate degree (60%) of sequence identity with venom serine proteases from Viperidae family. Previously, phylogenetic analysis has classified viperid SVSPs into three clusters, each of which corresponded to a major enzyme subtype: coagulating, kininogenase (KN) and plasminogen activator (PA) (Wang et al., 2001). However, when randomly picked SVSPs amino acid sequences from the GenBank database and phylogenetically analysed, the constructed phylogenetic tree could not be only classified into three subtypes as mentioned above (Fig. 4). Our results indicated that the present constructed phylogenetic tree is more complicated than that previously constructed (Wang et al., 2001). The evolutionary relationship of OhS1 with other SVSPs was analyzed by phylogenetic tree construction (Fig. 4). In the tree, OhS1 and serine protease 3 from T. flavoviridis forms a cluster whereas BmSP, NaSP and a serine protease from Lapemis hardwickii forms another cluster, indicating that elapid SVSPs of BmSP, NaSP and hydrophiid SVSPs were evolutionarily closely related, but OhS1 was distant from them. In summary, three serine proteases from different species of elapid snakes were cloned and reported.
Phylogenetic analysis based on the amino acid sequences of SVSPs indicated that SVSP might have a common ancestor. Acknowledgments This work was supported by grants from National Natural Science Foundation (30630014, 30570359, 30470380 and 30670412) and the grants of ‘‘Western Light’’ Projects. Dr. Lee also received a grant from Yunnan Science and Technology Commission (2005PY01-23)
Reference Bendtsen, J.D., Nielsen, H., von Heijne, G., Brunak, S., 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340, 783–795. Dekhil, H., Wisner, A., Marrakchi, N., El, Ayeb.M., Bon, C., Karoui, H., 2003. Molecular cloning and expression of a functional snake venom serine proteinase, with platelet aggregating activity, from the Cerastes cerastes viper. Biochemistry 42, 10609–10618. Deshimaru, M., Ogawa, T., Nakashima, K., Nobuhisa, I., Chijiwa, T., Shimohigashi, Y., Fukumaki, Y., Niwa, M., Yamashina, I., Hattori, S., Ohno, M., 1996. Accelerated evolution of crotalinae snake venom gland serine proteases. FEBS Lett. 397, 83–88. Du, X.Y., Clemetson, J.M., Navdaev, A., Magnenat, E.M., Wells, T.N., Clemetson, K.J., 2002. Ophioluxin, a convulxinlike C-type lectin from Ophiophagus hannah (King cobra) is a powerful platelet activator via glycoprotein VI. J. Biol. Chem. 277, 35124–35132. Fry, B.G., Vidal, N., Norman, J.A., Vonk, F.J., Scheib, H., Ramjan, S.F., Kuruppu, S., Fung, K., Hedges, S.B., Richardson, M.K., Hodgson, W.C., Ignjatovic, V., Summerhayes, R., Kochva, E., 2006. Early evolution of the venom system in lizards and snakes. Nature 439, 584–588. Higgins, D., Thompson, J., Gibson, T., Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Hung, C.C., Chiou, S.H., 2001. Fibrinogenolytic proteases isolated from the snake venom of Taiwan habu: serine proteases with kallikrein-like and angiotensin-degrading activities. Biochem. Biophys. Res. Commun. 281, 1012–1018. Itoh, N., Tanaka, N., Mihashi, S., Yamashina, I., 1987. Molecular cloning and sequence analysis of cDNA for batroxobin, a thrombin-like snake venom enzyme. J. Biol. Chem. 262, 3132–3135. Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5, 150–163. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
ARTICLE IN PRESS Y. Jin et al. / Toxicon 49 (2007) 1200–1207 Laing, G.D., Compton, S.J., Ramachandran, R., Fuller, G.L., Wilkinson, M.C., Wagstaff, S.C., Watson, S.P., Kamiguti, A.S., Theakston, R.D., Senis, Y.A., 2005. Characterization of a novel protein from Proatheris superciliaris venom: proatherocytin, a 34-kDa platelet receptor PAR1 agonist. Toxicon 46, 490–499. Lee, W.H., Zhang, Y., Wang, W.Y., Xiong, Y.L., Gao, R., 1995. Isolation and properties of a blood coagulation factor X activator from the venom of king cobra (Ophiophagus hannah). Toxicon 33, 1263–1276. Li, Z.Y., Yu, T.F., Lian, E.C., 1994. Purification and characterization of L-amino acid oxidase from king cobra (Ophiophagus hannah) venom and its effects on human platelet aggregation. Toxicon 32, 1349–1358. Markland, F.S., 1998. Snake venoms and the hemostatic system. Toxicon 36, 1749–1800. Matsui, T., Sakurai, Y., Fujimura, Y., Hayashi, I., Oh-Ishi, S., Suzuki, M., Hamako, J., Yamamoto, Y., Yamazaki, J., Kinoshita, M., Titani, K., 1998. Purification and amino acid sequence of halystase from snake venom of Agkistrodon halys blomhoffii, a serine protease that cleaves specifically fibrinogen and kininogen. Eur. J. Biochem. 252, 569–575. Murayama, N., Saguchi, K., Mentele, R., Assakura, M.T., Ohi, H., Fujita, Y., Camargo, A.C., Higuchi, S., Serrano, S.M., 2003. The unusual high molecular mass of Bothrops protease A, a trypsin-like serine peptidase from the venom of Bothrops jararaca, is due to its high carbohydrate content. Biochim. Biophys. Acta 1652, 1–6. Parry, M.A., Jacob, U., Huber, R., Wisner, A., Bon, C., Bode, W., 1998. The crystal structure of the novel snake venom
1207
plasminogen activator TSV-PA: a prototype structure for snake venom serine proteinases. Structure 6, 1195–1206. Perona, J.J., Craik, C.S., 1997. Evolutionary divergence of substrate specificity within the chymotrypsin-like serine protease fold. J. Biol. Chem. 272, 29987–29990. Serrano, S.M., Maroun, R.C., 2005. Snake venom serine proteinases: sequence homology vs. substrate specificity, a paradox to be solved. Toxicon 45, 1115–1132. Tan, N.H., Saifuddin, M.N., 1990. Isolation and characterization of a hemorrhagin from the venom of Ophiophagus hannah (king cobra). Toxicon 28, 385–392. Wang, Y.M., Wang, S.R., Tsai, I.H., 2001. Serine protease isoforms of Deinagkistrodon acutus venom: cloning, sequencing and phylogenetic analysis. Biochem. J. 354, 161–168. Zhang, Y., Lee, W.H., Xiong, Y.L., Wang, W.Y., Zu, S.W., 1994. Characterization of OhS1, an arginine/lysine amidase from the venom of king cobra (Ophiophagus hannah). Toxicon 32, 615–623. Zhang, Y., Wisner, A., Xiong, Y., Bon, C., 1995. A novel plasminogen activator from snake venom. Purification, characterization, and molecular cloning. J. Biol. Chem. 270, 10246–10255. Zhang, Y., Wisner, A., Maroun, R.C., Choumet, V., Xiong, Y., Bon, C., 1997. Trimeresurus stejnegeri snake venom plasminogen activator. Site-directed mutagenesis and molecular modeling. J. Biol. Chem. 272, 20531–20537. Zhu, Z., Liang, Z., Zhang, T., Zhu, Z., Xu, W., Teng, M., Niu, L., 2005. Crystal structures and amidolytic activities of two glycosylated snake venom serine proteinases. J. Biol. Chem. 280, 10524–10529.
Toxicon 49 (2007) 1200–1207 www.elsevier.com/locate/toxicon
Molecular cloning of serine proteases from elapid snake venoms Yang Jin1, Wen-Hui Lee1, Yun Zhang Biotoxin Units, Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China Received 27 October 2006; received in revised form 16 February 2007; accepted 19 February 2007 Available online 27 February 2007
Abstract Serine proteases are widely distributed in viperid snake venoms, but rare in elapid snake venoms. Previously, we have identified a fibrinogenolytic enzyme termed OhS1 from the venom of Ophiophagus hannah. The results indicated that OhS1 might be a serine protease, but there was no structural evidence previously. In the present study, the primary structure of OhS1 was determined by protein sequencing, in combination with RT-PCR and 50 -RACE methods. OhS1 precursor is composed of an 18-amino acid signal peptide, a 6-amino acid putative activation peptide and 236-amino acid mature protein. OhS1 homologues from Naja atra and Bungarus multicinctus were also cloned and reported. These elapid venom serine proteases exhibited 60% sequence identity with serine proteases from the snake venoms of the Viperidae and Colubridae family. Phylogenetic analysis indicated that snake venom serine protease might have a common ancestor. r 2007 Elsevier Ltd. All rights reserved. Keywords: Serine protease; Elapidae; Cloning
1. Introduction Snake venoms are complex mixtures of various molecules, most of which are peptide toxins, hydrolytic enzymes and non-lethal proteins possessing various biological functions. Serine proteases are among the best-characterized components of living organisms. Snake venom serine proteases (SVSPs) are classified into the trypsin family S1 of Clan SA. SVSPs possess an identical trypsin fold consisting of two b-barrels and the conserved catalytic triad, Ser195, His57 and Asp102 (Perona and Craik, 1997). These enzymes normally contain Corresponding author. Tel.: +86 871 5198515;
fax: +86 871 5191823. E-mail address: [email protected] (Y. Zhang). 1 These authors contributed equally to this work. 0041-0101/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2007.02.013
12 cysteine residues and a C-terminal extension which is the characteristic of SVSPs (Parry et al., 1998). SVSPs undergo fast adaptive evolution to generate variants of diverse functions (Deshimaru et al., 1996; Fry et al., 2006), including specifically degrading or activating blood components involved in coagulation and fibrinolysis (Markland, 1998; Serrano and Maroun, 2005; Zhang et al. 1995, 1997), activating kallikrein/kinin system (Hung and Chiou, 2001; Matsui et al., 1998) or affecting platelet aggregation (Laing et al., 2005; Dekhil et al., 2003). The king cobra (Ophiophagus hannah), which belongs to the Elapidae family, is a large poisonous snake inhabiting in south Asia and injects a large amount of venom once. Although the majority of the venom components are neurotoxins and phospholipase A2, it also contains high-molecular-weight
ARTICLE IN PRESS Y. Jin et al. / Toxicon 49 (2007) 1200–1207
proteins targeting at blood coagulation system, such as factor X activator, L-amino acid oxidase, metalloproteinase and platelet GPVI agonist (Lee et al., 1995; Li et al., 1994; Tan and Saifuddin, 1990; Du et al., 2002). However, there are few reports on the serine proteases from elapid snake venoms. Previously, we have isolated an arginine/lysine amidase termed OhS1 from the venom of king cobra. OhS1 is a single-chain protein with an apparent molecular weight of 43 kDa and possesses potent fibrinogenase and amidase activity. Its enzymatic activity could be completely inhibited by serine protease inhibitors, e.g., PMSF, DFP and soybean trypsin inhibitor, but not affected by EDTA (Zhang et al., 1994). Though these characteristics suggested that OhS1 is a serine protease, there was no structural information to confirm its identity. In this work, the cDNA of OhS1, as well as its homologues from two other elapid snakes were cloned and analyzed. 2. Materials and methods 2.1. Materials O. hannah crude venom was the stock of Kunming Institute of Zoology. Sephadex G-100 (superfine) and Resource Q column (1 ml) were products of Amersham Biosciences (Uppsala, Sweden). Human fibrinogen was purchased from Sigma Chemical (St. Louis, MO, USA). The RNeasy RNA isolation kit was a product of Qiagen (Valencia, CA, USA). Superscript II reverse transcription polymerase and 50 -RACE kit were products of Invitrogen. Agarose gel extraction kit, rTaq DNA polymerase, synthesized oligonucleotides and Escherichia coli JM109 competent cells were purchased from TaKaRa (Dalian, China). pGEM-T vector system was product of Promega Corporation Ltd. (Madison, WI, USA). All other chemicals were of analytic grade. 2.2. Isolation, purity examination and biological assay OhS1 was isolated according to our previous work (Zhang et al., 1994). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method described (Laemmli, 1970). In a separate experiment, the purity of final preparation was determined by high-performance liquid chromatography (HPLC) on a C4 reversed-
1201
phase (RP) column (ZORBAX, Agilent Technolgies, USA) using a linear gradient of 10–40% acetonitrile in 0.1% trifluoroacetic acid (TFA) run for 30 min. The preparation eluted from C4 column chromatography was subjected to ABI model 476A protein sequencer to determine the N-terminal amino acid sequence. Fibrinogenlytic and amidolytic activities were conducted as described previously (Zhang et al., 1994).
2.3. Venom gland RNA isolation and cloning of OhS1 Living O. hannah snake was collected in Guangxi province, China. Total RNA was isolated from the venom gland of O. hannah using a homogenizer and RNeasy RNA isolation kit according to the manufacture’s instruction. RNA purity and concentration were estimated by checking the optical density at 280 and 260 nm. RT-PCR and 50 -RACE methods were used to obtain the full-length cDNA of OhS1. Briefly, firststrand cDNAs were generated from total RNA using an adaptor-oligo(dT) primer (50 -GGC CAC GCG TCG ACT AGT AC(T)17-30 ). A forward degenerate primer (termed P1: 50 -ATH GGN GGN TTY GAR TGY AAY GA) was designed based on the determined amino-terminal sequence. A reverse primer (P2: 50 -GGC CAC GCG TCG ACT AGT AC-30 ) was designed according to the adaptoroligo(dT) primer. The PCR was carried out in a final volume of 50 ml containing 200 pmol of each primer (P1/P2), using 0.5 ml RT reaction mixture as DNA template. The cycling condition is as followed: 5 min at 94 1C, followed by 33 cycles of 15 s at 94 1C, 30 min at 52 1C and 2 min at 72 1C. The last cycle was followed by an extension step at 72 1C for 10 min. The PCR products were separated in a 1% agarose gel electrophoresis, and the target fragment was extracted and subcloned into pGEM-T vector. The ligation products were transformed into E. coli strain JM109. Five positive clones were sequenced and all the cDNAs were the same. Finally, two primers (P3: 50 -ACT GTT CAG CCT GAT CAA CAT GA-30 ; P4: 50 -AGA GGT GCG ATG TGT TCA CTA TA-30 ) were designed for 50 -RACE reactions based on the nucleotide sequence determined in the previous experiments. The reactions were performed according to the manufacture’s protocol. The longest DNA fragment was ligated into pGEM-T vector and applied to sequencing.
ARTICLE IN PRESS 1202
Y. Jin et al. / Toxicon 49 (2007) 1200–1207
2.4. Cloning of OhS1 homologues from Naja atra and Bungarus multicinctus Living N. atra and B. multicinctus snakes were collected in Hunan province, China. Total RNA extraction and first-strand cDNA synthesis were performed as described above. Forward primer (P5: 50 -GAG TGC AGA GCT GAA ACT ATG-30 ) was designed according to the 50 -untranslated region of OhS1 cDNA. Reverse primer (P6: 50 -ATT TCC TGC AAT AAT ACC CT-30 ) was designed according to coding region approaching to the termination code of OhS1. PCR reaction was conducted as follows. The reaction mixture was subjected to an initial incubation at 94 1C for 5 min before adding rTaq DNA polymerase. It was then subjected to 30 cycles of 15 s at 94 1C, 30 min at 52 1C and 2 min at 72 1C. The last cycle was followed by an extension step at 72 1C for 10 min. 2.5. Sequence analysis and phylogenetic tree construction Sequence similarity between OhS1 and other venom serine protease sequences was assessed using BLASTP. Multi-sequence alignment was conducted by CLUSTX (Higgins et al., 1994) and phylogenetic tree was constructed by neighbor-joining method with MEGA 3 (Kumar et al., 2004).
3. Results and discussion OhS1 was purified according to the method described previously (Zhang et al., 1994). The final preparation was found to be homogeneous estimated by both SDS-PAGE and HPLC RP C4 column (Fig. 1). It was revealed to be a single band with an apparent molecular weight of 43 kDa on the SDS-PAGE analysis under reducing conditions. The result is in accordance with that reported previously (Zhang et al., 1994). To further confirm the identity of OhS1 isolated this time. Fibrinogenolytic activity and amidolytic activity were examined. The enzyme gave a similar fibrinogendegrading profile and substrate specificity similar to that previously reported (Zhang et al., 1994). The OhS1 preparation eluted from C4 column was subjected to protein sequencing and the N-terminal sequence was determined to be IIGGFECNEYEHRSLVHLYNSSG. It shares great similarity with other snake venom thrombin-like enzymes. Based on the N-terminal sequence, a degenerate primer P1 was designed according to the fragment IGGFECNE. Using the RT-PCR and 50 -RACE methods, a full-length cDNA coding for OhS1 was obtained (Fig. 2). The obtained OhS1 cDNA contains a short (15-bp) 50 -untranslation region (50 -UTR), an open reading frame of 783bp and a 110-bp 30 -untranslation region (30 -UTR) containing
Fig. 1. Purity and molecular weight of OhS1. Purity of final preparation was determined by high-performance liquid chromatography (HPLC) on a C4 reversed-phase column using a linear gradient of 10–40% acetonitrile in 0.1% trifluoroacetic acid (TFA) run for 30 min. Insert: SDS-PAGE profile of purified OhS1 under reduced condition (R) and non-reduced condition (N). Molecular weight standards (M) were b-galactosidase (116.0 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45.0 kDa), lactate dehydrgenase (35.0 kDa), restriction endonuclease Bsp981 (25.0 kDa) from top to bottom, respectively.
ARTICLE IN PRESS Y. Jin et al. / Toxicon 49 (2007) 1200–1207
1203
Fig. 2. Nucleotide acid sequence and deduced amino acid sequence of OhS1. Putative signal peptide is in bold. Putative activation peptide is in bold and italic. The N-terminal sequence determined experimentally is boxed. Predicted N-glycosylation positions are indicated in dark.
a poly-adenylation signal, AATAAA (Fig. 2). The cDNA encodes a 260-amino acid precursor, comprising of a signal peptide, activation peptide and mature protein. The fragment containing the first 18 amino acid residues was predicted to be signal peptide by SignalP3.0 (Bendtsen et al., 2004). The six-residue fragment between the signal peptide and the mature enzyme is putative activation peptide. Among the serine proteases from viperid and crotalid snake venoms, they share a highly conserved activation peptide sequence: QR/KSSDR. Interestingly, the activation peptide sequence of OhS1 is VTPFDR. Only the last two residues are conserved comparing with other SVSPs. It is also different from that of colubrid SVSPs, though they share significant sequence identity with OhS1 (Fig. 3). The difference indicates that these homologues might have different activation mechanisms. The experimentally determined N-terminal sequence matched perfectly with the N-terminal
sequence of the predicted mature form of OhS1 (Fig. 2). The mature form of OhS1 is composed of 236 amino acid residues with a predicted molecular mass of 25968 Da, which is less than the apparent molecular weight of 43 kDa as judged by SDSPAGE. The apparent molecular weight of SVSPs varies from 25 to 70 kDa, though generally they are composed of around 230 amino acid residues. Many SVSPs are characterized as glycoproteins, and the carbohydrate content in SVSPs is responsible for the considerable difference in molecular mass. Five potential N-glycosylation sites were found in the sequence of OhS1 (Fig. 2), indicating that the carbohydrate moieties are responsible for its high molecular mass. The function of glycosylation in SVSPs is not well known. The carbohydrates in TSV-PA have been documented to have little influence on its amidolytic activity and plasminogen activation property (Parry et al., 1998); however, other work indicated that glycosylation of SVSPs
ARTICLE IN PRESS 1204
Y. Jin et al. / Toxicon 49 (2007) 1200–1207
Fig. 3. Sequence alignment of snake venom serine proteases. The sequences used in the alignment are OhS1 (GenBank accession number: EF080837), NaSP (GenBank accession number: EF080834), BmSP (GenBank accession number: EF080838), bothrombin from Bothrops jararaca (GenBank accession number: P81661), flavoxobin from Trimeresurus flavoviridis (GenBank accession number: P05620), TSV-PA from T. stejnegeri (GenBank accession number: Q91516), ancrod from Calloselasma rhodostoma (GenBank accession number: P47797), rCCPPP from Cerastes cerastes (GenBank accession number: CAD86932), KN-BJ2 from B. jararaca (GenBank accession number: O13069), Factor V activator from Macrovipera lebetina (Vr-FVA, GenBank accession number: Q9PT41), LhSP from Lapemis hardwickii (GenBank accession number: AAV98367), human thrombin (hThrombin, GenBank accession number: CAJ01369), bovine trypsin (bTrypsin, GenBank accession number: Q29463), serine protease from Philodryas olfersii (Po-Klk, GenBank accession number: AAZ75628) and serine protease from Varanus mitchelli (Vm-klk, GenBank accession number: AAZ75621). Cysteine residues are indicated by asterisks.
ARTICLE IN PRESS Y. Jin et al. / Toxicon 49 (2007) 1200–1207
has a stabilizing effect (Murayama et al., 2003) or contributes to resistance against protease inhibitors (Zhu et al., 2005).
1205
Partial cDNAs encoding serine proteases were cloned from the venom gland of N. atra and B. multicinctus by RT-PCR, with a pair of primers
Fig. 4. Phylogenetic tree of snake venom serine proteases. Venom serine proteases from snakes were aligned by CLUSTALX and phylogenetic tree was constructed by MEGA 3. The amino acid sequences were randomly picked from the GenBank database. The species name and the GenBank accession number are indicated in the tree. Bos taurus trypsin was used as out-group (Q29463).
ARTICLE IN PRESS 1206
Y. Jin et al. / Toxicon 49 (2007) 1200–1207
designed based on the nucleotide sequence of OhS1. The two predicted proteins, designed as NaSP and BmSP, shared a considerable high degree (99.6%) of sequence identity (Fig. 3), suggesting that this gene is highly conserved in both species. Mature OhS1 shared 80% amino acid sequence identity with predicted mature NaSP and BmSP, respectively. A significant difference between OhS1 and NaSP or BmSP is at the potential activation peptide domain. Unlike most SVSPs, NaSP and BmSP possess a considerable long activation peptide (Fig. 3). When comparing the whole amino acid sequence of OhS1 with that of NaSP and BmSP, lower identity degree of 62% is observed. Like trypsin, OhS1 has the conserved catalytic triad at the conserved position (Fig. 3). Generally, SVSPs contain 12 cysteine residues, 10 of which form disulfide bonds, homologue to trypsin (Itoh et al., 1987). Besides the 12 conserved cysteine residues, OhS1 has two additional cysteine residues (Fig. 3). The additional cysteine residues are also found in NaSP, BmSP and kallikrein-like proteases from a Colubridae snake, Philodryas olfersii. OhS1 also contains a C-terminal extension that is a typical motif of SVSPs. Sequence comparison of OhS1 with SVSPs revealed that OhS1 shared a moderate degree (60%) of sequence identity with venom serine proteases from Viperidae family. Previously, phylogenetic analysis has classified viperid SVSPs into three clusters, each of which corresponded to a major enzyme subtype: coagulating, kininogenase (KN) and plasminogen activator (PA) (Wang et al., 2001). However, when randomly picked SVSPs amino acid sequences from the GenBank database and phylogenetically analysed, the constructed phylogenetic tree could not be only classified into three subtypes as mentioned above (Fig. 4). Our results indicated that the present constructed phylogenetic tree is more complicated than that previously constructed (Wang et al., 2001). The evolutionary relationship of OhS1 with other SVSPs was analyzed by phylogenetic tree construction (Fig. 4). In the tree, OhS1 and serine protease 3 from T. flavoviridis forms a cluster whereas BmSP, NaSP and a serine protease from Lapemis hardwickii forms another cluster, indicating that elapid SVSPs of BmSP, NaSP and hydrophiid SVSPs were evolutionarily closely related, but OhS1 was distant from them. In summary, three serine proteases from different species of elapid snakes were cloned and reported.
Phylogenetic analysis based on the amino acid sequences of SVSPs indicated that SVSP might have a common ancestor. Acknowledgments This work was supported by grants from National Natural Science Foundation (30630014, 30570359, 30470380 and 30670412) and the grants of ‘‘Western Light’’ Projects. Dr. Lee also received a grant from Yunnan Science and Technology Commission (2005PY01-23)
Reference Bendtsen, J.D., Nielsen, H., von Heijne, G., Brunak, S., 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340, 783–795. Dekhil, H., Wisner, A., Marrakchi, N., El, Ayeb.M., Bon, C., Karoui, H., 2003. Molecular cloning and expression of a functional snake venom serine proteinase, with platelet aggregating activity, from the Cerastes cerastes viper. Biochemistry 42, 10609–10618. Deshimaru, M., Ogawa, T., Nakashima, K., Nobuhisa, I., Chijiwa, T., Shimohigashi, Y., Fukumaki, Y., Niwa, M., Yamashina, I., Hattori, S., Ohno, M., 1996. Accelerated evolution of crotalinae snake venom gland serine proteases. FEBS Lett. 397, 83–88. Du, X.Y., Clemetson, J.M., Navdaev, A., Magnenat, E.M., Wells, T.N., Clemetson, K.J., 2002. Ophioluxin, a convulxinlike C-type lectin from Ophiophagus hannah (King cobra) is a powerful platelet activator via glycoprotein VI. J. Biol. Chem. 277, 35124–35132. Fry, B.G., Vidal, N., Norman, J.A., Vonk, F.J., Scheib, H., Ramjan, S.F., Kuruppu, S., Fung, K., Hedges, S.B., Richardson, M.K., Hodgson, W.C., Ignjatovic, V., Summerhayes, R., Kochva, E., 2006. Early evolution of the venom system in lizards and snakes. Nature 439, 584–588. Higgins, D., Thompson, J., Gibson, T., Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Hung, C.C., Chiou, S.H., 2001. Fibrinogenolytic proteases isolated from the snake venom of Taiwan habu: serine proteases with kallikrein-like and angiotensin-degrading activities. Biochem. Biophys. Res. Commun. 281, 1012–1018. Itoh, N., Tanaka, N., Mihashi, S., Yamashina, I., 1987. Molecular cloning and sequence analysis of cDNA for batroxobin, a thrombin-like snake venom enzyme. J. Biol. Chem. 262, 3132–3135. Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5, 150–163. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
ARTICLE IN PRESS Y. Jin et al. / Toxicon 49 (2007) 1200–1207 Laing, G.D., Compton, S.J., Ramachandran, R., Fuller, G.L., Wilkinson, M.C., Wagstaff, S.C., Watson, S.P., Kamiguti, A.S., Theakston, R.D., Senis, Y.A., 2005. Characterization of a novel protein from Proatheris superciliaris venom: proatherocytin, a 34-kDa platelet receptor PAR1 agonist. Toxicon 46, 490–499. Lee, W.H., Zhang, Y., Wang, W.Y., Xiong, Y.L., Gao, R., 1995. Isolation and properties of a blood coagulation factor X activator from the venom of king cobra (Ophiophagus hannah). Toxicon 33, 1263–1276. Li, Z.Y., Yu, T.F., Lian, E.C., 1994. Purification and characterization of L-amino acid oxidase from king cobra (Ophiophagus hannah) venom and its effects on human platelet aggregation. Toxicon 32, 1349–1358. Markland, F.S., 1998. Snake venoms and the hemostatic system. Toxicon 36, 1749–1800. Matsui, T., Sakurai, Y., Fujimura, Y., Hayashi, I., Oh-Ishi, S., Suzuki, M., Hamako, J., Yamamoto, Y., Yamazaki, J., Kinoshita, M., Titani, K., 1998. Purification and amino acid sequence of halystase from snake venom of Agkistrodon halys blomhoffii, a serine protease that cleaves specifically fibrinogen and kininogen. Eur. J. Biochem. 252, 569–575. Murayama, N., Saguchi, K., Mentele, R., Assakura, M.T., Ohi, H., Fujita, Y., Camargo, A.C., Higuchi, S., Serrano, S.M., 2003. The unusual high molecular mass of Bothrops protease A, a trypsin-like serine peptidase from the venom of Bothrops jararaca, is due to its high carbohydrate content. Biochim. Biophys. Acta 1652, 1–6. Parry, M.A., Jacob, U., Huber, R., Wisner, A., Bon, C., Bode, W., 1998. The crystal structure of the novel snake venom
1207
plasminogen activator TSV-PA: a prototype structure for snake venom serine proteinases. Structure 6, 1195–1206. Perona, J.J., Craik, C.S., 1997. Evolutionary divergence of substrate specificity within the chymotrypsin-like serine protease fold. J. Biol. Chem. 272, 29987–29990. Serrano, S.M., Maroun, R.C., 2005. Snake venom serine proteinases: sequence homology vs. substrate specificity, a paradox to be solved. Toxicon 45, 1115–1132. Tan, N.H., Saifuddin, M.N., 1990. Isolation and characterization of a hemorrhagin from the venom of Ophiophagus hannah (king cobra). Toxicon 28, 385–392. Wang, Y.M., Wang, S.R., Tsai, I.H., 2001. Serine protease isoforms of Deinagkistrodon acutus venom: cloning, sequencing and phylogenetic analysis. Biochem. J. 354, 161–168. Zhang, Y., Lee, W.H., Xiong, Y.L., Wang, W.Y., Zu, S.W., 1994. Characterization of OhS1, an arginine/lysine amidase from the venom of king cobra (Ophiophagus hannah). Toxicon 32, 615–623. Zhang, Y., Wisner, A., Xiong, Y., Bon, C., 1995. A novel plasminogen activator from snake venom. Purification, characterization, and molecular cloning. J. Biol. Chem. 270, 10246–10255. Zhang, Y., Wisner, A., Maroun, R.C., Choumet, V., Xiong, Y., Bon, C., 1997. Trimeresurus stejnegeri snake venom plasminogen activator. Site-directed mutagenesis and molecular modeling. J. Biol. Chem. 272, 20531–20537. Zhu, Z., Liang, Z., Zhang, T., Zhu, Z., Xu, W., Teng, M., Niu, L., 2005. Crystal structures and amidolytic activities of two glycosylated snake venom serine proteinases. J. Biol. Chem. 280, 10524–10529.