Molecular evolution of myotoxic phospholipases A2 from snake venom

Toxicon 42 (2003) 841–854 www.elsevier.com/locate/toxicon

Molecular evolution of myotoxic phospholipases A2 from snake venom Motonori Ohnoa,*, Takahito Chijiwaa, Naoko Oda-Uedaa, Tomohisa Ogawab, Shosaku Hattoric a

b

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, Miyagi 981-8555, Japan c Institute of Medical Science, University of Tokyo, Oshima-gun, Kagoshima 894-1531, Japan

Abstract After two decades of study, we draw the conclusion that venom-gland phospholipase A2 (PLA2) isozymes, including PLA2 myotoxins of Crotalinae snakes, have evolved in an accelerated manner to acquire their diverse physiological activities. In this review, we describe how accelerated evolution of venom PLA2 isozymes was discovered. This type of evolution is fundamental for other venom isozyme systems. Accelerated evolution of venom PLA2 isozyme genes is due to rapid change in exons, but not in introns and the flanking regions, being completely opposite to the case of the ordinary isozyme genes. The molecular mechanism by which proper base substitutions had occurred in the particular sites of venom isozyme genes is a puzzle to be solved in future studies. It should be noted that accelerated evolution occurred until the isozymes had acquired their particular function and, since then, they have evolved with less frequent mutation, possibly for functional conservation. We also found that interisland mutations occurred in venom PLA2 isozymes. The relationships between mutation and its driving force are speculative and the real mechanism remains a mystery. q 2003 Elsevier Ltd. All rights reserved. Keywords: Phospholipases A2; Viperidae snake venoms; Myotoxic; cDNAs and genes; Accelerated evolution; Interisland mutation; Phylogeny

1. Preface Although snake venoms contain a number of bioactive proteins, phospholipase A2 (PLA2) isoforms constitute major toxic components. It is well known that snake venom PLA2s exhibit a variety of physiological activities in addition to intrinsic lipolytic action. From analysis of the cDNAs and genes encoding snake venom PLA2s, it became evident that they have evolved in an accelerated manner. Such accelerated evolution has rarely been known in general (ordinary) isozymes. Now it is thought that acquisition of diverse physiological activities of venom PLA2 isozymes is closely related to their accelerated evolution. Myotoxicity is one of the physiological activities exerted by snake venom PLA2s, and many PLA2s, especially those of basic nature, appear to be myotoxic. This article focuses on accelerated evolution of snake venom PLA2 isozymes, including * Corresponding author. Fax: þ 81-96-323-1331. E-mail address: [email protected] (M. Ohno). 0041-0101/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2003.11.003

myotoxic PLA2s, with particular emphasis on those from the venoms of Trimeresurus flavoviridis, T. gramineus and T. okinavensis, which belong to Crotalinae subfamily. At the same time, interisland mutation of venom PLA2 isozymes, which has recently been discovered in T. flavoviridis snakes inhabiting the southwestern islands (Amami-Oshima, Tokunoshima and Okinawa) of Japan, is described. The readers are also referred to the following related articles (Ohno et al., 1998, 2002; Gubensek and Kordis, 1997; Kordis and Gubensek, 2000).

2. Classification and amino acid sequences of snake venom PLA2s PLA2 [EC 3.1.1.4] catalyzes the hydrolysis of the 2-acyl ester bond of 3-sn-phosphoglycerides with the requirement of Ca2þto produce 3-sn-lysophosphoglycerides and fatty acids. The amino acid sequences of over 200 PLA2s, in

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Fig. 1. The aligned amino acid sequences of selected group II PLA2s from the venoms of T. flavoviridis (Tf) (A, Amami-Oshima; T, Tokunoshima; O, Okinawa), T. gramineus (Tg), T. okinavensis (To), T. mucrosquamatus (Tm), Crotalus atrox (Ca), C. durissus terrificus (Cd), Deinagkistrodon acutus (Da) and Agkistrodon halys pallas (Ag). They are classified into four types, PLA2 type, basic [Asp49]PLA2 type, [Lys49]PLA2 type and neuro[Asp49]PLA2 type (for details, refer to the latter pages). References: Tg PLA-I (Oda et al., 1991), Tg PLA-II (Fukagawa et al., 1992), Tg PLA-III and Tg PLA-IV (Fukagawa et al., 1993), Tg PLA-V (Nakai et al., 1995), Ca PLA2 (Randolph and Heinrikson, 1982), Cd crotoxin B (Bouchier et al., 1991), Ag agkistrotoxin (Kondo et al., 1989), Da[Lys49]PLA2 (Fan et al., 1999) and Da neurotoxic [Asp49]PLA2 (Wang et al., 1996a). The references for other PLA2s are somewhere in the text. Bovine pancreatic (Bp) [Asp49]PLA2 (Fleer et al., 1978) is included for alignment.

which about 170 are from snake venoms, had been determined up to 1997 (Danse et al., 1997). At present its number is still increasing. Most of them are classified into groups I and II based on the mode of disulfide pairings (Dufton and Hider, 1983). Group I PLA2s are found in

the venoms of Elapidae (Elapinae and Hydrophiinae) snakes, whereas group II PLA2s are in the venoms of Viperidae (Viperinae and Crotalinae) snakes. Group II PLA2s are divided into two subgroups, [Asp49]PLA2 forms and [Lys49]PLA2 forms (Maraganore et al., 1984; Maraganore

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and Heinrikson, 1986). PLA2s of both groups consist of 118– 124 amino acid residues and contain seven disulfide linkages. Group I PLA2s possess a disulfide bridge between halfcystines at positions 11 and 77 (the numbering according to consensus alignment in Fig. 1). Instead, group II PLA2s are characterized by a C-terminal extension containing a halfcystine at the C-terminal end linked to a half-cystine at position 50. The amino acid sequences of group II PLA2s from the venoms of T. flavoviridis, T. gramineus and other Crotalinae snakes are shown in Fig. 1.

3. Crystal structure of snake venom PLA2s After the crystal structure of bovine pancreatic [Asp49]PLA2, which belongs to group I, was established by Dijkstra et al. (1981), those of group I [Asp49]PLA2s from the venoms of Naja naja atra (Scott et al., 1990), N. n. naja (Fremont et al., 1993), and Notechis s. scutatus (Westerlund et al., 1992), and of group II [Asp49]PLA2s from the venoms of Crotalus atrox (Brunie et al., 1985), Agkistrodon halys blomhoffii (Tomoo et al., 1994), T. flavoviridis (Suzuki et al., 1995), A. halys pallas (Wang et al., 1996b) and Deinagkistrodon acutus (Gu et al., 2002), and those of group II [Lys49]PLA2s from the venoms of A. p. piscivorus (Holland et al., 1990; Scott et al., 1992) and Bothrops asper (Arni et al., 1995) have been solved. Group I [Asp49]PLA2s are normally present as monomers. Some of group II PLA2 s, such as C. atrox [Asp 49]PLA 2, T. flavoviridis PLA2 ([Asp49]PLA2), B. asper myotoxin II ([Lys49]PLA2) and D. acutus acidic [Asp49]PLA2, are dimeric. Even though the sources of PLA2s are different, their monomer unit has a similar scaffold.

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[Lys49]PLA2s, called BPI and BPII, are basic (pIs 10.2 and 10.3, respectively) and their myolytic activity is several times greater than that of PLA2 (Liu et al., 1990; Yoshizumi et al., 1990; Kihara et al., 1992). The amino acid sequences of BPI and BPII are identical except at position 67, Asp for BPI and Asn for BPII (Fig. 1). The difference between BPI and BPII was noted in contraction of smooth muscle preparations of guinea pig ileum and artery (Shimohigashi et al., 1995). BPII was 10 – 100 times more active than BPI (Fig. 2). A noncharged residue at position 67 is favorable for muscle contraction because PLA2 with Asn at position 67 causes contraction as strongly as BPII (Fig. 2). PLA-B, an [Asp49]PLA2 with pI 8.6, exhibits edema-inducing activity (Yamaguchi et al., 2001). Recently, the presence of neurotoxic [Asp49]PLA2 with pI 10.3, named PLA-N, was found in the venoms of Amami-Oshima and Tokunoshima T. flavoviridis (Chijiwa et al., 2003a). Apoptosis-inducing activity toward cancer cells such as HL-60 cells was observed for BPI and BPII but not for PLA2 (Oda-Ueda et al., unpublished work). As mentioned above, PLA2 isozymes from T. flavoviridis venom exhibit a variety of physiological activities. Since all the isozymes have the same tertiary structure, their diverse physiological activities have been acquired by proper amino acid substitutions at particular sites in the evolutionary process. This should be also the case for PLA2 isozymes of other snake venoms and for other isozyme systems of these venoms. In terms of myotoxicity, PLA2, BPI and BPII from T. flavoviridis venom are evidently myotoxic (Kihara et al., 1992). Although not examined in detail, PLA-B and PLA-N may be myotoxic because of their basic nature. Four myotoxic PLA2 isozymes of basic nature, called myotoxins I, II, III and IV, were isolated from B. asper venom

4. Diverse physiological activities of snake venom PLA2s Snake venom PLA2 isozymes are well known to exhibit a variety of physiological activities such as hemolysis (Kihara et al., 1992), myotoxicity (Gutie´rrez and Lomonte, 1997; Gopalakrishnakone et al., 1997), neurotoxicity (Bon, 1997; Gubensek et al., 1997; Fletcher and Rosenberg, 1997), anticoagulant activity (Evans and Kini, 1997), edemainducing activity (Vishwanath et al., 1987; Wang and Teng, 1990; Liu et al., 1991; Tan et al., 1991; Yamaguchi et al., 2001), cardiotoxicity (Fletcher et al., 1981) and platelet aggregating activity (Kini and Evans, 1997). These diverse physiological activities of PLA2 isozymes in one snake species can be seen in those from the venom of T. flavoviridis as a representative. We hitherto isolated five PLA2 isozymes from T. flavoviridis (Crotalinae) (Tokunoshima island) venom (Fig. 1). PLA2, an [Asp49]PLA2 with pI 7.9, is the most abundant (Ishimaru et al., 1980; Oda et al., 1990) and is highly hemolytic, in the presence of phospholipids, and myolytic (Kihara et al., 1992). Two

Fig. 2. Concentration-dependent contraction activity of T. flavoviridis venom PLA2 isozymes on the smooth muscle of guinea pig ileum. The activity are expressed as a percentage of the maximum contraction by 1.0 £ 10-5 M carbachol. PLA2 (A), BPII (X) and BPI (W).

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(Gutierrez et al., 1984; Francis et al., 1991; Kaiser et al., 1990; Diaz et al., 1995). Myotoxins I and III are [Asp49]PLA2s and myotoxins II and IV are [Lys49]PLA2s. It is likely that basic PLA2s are all myotoxic, whether their activities are high or low. Many other myotoxic PLA2s were isolated from the venoms of other snakes such as Agkistrodon p. piscivorus (Maraganore and Heinrikson, 1986), T. mucrosquamatus (Chen et al., 1994) and Vipera a. ammodytes (Krizaj et al., 1991). Ammodytin L from V. a. ammodytes has Ser instead of Asp or Lys at position 49. Myotoxic PLA2s are generally contained in a large quantity, particularly in the venoms of Viperidae snakes. Thus, PLA2 myotoxins play a central role as major toxic components for enforcement of strong damage to preys.

5. Structures of the cDNAs and genes encoding venom PLA2 isozymes Five cDNAs encoding T. flavoviridis (Tokunoshima) venom-gland PLA2 isozymes, PLA2, [Thr38]PLA2, PL-X0 , BPI and BPII, were cloned and sequenced (Oda et al., 1990; Ogawa et al., 1992). It was clearly indicated by Northern blot analysis with these cDNAs and their 50 or 30 untranslated regions (UTRs) as probes that the mRNAs coding for these PLA2 isozymes are expressed only in the venom gland but not in other tissues such as heart, lung, liver, pancreas, kidneys, gall bladder, testis and ovaries (Oda et al., 1990; Ogawa et al., 1992). Until now, 81 cDNAs and 20 genes encoding Viperidae group II PLA2s and 67 cDNAs and 26 genes encoding Elapidae group I PLA2s have been cloned (data not shown). The total number is more than 3 times that at 1998 (Ohno et al., 1998). T. flavoviridis venom-gland PLA2 isozyme cDNAs contain an open reading frame of 414 bp coding for 138 amino acid residues (Oda et al., 1990; Ogawa et al., 1992). It was noted that the 50 and 30 UTRs are more homologous than the protein-coding region, with nucleotide identities of 95% for the 50 UTR, 67% for the protein-coding region and 89% for the 30 UTR. Such greater sequence homology in the UTRs, as compared to the protein-coding region, has not been known in the cDNAs for other isozyme systems. Usually, greater sequence homology is observed in the protein-coding region than in the UTRs for general isozymes, as in various isoforms of a G-protein a-subunit family (Matsuoka et al., 1988; Strathman and Simon, 1990) and a protein kinase C family (Ohno et al., 1987; Kubo et al., 1987). The signal peptide-coding domain, encoding 16 amino acid residues, is exceptionally conserved (92% with only one substitution between amino acids with similar properties) when compared with the other portion of the protein-coding region. This may be because they share common roles in penetration into endoplasmic reticulum membranes and susceptibility to a common signal peptidase.

The nucleotide substitution rates at the first, second and third positions of the triplet codons in the protein-coding region were calculated to be 32.1, 30.0 and 28.6%, respectively. The high substitution rates at the first and second positions are unusual compared with other cases in which the third (silent) positions are more variable than the first and second positions. The substitutions at the first and second positions mostly cause amino acid change as being evident from the 64 codons of the genetic code. Thus, the high substitution rates at the first and second positions bring about production of isozyme proteins with numerous substituted amino acids and thus with a variety of physiological activities. It is also noted that the nucleotide substitutions spread over the entire range of the mature protein-coding region of T. flavoviridis PLA2 isozyme cDNAs. Such high rate of nucleotide substitutions was also observed in the mature protein-coding regions of T. flavoviridis and T. gramineus serine protease isozyme cDNAs (Deshimaru et al., 1996) and T. flavoviridis metalloprotease isozyme cDNAs (Nakai et al., unpublished work). To gain further insight into this evolutionary phenomenon of venom PLA2 isozymes, six genes encoding T. flavoviridis (Tokunoshima) venom PLA2 isozymes were isolated from its liver genomic DNA library and sequenced (Nakashima et al., 1993). Four genes, pgPLA 1a, pgPLA 2a, pgPLA 1b and pgPLA 2b, encoded [Asp49]PLA2s. pgPLA 1a and pgPLA 1b, as well as pgPLA 2a and pg PLA 2b, are arranged in tandem. pgPLA 1a and pgPLA 2a coded for PLA2, although there are five nucleotide substitutions between them, three in introns and two in exons, one of which causing the amino acid change Glu ! Asp in the signal peptide (Nakashima et al., 1994). pgPLA 1b and pgPLA 2b are also polymorphic with five nucleotide subtitutions only in introns. The product of pgPLA 1b and pgPLA 2b has not been isolated. Two genes, BP-I and BP-II, encode [Lys49]PLA2 isozymes BPI and BPII, respectively. The transcription initiation site was determined for pgPLA 1a and pgPLA 2a by primer extension analysis using total venom-gland RNAs and assigned to the adenosine residue located 204 nucleotides upstream from the translation initiation codon (ATG), and the TATA-like sequence (CATAAA) was found 34 nucleotide upstream of the translation initiation site (ATG). Each gene spanned 1.9 kb, consisted of four exons and three introns and encoded 138 amino acid residues, including the signal sequence of 16 amino acid residues. Four genes encoding T. gramineus (Taiwan) venom PLA2 isozymes were also cloned (Nakashima et al., 1995a). gPLA-I and gPLA-VI encoded [Asp49]PLA2s and gPLA-V and gPLA-VII coded for [Lys49]PLA2s. The genes spanned 1.9 kb, contained four exons and three introns and encoded 138 amino acid residues, including the highly conserved signal peptides of 16 amino acid residues. The common structure of these venom PLA2 isozyme genes is shown in Fig. 3. Dot blot analysis of T. flavoviridis genomic DNA with the protein-coding region of PLA2 cDNA showed that

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Fig. 3. A common structure of T. flavoviridis (Tokunoshima), T. gramineus (Taiwan) and T. okinavensis (Amami-Oshima) venom PLA2encoding genes. Four exons are indicated by boxes and the UTRs are hatched. The nucleotide position numbers represent those of pgPLA 1a encoding T. flavoviridis PLA2.

the number of genes encoding PLA2s highly (90%) and moderately (60%) similar to PLA2 are 4 – 8 and 16 – 32, respectively, per haploid genome when the genome size was assumed to be equal to that of human genome (Nakashima et al., 1993). T. flavoviridis PLA2 genes form a multigene family.

The genes encoding T. okinavensis (Amami-Oshima island) venom PLA2 isozymes were also cloned from its liver genomic DNA library (Nobuhisa et al., 1996). Only three genes, gPLA-o1, gPLA-o2 and gPLA-o3, were obtained. Their construct was identical to that of T. flavoviridis and T. gramineus PLA2 isozyme genes. However, some of

Fig. 4. Nucleotide sequences of the second exons and the second introns (approximately one-third to the 50 end) of T. flavoviridis PLA2 isozyme genes, pgPLA 1a, pgPLA 2a, pgPLA 1b, pgPLA 2b, BP-I and BP-II. The nucleotide positions were numbered from the transcription start site.

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T. okinavensis specimens contain inactivated genes (peudogenes) of gPLA-o2 and gPLA-o3 in which one base insertion or one base substitution, respectively, in the protein-coding region led to a stop codon. The number of PLA2 isozymeencoding genes of T. okinavensis seems much smaller than those of T. flavoviridis and T. gramineus. In this respect, a question arises as to whether T. okinavensis belongs to the same genus as T. flavoviridis and T. gramineus. The genes encoding mojave toxin ([Asp49]PLA2) from C. s. scutulatus venom (John et al., 1994) and human pancreatic [Asp49]PLA2 (Seilhamer et al., 1986) contain four exons similarly as above. On the other hand, the genes encoding ammodytoxin C from Vipera a. ammodytes venom (Kordis and Gubensek, 1996), human rheumatoid synovial fluid (nonpancreatic) [Asp49]PLA2 (Seilhamer et al., 1989; Kramer et al., 1989) and rat platelet [Asp49]PLA2 (Kusunoki et al., 1990) contain five exons. The highly conserved GT (donor site)/AG (acceptor site) structure commonly noted for introns (Breathnach et al., 1978) is seen for all the genes (Nakashima et al., 1993). The nucleotide sequences of the second exon and the second intron of six T. flavoviridis PLA2 isozyme genes are shown in Fig. 4 (Nakashima et al., 1993). The second exon is much more variable than the second intron. The same relationship is seen in other introns and in the protein-coding regions of other exons, except for the signal peptide-coding domain of the first exon. For instance, sectional homologies between pgPLA 1a and BP-II are 98.5% for the 50 -UTR, 97.5% for the signal-peptide domain (first exon), 93.8% (first intron), 67.7% (second exon), 92.8% (second intron), 82.1% (third exon), 96.9% (third intron), 75.2% for the protein-coding region and 91.5% for the 30 -UTR (fourth exon). Since it is known that introns are more variable than the protein-coding regions (exons) (Kimura, 1983), the structural features of venom PLA2 isozyme genes are anomalous.

6. Accelerated evolution of Crotalinae snake venom PLA2 isozyme genes The evolutionary significance was noted for the nucleotide sequences of Crotalinae snake (T. flavoviridis, T. gramineus and T. okinavensis) venom PLA2 isozyme genes. Mathematical analysis was conducted for relevant pairs of PLA2 isozyme genes. The numbers of nucleotide substitutions per site ðKN Þ for the noncoding regions, the numbers of nucleotide substitutions per synonymous site ðKS Þ; and the numbers of nucleotide substitutions per nonsynonymous site ðKA Þ for the protein-coding regions were computed according to the methods of Miyata and Yasunaga (1980) or Nei and Gojobori (1986), with correlations for multiple substitutions (Kimura, 1983). A synonymous site is a site of a codon at which a base substitution causes no amino acid change. A nonsynonymous

Table 1 KN =KS and KA =KS values for pairs of PLA2 isozyme genes within species and between species Pair of genes

KN

KS

KA

KN =KS

KA =KS

T. gramineus gPLA-I vs gPLA-V gPLA-I vs gPLA-VI gPLA-I vs gPLA-VII gPLA-V vs gPLA-VI gPLA-V vs gPLA-VII gPLA-VI vs gPLA-VII

0.0550 0.0635 0.0657 0.0539 0.0317 0.0620

0.263 0.174 0.301 0.325 0.024 0.371

0.296 0.103 0.331 0.290 0.063 0.324

0.209 0.365 0.218 0.166 0.133 0.167

1.13 0.594 1.10 0.902 2.63 0.873

T. gramineus and T. flavoviridis gPLA-I vs pgPLA 1a 0.0697 gPLA-I vs BP-I 0.0640 gPLA-V vs pgPLA 1a 0.0639 gPLA-V vs BP-I 0.0477 gPLAVI vs pgPLA 1a 0.0743 gPLAVI vs BP-I 0.0644 gPLAVII vs pgPLA 1a 0.0747 gPLAVII vs BP-I 0.0618

0.272 0.238 0.324 0.156 0.203 0.231 0.357 0.166

0.173 0.309 0.292 0.138 0.178 0.301 0.312 0.165

0.257 0.269 0.197 0.306 0.367 0.278 0.209 0.372

0.635 1.30 0.903 0.885 0.877 1.30 0.872 0.995

site is a site of a codon at which a base substitution causes an amino acid change. Table 1 shows the KN =KS and KA =KS values obtained for pairs of PLA2 isozyme genes within species and between species. The data show several characteristics. First, KN values for the introns (the noncoding regions) are one-fifth to one-third of KS values for all the pairs of the genes compared, indicating that the introns are unusually conserved as compared to the protein-coding regions. The high homology of introns may suggest that there are some functional constraints in the introns. However, the regions corresponding to introns of precursor RNAs involved no significant secondary structure (Nakashima et al., 1993) when analyzed by the GENAS system (Kuhara et al., 1984), according to the method of Zuker and Steigler (1981). It is likely that the introns have no significant role. Therefore, the absence of an apparent role for the introns suggested that the protein-coding regions of the exons have evolved at greater substitution rates than the introns. Second, KA =KS values are much greater than those reported for other isoprotein genes (Kimura, 1983; Nei, 1987). Although synonymous sites are known to be much more variable than nonsynonymous sites, because of much less functional constraint in the former (Kimura, 1983; Nei, 1987), this is not the case in the proteincoding regions of PLA2 isozyme genes of Trimeresurus genus. The KA =KS values of the protein-coding regions are close to or greater than one. Such high degrees of substitutions at the nonsynonymous sites indicate that the PLA2 isozyme-encoding genes have evolved in a way that brings about accelerated substitutions. Gene duplication is a ubiquitous feature of genome evolution and has been viewed as the mechanism for evolution of new gene functions (Walsh, 1995). The PLA2 multigenes are thought to have been formed by gene duplication, starting from a single ancestral gene, and

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the newly formed genes have acquired new functions by proper nucleotide substitutions at the nonsynonymous sites. Thus, it is natural to consider that functional diversification of venom-gland PLA2 isozymes has been brought about by accelerated evolution of their genes. Such evolution seems to be in no way explained by the neutral theory (Kimura, 1983). Analysis of six cDNAs encoding serine protease isozymes of T. flavoviridis (Tokunoshima) and T. gramineus (Taiwan) venoms (Deshimaru et al., 1996) and of three cDNAs encoding metalloprotease isozymes of T. flavoviridis (Tokunoshima) venom (Nakai et al., unpublished work) showed that accelerated evolution has occurred in these isozymes as well. It was recently found that Elapinae venom PLA2 isozymes (Chuman et al., 2000), which belong to group I PLA2 family, and a group of C-type lectins and Ctype lectin-like proteins in Viperidae snake venoms (Tani et al., 2002) have also evolved in an accelerated manner. Accelerated evolution is considered to be universal in snake venom-gland isozymes.

7. Mechanism of accelerated evolution of snake venom PLA2 isozyme genes A concept of accelerated evolution was led by several characteristics of the venom PLA2 isozyme genes: (1) the protein-coding regions, except for the signal sequence domain, are much more variable than the noncoding regions including introns, (2) the rates of nonsynonymous substitution are close to or greater than those of synonymous substitution in the protein-coding regions, and (3) the gene products exhibit diverse physiological activities. Two possibilities are considered for accelerated evolution of the genes. One is the rapid change in the protein-coding region while the noncoding region has changed at a rate

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similar to those of general genes. The other is the slower change in the noncoding region while the protein-coding region has changed at a rate similar to those of general genes. To examine which possibility is true, the KN values of introns for a pair of PLA2 isozyme genes were compared with those of introns for a pair of TATA box-binding protein (TBP) genes, which are assumed to be the representatives of general genes because TBP is a nonvenomous protein and its gene is assumed to exist in a single copy (Nakashima et al., 1995a). Two TBP genes were cloned from T. flavoviridis and T. gramineus liver genomic DNA libraries (Nakashima et al., 1995b). Both TBP genes spanned about 19 kb and consisted of eight exons and seven introns. Mathematical analysis of the nucleotide sequences of the two TBP genes gave the following values: KN ¼ 0:0365 for the combined sequences of the flanking regions, the UTRs and the introns, and KS ¼ 0:0095 and KA ¼ 0:000 for all the exons. Thus, KN =KS ¼ 3:84 and KA =KS ¼ 0:000: These values reveal that the characteristics of evolution obeyed the neutral theory (Kimura, 1983). The sectional KN ; KS and KA values for three introns and four exons of venom PLA2 isozyme gene pair, gPLA-VI and BP-I, were compared to those for seven introns and eight exons of TBP gene pair. The sectional KN values between gPLA-VI and BP-I were not significantly different from those between two TBP genes (Nakashima et al., 1995a). This shows that introns of venom PLA2 isozyme genes have evolved at rates similar to those of TBP genes. It can be concluded that introns of venom PLA2 isozyme genes have evolved at rates similar to those of general genes. Thus, accelerated evolution of venom PLA2 isozyme genes can be attributed only to the rapid change in the protein-coding region. The cDNAs encoding mammalian (nonvenomous) PLA2s from different origins were compared (Table 2) (Nakashima, 1995). The KA =KS values were in the range of 0.1– 0.3, indicating that no accelerated evolution occurred. They are evolutionarily general genes.

Table 2 KA =KS values for pairs of mammalian groups I and II PLA2 cDNAs Pair of cDNAs

KS

KA

KA =KS

Group Ia Rat pancreatic PLA2 vs human pancreatic PLA2 Rat pancreatic PLA2 vs bovine pancreatic PLA2 Rat pancretic PLA2 vs porcine pancreatic PLA2 Human pancreatic PLA2 vs bovine pancreatic PLA2 Human pancreatic PLA2 vs canine pancreatic PLA2 Bovine pancreatic PLA2 vs canine pancreatic PLA2 Canine pancreatic PLA2 vs porcine pancreatic PLA2

0.802 1.04 1.11 0.981 0.715 0.416 0.586

0.196 0.159 0.163 0.144 0.0869 0.143 0.098

0.171 0.153 0.147 0.147 0.122 0.343 0.168

Group II Rat platelet PLA2 vs human synovial PLA2

0.820

0.200

0.244

References: rat and canine pancreatic PLA2s (Ohara et al., 1986), porcine pancreatic PLA2 (Seilhamer et al., 1986), bovine pancreatic PLA2 (Tanaka et al., 1987) and rat platelet PLA2 (Kusunoki et al., 1990). a The remaining combinations gave the similar values.

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Table 3 KN =KS and KA =KS values for pairs of T. okinavensis PLA2 isozyme genes Pair of genes

KN

KS

KA

KN =KS

KA =KS

gPLA-o1 vs gPLA-o2 gPLA-o1 vs gPLA-o3 gPLA-o2 vs gPLA-o3

0.0712 0.0594 0.0491

0.234 0.237 0.175

0.334 0.285 0.268

0.334 0.285 0.280

1.426 1.203 1.531

The KS and KA values are much greater than KN values in Trimeresurus venom PLA2 isozyme genes, indicating that the protein-coding regions are much more variable than the noncoding regions. On the other hand, KS values are smaller than KN values in general genes as in the case of the TBP genes, indicating that the protein-coding regions are much less variable than the noncoding regions. These phenomena reveal that the protein-coding region of venom PLA2 isozyme genes have changed, after gene duplication from an ancestral gene, so as to code for PLA2 proteins carrying new physiological activities. Gene conversion was thought to be the mechanism that produces diversity in the genes. Whether or not this mechanism has been operative in the venom PLA2 isozyme genes was examined. T. flavoviridis and T. gramineus PLA2 isozyme genes form multigene families (Nakashima et al., 1993, 1995a). In contrast, only three PLA2 isozyme genes (gPLA-o1, gPLA-o2 and gPLAo3) were detected in T. okinavensis genome (Nobuhisa et al., 1996). Their genes consisted of four exons and three introns, similarly as T. flavoviridis and T. gramineus PLA2 isozyme genes. Mathematical analysis data for three pairs of the genes are shown in Table 3. The KN =KS values (0.30 in average) and KA =KS values (1.39 in average) indicate that they have evolved via accelerated evolution. If gene conversion occurred within only three genes, it would be

expected that all the genes converge into similar ones. If so, the genes with low KN =KS or high KA =KS values could not be produced. Thus, gene conversion cannot be the mechanism that brings about the diversity in the venom PLA2 isozyme genes. Ohta and Basten (1992) and Ohta (1993) reported that the mechanisms for generating hypervariability at the variable regions of the genes encoding kallikreins, their inhibitors, and the major histocompatibility complex (MHC) are due to gene conversion followed by natural selection or natural selection for point mutation. Hence, it is thought that natural selection for point mutation is the major mechanism to produce the diversity in the venom PLA2 isozyme genes.

8. Evolutionary relationships of Viperidae venom PLA2 isozymes Phylogenetic trees were constructed for the nucleotide sequences of 11 Viperidae group II PLA2 cDNAs (Ogawa et al., 1995) according to the one-parameter method (Jukes and Cantor, 1969) and to the neighbor-joining algorithm (Saitou and Nei, 1987). A evolutionary tree constructed from the combined sequences of the 50 and 30 UTRs and the signal peptide-coding region is shown in Fig. 5. This tree shows that there are two groups, Crotalinae PLA2s and Viperinae PLA2s, which are evidently divided. Crotalinae PLA2s can be further split into distinct subgroups, C. durissus terrificus crotoxins and T. flavoviridis PLA2 isozymes. The reliability of the phylogenetic tree was assessed by 1000 replicates bootstrapping with the oneparameter method, and the branchings between different species (V. a. ammodytes, C. durissus terrificus and T. flavoviridis) were supported by statistical confidence (73 probabilities). Thus, the phylogenetic tree which was

Fig. 5. Phylogenetic tree for 11 Viperidae snake venom group II PLA2s based on the combined nucleotide sequences of the 50 and 30 UTRs and the signal peptide regions of their cDNAs according to the one-parameter method (Jukes and Cantor, 1969) and the neighbor-joining algorithm (Saitou and Nei, 1987). The numerals at the nodes represent the bootstrap confidence. The horizontal branch lengths are drawn to scale and represent the numbers of nucleotide substitution per site. The cDNAs encoding PLA2, [Thr38]PLA2, PL-X0 , BPI and BPII are from T. flavoviridis (Crotalinae) (Tokunoshima) venom gland (Oda et al., 1990; Ogawa et al., 1992), those encoding crotoxins A and B are from C. durissus terrificus (Crotalinae) venom gland (Bouchier et al., 1991), and those encoding ammodytoxins B and C and ammodytins L and I2 from V. a. ammodytes (Viperinae) (Pungercar et al., 1991).

M. Ohno et al. / Toxicon 42 (2003) 841–854

constructed for the combined sequences of the 50 and 30 UTRs and the signal peptide-coding region of group II snake venom PLA2 cDNAs is consistent with the classification of species and subspecies from taxonomy. On the other hand, the phylogenetic tree constructed from the mature protein-coding region sequences of the cDNAs showed a random pattern and was apparently different from that made from the combined sequences of the 50 and 30 UTRs and the signal peptide-coding region (Ogawa et al., 1995) (data not shown). The random pattern is by no means able to divide PLA2s into Crotalinae and Viperinae subfamilies, but appears to reflect the relationships of molecular properties, such as acidic or basic. These phylogenetic observations suggest that the mature proteincoding region seems to have evolved through a process different from the UTRs and the signal peptide-coding region. It is likely that the random pattern of the phylogenetic tree constructed from the mature proteincoding region may be related to accelerated evolution of this region. A phylogenetic tree was constructed from the nucleotide sequences of the mature protein-coding region of the cDNAs encoding venom PLA2s of Trimeresurus genus (T. flavoviridis, T. gramineus and T. okinavensis) (Nobuhisa et al., 1996). As shown in Fig. 6, phylogenetic analysis classified the PLA2 isozymes into three groups. The first is acidic [Asp49]PLA2s including T. okinavensis PLA-o1 and T. gramineus PLA-I, the second corresponds to basic [Asp49]PLA2s including, T. okinavensis PLA-o2, which is

Fig. 6. Phylogenetic relationships of venom PLA2 isozymes of Trimeresurus genus based on the nucleotide sequences of the mature protein-coding regions of their genes according to the oneparameter method and the neighbor-joining algorithm.

849

encoded by the active gene for gPLA-o2, and T. flavoviridis PL-X0 , and the third corresponds to [Lys49]PLA2s, including T. okinavensis PLA-o3, which is encoded by active gene for gPLA-o3, and T. flavoviridis BPI and BPII. This observation suggests that three T. okinavensis PLA2 isozymes represent three fundamental PLA2 species with different physiological activities.

9. Interisland evolution of venom-gland PLA2 isozymes of T. flavoviridis snakes T. flavoviridis snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima and Okinawa. Amami-Oshima is the northernmost and Tokunoshima island is 30 km south of Amami-Oshima. Okinawa island is located a further 120 km south of Tokunoshima island. These islands are thought to have been separated by eustacy (change in sea level) in the orogenic stage 1 – 2 million years ago (Hoshino, 1975). Since then, ancestral T. flavoviridis species in the former Okinawa continent were scattered to these islands and have been kept isolated. We recently found in chromatographic analysis that myotoxic BPI and BPII, which are expressed abundantly in Amami-Oshima and Tokunoshima T. flavoviridis venoms, are completely missing from Okinawa T. flavoviridis venom (Chijiwa et al., 2000). Northern blot analysis and single-stranded conformational polymorphism-polymerase chain reaction (PCR) (Orita et al., 1989) for venom-gland mRNAs of T. flavoviridis from three islands indicated that BPI and BPII mRNAs are not expressed in Okinawa T. flavoviridis venom gland. When PCR was conducted for Okinawa T. flavoviridis genomic DNA species with a variety of primers, it became evident that although the upstream and downstream regions of the genes for BPI and/or BPII exist, most of the second exon at its 30 end and the first half of the second intron were lost. Analysis of the sequences containing polymorphisms (positions 236 and 1353) between the genes for BPI and BPII (Nakashima et al., 1993) confirmed that the upstream region of the gene for BPI down to the 50 moiety of the second exon is followed, with a possible insertion, by the downstream region of the gene for BPII starting from the middle portion of the second intron in the Okinawa T. flavoviridis genome (Fig. 7). Ancestral T. flavoviridis in the former Okinawa continent had genes for BPI and BPII that were arranged in tandem in this order on one chromosome. After separation into islands 1 – 2 million years ago, the genes for BPI and BPII were disrupted to form a pseudogene only in Okinawa T. flavoviridis. From the above results, it is inferred that Okinawa island was first separated from a former island and the rest was later disunited into Amami-Oshima and Tokunishima islands. The loss of BPI and BPII greatly decreases the venom toxicity of Okinawa T. flavoviridis because their myotoxic

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Fig. 7. A conceptual model showing the pseudogene derived from the genes encoding BPI and BPII in the Okinawa T. flavoviridis genome.

activity is several times stronger than that of PLA2 (Kihara et al., 1992) and the combined quantity of BPI and BPII is comparable to that of PLA2 in the venom. The lack of strongly toxic BPI and BPII would seem to be of great disadvantage to Okinawa T. flavoviridis, although it seems to thrive well enough. One possibility for the disappearance of strongly toxic BPI and BPII from Okinawa T. flavoviridis venom is that the prey species of Okinawa T. flavoviridis are partly or largely different from those of Amami-Oshima and Tokunoshima T. flavoviridis, such that strong venom toxicity might not be required for Okinawa T. flavoviridis. It seems that the lack of necessity for a strongly toxic venom gave rise to the inactivation or disappearance of genes encoding such highly toxic proteins. According to

a statistical investigation in Amami-Oshima, for example, T. flavoviridis snakes feed on rats (86%) and birds, reptiles and amphibians (14% in total) in the farmlands (Mishima, 1966). However, in Yambaru, a mountain area in the northern part of Okinawa, T. flavoviridis snakes mostly (approximately 90%) prey on Holst’s frogs (Rana holsti, a native animal designated by Okinawa Prefecture) inhabiting the streams in this area (K. Terada, personal communication). This natural, wild area seems to reflect the environment in Okinawa in which T. flavoviridis snakes lived in ancient times. It could be assumed that these feeding habits over a long period affected the venom components in Okinawa T. flavoviridis. This might be a sort of adaptation to the environment.

Fig. 8. Phylogenetic tree constructed for Crotalinae snake venom group II PLA2s based on their amino acid sequences (Fig. 1) and the neighborjoining algorithm. The numerals at the nodes represent the bootstrap confidence. The branch lengths are drawn to scale and represent the numbers of amino acid substitution per site.

M. Ohno et al. / Toxicon 42 (2003) 841–854

Recently we isolated a new PLA2 (an [Asp49]PLA2), named PL-Y, from Okinawa T. flavoviridis venom (Chijiwa et al., 2003b). This is different only at three positions from PLA-B from Tokunoshima T. flavoviridis venom (Yamaguchi et al., 2001). A new PLA2, named PLA-B0 , which is similar to PLA-B, was cloned from Amami-Oshima T. flavoviridis venom gland (Chijiwa et al., 2003b). Three T. flavoviridis venom basic [Asp49]PLA2 isozymes, PL-Y (Okinawa), PLA-B (Tokunoshima) and PLA-B0 (AmamiOshima), are identical in the N-terminal half but have one to four amino acid substitutions in the b1-sheet and its vicinity. Such interisland sequence diversities are due to isolation in different environments over a long period and appear to have been brought about by natural selection for point mutation in their genes. Otherwise, PLA2, a major PLA2, ubiquitously exists in the venoms of T. flavoviridis snakes from the three islands with one to three synonymous substitutions in their cDNAs. It is assumed that PLA2 gene is a prototype among T. flavoviridis venom PLA2 isozyme genes and has hardly undergone nonsynonymous mutation as a principal toxic component (Chijiwa et al., 2003b). Phylogenetic analysis based on the amino acid sequences revealed that T. flavoviridis PLA2 isozymes are clearly separated into three branches, PLA2 type, basic [Asp49]PLA2 type and [Lys49]PLA2 type (Chijiwa et al., 2003b). Basic [Asp49]PLA2-type isozymes may manifest their own particular toxic functions, which are different from the isozymes of PLA2 type and [Lys49]PLA2 type. More recently we isolated a novel, neurotoxic [Asp49 ]PLA2 , named PLA-N, from Amami-Oshima T. flavoviridis venom (Chijiwa et al., 2003a). The cDNA encoding PLA-N was also cloned from Tokunoshima T. flavoviridis venom gland. However, only the cDNA clones encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), were obtained from Okinawa T. flavoviridis venom-gland cDNA library. The substitution Lys (AAA)/Asn (AAC) was noted at position 116 (Fig. 1). This interisland mutation is reminiscent of the case of BPI

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Table 5 KA =KS values for pairs of the cDNAs encoding Crotalinae venom neurotoxic PLA2s Pair of cDNAs

PLA-N vs trimucrotoxin PLA-N vs agkistrotoxin PLA-N vs crotoxin B Trimucrotoxin vs agkistrotoxin Trimucrotoxin vs crotoxin B Agkistrotoxin vs crotoxin B

Mature protein-coding region KS

KA

KA =KS

0.0760 0.190 0.190 0.228 0.217 0.249

0.0387 0.0775 0.0775 0.0916 0.161 0.166

0.497 0.375 0.375 0.360 0.707 0.622

and BPII isozymes which are expressed in Amami-Oshima and Tokunoshima T. flavoviridis venom gland but not in Okinawa T, flavoviridis venom gland (Chijiwa et al., 2000). When a phylogenetic tree was constructed for Crotalinae snake venom PLA2s, including neurotoxic PLA2s, they were clearly separated into four branches, PLA2 type, basic [Asp49]PLA2 type, [Lys49]PLA2 type and neuro[Asp49]PlA2 type (Fig. 8) (Chijiwa et al., 2003a). When evolutionary calculation was made for the nucleotide sequences of T. flavoviridis PLA2s across the four branches, accelerated evolution was observed because KA =KS values were larger than one and KN =KS values were 0.26– 0.42 (Table 4). However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA2s, that is, those belonging to the same branch (Fig. 8), their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution because KA =KS values were 0.36– 0.71 (Table 5). It is highly likely that ancestral genes of neurotoxic PLA2s evolved in an accelerated manner until they had acquired neurotoxic function and, after that, they have evolved with less frequent mutation, possibly for functional conservation.

References Table 4 KA =KS and KN =KS values for pairs of the cDNAs encoding T. flavoviridis venom PLA2s Pair of cDNAs

PLA-N vs PL-X0 PLA-N vs PLA2 PLA-N vs BPI PLA-N vs PL-Y PL-Y vs PLA2 PL-Y vs BPI PL-X0 vs PLA2 PL-X0 vs BPI PLA2 vs BPI

50 -UTR KN

0.060 0.060 0.060 0.060 0.057 0.057 0.057 0.058 0.076

Mature protein-coding region KS

KA

KA =KS

KN =KS

0.144 0.229 0.237 0.144 0.181 0.162 0.181 0.162 0.229

0.189 0.237 0.278 0.193 0.226 0.234 0.226 0.231 0.283

1.36 1.04 1.22 1.40 1.29 1.54 1.30 1.51 1.30

0.417 0.262 0.253 0.417 0.315 0.352 0.315 0.358 0.332

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