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. Author manuscript; available in PMC: 2012 Mar 14.
Published in final edited form as: Toxicon. 2011 May 27;58(2):168–178. doi: 10.1016/j.toxicon.2011.05.014

Phylogenetic analysis of serine proteases from Russell's viper (Daboia russelli siamensis) and Agkistrodon piscivorus leucostoma venom

Pattadon Sukkapan a, Ying Jia b, Issarang Nuchprayoon a,c,*, John C Pérez b
PMCID: PMC3303153  NIHMSID: NIHMS361519  PMID: 21640745

Abstract

Serine proteases are widely found in snake venoms. They have variety of functions including contributions to hemostasis. In this study, five serine proteases were cloned and characterized from two different cDNA libraries: factor V activator (RVV-V), alpha fibrinogenase (RVAF) and beta fibrinogenase (RVBF) from Russell's viper (Daboia russelli siamensis), and plasminogen activator (APL-PA) and protein C activator (APL-C) from Agkistrodon piscivorus leucostoma. The snake venom serine proteases were clustered in phylogenetic tree according to their functions. KA/KS values suggested that accelerated evolution has occurred in the mature protein coding regions in cDNAs of snake venom serine proteases.

Keywords: Russell's viper, Agkistrodon piscivorus leucostoma, Snake venom serine protease, RT-PCR, cDNA

1. Introduction

Snake venoms are complex mixtures of molecules with various biological activities, which are used for capturing and/or digesting prey; however, these same molecules could have therapeutic value once characterized and cloned. Snake venoms contain various enzymes such as phospholipase A2, metalloproteases, serine proteases and l-amino acid oxidases, and non enzymatic peptides including C-types lectins and disintegrins (Cidade et al., 2006; Jia et al., 2008). These toxin families have possibly evolved through the gene recruitment strategy before the development of snake venom glands (Fry et al., 2006; Fry and Wuster, 2004). Gene recruitment processes include altered gene expression, mutations, gene duplication, and functional constraint (Soto et al., 2007).

Serine protease is a family of proteases which contains the “catalytic triad” active site: highly reactive serine, histidine and aspartic acid (Hedstrom, 2002a). They are widely found in eukaryotes, prokaryotes, arche, and viruses (Hedstrom, 2002a). In snake, they are present in venoms of the families Viperidae, Crotalidae, Elapidae and Colubridae. Snake venom serine proteases show stringent macromolecular substrate specificity that contrasts with the less specific activity of trypsin (Serrano and Maroun, 2005). Although the amino acid sequences of snake venom serine proteases share high similarity, these venom proteases exhibit different substrate specificity and function (Serrano and Maroun, 2005). A number of hemostasis-affecting snake venom serine proteases have been reported, including procoagulant, anticoagulant, platelet aggregating- and fibrinolytic proteases (Kini, 2005; Matsui et al., 2000; Serrano and Maroun, 2005).

The Russell's viper (Daboia russelli siamensis) is a medically important snake distributed in East and Southeast Asia, including Thailand (Chanhome et al., 1998; Warrell, 1989). The common clinical manifestation of Russell's viper bite is incoagulable blood associated with severe reduction of coagulation factor V, X and XIII (Warrell, 1989). Studies of Russell's viper venom gland cDNA indicates that phospholipase A2 isoforms were predominantly expressed (Nuchprayoon et al., 2001; Sai-Ngam et al., 2008). Two well-known procoagulant enzymes found in the Russell's viper venom have been characterized: factor X activator (RVV-X), a type IV metalloprotease, and factor V activator (RVV-V), a single chain serine protease, (Furie and Furie, 1976; Kisiel, 1979; Kisiel et al., 1976; Tokunaga et al., 1988).

RVV-V is a 29 kDa single chain serine protease, consisting of 236 amino acid residues and 6% carbohydrate (Kisiel, 1979; Tokunaga et al., 1988). RVV-V specifically cleaves the single peptide bond between Arg1545 and Ser1546, resulting in activation of factor V, a key component of the hemostatic system which acts as a co-factor in prothrombinase complex (Kalafatis et al., 2003; Keller et al., 1995; Rosing et al., 2001). Factor Va accelerates factor X-catalyzed prothrombin conversion by 300,000-fold (Mann and Kalafatis, 2003). The amino acid sequence of RVV-V has been reported (Tokunaga et al., 1988) and crystallized (Nakayama et al., 2009). RVV-V was also observed in a proteomic study of Russell's viper venom (Risch et al., 2009).

Agkistrodon piscivorus leucostoma, the western cottonmouth, is a venomous pit viper subspecies, geographically distributed from southern Illinois south to Alabama, west to Oklahoma and Central Texas in the United States (Jia et al., 2008). Expressed sequence tag (EST) analysis from venom gland cDNA library demonstrated that the venom of A. p. leucostoma is a rich source of serine proteases, including plasminogen activator and protein C activator (Jia et al., 2008). A parvalbumin and metalloproteases from this snake were also characterized (Jia et al., 2009; Jia and Perez, 2009, 2010).

In this study, cDNAs of RVV-V and two novel serine proteases from Russell's viper (D. r. siamensis) venom were cloned and characterized. To extend the knowledge of serine proteases, two proteases, plasminogen activator and protein C activator, from cDNA library of venom glands of A. p. leucostoma were also included in this study since their complete open reading frames were available instead of partial sequence. In addition, their functions were predictable by comparison with their homologues. The serine protease cDNAs in this study were analyzed by phylogenetic approach to understand the structure and function of snake venom proteins, as well as providing information for future studies.

2. Material and methods

2.1. cDNA libraries

The Russell's viper venom gland mRNA was obtained from previous study (Nuchprayoon et al., 2001). The cDNA of the APL-PA and APL-C was obtained from the cDNA library of A. p. leucostoma (Jia et al., 2008).

2.2. 5′ Rapid amplification of cDNA ends (5′ RACE)

To obtain the 5′ end nucleotide sequence of RVV-V, the 5′-end amplification of cDNA was performed using the 5′ RACE System kit (Invitrogen, CA, USA) according to the manufacturer's manual. Three gene specific primers (GSP) were designed from RVV cDNA library: 5′-CATTACAGATGAGCGGTCC-3′ (GSP1), 5′-CCGTGACATGTATCTCTGCCTCC-3′ (GSP2) and 5′-CCATGGATAAAGTGGTTCACACC-3′ (GSP3). The single strand cDNAs were reverse transcribed from 5 μg of Russell's viper gland mRNA using GSP1. After RNA digestion and purification, the cDNAs were tailed with polycytosine (poly-C) at the 5′ terminal and used in polymerase chain reaction (PCR) with kit-provided primer and GSP2. Nested amplification was performed using the diluted primary PCR product and GSP3. The amplified cDNA fragments were electrophoresed by 1% agarose gel and purified with QIAquick Gel Extraction Kit (Qiagen, CA, USA). Ligation of the PCR products into pGEM-T easy vector (Promega, MA, USA) was performed.

2.3. Plasmid preparation and DNA sequencing

To prepare plasmids for DNA sequencing, individual colonies were randomly picked from the Luria-Bertani (LB) with ampicilin plates and inoculated overnight at 37 °C. Plasmid DNAs were purified from the overnight cultures using the Sigma Plasmid Miniprep Kit (Sigma, CA, USA) according to the manufacture's instruction manual. Extracted plasmid DNAs were sent to Purdue Genomics Core Facility for automated sequencing for both directions using BigDye3.1 on an Applied Biosystems 9700 thermal cycler.

2.4. RT-PCR

To amplify novel serine proteases cDNA from Russell's viper glands, a forward primer (VSP-F: 5′-CCGCTTGGGTTATCTGATTAG-3′) and a reverse primer (VSP-R: 5′-GCACCTCACCCTAAAACAG-3′) were designed from the conserved sequences of the 5′- and 3′-untranslated region (UTR) from the cDNAs encoding RVV-V and snake venom serine preteinases from Genbank database. 5 μg of the Russell's viper gland mRNA was used in RT- PCR using SuperScript™ One-Step RT-PCR kit (Invitrogen, CA, USA). cDNA was synthesized by reverse transcription at 45 °C for 45 min. PCR amplification consisting of 35 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min followed by final extension at 72 °C for 7 min was performed. RT-PCR products were electrophoresed by 1% agarose gel and purified with QIA-quick Gel Extraction Kit (Qiagen, CA, USA). The purified RT-PCR products were subsequently cloned into the pGEM-T easy plasmid (Promega, MA, USA).

2.5. Sequence analysis

The open reading frame (ORF) in cDNA sequences was analyzed using BioEdit Sequence Alignment Editor version 7.0.9 (Hall, 1999). Nucleotide and amino acid sequences of 36 snake venom serine proteases, as shown in Table 1, were obtained from NCBI databases for phylogenetic analysis. Because a number of snake venom serine proteases, which have different functions, have been reported, the chosen serine proteases were selected regarding to their functions.

Table 1.

Snake venom serine proteases from GenBank database used in this study.

Serine proteases GenBank accession number
 Species
Protein DNA
VLFVA (factor V activator) Q9PT41 AF163973.1 Macrovipera lebetina
TLG2A (serine proteinase 2A) O13060 D67082.1 Trimeresurus gramineus
Serine protease KN4 homolog Q71QJ4 AF395763.1 Viridovipera stejnegeri
TjsvSPH B0ZT25 EU400543.1 Trimeresurus jerdonii
TLF2 O13057 D67079.1 Trimeresurus flavoviridis
Flavoxobin P05620 D67078.1 Trimeresurus flavoviridis
Catroxase II Q8QHK2 AF227154.1 Crotalus atrox
Haly-PA Q9YGJ8 AF017737.1 Gloydius blomhoffi brevicaudus
Serine beta-fibrinogenase (VLBF) Q8JH62 – Macrovipera lebetina
TLG3 (Venom serine proteinase 3) O13063 D67085.1 Trimeresurus gramineus
Salmobin O73800 AF056033.1 Gloydius halys
TLF3 O13058 D67080.1 Trimeresurus flavoviridis
LV-PA Q27J47 DQ396477.1 Lachesis muta muta
TSV-PA Q91516 U21903.1 Viridovipera stejnegeri
Acutobin Q9I8X2 AF159057.1 Deinagkistrodon acutus
TLG2C O13062 D67084.1 Trimeresurus gramineus
PTLE1 Q802F0 AY225505.1 Gloydius halys
CPI-enzyme 2 (capillary permeability-increasing enzyme-2) O42207 AF018568.1 Gloydius ussuriensis
KN-BJ 2 O13069 AB004067.1 Bothrops jararaca
Dav-KN Q9I8X0 AF159059.1 Deinagkistrodon acutus
PA-BJ P81824 – Bothrops jararaca
Serine alpha-fibrinogenase (VLAF) Q8JH85 AF528193.1 Macrovipera lebetina
ACC-C (protein C activator) P09872 – Agkistrodon contortrix contortrix
Dav-X Q9I8W9 AF159060.1 Deinagkistrodon acutus
Elegaxobin I P84788 – Protobothrops elegans
Ancrod P47797 L07308.1 Calloselasma rhodostoma
Contortrixobin P82981 – Agkistrodon contortrix contortrix
LM-TL P33589 – Lachesis muta muta
Acutin Q9YGS1 AF089847.1 Deinagkistrodon acutus
Bothrombin P81661 – Bothrops jararaca
Mucrofibrase Q91507 X83221.1 Protobothrops mucrosquamatus
Batroxobin AAA48552 J02684.1 Bothrops atrox
KR-E-1 P86171 Gloydius ussuriensis
Ohs1 ABN72544 EF080837 Ophiophagus hannah
Nasp ABN72541.1 EF080834 Naja atra
BmSP ABN72545 EF080838 Bungarus multicinctus
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Putative N-linked and O-linked glycosylation sites were predicted by NetNGlyc 1.0 and NetOGlyc 3.1 Server, respectively (Gupta et al., in preparation; Julenius et al., 2005). Multiple sequence alignment of amino acid sequences was performed using Clustal X version 2.0.11 (Larkin et al., 2007) and GeneDoc version 2.7 (Nicholas and Nicholas, 1997). The aligned amino acid sequences were edited by BioEdit (Hall, 1999). The nucleotide sequences alignment was buffered according to the amino acid sequence alignment using DAMBE version 5.1.5 (Xia and Xie, 2001). Phylogenetic analysis of the aligned amino acid or nucleotide sequences was performed with PHYLIP package version 3.69 (Felsenstein, 2010). Evolutionary distances were determined with F84 model for the aligned nucleotide sequences or JTT model for the aligned amino acid sequences. F84 model incorporates different rates of transition and transversion, and also allows for different frequencies of the four nucleotides. JTT model utilizes amino acid substitution matrices based on large protein databases. The distance matrices were subsequently used to construct the phylogenetic trees by the neighbor-joining method. Bootstrap estimates analysis was conducted in 1000 replicates. Numbers of non-synonymous substitutions per non-synonymous site (KA) and numbers of synonymous substitutions per synonymous site (KS) in protein coding regions, and numbers of nucleotide substitutions per site (KN) for the 3′ UTR of snake venom serine protease cDNAs were calculated by MEGA4 program using Nei and Gojobori (1986) method (Tamura et al., 2007). The one-tailed t-test was used for determine the signifcance of diferences between KA and KS for test of positive selection by the MEGA4 program.

3. Result and discussion

3.1. RVV-Vγ cDNA analysis

5′-RACE using RVV-V specific primers amplified 700 bp DNA fragments, which represented the 5′-untranslated region (UTR), pre- and pro-protein- encoding regions and N-terminal-encoding region of RVV-V cDNA. The complete sequence of the RVV-V cDNA (GenBank: HQ270463) was obtained by combination of the overlapping sequences between the 5′-RACE product and the cDNA library.

The RVV-V cDNA contained a start codon for methionine (ATG), stop codon (TGA), polyadenylation signal (AATAAA) and poly (A) tail (Fig. 1). The 5′- and the 3′-UTR were 192 and 606 base pairs, respectively. The 780 bp open reading frame (ORF) encoded signal peptides (54 base pairs, 18 amino acids), activation peptides (18 basepairs, 6 amino acids) and mature enzyme coding region (708 base pairs, 236 amino acids). The pre-pro peptide indicated that the RVV-V is expressed and secreted to the venom glands as a zymogen. The translated signal peptide sequence of the RVV-V was rich in hydrophobic amino acid residues which were highly conserved among snake venom serine proteases (Fig. 2). The catalytic triad, His57-Asp102-Ser195 (numbering based on that of trypsin), 12 conserved cysteine and one putative N-glycosylation site were presented (Figs. 1 and 2). Using Blastx and BioEdit program revealed that the deduced amino acid sequence of the mature RVV-V in this study was 99% identical with the RVV-Vγ since there was one amino acid substitution (H192K) (Fig. 2) (Tokunaga et al., 1988). This represented the genetic polymorphism between individual snakes from the same subspecies (siamensis).

Fig. 1.

Fig. 1

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Complete cDNA sequence of factor V activator (RVV-V) from Russell's viper cDNA. His-Asp-Ser residues of catalytic triad were boxed. Twelve conserved cysteines were bolded. The single underlined sequences indicated the signal and activation peptides. Polyadenylation signal was double underlined.

Fig. 2.

Fig. 2

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Multiple sequence alignment of amino acid sequences of snake venom serine proteinases. The alignment was performed by Clustal W program. Arrows indicate the boundaries of signal peptide, activation (pro-) peptide and mature protein. Catalytic triad residues were shown with solid diamond. Asterisks indicated the 12 conserved cysteins. Abbreviations: RVV-V: factor V activator from Russell's viper (from this study); RVV-Vg: RVV-V gamma; VLFVA: factor V activator from Macrovipera lebetina; RVAF: serine alpha-fibrinoginase from Russell's viper; VLAF: serine alpha-fibrinogenase from M. lebetina; RVBF: serine beta-fibrinoginase from Russell's viper; VLBF: serine beta-fibrinogenase from M. lebetina; APL-PA: plasminogen activator from A. p. leucostoma; TSV-PA: plasminogen activator from Viridovipera stejnegeri; APL-C: protein C activator from A. p. leucostoma; ACC-C: protein C activator from Agkistrodon contortrix contortrix; CPI-2: Capillary permeability-increasing enzyme 2 from Gloydius ussuriensis; Batroxobin from Bothrops atrox; Flavoxobin from Trimeresurus flavoviridis; Ohs1: serine proteinase from Ophiophagus hannah.

Since RVV-Vγ cDNA has not been reported, the cDNA of the RVV-V from this study was most identical with the cDNA of the factor V activator from M. lebetina (VLFVA) (94% identity) (Siigur et al., 1999, 1998). The identity of protein coding sequence, 5′- and 3′-UTR between the RVV-Vγ and the VLFVA cDNAs were 92, 95 and 97%, respectively. In the protein coding region, the identity was highest in the activation peptide-coding region (100%). The signal peptide- and the mature protein coding regions were 98% and 91% identical, respectively, compared with 94 and 83% identity in the translated amino acids.

Number of nucleotide substitutions per synonymous site (KS) for the mature protein coding region and numbers of nucleotide substitutions per site (KN) for the 3′ UTR of the RVV-Vγ cDNA were calculated in comparison with other snake venom serine proteases (Table 2). The KN values for the UTR were less than the KS values in all pairs, including the pair of the closely related RVV-Vγ and VLFVA. The KN/KS < 1 indicated that the mature protein coding region of the RVV-V cDNA evolved more rapidly than the conserved UTR. This observation was also found in serine proteases and phospholipase A2s from T. flavoviridis and Trimeresurus gramineus, and serine proteases from Deinagkistrodon actus (Deshimaru et al., 1996; Nakashima et al., 1995; Nikandrov et al., 2005; Tani et al., 2002).

Table 2.

KN and KS values of RVV-V cDNA compared with snake venom serine proteases from GenBank data base.

cDNA pairs KS (Mature) KN (3′UTR)
RVV-V vs. VLFVAa 0.085 0.012
RVV-V vs. TSV-PAb 0.267 0.078
RVV-V vs. CPI-2c 0.299 0.078
RVV-V vs. VLAFd 0.269 0.111
RVV-V vs. batroxobine 0.305 0.102
RVV-V vs. Flavoxobinf 0.308 0.089
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a

VLFVA: factor V activator from Macrovipera lebetina.

b

TSV-PA: Plasminogen activator from Viridovipera stejnegeri.

c

CPI-2: capillary permeability-increasing enzyme-2 from Gloydius ussuriensis.

d

VLAF: serine alpha-fibrinogenase from Macrovipera lebetina.

e

Batroxobin from Bothrops atrox.

f

Flavoxobin from Trimeresurus flavoviridis.

3.2. Amplification of novel serine proteases cDNA

According to the high conservation of the 5′- and 3′ UTR, RT-PCR was performed using the primers designed from the consensus regions from the 5′- and 3′ UTR of the RVV-Vγ and snake venom serine protease cDNAs. A RT-PCR product of approximately 800 bp was cloned and sequenced. To avoid mutation from PCR, several clones were sequenced. Similarly to the study of Siigur et al. (1999), only the RVV-Vγ cDNA was amplified while the alpha- and beta-like isoforms of RVV-V were not observed (Siigur et al.,1999; Tokunaga et al., 1988). Besides RVV-Vγ, two serine protease cDNAs were obtained, designed as Russell's viper alpha-fibrinogenase homolog (RVAF) (GenBank: HQ270464) and Russell's viper beta-fibrinogenase homolog (RVBF) (GenBank: HQ270465). Deduced amino acid sequence of RVAFand RVBF had highest identity with the serine alpha-fibrinogenase precursor (VLAF) (80%) and the serine beta-fibrinogenase precursor (VLBF) (85%), respectively, from M. lebetina (Siigur et al., 2003).

3.3. Sequence analysis of RVAF, RVBF, APL-PA and APL-C

The predicted pI values and molecular weight before glycosylation as well as putative glycosylation sites were predicted. RVAF had pI value of 9.2 and molecular weight of 25.8 kDa. OFR of the RVAF, encoded 258 amino acids, shared 80% amino acid identity and 90% nucleotide identity with VLAF. One putative N-linked glycosylation site (NXS/T) at the Asn44 and two O-linked glycosylation sites at the positionThr255 and Ser258 contributed to posttranslational modification and altered the molecular weight and pI (Siigur et al., 2003). The deduced 256 amino acid sequence of the RVBF was 85% identical with the VLBF and nucleotide sequence of both genes shared 92% identity. The RVBF had pI of 6.7, molecular weight of 25.40 kDa, 2 N-linked glycosylation sites (Asn78 and Asn101) and 1 O-linked glycosylation site (Thr 253). VLAF and VLBF hydrolyzed α- and β-chain of fibrinogen, respectively, inhibiting clotting function of fibrinogen by thrombin (Samel et al., 2002). The RVBF was previously found in the proteomic analysis of Russell's viper venom from Myanmar (Risch et al., 2009). The observed molecular weight of RVBF was approximately 60 kDa, which indicated the role of heavy glycosylation (Risch et al., 2009). To our knowledge, this is the first report of the presence of the alpha-fibrinogenase in Russell's viper venom. Expressions of the homologous factor V activators, fibrinogenase enzymes, as well as the factor X activators, the P-IV metal-loproteases, in the Russell's viper and M. lebetina venom gland indicated evolutionary relation between both species despite geographic difference (Chen et al., 2008; Siigur et al., 1999, 2004).

In addition to Russell's viper proteases, two serine proteases, APL-PA (GenBank: HQ270466) and APL-C (Gen-Bank: HQ270467), were obtained from venom gland cDNA library of A. p. leucostoma. APL-PA was a plasminogen activator homolog which showed 82% identity with TSV-PA, a plasminogen activator from V. stejnegeri (Zhang et al., 1995). APL-C was a protein C activator homolog, which was 88% identical with the protein C activator from A. c. contortrix (ACC-C) (Murakami and Arni, 2005).

Without glycosylation, APL-PA has pI value of 5.6 and molecular weight of 25.4 kDa. APL-PA contained only two O-linked glycosylation sites (Thr149 and Thr 255). Identity between APL-PA AND TSV-PA was 82% in amino acid sequence and 89% in nucleotide sequence. TSV-PA generated two-chain plasmin, a key enzyme in fibrinolysis, form plasminogen by selectively cleavage of plasminogen at the peptide bond Arg561-Val562, the same peptide bond cleaved by u-PA and t-PA (Zhang et al., 1997, 1995).

APL-C had predicted pI of 8.4 and molecular weight of 25.10 kDa. Three predicted N-linked glycosylation sites (Asn45, Asn 102 and Asn 153) and one O-linked glycosylation site (Thr148) were found. Amino acid sequences of ACC-L and ACC-C share 88% identity. However, nucleotide sequence is not available for the ACC-C since the amino acid sequence was obtained from direct protein sequencing (Kisiel et al., 1987; McMullen et al., 1989). ACC-C activated protein C by selectively cleavage of the heavy chain of protein C, generating activated protein C with amidolytic activity (Kisiel et al., 1987). ACC-C-activated protein C subsequently cleaved blood coagulation factors Va and VIIIa, leading to the prolongation of the activated partial thromboplastin time (Stocker et al., 1987).

3.4. Multiple sequence alignment

Multiple alignment of amino acid sequences of snake venom serine proteases was performed (Fig. 2). The hydrophobic residue-rich signal peptides, residue 1–18, were highly conserved among snake venom serine proteases. The catalytic triad of snake venom serine proteases, His57-Asp102-Ser195, and the surrounding regions were strongly conserved. Such His-Asp-Ser residues are strongly conserved in serine proteases involved in a wide variety of physiological processes, including blood coagulation, fibrinolysis and immune response (Hedstrom, 2002a, b). Substitution of His-57 by Arg-57 was found in the catalytic triad of TjsvSPH, a snake venom serine protease homolog (svSPH) protein from Trimeresurus jerdonii, which lacked arginine esterase, proteolytic activity and coagulant activity (Wu et al., 2008). All Viperidae proteases in the alignment contained 12 highly conserved cysteines, which formed 6 disulfide bridges: Cys7–Cys141, Cys28–Cys44, Cys120–Cys188, Cys152–Cys167, Cys178–Cys203 and Cys76–Cys234 (Serrano and Maroun, 2005; Siigur et al., 1999; Vitorino-Cardoso et al., 2006). Additionally, two additional cysteine residues, Cys75 and Cys82, was found in Ohs1, as well as in NaSP and BmSP, two Elapidae serine proteases from Naja atra and Bungarus multicinctus, respectively (Jin et al., 2007).

The amino acid sequence at residue 81–84, which is located in solvent-exposed loop (Zhang et al., 1997), represented activity-related characteristic (Fig. 2). The sequence FPNG found only in the RVV-V and the VLFVA may contribute to the factor V activation (Siigur et al., 1999). Both TSV-PA and APL-PA contained the charged peptide KDDE (residue 95–98). Substitution of D97V in TSV-PA resulted in 125-fold decrease in plasminogen activation, which might be the effect of the proximity of the DDE loop to the catalytic site (Zhang et al., 1997). Only the polar residue Ser81 was replaced by another polar amino acid, Asn81, in RVBF. The peptide NDTI was observed only in ACC-C and APL-C.

3.5. Phylogenetic analysis

The mature proteins-encoding nucleotide sequences (position 265–982 in Fig. 1), and mature amino acid sequences (position 25–260 in Fig. 2) of RVV-Vγ were used for construction of neighbor-joining phylogenetic tree (Fig. 3A) in comparison with that of RVAF, RVBF, APL-PA, APLC, as well as other 36 serine proteases from Viperidae and Elapid snakes, in which 32 amino acid sequences and 27 nucleotide sequences are available (Table 1). The sequence of human trypsin was used as outgroup. Only the bootstrap values of higher than 0.50 were shown on the tree. The analyzed serine proteases were clustered in the tree based on their functions: factor V activator, coagulant enzymes, plasminogen activators, kinin-releasing enzymes, inactive serine proteases, fibrinogenase enzymes, capillary permeability-increasing enzymes, protein C activators and Elapidae serine proteases. However, boostrap values at the base of functional clades were not significant for all nodes, such as plaminogen activators, kinin and coagulant. This indicated that the alternative hypothesis, in which clustering of such sequences was not based on their functions, could not be rejected. The serine proteases from Elapidae snakes were clustered separately from that from Viperidae snakes since early evolutionary speciation of colubroid snakes (Fry et al., 2006). The cluster of RVV-Vγ and VLFVA isolated from other Viperidae proteases with bootstrap value of 100%, suggesting that the factor V activator genes have evolved in independent way. The fibrinogenase cluster contained RVAF, RVBF, VLAF and VLBF with bootstrap value of 99%. Alpha and beta serine fibrinogenases share high similarity APL-PA was grouped in a cluster with other snake venom plasminogen activators, which had bootstrap value of 68%. APL-C and ACCC were uniquely clustered with bootstrap value of 100% since only ACC-C is the APL-C homologous protein available in GenBank data base. Parallel evolution of factor V activators and fibrinogenases enzymes indicated close relationship between Russell's viper and M. lebetina despite of geographic difference. The amino acid NJ tree for the snake venom serine proteases was confirmed by the DNA NJ tree, in which most serine proteases were grouped according to their functions (Fig. 3B). However, difference in tree topology between two trees was observed since some clusters and branches, such as the AC-C and batroxobin, were differently clustered. Since both trees were constructed based on genetic distance, the tree topology difference was according to different pattern in distance values between the translated amino acid and DNA sequences.

Fig. 3.

Fig. 3

Fig. 3

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Phylogenetic tree of snake venom serine proteinases, obtained from neighbor-joining analysis. The proteins analyzed in this study were underlined. Bootstrap values (1000 replicates) above 50% were shown at the node. The scale bar indicated substitutions per site. A) NJ tree based on amino acid sequence; B) NJ tree based on DNA sequence.

3.6. Analysis of KA/KS values

To investigate positive selection, ratio of the number of nucleotide substitutions per non-synonymous site (KA) and number of nucleotide substitutions per synonymous site (KS) of RVV-Vγ, RVAF, RVBF, APL-PA and APL-C were computed according to Nei and Gojobori method (Nei and Gojobori, 1986), as shown in Table 3. KA values indicate nucleotide substitutions that alter the corresponding amino acid compositions. KS values indicate nucleotide substitutions that do not alter the corresponding amino acid compositions. KA/KS values of the investigated genes compared with other Viperidae proteases were close to or greater than one. This indicated that accelerated evolution in the protein coding regions could be observed in serine proteases from Russell's viper and A. p. leucostoma venoms. Rapid evolution was also demonstrated in snake venom serine proteases from T. flavoviridis, T. gramineus and D. actus (Deshimaru et al., 1996; Nikandrov et al., 2005), as well as in snake venom metalloproteinase and disintegrins (Casewell et al., 2011; Soto et al., 2007), phospholipase A2 (Lynch, 2007; Soto et al., 2007) and Kunitz-BPTI protein (Zupunski et al., 2003). In contrast, KA/KS values of the Elapidae proteases were close to one, indicating that Elapidae serine proteases have evolved under neutral selection manner (Table 3). Slower evolutionary rates may consequently result in less diversity of Elapidae serine proteases than that of Viperidae, since a number of Viperidae serine proteases have been reported (Serrano and Maroun, 2005). It has been proposed that evolution of snake venom toxin families is a result of gene recruitment, in which alternate genes emerged from ancestor gene by altered gene expression, followed by mutations, gene duplication, and functional constraint (Fry and Wuster, 2004; Soto et al., 2007). According to this hypothesis, serine proteases were recruited before Colubroid radiation, and accelerated evolution has subsequently taken place especially in Viperidae serine proteases to gain a variety of functions, while some characteristics, such as the catalytic triad and disulfide bridges, have been retained.

Table 3.

KAa and KSb values of snake venom serine proteases. Only the pairs of Viperidae proteases with ratio higher than 1 were shown.

cDNA pairs KA KS KA/KS p value
RVV-V vs. RVAF 0.265 0.172 1.541 0.005
RVV-V vs. RVBF 0.234 0.157 1.490 0.013
RVV-V vs. VLAF 0.260 0.159 1.635 0.001
RVV-V vs. TSV-PA 0.228 0.165 1.382 0.034
RVV-V vs. KN-BJ2 0.249 0.179 1.391 0.024
RVV-V vs. CPI-2 0.253 0.180 1.406 0.020
RVAF vs. ACL-PA 0.222 0.149 1.490 0.016
RVAF vs. VL-FVA 0.244 0.166 1.470 0.012
RVAF vs. Flavoxobin 0.242 0.162 1.494 0.011
RVAF vs. Haly-PA 0.226 0.147 1.537 0.007
RVAF vs. RVS-2 vs. TLG3 0.195 0.140 1.393 0.026
RVAF vs. Acutobin 0.263 0.201 1.308 0.038
RVAF vs. Dav-KN 0.227 0.159 1.428 0.026
RVBF vs. ACL-PA 0.201 0.139 1.446 0.026
RVBF vs. VL-FVA 0.218 0.154 1.416 0.034
RVBF vs. Flavoxobin 0.225 0.165 1.364 0.039
RVBF vs. Haly-PA 0.199 0.127 1.567 0.010
RVBF vs. TLF3 0.153 0.102 1.500 0.038
RVBF vs. Acutobin 0.227 0.162 1.401 0.031
RVBF vs. TLG2C 0.216 0.153 1.412 0.023
ACL-PA vs. salmobin 0.184 0.119 1.546 0.008
ACL-PA vs. TLG2C 0.18 0.129 1.395 0.044
ACL-PA vs. PTLE1 0.183 0.123 1.488 0.021
ACL-PA vs. VLAF 0.222 0.150 1.480 0.014
ACL-PA vs. Dav-X 0.267 0.160 1.669 0.000
ACL-PA vs. Ancrod 0.276 0.194 1.423 0.011
ACL-PC vs. TLF3 0.162 0.108 1.500 0.022
ACL-PC vs. TSV-PA 0.195 0.137 1.423 0.029
ACL-PC vs. Acutobin 0.209 0.133 1.571 0.011
ACL-PC vs. KN-BJ2 0.182 0.110 1.655 0.004
OHS1 vs. NaSP 0.094 0.115 0.817 1.000
OHS1 vs. BmSP 0.092 0.109 0.844 1.000
NaSP vs. BmSP 0.002 0.002 0.100 1.000
OHS1 vs. RVV-V 0.257 0.277 0.928 1.000
NaSP vs. RVV-V 0.256 0.298 0.859 1.000
BmSP vs. RVV-V 0.255 0.288 0.885 1.000
Open in a new tab
a

KA: Numbers of non-synonymous substitutions per non-synonymous site.

b

KS: Number of nucleotide substitutions per synonymous site.

4. Conclusion

Five snake venom serine protease cDNAs from Russell's viper and M. lebetina were characterized. Two novel cDNAs were obtained from Rusell's viper gland transcripts using conserved primers. All five proteases contained the characteristics of snake venom serine proteases: highly conserved catalytic triad and 12 cysteine, which form six disulfide bonds. Accelerated evolution was also observed. Phylogenetic tree approach can be performed to classify these proteases according to their functions. Characterization of these proteases provides information for further studies, including specific activity assays and site-direct mutagenesis. These well-characterized proteases may have potential for medical applications such as development of diagnostic reagent or therapeutic agents.

Acknowledgments

This work was funded by the Royal Golden Jubilee (RGJ) Ph.D. scholarship Contract No. PHD/0061/2546, the National Research Council of Thailand, the National Natural Toxins Research Center at Texas A&M University-Kingsville and NIH/Viper Resource Center grant #5 P40 RR018300-07.

Abrreviations

pI

isoelectric point

RVV

Russell's viper venom

RVV-V

Russell's viper venom-factor V activator

KN

numbers of nucleotide substitutions per site

KS

number of nucleotide substitutions per synonymous site

KA

numbers of non-synonymous substitutions per non-synonymous site

Footnotes

Conflict of interest The authors declare that there are no conflicts of interest.

Data accessibility The multiple alignments used for construction of the phylogenetic trees were deposited at Dryad: doi:10.5061/dryad.tv8ph.

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