Venom phospholipases A
2of bamboo viper (Trimeresurus stejnegeri):
molecular characterization, geographic variations and evidence
of multiple ancestries
Inn-Ho TSAI*1, Ying-Ming WANG*, Yi-Hsuan CHEN*, Tein-Shun TSAI† and Ming-Chung TU†
*Institute of Biological Chemistry, Academia Sinica, Institute of Biochemical Sciences, National Taiwan University, P.O. Box 23-106, Taipei 107, Taiwan, and†Department of Biology, National Normal University, Taipei 107, Taiwan
Phospholipases A2(PLA2s) were purified from the Trimeresurus
stejnegeri venom obtained from various localities in Taiwan and three provinces in China, by gel filtration followed by reversed-phase HPLC. The precise molecular mass and N-terminal sequence of each PLA2 were determined. In addition
to the six previously documented PLA2isoforms of this species,
we identified ten novel isoforms. The venom gland cDNAs of individual specimens of the viper from four localities were used for PCR and subsequent cloning of the PLA2s. The molecular
masses and partial sequences of most of the purified PLA2s
matched with those deduced from a total of 13 distinct cDNA sequences of these clones. Besides the commonly known Asp49 or Lys-49 PLA2s of crotalid venoms, a novel type of PLA2with
Asn-49 substitution at the Ca2+-binding site was discovered. This type of PLA2 is non-catalytic, but may cause local oedema and
appears to be a venom marker of many tree vipers. In particular, we showed that T. stejnegeri displayed high geographic variations of the PLA2s within and between their Taiwanese and Chinese
populations, which can be explained by geological isolation and prey ecology. A phylogenetic tree of the acidic venom PLA2s of
this species and other related Asian vipers reveals that T. stejnegeri contains venom genes related to those from several sympatric pit vipers, including the genera Tropedolaemus and Gloydius be-sides the Trimeresurus itself. Taken together, these findings may explain the exceptionally high variations in the venom as well as the evolutionary advantage of this species.
Key words: Asn-49 variant, bamboo viper (Trimeresurus stejne-geri), geographic variation, phospholipase A2, phylogeny tree,
venom.
INTRODUCTION
Bamboo viper Trimeresurus stejnegeri is widely distributed in south China, Taiwan and northern areas of southeastern Asia. It is the most common cause of snakebites in Taiwan and North Vietnam. Although studies on its venom components have been continued for many years, recent studies revealed significant geo-graphic variations in the morphologies [1], diets [2], mitochon-drial DNA [3,4] and the venom components of this species [5,6]. According to nested clade analyses, two or three lineages of Taiwanese T. stejnegeri (formerly named Trimeresurus gramineus) are existent [5,6], but phylogeny does not appear to be the principal causal factor of its venom variations [6]. It was not clear whether or not the variations detected in the venom were the result of differential gene expression.
Combining protein chemistry and functional genomics in a pre-viously tested ecological framework [6], the present study aims to elucidate the venom evolution of T. stejnegeri using phospho-lipases A2 (PLA2s; EC 3.1.1.4) as a reference. The 14 kDa
PLA2s are common components in pit-viper venoms. They usually
display multiple isoforms with various functional specificities, including neurotoxic, myotoxic, oedema-inducing, anti-platelet or anti-coagulating activities [7]. Five PLA2s were previously
pur-ified from the pooled venom of Taiwanese T. stejnegeri and their full amino acid sequences were determined [8–10]. They are ap-parently different from the five PLA2s purified from a pooled
venom sample of Chinese T. stejnegeri [11].
Abbreviations used: CTs, ChineseT. stejnegeri; PLA2, phospholipase A2; PRP, platelet-rich plasma; Ts, TaiwaneseT. stejnegeri; for brevity we have
used single-letter codes for amino acids, e.g. D49 stands for Asp-49.
1 To whom correspondence should be addressed (e-mail bc201@gate.sinica.edu.tw).
The nucleotide sequence data forT. stejnegeri venom PLA2s will appear in EMBL Nucleotide Sequence Database under the accession numbers
AY211932–AY211944.
A recent survey by mass analyses of the PLA2s in 104
geographic venom samples of Taiwanese T. stejnegeri revealed that although an individual sample contains only 1–4 PLA2s,
a total of 22 distinct PLA2 isoforms were detected for all the
localities [6]. The structures and functions of these isoforms were not studied. The high diversity was rather exceptional, since other pooled viper venoms usually express less than six PLA2
isoforms [12,13]. In the present study, we purified the PLA2s from
individual juvenile and adult T. stejnegeri venoms from various localities in Taiwan, and pooled venoms from three provinces in China. The enzymic and pharmacological activities of the PLA2s
were characterized. The cDNAs encoding 13 novel PLA2s of this
venom species were also cloned and fully sequenced. The results help to understand better the structure–activity relationships and specificities of the PLA2s. Moreover, phylogeny relationships
between the acidic PLA2s in the venoms of T. stejnegeri and
several Asian pit vipers were analysed in an effort to trace the origin of the venom proteins.
EXPERIMENTAL Snakes and venoms
Individual T. stejnegeri venoms were collected from more than 12 regions in Taiwan. Pooled venoms of T. stejnegeri were also obtained from southern Fu-Jian, Zeh-Jiang and southern An-Huei in China. Venom powders of Trimeresurus albolabris and
216 I.-H. Tsai and others
Table 1 Primers used and theT. stejnegeriPLA2clones obtained in PCR
UTR, untranslated region.
Primer no. Nucleotide sequence Design rationale PLA2clone obtained when used with primer 1 in PCR
1 GCCTGCAGRACTTAGGCA Stop∼ 3-UTR, antisense None
2 TCTGGATTSAGGAGGATGAGG 5-UTR, sense Ts-K49a, Ts-R6, Ts-A3, Ts-A4 & Ts-A6, CTs-K49a, CTs-R6, CTs-A6
3 AGYCTNATNCARTTYGARAC Residues 1–7, sense Ts-A3, CTs-A3 4 AGYGTNATHGARTTNGGNAA Residues 1–7, sense CTs-K49b 5 ATGAAAGTGGCGGGGAGA Residues 10–15, sense Ts-A1 and Ts-A5 6 GGGAGGATGATHAARGARGA Residues 6–12, sense Ts-G6D49
Trimeresurus popeorum were purchased from Sigma (St. Louis, MO, U.S.A.). To obtain the venom glands, five adult T. stejnegeri were collected from four localities (Ilan, Sandemen and Manzhou in Taiwan and southern Fu-Jian in China), and a specimen of Tropedolaemus wagleri was purchased from Bali, Indonesia. Cloning and sequencing of PLA2
Venom of each specimen of T. stejnegeri was extracted 3 days before killing, and the fresh venom glands were dissected for RNA extraction. The cDNAs for the mRNA were subsequently synthesized [13]. Specific primers of 18–21 oligonucleotides were designed based on the highly conserved 5- and 3-untranslated regions [13,14] or specific N-terminal sequences of the purified PLA2 (Table 1). PCR was conducted using Super Taq DNA
polymerase to amplify the cDNAs encoding PLA2s [15]. A 0.4 kb
fragment was specifically amplified, as shown by 1% agarose gel electrophoresis. After being treated with polynucleotide kinase, the amplified DNA fragment was first inserted into the pGEM-T easy (Promega, Madison, WI, U.S.A.) and then transformed into the Escherichia coli strain JM 109. White transformants were screened and cDNA clones were selected. Both cDNA strands were sequenced by the dideoxynucleotide method [16].
Purification and characterization of venom PLA2s
Venoms of T. stejnegeri (5–15 mg) were dissolved in up to 0.2 ml of reagent-grade water. After repeated centrifugations at 20 000 g for 5 min, aliquots of 100 µl were injected into a gel-filtration column (Superdex75, HR10/30) on an FPLC system (Pharmacia). The column was pre-equilibrated and eluted at 1.0 ml/min with 0.1 M ammonium acetate (pH 6.2) at room temperature. Fractions containing PLA2activities were separately collected and
freeze-dried. They were further purified by reversed-phase HPLC with a Vydac C18column (4.5 mm× 250 mm) [13].
Purified PLA2s were dried in a vacuum-centrifuge device
(Labconco, U.S.A.). Their molecular masses were determined by electrospray ionization–MS on a mass spectrometer (API100, PerkinElmer). Protein sequences were determined by a gas-phase amino acid sequencer coupled with a phenylthiohydantoin amino acid analyser (model 120A; Applied Biosystems, Foster City, CA, U.S.A.).
Enzymic activities and other functional assays
Concentration of PLA2was determined by reading the absorbance
at 280 nm and assuming a molar absorption coefficient of 1.5 at 1.0 mg/ml of the protein. The hydrolytic activities of PLA2s
towards mixed micelles of L-dipalmitoyl phosphatidylcholine (99%, Avanti polar lipid, U.S.A.) and deoxycholate or Triton X-100 (Sigma) were assayed at pH 7.4 and 37◦C on a pH-stat apparatus (Radiometer, Denmark) [13].
For oedema test, a Wistar rat (approx. 200 g body mass) was anaesthetized by injection of sodium pentobarbital. The rat was then injected with a sterile solution of PLA2in saline buffer
on the hind-paw. Local swelling of the paw was monitored by a plethysmometer (type 7150, Ugo Basile) [14]. For platelet aggregation experiments, the anti-platelet activity of PLA2s was
assayed with PRP (platelet-rich plasma) prepared from a healthy human donor. The aggregation was initiated by the addition of 10 µM ADP and measured by an aggregometer (Payton, module 600B, Canada) at 37◦C [13].
The effect of venom PLA2s on blood coagulation was studied by
APTT (activated partial thromboplastin time) with a Hemostasis Analyzer (model KC1, Sigma Diagnostics, U.S.A.). The myotoxicities on mouse thigh and the direct haemolytic activities towards human erythrocytes of basic PLA2s were studied as
described previously [17].
Phylogenetic analysis of the acidic PLA2s ofT. stejnegeri venom
The sequences closely related to each of the acidic T. stejnegeri PLA2 isoforms were selected by BlastP search (NCBI). Our
unpublished sequences of the acidic PLA2s from the venoms of
several Asian pit vipers (T. wagleri, T. albolabris and T. popeorum) were also included in the dataset. Amino acid-sequence alignment was made using PILEUP program. Cladograms were constructed based on these sequences by neighbour-joining algorithm using PHYLIP program [18]; degree of confidence for the internal linage was determined by bootstrap methods [19].
RESULTS AND DISCUSSION
Purification and characterization of PLA2s
The monomeric and dimeric PLA2s in T. stejnegeri venom were
separated by gel-filtration column (Figure 1, fractions 1 and 2 respectively). From the T. stejnegeri samples of western Taiwan, an abnormally late peak (Figure 1, fraction 3) containing G6D49-PLA2was eluted. All PLA2s were further purified by HPLC, and
some examples are shown in Figure 2. The PLA2s were eluted
during HPLC in the following order: basic R6-PLA2, K49-PLA2,
weakly basic G6D49-PLA2 and the acidic PLA2s. Content of
each PLA2in the crude venom (%, w/w) was estimated from the
UV absorbance peak both during gel filtration and HPLC. Total content of PLA2s in each sample was found to vary between 1
and 24% (w/w), depending on the age of snake and locality (see Table 4).
After determining the N-terminal sequence and the molecular mass, each of the venom PLA2s was annotated based on the
abbre-viated species name (i.e. Ts for Taiwanese T. stejnegeri and CTs for Chinese T. stejnegeri) and its apparent structural subtype (e.g. A for acidic PLA2) [7]. We successfully identified a total of ten
Ts-PLA2s and seven CTs-PLA2s (Tables 2 and 3), including
Figure 1 Gel filtration of the venom samples ofT. stejnegeri
Elution profiles for theT. stejnegeri venoms from six representative localities are shown, and the diet subset (in parentheses) is indicated below each locality. The dissolved venom was eluted at a flow rate of 1.0 ml/min at room temperature (25◦C) on an FPLC system with a Superdex G75 (HR 10/30) column in equilibration with 0.1 M ammonium acetate (pH 6.2). PLA2-containing fractions
(indicated by bars) were pooled for further purification.
the pooled venom [8–10]. From a south Taiwanese T. stejnegeri, we also purified Tgr-PL-VI, which was previously cloned, but not isolated. However, we have not found Tgr-PL-IV (theoretical mass 13.778 kDa) [8] that differs from Ts-A1 by only one amino acid residue.
A recent report has identified a total of 22 distinct molecular masses of venom PLA2s in 104 Taiwanese T. stejnegeri samples
from 38 localities [6]. This is more than twice the number that we identified (Table 2). Overestimation of the number of venom PLA2s resulted, probably, from the limitation of
MALDI–TOF-MS (matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry) used in the previous survey (mass precision of approx. 0.06%) [6], and also from possible post-translational modifications of the proteins, such as oxidation of methionine and de-amidation of glutamine and asparagine residues.
Cloning and sequence determination
About 160 PLA2clones were obtained from the cDNAs prepared
individually from the venom glands of four T. stejnegeri speci-mens and sequenced. After comparing the cDNA sequences, we identified nine distinct clones encoding Ts-PLA2variants and six
distinct clones encoding CTs-PLA2variants (Table 1). We failed
to clone CTs-K49c, CTs-G6 and CTs-A1 from the venom glands of Fu-Jian specimen, although the three PLA2s were purified from
the venoms of Zhe-Jiang or An-Hui. Each specimen apparently expressed several, but not all, of the venom PLA2s. Notably, the
catalytic activity and amino acid sequence 1–32 of CTs-A1 are very similar to those of CTs-A3. A few additional PLA2cDNA
sequences (not shown) obtained probably encode pseudogenes [13].
The complete amino acid sequences of the acidic and basic PLA2deduced were aligned respectively in Figures 3(A) and 3(B)
according to a commonly used numbering system [20]. Assuming all the conserved cysteine residues in the PLA2s form disulphide
bonds, the mass and pI value of each PLA2were calculated from
the cDNA predicted protein sequence, and then matched one-to-one with the sequences of the purified PLA2s (Tables 2 and
3), whose masses were successfully confirmed with electrospray ionization–MS.
Catalytic activities and functions of the PLA2s
The in vitro enzymic activities of these PLA2s towards
zwitter-ionic and catzwitter-ionic micellar substrates were determined (Table 5). As expected, the catalytic activities of all the R6- and K49-PLA2s
of this venom species were hardly detectable (not shown), owing to the lack of D49 at their catalytic sites essential for Ca2+binding [21]. Ts-A5 differs from Ts-A1 by a substitution N1H, which causes a significant reduction of its enzymic activity especially when assayed with the mixed micellar substrate, containing Triton X-100. Notably, Ts-G6D49 is a potent enzyme and its specificity towards the micelles containing Triton X-100 is relatively high.
Previously, the acidic venom PLA2 of T. stejnegeri (Jian-Xi,
China) was reported to be a platelet aggregation inhibitor [22]. On the other hand, Ts-A2, but not Ts-A1 or A5, was found to induce contracture of guinea-pig ileum [8,9]. Figure 4 shows the anti-platelet activities of the purified acidic PLA2s using human
PRP; most of the PLA2s show moderate anti-platelet activities.
Although differing in a single substitution (A40P), Ts-A3 and CTs-A3 exhibit similar enzymic and anti-platelet activities. The enzymic activities of venom acidic PLA2s have been shown to be
218 I.-H. Tsai and others
Figure 2 Purification of the venom PLA2s by reversed-phase HPLC
After gel filtration and freeze-drying, the redissolved PLA2-containing fractions were purified
on a Vydac C18HPLC column with a gradient of B solvent (acetonitrile). Four representative
examples of the elution profiles are shown. The PLA2peaks were analysed by ESI–MS and
N-terminal sequencing and annotated accordingly.
Table 2 Molecular data and N-terminal sequences of all the PLA2s found
in TaiwaneseT. stejnegerivenoms
Residues at site 6 are shown in bold.
PLA2 Previous name* Mass pI N-terminal sequences
Ts-R6 13 689 9.4 HLLQLRKMIKKMTNKEPILSYGK Ts-K49a Tgr-PLA-V 13 892 9.5 SVIELGKMIFQETGKNPATSYGL
Tgr-PLA-VI 13 917 9.6 GVIELTKMIVQEMGKNALTSYSL Ts-K49b 13 931 9.4 GVIELTKMFVQEMGKNALTSYSL Ts-K49c 13 876 9.5 SVIELGKMIFQETGKNPATSYGL Ts-G6D49 13 805 7.8 SLLEFGRMIKEETGKNPLSSYFS Ts-A1 Tgr-PLA-I 13 734 4.8 HLMQFETLIMKVAGRSGVWYYGS Ts-A2 Tgr-PLA-II 13 779 5.0 NLLQFENMIRNVAGRSGIWWYSD Ts-A3 13 750 5.3 SLIQFETLIMKVAKKSGMFSYSA Ts-A4 13 905 5.5 HLLQFETMIIKMTKQTGLFSYSF Ts-A5 Tgr-PLA-III 13 711 4.7 NLMQFETLIMKVAGRSGVWYYGS Ts-A6 Tgr-PLA-VII 13 939 4.7 HLMQFENMIKKVTGRSGIWWYGS
* See [8–10].
essential, but not simply proportional to their anti-platelet effects [23]. Each acidic PLA2may have evolved with distinct specificity
towards platelets of different species [12]. To understand the specificities of each acidic PLA2, the platelets of various potential
preys of the species should be included in the study.
The hydrolytic activities of all the K49- or N49-PLA2s of
T. stejnegeri venoms were too low to be determined (results not
Table 3 Molecular data andN-terminal sequences of all the PLA2s found
in ChineseT. stejnegerivenoms
Ts-R6, Ts-A5 and Ts-A6 in this list were identical with those found in the Taiwanese venoms. The pI values were predicted from cDNA deduced protein sequences. Residues at site 6 are shown in bold.
PLA2 Mass pI N-terminal sequences
Ts-R6 13 689 9.4 HLLQLRKMIKKMTNKEPILSYGK CTs-R6 13 576 9.4 SLLQLRKMIKKMTNKEPILSYSK CTs-K49a 13 817 9.5 SLVQLGKMIFQETGKNPATSYGL CTs-K49b 13 771 9.4 SVIELGKMIFQETGKNPATSYGL CTs-K49c 13 512 – SVIELGKMILQETGKNPVTHYGA CTs-G6 13 758 – NLVQLGKMIFQETGKNPATSYGL CTs-A1 13 774 – SLIQFETLIMKVAGQSGMFSYSA CTs-A2 13 675 4.9 NLMQFELLIMKVAGRSGIVWYSD CTs-A3 13 776 5.3 SLIQFETLIMKVAKKSGMFSYSA Ts-A5 13 711 4.7 NLMQFETLIMKVAGRSGVWYYGS Ts-A6 13 939 4.7 HLMQFENMIKKVTGRSGIWWYGS
shown). However, local oedema was prominent within few hours after injection of 5–10 µg of these basic PLA2homologues/paw
(Figure 5). The oedematous potency of Ts-K49c was higher than that of Ts-K49a, whereas the potencies of Ts-R6 and CTs-R6 ranked between both the Ts-K49 isoforms. We also tested other functions of these basic PLA2homologues. The anti-coagulating
and myotoxic effects of the K49-PLA2s were approx. 4–8-fold
lower than those of trimucrotoxin (a myo-/neuro-toxin from Protobothrops mucrosquamatus [24]) and the anti-coagulating R6-PLA2from Gloydius [25] (results not shown). Inhibition of
platelet aggregation and direct haemolysis of washed human erythrocytes by Ts-R6 and CTs-R6 were found to be very weak.
The active enzyme Ts-G6D49-PLA2 induced fast and
sus-taining local oedema (Figure 5), and prolonged the coagulation time of human plasma (results not shown). A similar type of D49-PLA2 with myotoxicity has been found in Bothrops venom, and
its three-dimensional structure has recently been solved [26].
Structure–function relationships of the PLA2s
It is known that the venom acidic PLA2s from pitvipers Gloydius
[27] and Calloselasma [12] are potent platelet inhibitors. It has been shown that the residues E6, D114 or D115, W20, W21, Y113 and W119 of the PLA2s form a surface site to which
platelets may possibly bind [27]. The low or moderate anti-platelet effects of Ts-A1, Ts-A5 and CTs-A2 (Figure 4) may be owing to the lack of W20, W21 or D114 (Figure 3A). The relationships between membrane-disturbing effect and the basic residues of K49-PLA2s have been studied previously [28]. The lysine residues
at positions 116–119, 123, 127, 128, and the RRKPK sequences at positions 34–38 are, probably, responsible for the heparin-binding capability, oedematous effect (Figure 4) and local myonecrosis of these basic and non-catalytic PLA2homologues [14,28]. Among
the six isoforms of T. stejnegeri K49-PLA2s identified, the amino
acid sequences are at least 89% identical (Figure 3B), but only Ts-K49a and Ts-K49c contain all the 14 cysteine residues of group II PLA2s and are expressed abundantly in the venom.
Notably, unusual substitutions of C91H in K49a, CTs-K49b and Ts-CTs-K49b, as well as C105R in Tgr-PL-VI, apparently result in the low expression level or instability of these mutants (Table 4). Ts-K49b (cloned in the present study) differed from Tgr-PL-VI (cloned previously [29]) by only eight amino acid substitutions. It is known that seven disulphide bonds are con-served in all the viperid venom PLA2s except the disulphide
Figure 3 Multiple sequence alignments of the acidic (A) and basic (B) PLA2s ofT. stejnegerivenom
Single-letter codes for amino acids are used, and the numbering system follows that of Renetseder et al. [20]. The residues identical with those in the top line are denoted with dots, and the gaps are marked with hyphens. Special or function-related substitutions are boxed. The partial sequences of the acidic PLA2s fromT. albolabris (Talb-E6) and T. popeorum (Tpop-E6) venoms are also listed
220 I.-H. Tsai and others
Figure 4 Inhibition of platelet aggregation by the acidic PLA2s
ADP-induced aggregation of human platelets was studied after incubation of the acidic PLA2
with the freshly prepared PRP for 5 min at 37◦C. Dose-dependent inhibition of the aggregation was calculated by a comparison with the control (without adding PLA2). Data points are the
averages of 2–3 independent experiments.
Figure 5 Oedema-inducing activities of the basic PLA2s on rat paw
Time course of the swelling on the hind-paw was followed after injection of 10µg of the venom
protein dissolved in PBS. Volume of the paw was measured, and % swelling relative to original volume was the average from at least two independent experiments.
genus) [30]. The absence of C91-C62 may be a character of pri-mitive type of the venom group II PLA2s. It remains to be
investi-gated whether the missing C91-C62 is common in the venom K49-PLA2s of other arboreal vipers of the genus Trimeresurus sensu
stricto [3,4]. The disulphide bonds 50–131, 11–77 of the PLA2
contribute more to stability and enzyme activity than disulphide bonds 61–91 and 44–105; the last two contribute to the stability by 2.3 and 3.4 kcal/mol (1 cal≈ 4.184 J), respectively [31,32].
Ts-R6 and CTs-R6 are apparently novel types of group II PLA2
lacking a Ca2+-binding site. Their relatively low molecular masses could be attributed to a distinct gap at position 92 and the presence of many glycine, serine and threonine substitutions. As compared with the K49-PLA2s, both R6-PLA2s contain less basic residues
at positions 115–128 but more basic residues at the N-terminal regions (e.g. positions 6, 7, 10, 11 and 24), and have several
Table 4 Geographic variations of theT. stejnegerivenom PLA2s
Protein content of each PLA2in crude venom (%, w/w) was estimated based on relativeA280
areas of the protein peaks in both Figures 1 and 2. (A) Individual venoms of adult TaiwaneseT. stejnegeri
Locality (diet subset) Ts-PLA2isoform Content (%, w/w)
Taipei (N) R6 5–6 K49a 4–7 G6D49 3–4 A3 5–8 Miaoli (W) R6 10–12 G6D49 5–6 A3 6–8 Kao-Ping (SW) R6 5–8 K49a 2–10 G6D49 5–9 A3 2–4 Ilan (NE) R6 2 K49a 9 A3 3
Taitung (SE) K49a 10–15 Orchid Island (Is) K49c 12
A6 3
(B) Venoms of juvenile TaiwaneseT. stejnegeri
Locality (diet subset) Ts-PLA2isoform Content (%, w/w)
Miaoli (W) R6 8 G6D49 3 A3 4 A1 4 Kao-Ping (SW) R6 5 G6D49 1 A3 2 A1 2
Orchid Island (Is) K49c 1 (C) Pooled venoms of adult ChineseT. stejnegeri
Provinces PLA2isoforms Content (%, w/w)
Zhe-Jiang CTs-K49c 7 CTs-R6 11 Ts-A5 1 CTs-A2 1 CTs-A3 4 An-Hui Ts-R6 2 CTs-R6 7 CTs-G6 1 CTs-A1 6 CTs-A2 3 Fu-Jian CTs-R6 0.5 CTs-A1 0.5 CTs-A2 4
acidic substitutions at positions 71–86 that may confer specific pharmacological properties [21]. Since a similar type of R6-PLA2 was found in the venom of other arboreal Trimeresurus
(e.g. T. albolabris and T. popeorum; results not shown), the pharmacological effects of the PLA2s towards the major prey,
frogs, are currently being investigated. Geographic variations of the venom PLA2s
Subtropical Taiwan is climatically and ecologically diverse, owing to a central mountain range approx. 4000 m above sea level extending across the country from north to south. According to a
previous survey [2], the content of frogs in the diet of adult Taiwanese T. stejnegeri could be separated into five diet subsets: (N), northern subset containing 100% frogs; (W), western subset containing 92% frogs; (SW), southwestern subset containing 85% frogs; (E), eastern subset containing 78–80 % frogs; and (Is) offshore Island subset containing 47–52% frogs. Our results of the PLA2s variations of the Taiwanese T. stejnegeri were
empiri-cally grouped according to these five subsets (Tables 4A and 4B). At least 3–5 individual samples were analysed for each locality or diet subset to ensure that the results were representative. The results reveal that most of the venoms from western Taiwan contain Ts-R6, Ts-G6D49 and Ts-A3, whereas the venoms from eastern Taiwan contain Ts-K49a. Thus, the differences in the venom between the eastern and the western groups are obvious [6].
Our results in Table 4(A) are not contradicted by the findings of previous surveys of the geographic variations of T. stejnegeri venom. For example, of the ten samples from Luku (subset W) previously analysed [6], PLA2s with molecular masses similar
to those of Ts-R6, Ts-G6D49 and Ts-A3 were frequently found, and the molecular masses matching those of Ts-A6 and Ts-K49c were reported for the venom from two offshore populations [6]. Differing in a single substitution of L92P, the oedema-inducing activity of Ts-K49c for the rats is more prolonged and 2-fold higher than that of Ts-K49a (Figure 5). The abundant Ts-K49c in the venoms from two offshore islands would probably have evolved from Ts-K49a in the eastern populations by a founder effect [6]. The isoforms Ts-A2 and A5 were found only in a few samples collected in southwestern Taiwan in the vicinity of where the pooled T. stejnegeri venoms were obtained for previous studies [8–10] (C. C. Chang, personal communication).
Taiwan and China are separated by Taiwan Strait. Land bridges have connected Taiwan to the Asian continent possibly 2–3 times during the Pliocene and Pleistocene, and therefore made the exchanges of T. stejnegeri between Taiwan and China possible [6]. Among the eight distinct PLA2s purified from the samples
from three Chinese provinces (Table 2), two (Ts-R6 and Ts-A5) were identical with those from Taiwanese venom, but of very low venom content (Table 4C). The present-day T. stejnegeri on both sides of Taiwan Strait differ significantly in their venom genes, due to geological separation and a rapid venom evolution. Notably, the K49- and G6D49-PLA2s are weakly expressed in the
Chinese samples. Its ecological meaning is not clear, since the diets of Chinese T. stejnegeri have not been investigated. Ontological variations of the venom PLA2s
The venoms were collected from juvenile T. stejnegeri below 5 months from three locations in Taiwan. By purification and identification of the venom PLA2s, the ontological variations of
the PLA2s are shown in Table 4(B). The venom from juvenile
vipers was particularly abundant in Ts-R6 and Ts-A1, whereas Ts-A1 was rarely found in the venom of adult viper. Notably, K49-PLA2s were rarely expressed in the venom of juvenile vipers,
possibly correlated with a diet lacking rodents. Possible ecological correlations of the venom variations
To interpret the venom variations, both the functional specificities of the venom proteins and the ecological factors for the viper need to be examined. It appears that the presence of oedematous and myotoxic K49-PLA2s correlates with the rodent-rich diet of
pit vipers. The venoms from eastern Taiwan and the offshore islands are abundant in K49-PLA2s, the T. stejnegeri in these
localities have been reported to consume relatively more
mam-Table 5 Enzymic activities of purified Asp-49 PLA2s ofT. stejnegerivenom
towards micellar substrates
Initial rates were measured by pH-stat method at 37◦C and pH 7.4. dPPC, dipalmitoyl L-phosphatidylcholine.
Specific activity (µmol · mg−1· min−1)
PLA2 dPPC+ deoxycholate dPPC+ Triton X-100
Ts-G6D49 308+− 8 372+− 2 Ts-A1 406+− 32 246+− 3 Ts-A2 254+− 4 81+− 7 Ts-A3 573+− 53 215+− 15 Ts-A5 376+− 28 44+− 6 Ts-A6 178+− 20 101+− 5 CTs-A1 444+− 9 147+− 10 CTs-A2 457+− 46 352+− 9 CTs-A3 681+− 17 133+− 3
mals than in other localities [2]. Moreover, the higher specificity of Ts-K49c than Ts-K49a for rodents (Figure 5) is also in accordance with the diet of offshore island populations [2,6]. Abundance of K49-PLA2s in many South American Bothrops venoms [14,27]
is also consistent with the mammal-rich diets of the snakes [34]. On the other hand, the frog-dominant diet [2] is often associated with the lack of venom K49-PLA2in the juveniles of the northern
and western populations of Taiwanese T. stejnegeri (Table 4A). Similarly, the unusual absence of K49-basic protein in the venom has been reported for the T. flavoviridis on Okinawa Island with a frog diet [33].
The acidic PLA2s in the viper venoms have, in general,
evol-ved more isoforms than the basic PLA2s [12,13]. Whether the
hydrolytic activities of acidic venom PLA2s contribute to the
di-gestion of preys [35] is not clear, and have been shown to be inhibitors of platelet aggregation. The presence of multiple iso-forms of T. stejnegeri acidic PLA2s is probably an adaptation to
diverse preys with different platelet properties [13]. PLA2cladogram and possible ancestry ofT. stejnegeri
Apparently, the venom PLA2variations may be attributed to the
differential expression of a set of about nine PLA2 isoforms in
either the Taiwanese or Chinese T. stejnegeri (Table 5). We look deeper into this unusually high polymorphism by the phylogenetic analysis based on the sequences of venom acidic PLA2s, which are
the common venom components of most pit vipers [6,7,31,36]. The resultant tree displays a polyphyletic character for the PLA2s
from T. stejnegeri, and the bootstrap values are high when the PLA2of Protobothrops venom is assigned as outgroup (Figure 6).
Interestingly, Ts-A1, -A5 and -A6 are linked with the acidic venom PLA2s of Tropedolaemus, Ts-A2 and CTs-A2 are linked
with those from other tree vipers of the same genus (T. sensu stricto), and Ts-A3 and CTs-A3 are linked with those of the Chinese Gloydius (formerly named Agkistrodon). All these related genera or species are viviparous, like T. stejnegeri [37], and are probably sympatric to T. stejnegeri [38].
Since T. stejnegeri is not at a basal position in the mitochondrial DNA phylogeny tree of pit vipers [3], and the other venom species studied so far contained only a few highly similar acidic PLA2isoforms [7,12,30], it seems less probable that most of the
venom genes of the other pit vipers were lost during evolution, whereas those in T. stejnegeri were conserved. This can be best explained by the theory that T. stejnegeri was derived probably from interbreeding between several pit-viper species in the same or different genera, e.g. Gloydius (Figure 6). Merger of the
222 I.-H. Tsai and others
Figure 6 Phylogenetic relationships of the acidic PLA2s from venoms ofT. stejnegeriand selected Asian pit vipers
Data used for phylogenetic analysis include all the amino acid sequences listed in Figure 3(A) (indicated in boldface) and the complete sequences of the E6-PLA2s ofT. wagleri (Twg-E6a–c, results
not shown),Calloselasma rhodostoma [12] and Gloydius halys [31]. Venom sources and GenBank®accession numbers are: Ghb-E6 ofGloydius b. bravicaudus (P14418); GhP-E6 of G. halys
Pallas (AF015246); Crh-E6 isoforms of C. rhodostoma (AF104067-70); Tf-PLA2 (AB072175) of Trimeresurus flavoviridis; and Tmu-PL-I (X77 088) of Trimeresurus mucrosquamatus, which was assigned as the outgroup for making the phylogeny tree using the computer program. Values are calculated bootstrap values, indicating the confidence level of the branching.
venom genes from the ancestral species and subsequent selective expression of some of the genes through natural selection may have increased the diversity of the present-day T. stejnegeri venom.
The rich isoforms were found not only in the acidic PLA2s
but also in the K49-PLA2s (Tables 2 and 3) and other venom
protein families (e.g. the serine proteases; I.-H. Tsai and Y.-M. Wang, unpublished work). The interbreeding between present-day vipers of the same genus have been documented [39,40] and those between different genera have been rare [41] but may have happened by some means when the genetic gaps were not as wide as the present. The interbreeding must have occurred before the geographic separation of Taiwan from China, since the orthologous T. stejnegeri PLA2s in each branch of the phylogeny
tree (Figure 6) are existent in the venoms from both areas. Namely, Ts-A5 is found in the venoms of both Chinese and Taiwanese T. stejnegeri, CTs-A3 differs from Ts-A3 by only a substitution of A40P, and CTs-A2 is 84% identical with Ts-A2 (Figure 3). Conclusion
The venom proteins are heritable [6] and have been known to adapt a positive Darwinian evolution and accelerated mutations [29]. The intra-species variations of the venom proteins in T. stejnegeri reflect a dynamic adaptation and evolution of the venom. The present study draws five important conclusions: (1) T. stejnegeri is especially rich in venom genes, and the venom variations within the population of T. stejnegeri in Taiwan or China could be explained by differential expression of a set of genes in response to ecological and ontological conditions; (2) different sets of venom genes are present in Chinese and Taiwanese T. stejnegeri,
suggesting a long history of separation of both populations; (3) a new type of oedematous R6N49-PLA2s has been discovered, and
it appears to be a marker protein in the venoms of Asian arboreal pit vipers; (4) some of the T. stejnegeri venom proteins are related to those from the same genus, but some are related more to those from other genera, suggesting possibly multiple ancestors for this species; and (5) increased venom diversity is likely to confer an adaptation advantage for the snake, as testified by the high density and wide distribution of T. stejnegeri.
We thank Professor Q. C. Wang of Fu-Jian Medical University and Professor Yu-Yen Shu of Guan-Xi Medical University for donating the ChineseT. stejnegerivenom glands and venom, respectively, and Mr Chia-Wei Chu for collecting the venoms from Orchid Island and Taitung. This research was supported by a grant from National Science Council, Taiwan.
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Received 3 June 2003/21 August 2003; accepted 5 September 2003
