Amino acid sequence of a basic aspartate-49-phospholipase A2 from Trimeresurus flavoviridis venom and phylogenetic analysis of Crotalinae venom phospholipases A2

Toxicon 46 (2005) 185–195 www.elsevier.com/locate/toxicon

Amino acid sequence of a basic aspartate-49-phospholipase A2 from Trimeresurus flavoviridis venom and phylogenetic analysis of Crotalinae venom phospholipases A2 Takahito Chijiwaa, Kazuki Abea, Tomohisa Ogawab, Nikolai N. Nikandrovc, Shosaku Hattorid, Naoko Oda-Uedaa, Motonori Ohnoa,* a Department of Applied Life Science, Faculty of Engineering, Sojo University, Kumamoto 860-0082, Japan Department of Biomolecular Science, Graduate School of Life Science, Tohoku University, Sendai 981-8555, Japan c Division of Biological Resources and Environmental Science, Graduate school of Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan d Institute of Medical Science, University of Tokyo, Oshima-gun, Kagoshima 894-1531, Japan b

Received 4 March 2005; accepted 4 April 2005 Available online 20 June 2005

Abstract Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima and Okinawa. A phospholipase A2 (PLA2) of basic nature (pI 8.5) was isolated from the venom of Amami-Oshima T. flavoviridis. Its amino acid sequence determined by the ordinary procedures was completely in accord with that predicted from the nucleotide sequence of the cDNA previously cloned from Amami-Oshima T. flavoviridis venom gland, which was named PLA-B 0 . It consists of 122 amino acid residues and has aspartate at position 49. It induced edema in a mouse footpad assay and caused necrosis in mouse skeletal muscles. PLA-B 0 is similar in sequence to PLA-B (Tokunoshima) and PL-Y (Okinawa), both basic [Asp49]PLA2s, with a few amino acid substitutions, indicating occurrence of interisland mutation. Although PLA2s of Crotalinae subfamily were phylogenetically classified into four types, PLA2 (acidic or neutral [Asp49]PLA2) type, basic [Asp49]PLA2 type, neurotoxic [Asp49]PLA2 type and [Lys49]PLA2 type, it was ascertained that PLA2s of PLA2 type and [Lys49]PLA2 type are most essential as toxic components for Crotalinae snake venoms and that basic [Asp49]PLA2-type PLA2s are uniquely contained only in the venoms of T. flavoviridis species. Prediction of physiological activities of some PLA2s was made based on their location in the phylogenetic tree. Relationship of divergence of PLA2s via accelerated evolution followed by less rapid mutation and physiological activities was discussed. q 2005 Elsevier Ltd. All rights reserved. Keywords: Phospholipase A2; Amino acid sequence; Trimeresurus flavoviridis; Crotalinae subfamily; Evolution; Phylogenetic tree

1. Introduction Phospholipase A2 (PLA2, EC 3.1.1.4) catalyses the hydrolysis of the 2-acyl ester bond of 3-sn-phosphoglycerides with the requirement of Ca2C to produce * Corresponding author. Tel.: C81 96 326 3111; fax: C81 96 323 1331. E-mail address: [email protected] (M. Ohno).

0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2005.04.004

3-sn-lysophosphoglycerides and fatty acids (Dijkstra et al., 1981, 1983). Snake venoms contain PLA2 isoforms as major toxic components. Snake venom PLA2s are classified into groups I and II based on the mode of disulfide pairings (Dufton and Hider, 1983). Group I PLA2s are found in Elapidae (Elapinae and Hydrophiinae) venoms, while group II PLA2s are in Viperidae (Viperinae and Crotalinae) venoms. Group II PLA2s are divided into two subgroups, [Asp49]PLA2 forms and [Lys49]PLA2

forms (Maraganore et al., 1984; Maraganore and Heinrikson, 1993). They share the similar scaffold (Brunie et al., 1985; Renetseder et al., 1985; Holland et al., 1990; Suzuki et al., 1995). Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan, namely, AmamiOshima, Tokunoshima and Okinawa. Four PLA2 isozymes consisting of 122 amino acid residues were isolated from Tokunoshima T. flavoviridis venom: neutral [Asp49]PLA2, called PLA2 (highly lipolytic and myolytic) (Oda et al., 1990; Kihara et al., 1992); basic [Asp49]PLA2, called PLA-B (edema-inducing) (Yamaguchi et al., 2001); and two [Lys49]PLA2s, called BPI and BPII (extremely low lipolytic but strongly myolytic) (Yoshizumi et al., 1990; Liu et al., 1990; Kihara et al., 1992). PLA2 is also contained in Amami-Oshima and Okinawa T. flavoviridis venoms (Chijiwa et al., 2003a). Recently, an [Asp49]PLA2 which is more basic than PLA-B and neurotoxic was isolated from Amami-Oshima T. flavoviridis venom and named PLA-N (Chijiwa et al., 2003b). PLA-N is also expressed in Tokunoshima T. flavoviridis venom gland (Chijiwa et al., 2003b). However, it was found that Okinawa T. flavoviridis venom gland expresses one amino acid-substituted PLA-N homologue, named PLA-N(O) (Chijiwa et al., 2003b). As to basic [Asp49]PLA2, Okinawa T. flavoviridis venom contains PL-Y which is similar in sequence to PLA-B from Tokunoshima T. flavoviridis venom (Chijiwa et al., 2003a). It is emphasized that BPI and BPII abundantly expressed in Amami-Oshima and Tokunoshima T. flavoviridis venoms are completely missing in Okinawa T. flavoviridis venom (Chijiwa et al., 2000). These phenomena revealed that interisland evolution has occurred in venom PLA2 isozymes of T. flavoviridis. In the present study, we newly isolated a basic [Asp49]PLA2, named PLA-B 0 , from Amami-Oshima T. flavoviridis venom and its amino acid sequence was determined. Its edema-inducing and necrotic activities were assayed. Phylogenetic trees of PLA2s from T. flavoviridis species (Amami-Oshima, Tokunoshima and Okinawa), of PLA2 s from Trimeresurus genus (T. gramineus, T. mucrosquamatus, T. jerdonii, T. stejnegeri and T. flavoviridis) and of PLA2s from four genera (Agkistrodon, Bothrops, Crotalus and Trimeresurus) of Crotalinae subfamily were constructed. Discussion is made for their evolutionary divergence and its relationship with acquisition of physiological activities.

3 5 2 3 1 1 100

0

200 Fraction Number

Conductivity / mho × 103

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A280

186

400

300

Fig. 1. Chromatography of the second peak proteins (2.90 g) from Sephadex G-100 chromatography of Amami-Oshima T. flavoviridis venom (5.0 g) on a carboxymethyl cellulose (CM52) column. The proteins were loaded on a CM52 column (2.8!50 cm) equilibrated with 0.02 M ammonium acetate buffer (pH 6.8) and eluted with a linear concentration gradient to 0.6 M ammonium acetate (pH 6.8) over 3 l. The flow rate was 28 ml/h and 7 ml fractions were collected. Fractions indicated by a bar were pooled, dialyzed against water and lyophilized to give 38 mg of PLA-B 0 .

after 2-fold dilution with water. 4-Vinylpyridine was purchased from Nacalai Tesque (Kyoto, Japan). Achromobacter protease I and chymotrypsin were obtained from Wako Pure Chemicals (Osaka, Japan). 203 118 82

50.4

33.4

26.7

19.6

7.4

2. Materials and methods 2.1. Materials The venom was collected from various individuals of Amami-Oshima T. flavoviridis and immediately lyophilized

1

2

3

4

Fig. 2. SDS-PAGE of PLA-B 0 together with PLA-B and PL-Y with 15% polyacrylamide gel. lane 1, molecular weight marker; lane 2, PLA-B 0 ; lane 3, PLA-B; lane 4, PL-Y.

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2.2. Purification Two chromatographic steps consisting of Sephadex G-100 and carboxymethyl cellulose (Whatman CM52) were employed for purification of PLA-B 0 from the crude Amami-Oshima T. flavoviridis venom. 2.3. Polyacrylamide gel electrophoresis Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was carried out according to the method of Laemmli (1970) with 15% polyacrylamide gel. Two-dimensional polyacrylamide gel electrophoresis was conducted in the range of pH 7–10 with reference of pI marker Biolyte according to the instruction manual of Bio-Rad. 2.4. Physiological activities PLA-B 0 , PLA-B and crude Amami-Oshima T. flavoviridis venom (5 mg each) in phosphate buffered saline (PBS) (5 ml) were injected into the left footpad of mice. As a reference, PBS (50 ml) was injected into the right footpad. After 6 h,

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both legs were dissected at the genu and weighed. The experiment was done in triplicate. Mice (25–27 g) were injected intramuscularly in the hind thigh with 20 mg of PLA-B 0 and PLA-B dissolved in PBS (20 ml). Control mice were injected with 20 ml of PBS alone. After 24 h, mice were sacrificed and the histological samples were cut from the thigh and immersed in 10% formaldehyde. The fixed samples were processed for paraffin embedded sectioning and hematoxyline-eosin stain was applied for light microscopic examination. 2.5. Sequence analysis PLA-B 0 was reduced and S-carboxymethylated by the method of Stone et al. (1989) or S-pyridylethylated by the method of Cavins and Friedman (1970). S-Carboxymethylated (CM)-PLA-B 0 was digested with Achromobacter protease I (1% by weight) in 0.1 M Tris–HCl (pH 8.5) at 37 8C for 5 h. S-Pyridylethylated (PE)-PLA-B 0 was digested with chymotrypsin (0.4% by weight) in 0.05 M phosphate buffer (pH 7.0) at 37 8C for 2 h. Peptides were fractionated by HPLC on a VYDAC C18 column. The amino acid sequences of the N-terminal moiety of S-CM-PLA-B 0 and of

Fig. 3. The amino acid sequence of PLA-B 0 . S-CM-PLA-B 0 and the peptides obtained by digesting S-CM-PLA-B 0 and S-PE-PLA-B 0 with Achromobacter protease I and chymotrypsin, respectively, were sequenced by Edman degradation. Peptides were designated as follows. Letters indicate the type of cleavage: C, chymotrypsin; K, Achromobacter protease I. Arabic numerals represent the number of peptide in the order of elution on an HPLC column. The nucleotide sequence of the cDNA coding for PLA-B 0 (Chijiwa et al., 2003a) was aligned above the amino acid sequence.

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Fig. 4. The aligned amino acid sequences of PLA2s of Crotalinae subfamily (Trimeresurus, Agkistrodon, Bothrops and Crotalus). Abbreviations: Tf, Trimeresurus flavoviridis. A, AmamiOshima; T, Tokunoshima; O, Okinawa; Tg, T. gramineus; Tj, T. jerdonii; Tm, T. mucrosquamatus; To, T. okinavensis; Ts. T. stejnegeri. Aa, Agkistrodon acutus; Ab, A. halys blomhoffii; Ac, A. contortrix laticinctus; Al, A. halys palles; Ap, A. piscivorus piscivorus; Ba, Bothrops asper; Bc, B. pictus; Bi, B. insularis; Bj, B. jararaca; Bm, B. moojeni; Bn, B. nummifer; Bp, B. pirajai; Bs, B. schlegelii; Bu, B. jararacussu; Ca, Crotalus atrox; Cd, C. durissus terrificus; Cm, C. adamanteus; Cs, C. scutulatus scutulatus. References: 1, this paper; 2, Yamaguchi et al. (2000); 3, Chijiwa et al. (2003a); 4, Ogawa et al. (1992); 5, Oda et al. (1990); 6 Oda et al. (1991); 7, Fukagawa et al. (1992); 8, Fukagawa et al. (1993); 9, Tsai et al. (2003) (Q91506); 10, Nobuhisa et al. (1996); 11, Li et al. (2003); 12, Tomoo et al. (1989); 13, Maraganore et al. (1984); 14, Quintana et al. (2004) (Q918F8); 15, Junqueira-de-Azevedo and Ho (2003) (Q8QG87); 16, Randolph and Heinrikson (1982); 17, Aird et al. (1990); 18, Heinrikson et al. (1977); 19, Bieber et al. (1990); 20, Chijiwa et al. (2003b); 21, Lu et al. (2002); 22, Tsai et al. (1995); 23, Kondo et al. (1989); 24, Kaiser et al. (1990); 25, Serrano et al. (1999); 26, Bouchier et al. (1988); 27, Bouchier et al. (1991); 28, Yoshizumi et al. (1990); 29, Liu et al. (1990); 30, Nakai et al. (2001); 31, Liu et al. (1991); 32, Tsai et al. (2001); 33, Selistre-de-Araujo et al. (1996); 34, Maraganore and Heinrikson (1993); 35, Francis et al. (1991); 36, Pescatri et al. (2002) (Q9PVE3); 37, Soares et al. (2000); 38, De Azevedo et al. (1997); 39, Angulo et al. (2002); 40, Toyama et al. (1998); 41, Toyama et al. (2000); 42, Angulo et al. (1997); 43, Cintra et al. (2001); 44, Seilhamer et al. (1989); 45, Kusunoki et al. (1990)

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the peptides fractionated were analysed on an Applied Biosystems Procisee protein sequencing system. 2.6. Phylogenetic analysis

Fig. 4 (continued)

A phylogenetic tree of PLA2s from T. flavoviridis species (Amami-Oshima, Tokunoshima and Okinawa) was first constructed based on their amino acid sequences and by the neighbor-joining algorithm (Saitou and Nei, 1987). Alignment was made by the CLUSTAL W program (Thompson et al., 1994). The degrees of confidence for internal lineages in a phylogenetic tree were determined by the bootstrap confidence (Felsenstein, 1985) using the Kimura’s method (1969) to compute a distance matrix with 1000 replicates. Non-venomous group II PLA2s from human rheumatoid arthritic synovial fluid (Seilhamer et al., 1989) and rat platelet (Kusunoki et al., 1990) were employed as the outgroups. Furthermore, phylogenetic trees of PLA2s from Trimeresurus genus (T. gramineus. T. mucrosquamatus, T. jerdonii, T. stejnegeri and T. flavoviridis) and of PLA2s from four genera (Agkistrodon, Bothrops, Crotalus and Trimeresurus) of Crotalinae subfamily were constructed to search their evolutionary relationships.

3. Results and discussion The proteins (2.90 g) obtained from the second peak (a middle molecular mass fraction) of Sephadex G-100 chromatography of the crude Amami-Oshima T. flavoviridis venom (5.0 g) were fractionated on a CM52 column as shown in Fig. 1. The fractions indicated by a bar were pooled, dialysed against water and lyophilized to give 38 mg which was named PLA-B 0 . PLA-B 0 gave a single band on SDS-PAGE and its molecular weight was estimated to be 17,000 (Fig. 2). Its pI was estimated to be 8.9 with twodimensional polyacryamide gel electrophoresis with 7–10 pH gradient (data not shown). When PLA-B 0 and PBS were injected into the left and right footpads, respectively, of mice, a swelling was observed in the left leg. The weight increase of the left leg was 32% in average after 6 h of injection. Injection of PLA-B and the crude Amami-Oshima T. flavoviridis venom also caused swelling and the weight increases were 22% for the former and 37% for the latter. PLA-B 0 is fairly stronger than PLA-B in edema-inducing. When PLA-B 0 and PLA-B was injected into skeletal muscle of upper limb of mice, it caused myolytic necrosis (data not shown). Infiltration of polymorphonuclear cells and edema in stromal tissues were also observed. The necrotic activity of PLA-B 0 was somewhat weaker than that of PLA-B. Only a little hemorrhage was detected in both. These changes were not produced by PBS alone. The N-terminal 29 residues were first determined for S-CM-PLA-B 0 . S-CM-PLA-B 0 and S-PE-PLA-B 0 were digested with Achromobacter protease I and chymotrypsin,

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respectively, under the conditions described in Materials and methods section. The peptides were fractionated by HPLC (data not shown) and sequenced. The total amino acid sequence was determined by taking overlaps of the sequences of the peptides as shown in Fig. 3. This sequence is completely in accord with that predicted from the nucleotide sequence of the cDNA previously cloned from Amami-Oshima T. flavoviridis venom gland (AB087496) (Chijiwa et al., 2003a). The amino acid sequences of PLA-B 0 (Amami-Oshima), PLA-B (Tokunoshima) and PL-Y (Okinawa), which are all basic [Asp49]PLA2s, are aligned in Fig. 4 together with those of PLA2s from the venoms of four genera (Trimeresurus, Agkistrodon, Bothrops and Crotalus) of Crotalinae subfamily. The difference between PLA-B 0 and PLA-B is noted at position 52, Glu for PLA-B 0 and Gly for PLA-B (the numbering according to Fig. 4). There are three substitutions between PLA-B 0 and PL-Y at positions 70 (Ser/Leu), 78 (Glu/Gly) and 87 (Glu/Val). Since PL-Y is reported to be unable to induce edema (Chijiwa et al., 2003a), such substitution(s) may affect edema-inducing potency, however, the details are unknown at the moment. In Fig. 5 is shown the phylogenetic tree constructed for PLA2s from T. flavoviridis species of Amami-Oshima, Tokunoshima and Okinawa based on their amino acid sequences. They are separated into four types, PLA2 (neutral [Asp49]PLA2) type, basic [Asp49]PLA2 type, neurotoxic [Asp49]PLA2 type and [Lys49]PLA2 type. This branching pattern is clearly reflecting their physiological properties. It is noted that basic [Asp49]PLA2s such as PLA-B 0 , PLA-B and PL-Y form an independent branch

which is clearly separated from neurotoxic [Asp49]PLA2s. Human rheumatoid arthritic fluid PLA2 and rat platelet PLA2 employed as the outgroups are located distantly far from the four types of venomous PLA2s, indicating that PLA2 species from the snake venoms are evolutionarily the least related to non-venomous PLA2s. The phylogenetic tree made for PLA2s from Trimeresurus genus (T. gramineus, T. mucrosquamatus, T. jerdonii, T. stejnegeri and T. flavoviridis) is shown in Fig. 6. In the four branches, PLA2s from each species tend to form clusters. It is evident from the branching pattern that T. flavoviridis is phylogenetically in a close relationship with T. mucrosquamatus rather than T. gramineus. Jerdoxin (Lu et al., 2002) from T. jerdonii venom is located between neurotoxic PLA2s like trimucrotoxin (T. mucrosquamatus) (Tsai et al., 1995) and PLA-N and PLA-N(O) (T. flavoviridis) (Chijiwa et al., 2003b) and basic [Asp49]PLA2s. Although jerdoxin was reported to have edema-inducing activity (Lu et al., 2002), it seems likely that it exhibits neurotoxic activity too. Further addition of PLA2s from Agkistrodon, Bothrops and Crotalus genera was made in Fig. 7. Here, it is also noted that PLA2s from each genus tend to form clusters in the branches, indicating that they are evolutionarily in proximity to one another. B. asper myotoxin I (Kaiser et al., 1990) and B. jararaca PLA2 (Serrano et al., 1999) phylogenetically belong to neurotoxic [Asp49]PLA2 group (Fig. 7). Although their neurotoxicity has not been reported, it is probable that they are neurotoxic. Prediction of physiological activities of PLA2s based on phylogenetic

Basic [Asp49]PLA2 type Tf PLA-B' (A) Tf PL-X' (T) 997 Tf PLA-N (A,T) Tf PLA-N (O)

1000

Tf PLA-B (T) Tf PL-Y (O) 982 1000

0.1

976

Neurotoxic [Asp49]PLA2 type

983 983 1000

Outgroup HRAF PLA2

Tf PLA2 Tf [Thr37]PLA2

PLA2 type

1000

1000 Tf BPI (A,T) Tf BPII (A,T)

RP PLA2

[Lys49]PLA2 type Fig. 5. Phylogenetic tree constructed for PLA2 s from T. flavoviridis species of Amami-Oshima (A), Tokunoshima (T) and Okinawa (O) based on their amino acid sequences. Human rheumatoid arthritic fluid (HRAF) PLA2 and rat platelet (RP) PLA2 were employed as the outgroups.

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PLA2 type

191

ToPLA-01

Tf [Thr37]PLA2 Tf PLA2 1000 TmPLA2 TgPLA-I TgPLA-IV TgPLA-III

1000

676 999

TsPLA2 TgPLA-II 537

616

945

873 764

[Lys49]PLA2 type Tf BPII (A,T) 1000 Tf BPI (A,T)

966

Basic [Asp49]PLA2 type Tf PLA-B' (A) 1000 535 Tf PL-Y (O) 667Tf PL-X' (T) Tf PLA-B (T)

790

519 1000

TmK49PLA2

TgPLA-V ToPLA-03 1000

Tjjerdoxin 998 Tf PLA-N (O ) (O) Tf PLA-N (A,T) TmTrimcrotoxin

0.1

Neurotoxic [Asp49]PLA2 type

Fig. 6. Phylogenetic tree constructed for PLA2 s from Trimeresurus genus (T. gramineus, T. mucrosquamatus, T. jerdonii, T. stejnegeri and T. flavoviridis) based on their amino acid sequences.

analysis might be useful for further investigation of their true activities. When gazing at the phylogenetic tree of PLA2s from four genera (Agkistrodon, Bothrops, Crotalus and Trimeresurus) of Crotalinae subfamily (Fig. 7), it is perceived that the numbers of PLA2-type [Asp49]PLA2s and [Lys49]PLA2s are much greater than those of neurotoxic [Asp49]PLA2s and basic [Asp49]PLA2 s. This indicates that PLA2-type [Asp49]PLA2s and [Lys49]PLA2s are essential toxic components of Crotalinae venoms. Neurotoxic [Asp49]PLA2 appears to be not necessarily contained in the venoms of all

the genera or species. It is worthy to note that although the number of basic [Asp49]PLA2-type PLA2s is limited, PLA2s of this type are all from T. flavoviridis species. Previously, we showed that T. flavoviridis venom PLA2 isozyme genes have evolved in an accelerated manner to acquire diverse physiological activities of their products (Nakashima et al., 1993, 1995; Ohno et al., 1998, 2002, 2004). Accelerated evolution was also found for T. okinavensis venom PLA2 isozymes (Nobuhisa et al., 1996) and T. flavoviridis and T. gramineus venom serine proteases (Deshimaru et al., 1996). Such evolution seems

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PLA2 type

Tf [Thr37]PLA2 (T) Tf PLA2 (A,T,O)

CmPLA2 -a

1000

TmPLA2

1000

ToPLA-01 ApD49PLA2

CaPLA2 CdCrotapotin

BcPLA2 AbPLA2 TgPLA-IV TgPLA-I 995TgPLA-III

CsMtx-a

1000

603

271

822

TgPLA-II TsPLA2

922 841

266 934

84

420

[Lys49]PLA2 type

BiPLA2

352

16

Basic [Asp49]PLA2 type 816

BmMyotoxin I BaMyotoxin III

444 1000

636

BpPrTx-II 974 BpPrTx-I 871 736 BuBothropstoxin 958 BaMyotoxin II 698 BmMyotoxin II

777

819 500

873 604 343

ApK49PLA2 1000

Tf PL-X' (T) Tf PL-Y (O) Tf PLA-B' (A) Tf PLA-B (T)

716

515 471

778 564

TjJerdoxin

AcMyotoxin

1000

999

CaCaxK49 BnMyotoxin II

1000

1000

TmTrimucrotoxin

Tf PLA-N (A,T) Tf PLA-N (O) (O)

BjPLA2

BsMyotoxin II AaK49II TgPLA-V ToPLA-03 1000

999

BaMyotoxin I

AlAgkistrotoxin CdCB2 CdCB1

Neurotoxic [Asp49]PLA2 type

Tf BPI (A,T) Tf BPII (A,T) TmK49PLA2

0.1

Fig. 7. Phylogenetic tree constructed for PLA2s from Trimeresurus, Agkistrodon, Bothrops and Crotalus genera of Crotalinae subfamily based on their amino acid sequences.

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universal for venom isozyme systems. Thus, it is natural to consider that venom PLA2s of Crotalinae subfamily snakes have evolved in an accelerated manner. However, we recently found that the genes encoding neurotoxic [Asp49]PLA2s, PLA-N (Chijiwa et al., 2003b), trimucrotoxin (Tsai et al., 1995), agkistrotoxin (Kondo et al., 1989) and crotoxin B (Bouchier et al., 1988) from T. flavoviridis, T. mucrosquamatus, A. halys pallas and C. durissus terrificus, respectively, evolved in an accelerated manner until they had acquired neurotoxic function, but after that they have evolved under less frequent mutation (Chijiwa et al., 2003b). It can be said that divergence of PLA2s of Crotalinae subfamily snakes into the four types, PLA2 type, basic [Asp49]PLA2 type, neurotoxic [Asp49]PLA2 type and [Lys49]PLA2 was brought about by accelerated evolution of their genes. Although less rapid evolution has been occurring between PLA2s in the same branch (or subbranch), this not necessarily means that PLA2s in the same branch (or sub-branch) show the same physiological activity. For example, BPI and BPII, T. flavoviridis [Lys49]PLA2s with only one amino acid substitution Asp/ Asn at position 58 (Fig. 4), exhibited the same level of myolytic activity (Kihara et al., 1992) but BPII was 10–100 times more potent than BPI in guinea pig ileum contracting activity (Shimohigashi et al., 1995). The difference in physiological activity between both PLA-B 0 and PLA-B (edema inducible) and PL-Y (edema hardly inducible) shows also the case. It is likely that even evolution at a slower rate is related to production of new or modified physiological activities.

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