vipers Trimeresurus puniceus and Trimeresurus borneensis
Ying-Ming Wang, Hao-Fan Peng and Inn-Ho TsaiInstitute of Biological Chemistry, Academia Sinica and Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
For a long time, the genus Trimeresurus (sensu lateral) has been known to consist of over 40 species of Asian pit vipers. It is now divided into four recognized gen-era: Trimeresurus, Ovophis, Protobothrops and Trop-idolaemus[1]. The arboreal Trimeresurus (sensu stricto) are indigenous to south and south-eastern Asia. It should be noted that data from morphological and mtDNA analyses suggest that Trimeresurus (sensu stricto) is possibly derived from more than one ances-tral species and should be considered as a polyphyletic group [1–3]. However, their venom components have not been well studied except for those of the green
bamboo vipers Trimeresurus stejnegeri [4]. Distin-guished by a yellow–brown skin color, Trimeresurus puniceus and Trimeresurus borneensis only inhabit Sumantra, Java and adjacent areas [5]. Previous phylo-genetic studies suggest that both species are primitive and closely related to several Trimeresurus species in the Indian subcontinent [1].
Snake venoms are characteristic, with specific phar-macological activities and bioavailability, and thus have potential for medical applications. In addition, investigating the diversity of venom proteins may help us to understand snake systematics and their venom Keywords
phospholipase A2; phylogenetic analysis; snake venom; Trimeresurus borneensis; Trimeresurus puniceus
Correspondence
I.-H. Tsai, Institute of Biological Chemistry, Academia Sinica and Institute of
Biochemical Sciences, National Taiwan University, PO Box 23-106, Taipei, Taiwan 10798
Fax: +886 223635038
E-mail: bc201@gate.sinica.edu.tw
Note
Novel cDNA sequences encoding PLA2s have been submitted to EMBL Databank and are available under accession numbers: AY355171 to AY355175 for K49a, Tpu-K49b, Tpu-G6D49, Tpu-E6a and Tpu-E6b; AY355177 to AY355179 for Tbo-K49, Tbo-E6 and Tbo-G6D49, respectively.
(Received 3 March 2005, revised 25 March 2005, accepted 11 April 2005)
doi:10.1111/j.1742-4658.2005.04715.x
To explore the venom diversity of Asian pit vipers, we investigated the structure and function of venom phospholipase A2 (PLA2) derived from
two primitive tree vipers Trimeresurus puniceus and Trimeresurus borneen-sis. We purified six novel PLA2s from T. puniceus venom and another three
from T. borneensis venom. All cDNAs encoding these PLA2s except one
were cloned, and the molecular masses and N-terminal sequences of the purified enzymes closely matched those predicted from the cDNA. Three contain K49 and lack a disulfide bond at C61–C91, in contrast with the D49-containing PLA2s in both venom species. They are less thermally
stable than other K49-PLA2s which contain seven disulfide bonds, as
indi-cated by a decrease of 8.8C in the melting temperature measured by CD spectroscopy. The M110D mutation in one of the K49-PLA2s apparently
reduced its edematous potency. A phylogenetic tree based on the amino-acid sequences of 17 K49-PLA2s from Asian pit viper venoms illustrates
close relationships among the Trimeresurus species and intergeneric segre-gations. Basic D49-PLA2s with a unique Gly6 substitution were also
puri-fied from both venoms. They showed edema-inducing and anticoagulating activities. It is notable that acidic PLA2s from both venoms inhibited blood
coagulation rather than platelet aggregation, and this inhibition was only partially dependent on enzyme activity. These results contribute to our understanding of the evolution of Trimeresurus pit vipers and the struc-ture–function relationships between various subtypes of crotalid venom PLA2.
Abbreviations
PLA2, phospholipase A2; Tbo, Trimeresurus borneensis; Tpu, Trimeresurus puniceus; Tst, Trimeresurus stejnegeri; APTT, activated partial thromboplastin time.
protein evolution. Phospholipase A2 (PLA2; EC
3.1.1.4) is one of the most widely studied snake venom proteins because of its abundance, small size, and structural stability. The amino-acid sequences of 290 snake venom PLA2s have been determined, and many
of their 3D structures resolved [6–8]. PLA2s of pit
viper venoms have evolved into several subtypes, each playing distinct functional roles such as platelet aggre-gation inhibitor [9], neurotoxin [10], anticoagulant [11], and myotoxin [12].
In this study, we purified the PLA2s from T.
puni-ceus and T. borneensis venoms, cloned the cDNAs, and solved their full amino-acid sequences. The func-tions of these purified PLA2s and the effects of a
missing disulfide bond at C61–C91 were investigated. On the basis of amino-acid sequences of orthologous venom PLA2s, we built a phylogenetic tree to study
evolutionary relationships among the Asian pit vipers.
Results
Purification and characterization of venom PLA2
The results of gel filtration indicated that T. puniceus (Tpu) venom contained abundant high molecular mass components, whereas T. borneensis (Tbo) venom was relatively rich in smaller proteins (< 20 kDa) (Fig. 1). By RP-HPLC of fraction II (Fig. 1, corresponding to 14 2 kDa), we purified six PLA2isoforms from Tpu
venom (Fig. 2A) and two from Tbo venom (Fig. 2B). They were designated K49-PLA2, G6D49-PLA2 and
E6-PLA2 according to substitutions at residue 6 or
49 as previously suggested [4,13]. The PLA2s were
eluted in the order K49-PLA2s, weakly basic
G6D49-PLA2, and finally acidic E6D49-PLA2s. Unexpectedly,
G6D49-PLA2s of both Tpu and Tbo venom were eluted
in fraction III (Figs 1 and 2C), suggesting their affinity for the column. Unlike previous experience with pit viper venoms [4,14], we did not find dimeric PLA2s in
this two venom species.
The protein content of each PLA2 in the crude
venom (%, w⁄ w) was estimated from the relative UV absorbance during the two chromatographic steps. Molecular mass and N-terminal sequence of the puri-fied PLA2s were determined. The results are
summar-ized along with the predicted pI values in Table 1. The total protein content of Tpu and Tbo venom PLA2s
was 19% and 27%, respectively. The activities of the purified enzymes with micellar substrates are shown in Table 2. Consistent with previous reports [4,12], the catalytic activity of K49-PLA2s was hardly
detectable.
Cloning and full sequencing of the PLA2s
Cloning of venom PLA2s has been facilitated by PCR
using properly designed primers and conditions [4,15]. Five and three distinct cDNAs encoding the venom PLA2s from Tpu and Tbo, respectively, were identified
after more than 60 cDNA clones for each species were sequenced. All the deduced PLA2 sequences consist of
a signal peptide of 16 amino-acid residues followed by an enzyme domain of 122 residues. Assuming that all the conserved cysteine residues in PLA2 form disulfide
bonds, the mass and pI value of each cloned PLA2
were calculated. Exact matches were found for eight PLA2s purified from both venoms (Table 1). In
addi-tion, complete amino-acid sequences of the basic (Fig. 3A,B) and acidic (Fig. 3C) PLA2 were aligned
with closely related or similar sequences, respectively. However, we failed to clone Tpu-E6c in spite of a great number of clones selected for sequencing or Fig. 1. Gel filtration of the crude venom. Dissolved venom of T. pu-niceus or T. borneensis was loaded on to a Superdex G75 (HR10⁄ 30) column on a FPLC system. The elution was carried out with equilibration buffer, 0.1M ammonium acetate (pH 6.4), at a flow rate of 1.0 mLÆmin)1. Fractions I–III (shown by bars) were pooled separately.
using alternative primers based on its N-terminal resi-dues 1–9 (AAYCTNCTNCARTTYGARATGATGAT) or residues 5–11 (TTYGARATGATGATHYTNAA). We therefore used peptide mass spectra fingerprinting to analyze the peptides derived from trypsin digestion of reduced and alkylated Tpu-E6c. By the rationale that more acidic PLA2 was eluted later in the
RP-HPLC, a hypothetical sequence for Tpu-E6c was deduced from the peptide mass spectra fingerprint data by assuming the presence of D70–E71, which hampers the cleavage at the K69–D70 bond in Tpu-E6c
(Table 3). The calculated mass (13 794.39 Da) of the hypothetical sequence for Tpu-E6c in Fig. 3C matched that obtained from ESI-MS of the purified protein (13 792.8 ± 4.1 Da, Table 1).
CD and stability of K49-PLA2
The CD spectra of Tpu-K49a and Tst-K49a [4] at 27C were very similar (Fig. 4A). Based on computer analyses of the two spectra, the calculated contents of a-helices, b-sheets and b-turns were 34%, 18% and 22%, respectively. The molar ellipticities at 222 nm, which reflect the helical contents of the proteins, were also measured at various temperatures between 20C and 80C to evaluate the thermal stabilities. One melt-ing temperature was observed for each protein, i.e. 54.3C for Tpu-K49a and 63.1 C for Tst-K49 (Fig. 4B).
Functional studies
Local edema was obvious on the foot a few hours after injection of the basic venom PLA2s (Fig. 5). The
ede-matous potencies of Tpu-K49a and Tbo-K49 were similar to that of the CTs-K49c isoform [4], whereas Tpu-K49b was 50% less potent. Tpu-G6D49 was also capable of inducing fast and sustained local edema. The inhibition of ADP-induced platelet aggre-gation by acidic E6-PLA2s or the weak basic G6-PLA2
from both venoms was also studied using platelet rich plasma prepared from human and rabbit blood. Inhibi-tion was not large: 15–25% at 5–10 lg PLA2 per ml
platelet-rich plasma (data not shown).
Significantly, some of the E6-PLA2s and
G6D49-PLA2s prolonged the blood coagulation time in a
dose-dependent manner (Table 4). A strongly anticoag-ulating R6-PLA2 purified from Protobothrops
tokaren-sisvenom [13] served as a positive control. During the measurement of activated partial thromboplastin time (APTT), the anticoagulating effect of Tpu-E6a was not affected by increasing the preincubation time from 1 min to 10 min. We then used His48-methylated and inactivated Tpu-E6a to study the dependence of the anticoagulation effect on enzyme activity. After 1 h and 4 h of treatment with the affinity label, the enzy-matic activity remaining was 14% and 5%, respect-ively. After 6 h of treatment and with < 4% of the original hydrolytic activity, the methylated PLA2
retained 35% of the original anticoagulation activity. Native Tpu-G6D49 and Tbo-G6D49 also prolonged the blood coagulation time (Table 4). The former was twice as potent as the latter although their enzymatic activities were about the same.
Fig. 2. Purification of PLA2s by RP-HPLC. Lyophilized pooled frac-tions II and III from gel filtration were redissolved and fractionated on a C8-Vydac HPLC column with a gradient of B solvent (dashed lines). The PLA2 peaks were assessed by ESI-MS and enzyme assay. Annotations of the PLA2s are the same as those shown in Table 1.
Molecular phylogeny of venom K49-PLA2s from
Asian pit vipers
A phylogenetic tree was built to study the structural relationships among venom K49-PLA2s from 10 Asian
pit viper venom species (Fig. 6). The outgroup in this tree was a K49-PLA2 (Bothropstoxin-I) from the
venom of a New World species Bothrops jararacussu [16].
Discussion
In contrast with all PLA2s previously purified from
Crotalinae venom [6–8], PLA2s containing six disulfide
bonds have been isolated from the venom of two Afri-can Viperinae, Bitis gabonica [17] and Bitis nasicornis [18]. Unlike the D49-PLA2s from Tpu and Tbo
ven-oms, Tpu-K49a, Tpu-K49b and Tbo-K49 contain only six disulfide bonds, although K49-PLA2s missing
resi-due C91 but retaining C61 were cloned from the venom glands of T. stejnegeri, but the proteins could not be found in the venom. In fact, all the K49-PLA2s
purified from different geographic venom samples of T. stejnegeri contain seven disulfide bonds [4]. This is
the first report on venom K49-PLA2s with six disulfide
bonds. As the numbers of disulfide bonds in many secreted protein families are increasing through evolu-tion (e.g. the serine protease family [19]), Tpu and Tbo may be considered as relatively primitive among the Trimeresurus species, as also suggested by the phylo-genetic analysis of their mtDNA [1,2].
It is widely accepted that disulfide bonds play an important role in maintaining conformational stability and tolerance to environmental factors such as heat, proteolytic enzymes and detergent [20]. Disulfide bonds at 50–131 and 11–77 of the secreted PLA2 contribute
significantly to conformational stability, whereas the disulfide bond at 61–91 contributes much less [21]. Mutagenesis of C61–C91 resulted in a decrease of 2.3 kcalÆmol)1 (9.63 kJÆmol)1) of unfolding free energy and lowered hydrolytic activity in the case of bovine pancreatic PLA2, or 11 kJÆmol)1decrease of unfolding
free energy in the case of porcine pancreatic PLA2
[22]. It was found that the melting temperature of Tpu-K49 was 8.8C lower than that of Tst-K49 (Fig. 5). This temperature reduction is consistent with that observed in a mutagenesis study of T4 lysozyme [23].
The K49-PLA2s display several Ca2+-independent
activities, including myotoxicity, bactericidal and edema-inducing effects [12]. These activities are poss-ibly related to certain conserved residues which are unique to the K49-PLA2 family, but absent from the
D49-PLA2s, including L5, Q11, E12, N28, R34, K49,
K53, W77, K80, V102, K115, K117, K123, K127 and K128 (Fig. 3A). The presence of the bulky amino acids V, M or F at position 102 possibly prevents the access of phospholipids to the active site [24]. A common heparin-binding motif is present near the C-termini of all the K49-PLA2s (Fig. 3A and [25,26]). Notably,
sequences of Tpu-K49a and Tpu-K49b differed by only two substitutions, i.e. R72 and M110 in Table 1. Inventory of PLA2 purified from T. puniceus and T. borneensis venom. Values of pI were predicted from protein sequences deduced from the cDNA sequences. Molecular masses were determined by ESI-MS.
PLA2
Protein
(%) pI
Molecular mass
(Da) N-Terminal sequences 1–23
Tpu-K49a 3.5 9.3 14221.5 ± 1.7 SVIQLGKMILQETGKNPVKYYGA Tpu-K49b 3 9.1 14112.8 ± 2.7 SVIQLGKMILQETGKNPVKYYGA Tpu-G6D49 1.5 8.4 13912.8 ± 2.2 SLLEFGRMIKEETGKNPLFSYIS Tpu-E6a 3.5 5.3 13723.2 ± 5.7 NLLQFELMIKKMSGRSGIRWYSD Tpu-E6b 1.5 4.5 13978.8 ± 2.2 HLMQFETMIMKVAGRSGVWWYGS Tpu-E6c 4 4.7 13792.8 ± 4.1 NLLQFEMMILKMAGRSGIRWYSD Tbo-K49 10 9.0 14034.0 ± 5.3 SVIELGKMILQETGKNPVTYYSA Tbo-G6D49 3 8.4 13959.6 ± 0.9 SLLEFGRMIKEETGKNPLFSYIS Tbo-E6 14 5.3 13723.0 ± 3.6 NLLQFEMMINKMAGRSGIRWYSD
Table 2. Enzymatic activities of venom D49-PLA2s with micellar substrates. Hydrolysis of L-dipalmitoyl phosphatidylcholine was measured at pH 7.4, 37C in the presence of 3 mMdeoxycholate or 6 mMTriton X-100, 10 mMCaCl2and 0.1MNaCl.
Purified PLA2
Specific activity (lmolÆmg)1Æmin)1) +Deoxycholate +Triton X-100 Tpu-G6D49 279 ± 35 353 ± 30 Tpu-E6a 661 ± 11 393 ± 14 Tpu-E6b 188 ± 11 69 ± 1 Tpu-E6c 504 ± 11 446 ± 40 Tbo-G6D49 272 ± 16 509 ± 7 Tbo-E6 701 ± 17 471 ± 45
Fig. 3. Alignment of the amino-acid sequences of three subtypes of PLA2s. (A) K49-PLA2s. (B) G6D49-PLA2s. (C) E6-PLA2s. Single-letter codes of amino acids and the numbering system of Renetseder et al. [43] were used. Residues identical with those in the top line were denoted with dots, and gaps were marked with hyphens. New seq-uences and special substitutions are shown in bold. Heparin-binding motifs are boxed. GenBank (SwissProt) accession numbers for the PLA2s and the species are: Ts-A2 (P81478), Ts-A6 (P70088), Ts-A5 (P81480), Ts-K49c (AY211936), Ts-K49a (AY211935), CTs-K49c (AY211938), Ts-G6D49 (AY211944) and CTs-A2 [4] from T. stejne-geri; Tmv-K49 (X77647) from Protobothrops mucrosquamatus; Dav-K49b (AF269132) from Deinagkistrodon acutus; Tfl-BPI (P20381) from Protobothrops flavoviridis; Bpir-G6D49 (1GMZ_A) from Bothrops pirajai; Bj-D1G6 (AY185201), Bj-S1G6(AY145836) from Bothrops jararacussu.
Tpu-K49a and S72 and D110 in Tpu-K49b. The higher basicity and hydrophobicity at these two posi-tions explain the twofold higher edema-inducing activ-ity of Tpu-K49a than Tpu-K49b (Fig. 5).
So far, up to 10 3D structures of the K49-PLA2
family have been solved by X-ray crystallography [24,27,28]. In common with other structures of
D49-PLA2s, the K49-PLA2s consist of three a-helices,
two antiparallel b-strands, and a few connecting loops. Two long a-helices are interlocked by disulfide bonds at C44–C105 and C51–C98 which form a rigid plat-form stabilizing the overall structure. Despite the dif-ference in one disulfide bond, the amino-acid sequence of Tpu-K49a was 80% identical with that of Ts-K49a from T. stejnegeri venom [4]. Potential hep-arin-binding motifs at positions 115–119 and 35–39 or 69–72 of most of the K49-PLA2s [12,26] were also
con-served (Fig. 3A). Therefore, the pharmacological acti-vities of these six-disulfide-bonded K49-PLA2s were
Table 3. Comparison of molecular mass (Da) of the tryptic peptides of Tpu-E6c (MMT) determined by peptide mass spectra fingerprint-ing with the calculated molecular mass (MMC). Sequences that differed from those of Tpu-E6a are shown in bold. Segment num-bering follows that in Fig. 3C.
Peptide sequence Position MMT MMC
NLLQFEMMILK 1–11 1380.66 1379.82 WYSDYGCYCGK 21–31 1458.80 1460.51 GGHGQPQDATDR 32–43 1239.56 1238.55 CCFVHDCCYGK 44–54 1509.78 1509.48 VSGCDPKDEFYK 55–74 1466.76 1464.65 YSSDNNDIVCGGNNPCLK 75–93 2028.96 2028.83 EICECDR 94–100 982.45 983.34 DAAICFR 101–107 853.93 853.40 DNLSTYNNK 108–117 1067.9 1068.49 YWNVPSETCQVESEPC 118–133 1987.11 1986.77
Fig. 4. CD spectra and conformational stability. (A) CD spectra of the K49-PLA2s with six and seven disulfide bonds. (B) Changes in helical content of the PLA2s during thermal denaturation as fol-lowed by molar ellipticity [h] at 222 nm. Melting temperatures were calculated from the reflection points.
Fig. 5. Time course of rat foot edema induced by the PLA2s. A rat foot was injected with 10 lg purified venom PLA2in 100 lL sterile NaCl⁄ Pi. The control group received only NaCl⁄ Pi. Swelling or size of the foot was measured with a plethysmometer. Experiments were performed in duplicate, and data points were averaged results.
Table 4. Anticoagulant activities of purified venom D49-PLA2s. APTT was measured twice (final volume 150 lL). Results shown are mean ± SEM.
PLA2 Dose (lg) Coagulation time (s) Control 0 29.0 ± 1.0 Tpu-E6a 3.0 78.7 ± 2.5 1.0 55.7 ± 1.1 0.3 48.0 ± 0.5 Methyl-Tpu-E6aa 3.0 55.2 ± 0.7 Tpu-E6c 3.0 44.3 ± 0.2 Tpu-G6D49 3.0 48.3 ± 1.1 1.0 42.2 ± 0.3 0.3 35.1 ± 0.5 Tbo-E6 3.0 43.4 ± 0.3 Tbo-G6D49 3.0 45.3 ± 0.3 2.0 42.3 ± 0.1 1.0 38.6 ± 0.4 Pto-R6-PLA2 0.42 73.5 ± 2.0 0.10 47.0 ± 1.0 a
nearly the same as other K49-PLA2s at ambient
tem-perature.
Both K49-PLA2 and E6-PLA2s are marker proteins
of pit viper venoms [13,14]. A previous phylogenetic tree of the K49-PLA2s showed separate clusters for the
venom proteins from the Old World pit vipers and the New World pit vipers [15]. Herein we focus on the evolutionary relationships among K49-PLA2s of Old
World pit vipers (Fig. 6). The robustness of this clado-gram is supported by high bootstrap values at most nodes. It is notable that most Trimeresurus species, including T. borneensis, T. puniceus and T. stejnegeri, are linked. Clustering of these Trimeresurus species in the cladogram may be attributed to a unique deletion at residue 89, specific conservation of V2, R35, I69, F106 and N121, and a charged residue 118 in their K49-PLA2s, in contrast with those from other Asian
pit vipers (Fig. 3A). Whether and how these structural diversities affect K49-PLA2function are not clear.
The G6D49-PLA2s have so far been found only in
the venom of a few venomous genera, including T. ste-jnegeri [4] and South American Bothrops (Fig. 3B). These PLA2 isoforms were potent, with specificity for
micelles containing Triton X-100 (Table 2). Under weakly acidic conditions, Tpu-G6D49 and Tbo-G6D49 were eluted from the Superdex gel-filtration column later than expected, as has also been reported for the purification of a few other basic venom PLA2s [28].
The enzymes are capable of inducing local edema (Fig. 6) and are more potent anticoagulants than K49-PLA2s (Table 4). A previous study showed that a
G6D49-PLA2 (i.e. myotoxin MT-III) from Bothrops
asper venom increased mouse vascular permeability and induced edema and inflammation in vivo [29].
The mechanism behind the anticoagulation effect of PLA2 is probably its binding via basic residues to
coagulation factors in the prothrombinase complex, thus inhibiting thrombin activation [30,31]. But which
residues are crucial for hindering the prothrombinase is puzzling. Despite differing by only two amino-acid substitutions at 115–119 (Fig. 3B), Tbo-G6D49 was 50% less potent than Tpu-G6D49, suggesting that this interface-recognition region affects the anticoagulating activity. Moreover, by careful sequence comparison (Fig. 3C), we noticed that basic residues K10, R16, R20, and K69 in Tpu-E6a, Tbo-E6 and Pto-R6 PLA2s
[13] possibly contribute to the anticoagulation activity (Table 4). Some of these residues have been suggested to be important for the anticoagulation effect of cro-talid venom PLA2s [13,30,31]. However, venom PLA2s
from elapid snakes or true vipers may have different anticoagulating sites [32].
Multiple acidic E6-PLA2s are present in the venom
of many pit vipers, and each enzyme may play differ-ent roles [4,33]. Many of them have been found to affect platelet function [9,34]. However, we found that the acidic PLA2s of Tpu and Tbo inhibit platelet
aggregation only relatively weakly. We also found that Tpu-E6a at a concentration of 0.1–1 lm significantly prolonged the blood coagulation time. After methyla-tion at His48 and inactivamethyla-tion, Tpu-E6a retained con-siderable anticoagulation activity (Table 4). Moreover, the APTT was hardly affected by the duration of the Tpu-E6a preincubation time. In fact, many strongly anticoagulating venom PLA2s show low hydrolytic
activity [30,35]. It has also been shown that an acidic PLA2, Cvv-E6f, from Crotalus v. viridis venom induced
severe edema [33]. Therefore, acidic E6-PLA2s
prob-ably have evolved with more diversity than previously recognized. Their target proteins remain the challenge for future investigations.
The morphologies of T. puniceus and T. borneensis are remarkably similar. Previous phylogenetic analyses suggested a close relationship between T. puniceus, T. borneensisand the cogeneric species in southern Asia (e.g. Trimeresurus trigonocephalus and Trimeresurus Fig. 6. Phylogenetic analysis of K49 PLA2s
from Asian pit viper venoms. Dataset used were 17 complete amino-acid sequences of K49 PLA2s, including those from the venom of Tropidolaemus wagleri and Ovophis graci-lis (I.-H. Tsai, Y.-M. Wang & C.M. Tu, unpub-lished data). The isoforms from T. stejnegeri venom are denoted with asterisks. In addi-tion to those shown in the legend of Fig. 3, accession numbers of the K49-PLA2s are: Ts-K49b (AY211937), CTs-K49a (AY211934), Tgr-PLVII (P70089), Ook-K49 (Q92152), and Bothropstoxin-I (Q90249).
malabaricus) [1,2]. All three subtypes of venom PLA2,
K49, E6 and G6D49 (Fig. 3A–C), are present in T. puniceus, T. borneensis and T. stejnegeri. The amino-acid sequence of Tbo-K49 differs from that of Tpu-K49 by only four substitutions (Fig. 3A), and Tbo-E6 is structurally very similar to Tpu-E6a and Tpu-E6c, while the sequence of Tpu-E6b is 92% identical with that of Ts-A6 of T. stejnegeri (Fig. 3C), and their speci-fic hydrolytic activities were very similar and relatively low (Table 2 and [4]). The cladogram in Fig. 6 also supports the previous conclusion that cogeneric species contain similar venom PLA2s [13]. However, basic
R6-PLA2s, which are present in venoms of T. stejnegeri [4]
and Trimeresurus popeorum (our unpublished data), are absent in venoms of T. puniceus and T. borneensis. Thus, present day arboreal Trimeresurus are probably derived from more than one ancestral species, or it is not a monophyletic genus [1–3].
The venom of T. borneensis used in this study was collected from a single specimen whereas that of T. puniceus was pooled venom. As intraspecies varia-tions of acidic E6-PLA2s of pit viper venom may be
common [4,33], the three isoforms of E6-PLA2s
puri-fied from the T. puniceus venom may be combined contributions from different snakes. Tpu-E6c is prob-ably an ortholog of Tpu-E6a (Fig. 3C) and is possibly absent or hardly expressed at all in the snake we killed. Why these E6-PLA2s do not form homodimers is not
certain, but it may be related to the lack of Pro113 [34]. It appears that the presence of K69 in a PLA2 is
not a sufficient condition for forming dimers [36]. In conclusion, full sequencing and phylogenetic ana-lyses of the venom PLA2s of two primitive species
T. puniceus and T. borneensis confirms their close rela-tionship to the cogeneric T. stejnegeri (Figs 3 and 6). However, the venom diversities of T. puniceus and T. borneensisPLA2s are not as great as those observed
with T. stejnegeri [4]. We also show the presence of un-usual K49-PLA2s with six pairs of disulfide bonds and
rare basic G6D49-PLA2s in these venoms. Their acidic
PLA2s showed significant anticoagulating effects. This
study on the diversity of venom PLA2s also helps us to
understand the structure–function relationships of the venom protein isoforms and the evolution of pit vipers.
Experimental procedures
Venoms and other materials
A live specimen and pooled venom powder of T. puniceus were purchased from Ramba Reptile Park, Bali, Indonesia. A live specimen of T. borneensis was purchased from Glades Herp Inc. (Fort Myers, FL, USA). Venom was
col-lected from the snakes 2 days before the venom glands were removed and the snake killed. All measures were taken to minimise pain. NIH guidelines for animal experiments were followed. The glands were immediately preserved in RNA-later solution (Ambion, Austin, TX, USA) until ready for RNA extraction. The mRNA extraction and the cDNA synthesis kits were purchased from Stratagene (La Jolla, CA, USA). Modification and restriction enzymes were from Promega. Synthetic l-dipalmitoyl glycerophosphocholine was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Other chemicals were from Merck and Sigma.
Purification and enzymatic activities of venom PLA2
Crude venom (15 mg) was dissolved in 100 lL buffer and centrifuged at 15 000 g for 5 min to remove insoluble mater-ial. The supernatant was injected into a gel-filtration column (Superdex G75, HR10⁄ 30) on an FPLC system. Venom pro-teins were eluted in 0.1 m ammonium acetate at room tem-perature. Fractions containing PLA2activities were pooled and freeze-dried. They were further purified by RP-HPLC using a column of silica gel (Vydac C8, 4.5 mm· 250 mm; Hesperia, CA, USA) equilibrated with 0.07% aqueous tri-fluoroacetic acid (solvent A), and eluted with a 25–45% lin-ear gradient of acetonitrile containing 0.07% trifluoroacetic acid (solvent B). Purified PLA2s were dried in a vacuum-cen-trifuge device (Labconco, Kansas City, MO, USA).
The concentration of PLA2 was determined from the A280, assuming an absorption coefficient of 1.5 at 1.0 mgÆmL)1. The hydrolytic activities of PLA2 towards mixed micelles of L-dipalmitoyl phosphatidylcholine and deoxycholate or Triton X-100 were assayed in 2.5 mL solu-tion at pH 7.4 and 37C, on a pH-stat apparatus (RTS 822; Radiometer, Copenhagen, Denmark). The reaction rate was corrected for the nonenzymatic spontaneous rate.
Amino-acid sequence and molecular mass of PLA2
The N-terminal sequences of purified PLA2s were deter-mined using a gas-phase amino-acid sequencer coupled with a phenylthiohydantoin amino-acid analyzer (model 477A; Perkin-Elmer, Foster City, CA, USA). The molecular mas-ses of the PLA2s [dissolved in 0.1% (v⁄ v) acetic acid with 50% (v⁄ v) CH3CN] were analyzed by ESI-MS on a mass spectrometer (model API100; Perkin-Elmer) equipped with the computer software biomultiview 1.2.
For peptide mass spectra fingerprinting, PLA2 was reduced with dithioerythritol and alkylated with iodoaceta-mide in the dark. Alkylated PLA2 was digested overnight with sequencing grade, modified trypsin (Promega, Madi-son, WI, USA). Enzyme digestion was stopped with acid before injection into the nanoLC-MS⁄ MS system, which
comprising a four-pumping Ultra-PlusTMII system (Micro-Tech Scientific, Vista, CA, USA) connected to the Q-Tof UltimaTM API mass spectrometer in place of the Micro-mass CapLCTM system. The masses of peptides obtained were sorted and matched to the calculated molecular mas-ses of the most possible fits predicted from a known ortho-logous PLA2sequence.
Cloning and sequence determination
RNA was isolated from venom glands, and the cDNA to mRNA was prepared using a kit [10,15]. To amplify and clone venom PLA2s, PCR [37] was conducted using SuperTaq DNA polymerase with a pair of mixed-base oligonucleotide primers (primer 1: 5¢-TCTGGATTSAGG
AGGATGAGG-3¢; primer 2: 5¢-GCCTGCAGAGACT
TAGCA-3¢), which were designed according to the highly conserved cDNA regions of the group-II venom PLA2s [38]. In addition, another primer (5¢-CAYCTNATGC ARTTYGARAC-3¢) was designed to replace primer 1 based on the amino-acid sequences 1–7 of Tpu-E6b, to make the amplification successful. Fragments of 0.4 kb were specifically amplified by PCR as shown by electro-phoresis of the products on a 1% agarose gel.
After treatment with polynucleotide kinase, the amplified DNA was inserted into the pGEM-T easy vector (Prome-ga). It was then transformed into Escherichia coli strain JM109. White transformants were picked up to select the cDNA clones. The DNA Sequencing System (model 373A) and the Taq-Dye-Deoxy terminator-cycle sequencing kit (PE Applied Biosystems, Foster City, CA, USA) were used to determine the sequences [39]. All the cDNA sequences reported were cloned at least twice, and both nucleotide strands were sequenced.
CD and thermal stability
The concentration of venom protein in phosphate-buffered saline (NaCl⁄ Pi)⁄ NaF-saturated buffer (pH 7.4) was deter-mined by the UV absorbance at 280 nm and adjusted to 0.3 mgÆmL)1. CD measurements were carried out on a J720 spectropolarimeter (Jasco, Tokyo, Japan) under constant flushing of nitrogen at 27C. All results were the average of five scanning measurements. Thermal stability of the protein in the NaCl⁄ Pi⁄ NaF buffer was investigated by measuring the ellipticity at 222 nm with stepwise tempera-ture increments of 0.5C from 20 C to 80 C using a thermostatically controlled sample holder.
Edema induction and effects on platelets and blood coagulation
For testing of the edematous effect of venom PLA2, Wistar rats (male, 200 g body weight) were anaesthetized with
sodium pentobarbital. One of the hind feet was injected with 10 lg purified PLA2 in 100 lL sterile NaCl⁄ Pi, and the other received NaCl⁄ Pionly. The size of the foot was measured at several intervals with a plethysmometer (type 7150; Ugo Basile, Comerio, Italy), and the time course of the swelling was recorded [4,15].
Blood was collected from rabbit and healthy human donors. Dose-dependent inhibition of ADP-induced aggre-gation of platelet-rich plasma by purified PLA2 was meas-ured with an aggregometer (model 600B; Payton, Scarbrough, Ont, Canada) at 37C after the addition of 10 lm ADP [4]. The effects of PLA2s on blood coagulation time (i.e. APTT) were studied using a Hemostasis Analyzer (model KC1; Sigma Diagnostics). To inactivate PLA2, methylation of His48 at the active site was performed by incubating purified 0.14 mm PLA2 in 0.1 m sodium phos-phate buffer (pH 7.9) with 2.86 mm methyl p-nitrobenzene-sulfonate and 9% (v⁄ v) acetonitrile at 25 C [40]. The remaining catalytic and anticoagulating activities were measured.
Phylogenetic analysis of K49-PLA2s
Phylogenetic analysis was based on the 17 available amino-acid sequences of venom K49-PLA2s from Old World pit vipers. Our unpublished amino-acid sequences of K49-PLA2 from venom glands of Tropidolaemus wag-leri [14] and Ovophis gracilis were also included in the dataset. Multiple alignments of the sequences were made using the pileup program and neighbor-joining methodo-logy. Then the tree was built by the program phylip (http://www.evolution.genetics.washington.edu./phylip.html) [41]. The degree of confidence of the lineage at each node was determined by bootstrap analyses of 1000 replicates [42].
Acknowledgements
We thank Ms Yi-Hsuan Chen for preparing Tst-K49 and Pto-R6-PLA2, and Dr Yuh-Ling Chen for
collect-ing venom glands. Proteomic MS analyses were per-formed by the Core Facilities for Proteomics Research at the Institute of Biological Chemistry, Academia Sinica. The work was supported by grant NSC91-2311-B001-114 from the National Science Council, Taiwan.
References
1 Malhotra A & Thorpe RS (2000) A phylogeny of the Trimeresurus group of pit vipers: new evidence from a mitochondrial gene tree. Mol Phylog Evol 16, 199– 211.
2 Creer S, Malhotra A & Thorpe RS (2003) Assessing the phylogenetic utility of four mitochondrial genes and a nuclear intron in the Asian pit viper genus, Trimere-surus; separate, simultaneous, and conditional data com-bination analyses. Mol Biol Evol 20, 1240–1251. 3 Giannasi N, Malhotra A & Thorpe RS (2001) Nuclear
and mtDNA phylogenies of the Trimeresurus complex: implications for the gene versus species tree debate. Mol Phylog Evol 19, 57–66.
4 Tsai IH, Wang YM, Chen YH, Tsai TS & Tu MC (2004) Venom phospholipases A2 of bamboo viper (Tri-meresurus stejnegeri): molecular characterization, geo-graphic variations and evidence of multiple ancestries. Biochem J 377, 215–223.
5 David P & Vogel G (1996) Snakes of Sumantra: Annotated Checklist and Key with Natural History Notes, pp. 160–167. Chimaira, Frankfurt am Main, Germany.
6 Danse JM, Gasparini S & Menez A (1997) Molecular biology of snake venom phospholipase A2. In Venom Phospholipase A2 Enzyme: Structure, Function and Mechanism(Kini RM, ed), pp. 29–71. Wiley, London. 7 Francis BR, Meng J & Kaiser II (1998) Classification of
snake venom group II Phospholipases A2 according to amino acid sequences. In Enzymes from Snake Venom (Bailey GS, ed), pp. 503–544. Alaken Inc, Fort Collins, CO.
8 Scott D & Sigler PB (1994) Structure and catalytic mechanism of secretory phospholipase A2. Adv Protein Chem 45, 53–88.
9 Kini RM & Evans HJ (1997) Effect of phospholipase A2 enzymes on platelet aggregation. In Venom Phos-hpholipase A2 Enzyme: Structure, Function and Mechanism (Kini RM, ed), pp. 369–387. Wiley, London.
10 Tsai IH & Wang YM (1998) Effect of site directed mutagenesis on the activity of recombinant trimucro-toxin, a neurotoxic phospholipase from Trimeresurus mucrosquamatusvenom. Toxicon 36, 1591–1597. 11 Mounier CM, Luchetta P, Lecut C, Koduri RS, Faure
G, Lambeau G, Valentin E, Singer A, Ghomashchi F, Beguin S, et al. (2000) Basic residues of human group IIA phospholipase A2 are important for binding to fac-tor Xa and prothrombinase inhibition. Eur J Biochem 267, 4960–4969.
12 Lomonte B, Pizarro-Cerda J, AnguloY, Gorvel JP & Moreno E (1999) Tyr fi Trp-substituted peptide 115–129 of a Lys49 phospholipase A2 expresses enhanced membrane-damaging activities and reprodu-ces its in vivo myotoxic effect. Biochim Biophys Acta 1461, 19–26.
13 Tsai IH, Chen YH & Wang YM (2004) Comparative proteomics and subtyping of venom phospholipases A2 and disintegrins of Protobothrops pit vipers. Biochim Biophys Acta 1702, 111–119.
14 Wang YM, Liew YF, Chang KY & Tsai IH (1999) Purification and characterization of the venom phospho-lipases A2 from four monotypic Crotalinae snakes. J Nat Toxins 8, 331–340.
15 Tsai IH, Chen YH, Wang YM, Tu MC & Tu T (2001) Purification, sequencing, and phylogenetic analyses of novel Lys-49 phospholipase A2 from the venoms of rat-tlesnakes and other pit vipers. Arch Biochem Biophys 394, 236–244.
16 Ward RJ, Monesi N, Arni RK, Larson RE & Pac¸o-Larson ML (1995) Sequence of a cDNA encoding bothropstoxin I, a myotoxin from the venom of Bothrops jararacussu. Gene 156, 305–306.
17 Botes DP & Viljoen CC (1974) Bitis gabonica venom: the amino acid sequence of phospholipase A. J Biol Chem 249, 3827–3835.
18 Joubert FJ, Townshend GS & Botes DP (1983) Purifica-tion, some properties of two phospholipases A2 (CM-I and CM II) and the amino-acid sequence of CM-II from Bitis nasicornis (Horned Adder) venom. Hoppe-Seyler’s Z Physiol Chem 326, 1717–1726.
19 Topfer-Petersen E, Calvete J, Schafer W & Henschen A (1990) Complete localization of the disulfide bridges and glycosylation sites in boar sperm acrosin. FEBS Lett 275, 139–142.
20 Freedman RB (1995) The formation of protein disulfide bonds. Curr Opin Struct Biol 5, 85–91.
21 Zhu H, Dupureur CM, Zhang X & Tsai MD (1995) Phospholipases A2 engineering. The role of disulphide bonds in structure, conformational stability, and cataly-tic function. Biochemistry 34, 15307–15314.
22 Janssen MJW, Verheij HM, Slotboom AJ & Egmond MR (1999) Engineering the disulphide bond patterns of secretory phospholipases A2 into porcine pancreatic iso-zyme. Eur J Biochem 261, 197–207.
23 Matsumura M, Becktel WJ, Levitt M & Matthews BW (1989) Stabilization of phage T4 lysozyme by engineered disulphide bonds. Proc Natl Acad Sci USA 86, 6562–6566. 24 Liu Q, Huang Q, Teng M, Weeks CM, Jelsch C, Zhang R & Niu L (2003) The crystal structure of a novel, inactive, lysine 49 PLA2 from Agkistrodon acutus venom. J Biol Chem 278, 41400–41408.
25 Ward RJ, de Azevedo WF Jr & Arni RK (1998) At the interface: crystal structures of phospholipases A2. Toxi-con 36, 1623–1633.
26 Lomonte B, Yamileth A & Caldero´n L (2003) An over-view of lysine-49 phospholipase A2 myotoxins from cro-talid snake venoms and their structural determinants of myotoxic action. Toxicon 42, 885–901.
27 Francis B, Schmidt J, Yang Y, Orlando R & Kaiser II (1995) Anions and the anomalous gel filtration behavior of notexin and scutoxin. Toxicon 33, 779–789.
28 Arni RK, Fontes MRM, Barberato C, Gutie´rrez JM, Dı´az C & Ward RJ (1999) Crystal structure of myotoxin II, a monomeric Lys49-phospholipase A2 homologue
isolated from the venom of Cerrophidion (Bothrops) godmani. Arch Biochem Biophys 366, 177–182.
29 Zuliania JP, Fernandesa CM, Zamunera SR, Gutie´rrez JM & Teixeira CFP (2005) Inflammatory events induced by Lys-49 and Asp-49 phospholipases A2 isolated from Bothrops aspersnake venom: role of catalytic activity. Toxicon 45, 335–346.
30 Mounier C, Franken PA, Verheij HM & Bon C (1996) The anticoagulant effect of the human secretory phos-pholipase A2 on blood plasma and on a cell-free system is due to a phospholipid-independent mechanism of action involving the inhibition of factor Va. Eur J Bio-chem 237, 778–785.
31 Evans HJ & Kini RM (1997) Anticoagulant effects of phospholipases. In Venom Phospholipase A2 Enzymes: Structure, Function and Mechanism(Kini RM, ed), pp. 353–368. Wiley, London.
32 Carredano E, Westerlund B, Persson B, Saarinen M, Ramaswamy S, Eaker D & Eklund H (1998) The three-dimensional structures of two toxins from snake venom throw light on the anticoagulant and neurotoxic sites of phospholipase A2. Toxicon 36, 75–92.
33 Tsai IH, Wang YM, Chen YH & Tu AT (2003) Geographic variations, cloning and functional analyses of the venom acidic phospholipases A2 of Crotalus viridis viridis. Arch Biochem Biophys 411, 289–296.
34 Welches W, Reardon I & Heinrikson RL (1993) An examination of structural interactions presumed to be of importance in the stabilization of phospholipase A2 dimers based upon comparative protein sequence analy-sis of a monomeric and dimeric enzyme from the venom of Agkistrodon p. piscivorus. J Protein Chem 12, 187–193.
35 Inada M, Crowl RM, Bekkers AC, Verheij H & Weiss J (1994) Determinants of the inhibitory action of purified 14-kDa phospholipases A2 on cell-free prothrombinase complex. J Biol Chem 269, 26338–26343.
36 Nakamura S, Nakai M, Nakashima K, Ogawa T, Shimohigashi Y, Ohno M, Kihara H, Yamane T & Ashida T (1996) Roles of lysine-69 in dimerization and activity of Trimeresurus flavoviridis venom aspartate-49-phospholipase A2. J Mol Recognit 9, 23–30.
37 Mullis KB & Faloona F (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155, 335–350.
38 Ogawa T, Kitajima M, Nakashima KI, Sakaki Y & Ohno M (1995) Molecular evolution of group II phos-pholipase A2. J Mol Evol 41, 867–877.
39 Maniatis T, Fritsch EF & Sambrook J (1989) Molecular Cloning: A Laboratory Manual.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 40 Verheij HM, Volwerk JJ, Jansen EHJM, Puyk WC,
Dijkstra BW, Drenth J & De Haas GH (1980) Methyla-tion of histidine-48 in pancreatic phospholipase A2: role of histidine and calcium ion in the catalytic mechanism. Biochemstry 19, 743–750.
41 Felsenstein J (1992) phylip: the PHYLogeny Inference Package, Version 3.573. Computer Program Distributed by the. University of Washington Department of Genet-ics, Seattle.
42 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. 43 Renetseder R, Brunie S, Dijkstra BW, Drenth J & Sigler
PB (1985) A comparison of the crystal structures of phospholipase A2 from bovine pancreas and Crotalus atroxvenom. J Biol Chem 260, 11627–11636.
