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Regional and accelerated molecular evolution in group I snake venom gland phospholipase A2 isozymes
Toxicon 38 (2000) 449±462 www.elsevier.com/locate/toxicon
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Regional and accelerated molecular evolution in group I snake venom gland phospholipase A2 isozymes Yoshiro Chuman a, Ikuo Nobuhisa a, Tomohisa Ogawa a, Masanobu Deshimaru a, Takahito Chijiwa a, Nget-Hong Tan b, Yasuyuki Fukumaki c, Yasuyuki Shimohigashi a, FreÂdeÂric Ducancel d, Jean-Claude Boulain d, Andre MeÂnez d, Motonori Ohno e,* a
Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka 812, Japan b Department of Biochemistry, University of Malaya, Kuala Lumpur, Malaysia c Institute of Genetic Information, Kyushu University, Higashi-ku, Fukuoka 812, Japan d DeÂpartement d'Ingenierie et d'Etudes des ProteÂines, CEA Saclay, 91191Gif-sur-Yvette, France e Kumamoto Institute of Technology, Kumamoto 860, Japan Received 2 March 1999; accepted 1 June 1999
Abstract In accordance with detection of a few phospholipase A2 (PLA2) isozyme genes by Southern blot analysis, only two cDNAs, named NnkPLA-I and NnkPLA-II, encoding group I PLA2s, NnkPLA-I and NnkPLA-II, respectively, were isolated from the venom gland cDNA library of Elapinae Naja naja kaouthia of Malaysia. NnkPLA-I and NnkPLAII showed four amino acid substitutions, all of which were brought about by single nucleotide substitution. No existence of clones encoding CM-II and CM-III, PLA2 isozymes which had been isolated from the venom of N. naja kaouthia of Thailand, in Malaysian N. naja kaouthia venom gland cDNA library was veri®ed by dot blot hybridization analysis with particular probes. NnkPLA-I and NnkPLA-II diered from CM-II and CM-III with
Abbreviations: PLA2 phospholipase A2; UTR, untranslated region; RT-PCR, reverse transcriptionpolymerase chain reaction; SSC, standard saline citrate; NnkPLA, Naja naja kaouthia venom gland PLA2; NnkPLA, cDNA encoding NnkPLA; bp, base pair(s). * Corresponding author. Fax: +81-96-326-3000. E-mail address: [email protected] (M. Ohno) 0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 9 ) 0 0 1 6 5 - 8
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four and two amino acid substitutions, respectively, suggesting that their molecular evolution is regional. The comparison of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s indicated that the 5 '- and 3'-untranslated regions are more conserved than the mature protein-coding region and that the number of nucleotide substitutions per nonsynonymous site is almost equal to that per synonymous site in the protein-coding region, suggesting that accelerated evolution has occurred in group I venom gland PLA2s possibly to acquire new physiological functions. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Naja naja kaouthia venom gland; Group I PLA2 isozymes; cDNA cloning; Regional variation; Accelerated evolution
1. Introduction Phospholipase A2 (PLA2) [EC 3.1.1.4] catalyzes the hydrolysis of the 2-acyl ester linkage of 3-sn-phosphoglycerides with the requirement of Ca2+ (Dijkstra et al., 1981,1983). Snake venom PLA2s are classi®ed into groups I and II based on the mode of disul®de bond pairings (Dufton and Hider, 1983). In addition to the six disul®de bonds shared between groups I and II PLA2s, group I PLA2s construct the 7th disul®de bond between cysteines at positions 11 and 69. Instead, group II PLA2s have a C-terminal extension containing Cys-122 forming a disul®de bond with Cys-50. Viperidae (Viperinae and Crotalinae) snake venom PLA2s belong to a group II PLA2 family. By contrast, group I PLA2s are found in Elapidae (Elapinae and Hydrophiinae) snake venoms. Not only the amino acid sequences but also the three dimensional structures are known to be similar between groups I and II PLA2s (Brunie et al., 1985; Wery et al., 1991; Suzuki et al., 1995). Analysis of the nucleotide sequences of cDNAs and genes encoding venom gland PLA2 isozymes of Crotalinae snakes such as Trimeresurus ¯avoviridis, T. gramineus (Ogawa et al., 1992,1995,1996; Nakashima et al., 1993,1995) and T. okinavensis (Nobuhisa et al., 1996) indicated that the protein-coding region except for the signal peptide-coding domain is much more variable than the noncoding regions including introns and that nucleotide substitutions causing amino acid change have occurred equally to or more frequently than those causing no amino acid change in the mature protein-coding region. The same structural features have also been noted for genes encoding venom gland PLA2 isozymes of Crotalus scutulatus scutulatus (Crotalinae) (John et al., 1994) and Vipera ammodytes (Viperinae) (Kordis and Gubensek, 1996). These ®ndings revealed that Viperidae snake venom gland PLA2s have evolved in an accelerating manner to gain diverse physiological activities (Kihara et al., 1992; Shimohigashi et al., 1996; Ohno et al., 1998). Therefore, it is of great interest to examine whether accelerated evolution has occurred in group I snake venom gland PLA2s as has been observed for group II snake venom gland PLA2s.
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Two group I PLA2 isozymes, called CM-II and CM-III, have been isolated from the venom of Naja naja kaouthia (Elapinae, Siamese cobra) of Thailand and sequenced (Francois and Nico, 1980). For the reason, in the work reported here, cDNAs encoding PLA2s in the venom gland of N. naja kaouthia of Malaysia have been cloned. The amino acid sequences deduced were compared with those of CM-II and CM-III in terms of possibility of regional variation. The nucleotide sequences were analyzed together with those for other group I snake venom gland PLA2 cDNAs in order to assess the mode of molecular evolution. 2. Materials and methods 2.1. Materials Specimens of N. naja kaouthia were collected in central Malaysia. Oligonucleotide primers were custom-synthesized by Biologica Co. (Nagoya, Japan). Restriction enzymes EcoRI, NotI and BamHI were purchased from Takara Shuzo Co. (Siga, Japan). Radio-labeled nucleotides [g-32P]dATP (167 TBq/mmol) and [a-32P]dCTP (111 TBq/mmol) were obtained from ICN Biomedicals Inc. (Costa Mesa, CA). [a-35S]dATP was from Amersham (Buckinghamshire). 2.2. Construction of cDNA library The venom glands were excised from a mature N. naja kaouthia snake. Poly(A)+RNAs were puri®ed by using Daynabeads mRNA puri®cation kit (Daynal, Lake Success, NY) according to the manufacturer's protocol. Venom gland cDNA library was constructed with cDNA synthesis kit and lgt 10 cloning system (Amersham). cDNA ®rst strand was synthesized with poly(A)+RNA and oligo(dT)12±18 primer. Linkers containing a NotI restriction site and an EcoRI site were added to cDNAs at the 5' and 3 ' ends, respectively. These cDNAs were inserted into l ExCell vector (Pharmacia, Upsala) at NotI and EcoRI sites. Escherichia coli NM522 was transformed with the recombinant plasmids to construct the cDNA library. Completed library contained 6 105 primary recombinants. 2.3. cDNA cloning In order to obtain partial fragment(s) of cDNA(s) encoding PLA2(s) of Malaysian N. naja kaouthia venom gland, reverse transcription-polymerase chain reaction (RT-PCR) was carried out for its venom gland mRNAs with primers designed by choosing the sequences conserved between CM-II and CM-III, PLA2 isozymes of Thailand N. naja kaouthia venom (Francois and Nico, 1980), and composed of the amino acid residues with fewer degenerated codons. These are the sense primer 5 '-TAT/C CAA/C TTA/C AAA/G AAT/C ATG AT-3 ' (OG1;
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corresponding to the amino acid sequence, 3Tyr±Gln±Phe±Lys±Asn±Met±Ile9) and the antisense primer 3 '-ACA/G TTT/C TTA/G CCT/C/A/G/TTA/G TTA/ G CG-5 ' (OG2; corresponding to the sequence, 78Cys±Lys±Asn±Gly±Asn±Asn± Ala84). The ampli®ed DNAs (D1) were sequenced by the dideoxy chain termination method (Sanger et al., 1977) after subcloning into Bluescript II SK+ vector, radio-labeled with the Multiprime DNA labeling system (Amersham) using [a-32P]dCTP by the random priming method (Feinberg and Vogelstein, 1983) and employed for screening of the cDNA library by the plaque hybridization method (Sombrook et al., 1989). Hybridization was carried out at 658C for 12 h in a mixture of 5 standard saline citrate (1 SSC: 1.0 M sodium chloride, 0.1 M sodium citrate), 50 mM sodium phosphate pH 6.5, 0.5% SDS, 2 Denhardt's solution (100 Denhardt's solution: 0.1% Ficoll 400, 0.1% polyvinyl-pyrrolidone, 0.1% bovine serum albumin), 50% formamide and 100 mg/ml sonicated salmon sperm DNA. The membranes were ®nally washed twice for 30 min at 658C in 0.2 SSC and 0.1% SDS. Two cDNA clones, named NnkPLA-I and NnkPLA-II, encoding PLA2s, NnkPLA-I and NnkPLA-II, were thus obtained and sequenced. The cDNA library was also screened with probe D2 which contains the 3 ' moiety of the protein-coding region and the 3'-untranslated region (UTR), being prepared by PCR for NnkPLA-I-inserted Bluescript II SK+ vector with the sense primer 5 '-TATCGACCTCAA-GGCACG-3 ' (OG3; corresponding to the sequence, 112Ile± and the antisense primer 5'Asp±Leu±Lys±Ala±Arg117) TAATACGACTCACTATAGGG-3 ' (T7 primer: OG4) of Bluescript II SK+ vector. 2.4. Dot blot hybridization analysis In order to amplify all possible cDNAs encoding PLA2 isozymes other than NnkPLA-I and NnkPLA-II, PCR was conducted against cDNA library. Primers utilized were 5 '-CTTCACCACGGACAGATG-3 ' (OG5) and 5'GCCATGTGCAGGTTAGTAA-3 ' (OG6), segments of the 5'- and 3 '-UTRs, respectively, being common between NnkPLA-I and NnkPLA-II. The PCR products (D3) were subjected to hybridization at 488C for 12 h with oligonucleotide (OG7) corresponding to a part of the nucleotide sequences deduced from CM-II and CM-III and with oligonucleotide (OG8) speci®c to NnkPLA-I and NnkPLA-II (see Results section). These probes were endlabeled with [g-32P]dATP by using T4 polynucleotide kinase (Takara). The membranes were washed in 2 SSC for 15 min at intervals of 28C from 50 to 608C and analyzed by a BAS 2000 Bio imaging analyzer (Fuji Film Co., Tokyo). 2.5. Southern blot analysis Genomic DNAs (15 mg) extracted from the liver of Malaysian N. naja kaouthia were digested with EcoRI and BamHI, respectively, electrophoresed on 0.7% agarose gels and transferred onto Hybond-N+ nylon membranes (Amersham). The membranes were hybridized with three probes derived from NnkPLA-I,
Fig. 1. The nucleotide sequences of NnkPLA-I and NnkPLA-II together with those of BmPLA2 (Bungarus multicinctus ) (Danse et al., 1990), LlPLA2 (Laticauda laticauda ) (Guignery-Frelat et al., 1987) and NsPLA2 (Notechis scutstus ) (Ducancel et al., 1988a) which encode group I venom gland PLA2s. The nucleotides of the protein-coding region are shown by uppercase letters and those of the noncoding region by lowercase letters. Dots indicate the same nucleotide residue as in NnkPLA-I. A mark W denotes the start site of the mature protein.
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Fig. 2. The amino acid sequences of NnkPLA-I and NnkPLA-II deduced from the nucleotide sequences of their cDNAs together with those of CM-II and CM-III analyzed from PLA2 proteins isolated from the venom of Naja naja kaouthia of Thailand. The regions of a-helices (a1±a4), b-wing (b 1) and loops 1±5 are indicated.
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namely, the coding-region probe (nucleotides 103±349), the 3 '-UTR probe (nucleotides 420±640) and the full-length probe (nucleotides 1±640) which were internallabeled with [a-32P]dCTP. The membranes were washed in 2 SSC and 0.1% SDS at 658C and analyzed by the imaging analyzer. 2.6. Evolutionary calculation For assessment of evolutionary characteristics of genes encoding group I snake venome gland PLA2s, the nucleotide sequences of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s were compared and the number of nucleotide substitutions per site (KN) in the 5'- and 3'-UTRs and the numbers of nucleotide substitutions per synonymous site (KS) and per nonsynonymous site (KA) in the protein-coding region were computed for pairs of cDNAs according to the method of Nei and Gojobori (1986) using ODEN package developed by Ina (National Institute for Genetics, Mishima, Japan). The nucleotide sequence data reported in this paper are available from DDBJ, EMBL and GenBank databases with accession numbers of AB011388 and AB011389. 3. Results Partial fragment(s) of cDNA(s) encoding PLA2(s) of Malaysian N. naja kaouthia venom gland were ®rst prepared by RT-PCR for its venom gland mRNAs with primers OG1 and OG2 designed based on the partial amino acid sequences of CM-II and CM-III as described above. The resulting products (D1) consisted of 246 bp which coded for the residues 3±84 of PLA2(s). Screening of Malaysian N. naja kaouthia venom gland cDNA library with D1 by the plaque hybridization method gave 41 positive clones and their sequences were determined. Only two PLA2 cDNAs, NnkPLA-I and NnkPLA-II, were identi®ed. On the other hand, screening of the cDNA library with D2 probe, which contains the 3' moiety of the protein-coding region and the 3 '-UTR, gave 24 positive clones. However, these were identi®ed as either NnkPLA-I or NnkPLA-II. In Fig. 1 are shown the nucleotide sequences of NnkPLA-I and NnkPLA-II which consist of a 5 '-UTR of 16 or 18 bp, an open reading frame of 438 bp, a stop codon and a 3 '-UTR of 163 bp. Both cDNA clones encoded proteins of 146 amino acids, including the signal peptide of 27 amino acids (Fig. 2). Their amino acid sequences indicated that they belong to a group I PLA2 family. The amino acid sequences of NnkPLA-I and NnkPLA-II were partly dissimilar to those of CM-II and CM-III, respectively (Fig. 2). Therefore, all possible cDNAs encoding PLA2s were ampli®ed by PCR against cDNA library with primers, OG5 and OG6, from the 5 '- and 3'-UTRs common to both NnkPLA-I and NnkPLA-II and possibly to cDNA clones, if any, of CM-II and CM-III. The ampli®ed DNAs (D3) were employed for dot blot hybridization to search cDNAs encoding CM-II and CM-III. The probes utilized were 5'-
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CAACTGCTATGATGAAGCCGA-3 ' (OG7) which corresponds to residues 48± 55 of CM-II and CM-III and 5 '-CAACTGCTATAATGAAGCCGA-3 ' (OG8) which corresponds to residues 48±55 of NnkPLA-I and NnkPLA-II. One of the major dierences between NnkPLA-I and NnkPLA-II and CM-II and CM-III was noted at position 52: Asn for NnkPLA-I and NnkPLA-II and Asp for CM-II and CM-III which are brought about by one nucleotide substitution A to G. The oligonucleotide OG8 speci®c to NnkPLA-I and NnkPLA-II hybridized to D3, in addition to NnkPLA-I and NnkPLA-II, even at 608C (Fig. 3). Although the oligonucleotide OG7 for cDNA clones, if any, of CM-II and CM-III hybridized to D3 at 508C, it was washed out at 608C in any cases (Fig. 3). Hybridization of OG7 and OG8 was not observed for vector pTV118N employed as a control. These results revealed that cDNAs encoding NnkPLA-I and NnkPLA-II de®nitely exist in the cDNA library, but cDNAs corresponding to CM-II and CM-III are not present (Fig. 2). The presence of only two PLA2 isozymes in N. naja kaouthia venom is in
Fig. 3. Dot blot hybridization of NnkPLA-I, NnkPLA-II and PLA2 cDNAs (D3) ampli®ed by PCR from N. naja kaouthia (Malaysia) venom gland cDNA library (see Materials and methods section) with probe OG8 (5'-CAACTGCTATAATGAAGCCGA-3') speci®c for NnkPLA-I and NnkPLA-II (A) and with probe OG7 (5'-CAACTGCTATGATGAAGCCGA-3 ') speci®c for cDNAs, if any, of CM-II and CM-III (B). After hybridization at 488C for 12 h, the membranes were washed at 28C intervals from 50 to 608C. The hybridization pro®les at 50 and 608C are shown. a1, NnkPLA-I; a2, NnkPLA-II; b1, D3; and b2, vector pTV118N as a negative control.
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contrast to the fact that Crotalidae snake venoms contain more PLA2 isozymes (Ogawa et al., 1992,1995,1996; Nakashima et al., 1993,1995; Nobuhisa et al., 1996). In order to assess how many PLA2 isozyme genes exist in N. naja kaouthia genome, Southern blot analysis was conducted for its genomic DNAs extracted from its liver and digested with restriction enzymes EcoRI and BamHI. As shown in Fig. 4, only two bands were detected when the coding-region probe (nucleotides 103±349) which corresponds to the mature protein-coding region of NnkPLA-I was employed. A similar result was obtained for the full-length probe (nucleotides 1±640). Even when the 3 '-UTR probe (nucleotides 420±640) was used, the number of bands detected was not likely to be increased (Fig. 4). These results strongly suggested that N. naja kaouthia genome contains only two PLA2 isozyme genes. When the nucleotide sequences of NnkPLA-I and NnkPLA-II were compared, six nucleotide substitutions were noted, two in the signal peptide domain and four in the open reading frame. All the substitutions were found to occur at nonsynonymous site. Proÿ12, Pheÿ8, Asn15, Arg59, Asp81 and Asp83 in NnkPLA-I are replaced by Serÿ12, Serÿ8, Ser15, Gly59, Gly81 and Asn83, respectively, in NnkPLA-II (the positions with minus number indicate those in the signal peptide). The nucleotide sequences of NnkPLA-I and NnkPLA-II were compared with
Fig. 4. Southern blot analysis of Malaysian N. naja kaouthia genomic DNAs. Three probes, the codingregion probe (nucleotides 103±349), the 3'-UTR probe (nucleotides 420±640) and the full-length probe (nucleotides 1±640), were prepared from NnkPLA-I (A). Genomic DNAs extracted from the liver were digested with EcoRI and BamHI, respectively, electropheresed on 0.8% agarose gels, transferred onto Hybond-N+ nylon membranes and hybridized with the probes (B).
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those of cDNAs, BmPLA2, NsPLA2, LlPLA2 and AlPLA2, which encode group I snake venom gland PLA2s, BmPLA2 (Bungarus multicinctus ) (Danse et al., 1990), NsPLA2 (Notechis scutatus ) (Ducancel et al., 1988a), LlPLA2 (Laticauda laticauda ) (Guignery-Frelat et al., 1987) and AlPLA2 (Aipysurus laevis ) (Ducancel et al., 1988b), respectively (the nucleotide sequences of BmPLA2, LlPLA2 and NsPLA2 are in Fig. 1). It was found that the 5'- and 3 '-UTRs (86±87%) are more conserved than the protein-coding regions (74%), being in contrast to ordinary genes in which the noncoding regions are more variable than the protein-coding region (Kubo et al., 1987; Ohno et al., 1987; Matsuoka et al., 1988; Strathmann and Simon, 1990). Thus, the signi®cance of group I snake venom gland PLA2 isozymes in molecular evolution was evaluated by mathematical analysis of the nucleotide sequences of NnkPLA-I, NnkPLA-II, BmPLA2, NsPLA2, LlPLA2 and AlPLA2. The KN values for the 5 '- and 3 '-UTRs and the KS and KA values for the protein-coding region were computed for all the pairs of cDNAs. Table 1 shows the data for 11 pairs. The values between NnkPLA-I and NnkPLA-II was excluded because these have the highly homologous sequences. These genes appear to have diverged quite recently. The following two characteristics are prominent from the data. First, KN/KS values are smaller than 1, indicating that nucleotide substitutions have occurred in the protein-coding region more frequently than in the UTRs. Second, KA/KS values are close to 1, indicating that nucleotide substitutions in the protein-coding region tend to cause amino acid change. These facts indicate that group I snake venom gland PLA2s have evolved in an accelerating manner.
Table 1 The KS, KA, KN, KN/KS and KA/KS values for 11 pairs of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2 isozymesa Pair of cDNAs
KS
KA
KN
KN/KS
KA/KS
NnkPLA-I vs. BmPLA2 NnkPLA-I vs. NsPLA2 NnkPLA-I vs. LlPLA2 NnkPLA-I vs. AlPLA2 NnkPLA-II vs. BmPLA2 NnkPLA-II vs. NsPLA2 NnkPLA-II vs. LlPLA2 NnkPLA-II vs. AlPLA2 BmPLA2 vs. NsPLA2 BmPLA2 vs. LlPLA2 NsPLA2 vs. LlPLA2
0.268 0.355 0.463 0.364 0.275 0.356 0.466 0.369 0.391 0.413 0.216
0.277 0.346 0.428 0.378 0.274 0.345 0.408 0.377 0.346 0.415 0.241
0.162 0.152 0.166 0.150 0.162 0.152 0.166 0.150 0.089 0.149 0.077
0.604 0.432 0.356 0.411 0.588 0.426 0.356 0.406 0.227 0.361 0.356
1.032 0.974 0.925 1.040 0.996 0.970 0.876 1.022 0.885 1.005 1.245
a Other combinations other than a pair of NnkPLA-I and NnkPLA-II gave the similar values as listed in each column.
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4. Discussion Screening of Malaysian N. naja kaouthia venom gland cDNA library by probe D1, DNA fragment(s) prepared by RT-PCR for its venom gland mRNAs with primers OG1 and OG2, gave two PLA2 cDNAs, NnkPLA-I and NnkPLA-II. No additional clones were detected even when probe D2 containing the 3' moiety of the protein-coding region and the 3 '-UTR was employed. Similarly as this fact, Southern blot analysis (Fig. 4) implied that N. naja kaouthia genome codes for only two PLA2 isozymes. NnkPLA-I diers from NnkPLA-II in sequence only at four positions, 15, 59, 81 and 83 (Fig. 2). The sequences of NnkPLA-I and NnkPLA-II are similar to those of CM-II and CM-III, respectively. Asn-52, Gly-80, Asp-81 and Asp-83 of NnkPLA-I are replaced by Asp, Asn, Gly and Asn, respectively, in CM-II. On the other hand, NnkPLA-II diers from CM-III only at two positions. Asp-20 and Asn-52 of NnkPLA-II are substituted by Asn and Asp, respectively, in CM-III. cDNA cloning and dot blot hybridization (Fig. 3) showed that no clones corresponding to CM-II and CM-III exist in Malaysian N. naja kaouthia venom gland cDNA library. This reveals that PLA2s from Malaysian N. naja kaouthia venom are distinct from those of Thailand N. naja kaouthia venom. Such geographical variation of venom components within the same snake species has been known (Chippaux et al., 1991; Daltry et al., 1996; Tsai et al., 1996). For instance, according to Tsai et al. (1996), Russell's viper PLA2s can be separated into two types; one having Asn (Thailand, Taiwan, Burma and Pakistan) and the other Ser at the N-terminus (Southern India and Sri Lanka). Pair of CM-II and NnkPLA-I and that of CM-III and NnkPLA-II appear to be in orthologous relationship in evolution. All snake venom PLA2s whose crystal structures have been clari®ed possess a common scaold constructed from four a-helices and a b-sheet which are connected by ®ve loops (Fig. 2) (Ohno et al., 1998). The four amino acid substitutions between NnkPLA-I and NnkPLA-II are found only in the loop regions. Even in CM-II and CM-III, three of four substitutions occur in the loop regions. Diverged biological functions of proteins appear to be exerted or carried in the loop region. Loop 2 of PLA2s are known to construct a part of the Ca2+ binding site (Dijkstra et al., 1981,1983). Substitutions between NnkPLA-I and NnkPLA-II occur at loops 3 and 4 which are believed to construct a lipid-water interface recognition site. Although two Lys-49-PLA2s, called BPI and BPII, from T. ¯avoviridis venom show only one amino acid replacement (Asp/Asn) at position 67 in loop 3, they exhibited considerably dierent potencies in enzymatic activity and in contraction of the isolated muscle tissue (Shimohigashi et al., 1995,1996; Ohno et al., 1998). It is thus likely that NnkPLA-I and NnkPLA-II, or CM-II and CM-III, have distinct physiological functions due probably to dierent surface interactions. Analysis of the nucleotide sequences of NnkPLA-I, NnkPLA-II, BmPLA2, NsPLA2, L1PLA2 and A1PLA2, all of which encode group I snake venom gland PLA2s, showed that the protein-coding region except for the signal peptide-coding
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domain is more variable than the 5 '- and 3 '-UTRs (Fig. 1) and that nonsynonymous nucleotide substitutions are almost equal to synonymous nucleotide substitutions in the protein-coding region (Table 1). These features are the same as have been observed for genes encoding group II Viperidae snake venom gland PLA2 isozymes which are thought to have evolved in an accelerating manner (Nakashima et al., 1993,1995; John et al., 1994; Ogawa et al., 1995,1996; Kordis and Gubensek, 1996; Nobuhisa et al., 1996). This indicates that group I snake venom gland PLA2 isozymes have also evolved in an accelerating manner, possibly to gain new physiological functions. Thus, accelerated evolution is a common phenomenon for PLA2s in both Viperidae and Elapidae snake venom glands. It has recently been established that serine proteases of Crotalinae snake venom glands have evolved in an accelerating manner (Deshimaru et al., 1996). It may be said that accelerated evolution is universal for plural protein families in snake venom glands.
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Kordis, D., Gubensek, F., 1996. Ammodytoxin C gene helps to elucidate the irregular structure of Crotalinae group II phospholipase A2 genes. Eur. J. Biochem. 240, 83±90. Kubo, K., Ohno, S., Suzuki, K., 1987. Primary structures of human protein kinase C bI and bII dier only in their C-terminal sequences. FEBS Lett. 223, 138±142. Matsuoka, M., Itoh, H., Kozasa, T., Kaziro, Y., 1988. Sequence analysis of cDNA and genomic DNA for a putative pertussis toxin-insensitive guanine nucleotide-binding regulatory protein a subunit. Proc. Natl. Acad. Sci. USA 85, 5384±5388. Nakashima, K., Ogawa, T., Oda, N., Hattori, M., Sakaki, Y., Kihara, H., Ohno, M., 1993. Accelerated evolution of Trimeresurus ¯avoviridis venom gland phospholipase A2 isozymes. Proc. Natl. Acad. Sci. USA 90, 5964±5968. Nakashima, K., Nobuhisa, I., Deshimaru, M., Nakai, M., Ogawa, T., Shimohigashi, Y., Fukumaki, Y., Hattori, M., Sakaki, Y., Hattori, S., Ohno, M., 1995. Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes. Proc. Natl. Acad. Sci. USA 92, 5605±5609. Nei, M., Gojobori, T., 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3, 418±426. Nobuhisa, I., Nakashima, K., Deshimaru, M., Ogawa, T., Shimohigashi, Y., Fukumaki, Y., Sakaki, Y., Hattori, S., Kihara, H., Ohno, M., 1996. Accelerated evolution of Trimeresurus okinavensis venom gland phospholipase A2 isozyme encoding genes. Gene 172, 267±272. Ogawa, T., Oda, N., Nakashima, K., Sasaki, H., Hattori, M., Sakaki, Y., Kihara, H., Ohno, M., 1992. Unusually high conservation of untranslated sequences in cDNAs for Trimeresurus ¯avoviridis phospholipase A2 isozymes. Proc. Natl. Acad. Sci. USA 89, 8557±8561. Ogawa, T., Nakashima, K., Oda, N., Shimohigashi, Y., Ohno, M., 1995. Trimeresurus ¯avoviridis venom gland phospholipase A2 isozymes genes have evolved via accelerated substitutions. J. Mol. Recogn. 8, 40±46. Ogawa, T., Nakashima, K., Nobuhisa, I., Deshimaru, M., Shimohigashi, Y., Fukumaki, Y., Sakaki, Y., Hattori, S., Ohno, M., 1996. Accelerated evolution of snake venom phospholipase A2 isozymes for acquisition of diverse physiological functions. Toxicon 34, 1229±1236. Ohno, S., Kawasaki, H., Imajoh, S., Suzuki, K., Inagaki, M., Yokokura, H., Sakoh, T., Hidaka, H., 1987. Tissue-speci®c expression of three distinct types of rabbit protein kinase C. Nature 325, 161± 166. Ohno, M., MeÂnez, R., Ogawa, T., Danse, J.M., Shimohigashi, Y., Fromen, C., Ducancel, F., ZinnJustin, S., Le Du, M.H., Boulain, J.-C., Tamiya, T., MeÂnez, A., 1998. Molecular evolution of snake toxins: is the functional diversity of snake toxins associated with a mechanism of accelerated evolution? In: Moldave, K. (Ed.), Progress in Nucleic Acid Research and Molecular Biology, 59. Academic Press, New York, pp. 307±364. Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463±5467. Shimohigashi, Y., Tani, A., Matsumoto, H., Nakashima, K., Yamaguchi, Y., Oda, N., Takano, Y., Kamiya, H., Kishino, J., Arita, H., Ohno, M., 1995. Lysine-49-phospholipases A2 from Trimeresurus ¯avoviridis venom are membrane-acting enzymes. J. Biochem. (Tokyo) 118, 1037±1044. Shimohigashi, Y., Tani, A., Yamaguchi, Y., Ogawa, T., Ohno, M., 1996. Discriminatory recognition of membrane phospholipids by lysine-49-phospholipases A2 from Trimeresurus ¯avoviridis venom. J. Mol. Rocogn. 9, 639±643. Sombrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular cloning, a laboratory manual, 2. Cold Spring Harbor Laboratory Press, New York, pp. 9.16±9.19. Strathmann, M., Simon, M.I., 1990. G protein diversity: A distinct class of a subunits is present in vertebrates and invertebrates. Proc. Natl. Acad. Sci. USA 87, 9113±9117. Suzuki, A., Matsueda, E., Yamane, T., Ashida, T., Kihara, H., Ohno, M., 1995. Crystal structure analysis of phospholipase A2 from Trimeresurus ¯avoviridis (Habu snake) venom at 1.5 AÊ resolution. J. Biochem. (Tokyo) 117, 730±740. Tsai, I.H., Lu, P.J., Su, J.C., 1996. Two types of Russell's viper revealed by variation in phospholipases A2 from venom of the subspecies. Toxicon 34, 99±109. Wery, J.-P., Schevitz, R.W., Clawson, D.K., Bobbitt, J.L., Dow, E.R., Gamboa, G., Goodson Jr, T.,
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Short communication
Regional and accelerated molecular evolution in group I snake venom gland phospholipase A2 isozymes Yoshiro Chuman a, Ikuo Nobuhisa a, Tomohisa Ogawa a, Masanobu Deshimaru a, Takahito Chijiwa a, Nget-Hong Tan b, Yasuyuki Fukumaki c, Yasuyuki Shimohigashi a, FreÂdeÂric Ducancel d, Jean-Claude Boulain d, Andre MeÂnez d, Motonori Ohno e,* a
Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka 812, Japan b Department of Biochemistry, University of Malaya, Kuala Lumpur, Malaysia c Institute of Genetic Information, Kyushu University, Higashi-ku, Fukuoka 812, Japan d DeÂpartement d'Ingenierie et d'Etudes des ProteÂines, CEA Saclay, 91191Gif-sur-Yvette, France e Kumamoto Institute of Technology, Kumamoto 860, Japan Received 2 March 1999; accepted 1 June 1999
Abstract In accordance with detection of a few phospholipase A2 (PLA2) isozyme genes by Southern blot analysis, only two cDNAs, named NnkPLA-I and NnkPLA-II, encoding group I PLA2s, NnkPLA-I and NnkPLA-II, respectively, were isolated from the venom gland cDNA library of Elapinae Naja naja kaouthia of Malaysia. NnkPLA-I and NnkPLAII showed four amino acid substitutions, all of which were brought about by single nucleotide substitution. No existence of clones encoding CM-II and CM-III, PLA2 isozymes which had been isolated from the venom of N. naja kaouthia of Thailand, in Malaysian N. naja kaouthia venom gland cDNA library was veri®ed by dot blot hybridization analysis with particular probes. NnkPLA-I and NnkPLA-II diered from CM-II and CM-III with
Abbreviations: PLA2 phospholipase A2; UTR, untranslated region; RT-PCR, reverse transcriptionpolymerase chain reaction; SSC, standard saline citrate; NnkPLA, Naja naja kaouthia venom gland PLA2; NnkPLA, cDNA encoding NnkPLA; bp, base pair(s). * Corresponding author. Fax: +81-96-326-3000. E-mail address: [email protected] (M. Ohno) 0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 9 ) 0 0 1 6 5 - 8
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four and two amino acid substitutions, respectively, suggesting that their molecular evolution is regional. The comparison of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s indicated that the 5 '- and 3'-untranslated regions are more conserved than the mature protein-coding region and that the number of nucleotide substitutions per nonsynonymous site is almost equal to that per synonymous site in the protein-coding region, suggesting that accelerated evolution has occurred in group I venom gland PLA2s possibly to acquire new physiological functions. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Naja naja kaouthia venom gland; Group I PLA2 isozymes; cDNA cloning; Regional variation; Accelerated evolution
1. Introduction Phospholipase A2 (PLA2) [EC 3.1.1.4] catalyzes the hydrolysis of the 2-acyl ester linkage of 3-sn-phosphoglycerides with the requirement of Ca2+ (Dijkstra et al., 1981,1983). Snake venom PLA2s are classi®ed into groups I and II based on the mode of disul®de bond pairings (Dufton and Hider, 1983). In addition to the six disul®de bonds shared between groups I and II PLA2s, group I PLA2s construct the 7th disul®de bond between cysteines at positions 11 and 69. Instead, group II PLA2s have a C-terminal extension containing Cys-122 forming a disul®de bond with Cys-50. Viperidae (Viperinae and Crotalinae) snake venom PLA2s belong to a group II PLA2 family. By contrast, group I PLA2s are found in Elapidae (Elapinae and Hydrophiinae) snake venoms. Not only the amino acid sequences but also the three dimensional structures are known to be similar between groups I and II PLA2s (Brunie et al., 1985; Wery et al., 1991; Suzuki et al., 1995). Analysis of the nucleotide sequences of cDNAs and genes encoding venom gland PLA2 isozymes of Crotalinae snakes such as Trimeresurus ¯avoviridis, T. gramineus (Ogawa et al., 1992,1995,1996; Nakashima et al., 1993,1995) and T. okinavensis (Nobuhisa et al., 1996) indicated that the protein-coding region except for the signal peptide-coding domain is much more variable than the noncoding regions including introns and that nucleotide substitutions causing amino acid change have occurred equally to or more frequently than those causing no amino acid change in the mature protein-coding region. The same structural features have also been noted for genes encoding venom gland PLA2 isozymes of Crotalus scutulatus scutulatus (Crotalinae) (John et al., 1994) and Vipera ammodytes (Viperinae) (Kordis and Gubensek, 1996). These ®ndings revealed that Viperidae snake venom gland PLA2s have evolved in an accelerating manner to gain diverse physiological activities (Kihara et al., 1992; Shimohigashi et al., 1996; Ohno et al., 1998). Therefore, it is of great interest to examine whether accelerated evolution has occurred in group I snake venom gland PLA2s as has been observed for group II snake venom gland PLA2s.
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Two group I PLA2 isozymes, called CM-II and CM-III, have been isolated from the venom of Naja naja kaouthia (Elapinae, Siamese cobra) of Thailand and sequenced (Francois and Nico, 1980). For the reason, in the work reported here, cDNAs encoding PLA2s in the venom gland of N. naja kaouthia of Malaysia have been cloned. The amino acid sequences deduced were compared with those of CM-II and CM-III in terms of possibility of regional variation. The nucleotide sequences were analyzed together with those for other group I snake venom gland PLA2 cDNAs in order to assess the mode of molecular evolution. 2. Materials and methods 2.1. Materials Specimens of N. naja kaouthia were collected in central Malaysia. Oligonucleotide primers were custom-synthesized by Biologica Co. (Nagoya, Japan). Restriction enzymes EcoRI, NotI and BamHI were purchased from Takara Shuzo Co. (Siga, Japan). Radio-labeled nucleotides [g-32P]dATP (167 TBq/mmol) and [a-32P]dCTP (111 TBq/mmol) were obtained from ICN Biomedicals Inc. (Costa Mesa, CA). [a-35S]dATP was from Amersham (Buckinghamshire). 2.2. Construction of cDNA library The venom glands were excised from a mature N. naja kaouthia snake. Poly(A)+RNAs were puri®ed by using Daynabeads mRNA puri®cation kit (Daynal, Lake Success, NY) according to the manufacturer's protocol. Venom gland cDNA library was constructed with cDNA synthesis kit and lgt 10 cloning system (Amersham). cDNA ®rst strand was synthesized with poly(A)+RNA and oligo(dT)12±18 primer. Linkers containing a NotI restriction site and an EcoRI site were added to cDNAs at the 5' and 3 ' ends, respectively. These cDNAs were inserted into l ExCell vector (Pharmacia, Upsala) at NotI and EcoRI sites. Escherichia coli NM522 was transformed with the recombinant plasmids to construct the cDNA library. Completed library contained 6 105 primary recombinants. 2.3. cDNA cloning In order to obtain partial fragment(s) of cDNA(s) encoding PLA2(s) of Malaysian N. naja kaouthia venom gland, reverse transcription-polymerase chain reaction (RT-PCR) was carried out for its venom gland mRNAs with primers designed by choosing the sequences conserved between CM-II and CM-III, PLA2 isozymes of Thailand N. naja kaouthia venom (Francois and Nico, 1980), and composed of the amino acid residues with fewer degenerated codons. These are the sense primer 5 '-TAT/C CAA/C TTA/C AAA/G AAT/C ATG AT-3 ' (OG1;
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corresponding to the amino acid sequence, 3Tyr±Gln±Phe±Lys±Asn±Met±Ile9) and the antisense primer 3 '-ACA/G TTT/C TTA/G CCT/C/A/G/TTA/G TTA/ G CG-5 ' (OG2; corresponding to the sequence, 78Cys±Lys±Asn±Gly±Asn±Asn± Ala84). The ampli®ed DNAs (D1) were sequenced by the dideoxy chain termination method (Sanger et al., 1977) after subcloning into Bluescript II SK+ vector, radio-labeled with the Multiprime DNA labeling system (Amersham) using [a-32P]dCTP by the random priming method (Feinberg and Vogelstein, 1983) and employed for screening of the cDNA library by the plaque hybridization method (Sombrook et al., 1989). Hybridization was carried out at 658C for 12 h in a mixture of 5 standard saline citrate (1 SSC: 1.0 M sodium chloride, 0.1 M sodium citrate), 50 mM sodium phosphate pH 6.5, 0.5% SDS, 2 Denhardt's solution (100 Denhardt's solution: 0.1% Ficoll 400, 0.1% polyvinyl-pyrrolidone, 0.1% bovine serum albumin), 50% formamide and 100 mg/ml sonicated salmon sperm DNA. The membranes were ®nally washed twice for 30 min at 658C in 0.2 SSC and 0.1% SDS. Two cDNA clones, named NnkPLA-I and NnkPLA-II, encoding PLA2s, NnkPLA-I and NnkPLA-II, were thus obtained and sequenced. The cDNA library was also screened with probe D2 which contains the 3 ' moiety of the protein-coding region and the 3'-untranslated region (UTR), being prepared by PCR for NnkPLA-I-inserted Bluescript II SK+ vector with the sense primer 5 '-TATCGACCTCAA-GGCACG-3 ' (OG3; corresponding to the sequence, 112Ile± and the antisense primer 5'Asp±Leu±Lys±Ala±Arg117) TAATACGACTCACTATAGGG-3 ' (T7 primer: OG4) of Bluescript II SK+ vector. 2.4. Dot blot hybridization analysis In order to amplify all possible cDNAs encoding PLA2 isozymes other than NnkPLA-I and NnkPLA-II, PCR was conducted against cDNA library. Primers utilized were 5 '-CTTCACCACGGACAGATG-3 ' (OG5) and 5'GCCATGTGCAGGTTAGTAA-3 ' (OG6), segments of the 5'- and 3 '-UTRs, respectively, being common between NnkPLA-I and NnkPLA-II. The PCR products (D3) were subjected to hybridization at 488C for 12 h with oligonucleotide (OG7) corresponding to a part of the nucleotide sequences deduced from CM-II and CM-III and with oligonucleotide (OG8) speci®c to NnkPLA-I and NnkPLA-II (see Results section). These probes were endlabeled with [g-32P]dATP by using T4 polynucleotide kinase (Takara). The membranes were washed in 2 SSC for 15 min at intervals of 28C from 50 to 608C and analyzed by a BAS 2000 Bio imaging analyzer (Fuji Film Co., Tokyo). 2.5. Southern blot analysis Genomic DNAs (15 mg) extracted from the liver of Malaysian N. naja kaouthia were digested with EcoRI and BamHI, respectively, electrophoresed on 0.7% agarose gels and transferred onto Hybond-N+ nylon membranes (Amersham). The membranes were hybridized with three probes derived from NnkPLA-I,
Fig. 1. The nucleotide sequences of NnkPLA-I and NnkPLA-II together with those of BmPLA2 (Bungarus multicinctus ) (Danse et al., 1990), LlPLA2 (Laticauda laticauda ) (Guignery-Frelat et al., 1987) and NsPLA2 (Notechis scutstus ) (Ducancel et al., 1988a) which encode group I venom gland PLA2s. The nucleotides of the protein-coding region are shown by uppercase letters and those of the noncoding region by lowercase letters. Dots indicate the same nucleotide residue as in NnkPLA-I. A mark W denotes the start site of the mature protein.
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Fig. 2. The amino acid sequences of NnkPLA-I and NnkPLA-II deduced from the nucleotide sequences of their cDNAs together with those of CM-II and CM-III analyzed from PLA2 proteins isolated from the venom of Naja naja kaouthia of Thailand. The regions of a-helices (a1±a4), b-wing (b 1) and loops 1±5 are indicated.
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namely, the coding-region probe (nucleotides 103±349), the 3 '-UTR probe (nucleotides 420±640) and the full-length probe (nucleotides 1±640) which were internallabeled with [a-32P]dCTP. The membranes were washed in 2 SSC and 0.1% SDS at 658C and analyzed by the imaging analyzer. 2.6. Evolutionary calculation For assessment of evolutionary characteristics of genes encoding group I snake venome gland PLA2s, the nucleotide sequences of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s were compared and the number of nucleotide substitutions per site (KN) in the 5'- and 3'-UTRs and the numbers of nucleotide substitutions per synonymous site (KS) and per nonsynonymous site (KA) in the protein-coding region were computed for pairs of cDNAs according to the method of Nei and Gojobori (1986) using ODEN package developed by Ina (National Institute for Genetics, Mishima, Japan). The nucleotide sequence data reported in this paper are available from DDBJ, EMBL and GenBank databases with accession numbers of AB011388 and AB011389. 3. Results Partial fragment(s) of cDNA(s) encoding PLA2(s) of Malaysian N. naja kaouthia venom gland were ®rst prepared by RT-PCR for its venom gland mRNAs with primers OG1 and OG2 designed based on the partial amino acid sequences of CM-II and CM-III as described above. The resulting products (D1) consisted of 246 bp which coded for the residues 3±84 of PLA2(s). Screening of Malaysian N. naja kaouthia venom gland cDNA library with D1 by the plaque hybridization method gave 41 positive clones and their sequences were determined. Only two PLA2 cDNAs, NnkPLA-I and NnkPLA-II, were identi®ed. On the other hand, screening of the cDNA library with D2 probe, which contains the 3' moiety of the protein-coding region and the 3 '-UTR, gave 24 positive clones. However, these were identi®ed as either NnkPLA-I or NnkPLA-II. In Fig. 1 are shown the nucleotide sequences of NnkPLA-I and NnkPLA-II which consist of a 5 '-UTR of 16 or 18 bp, an open reading frame of 438 bp, a stop codon and a 3 '-UTR of 163 bp. Both cDNA clones encoded proteins of 146 amino acids, including the signal peptide of 27 amino acids (Fig. 2). Their amino acid sequences indicated that they belong to a group I PLA2 family. The amino acid sequences of NnkPLA-I and NnkPLA-II were partly dissimilar to those of CM-II and CM-III, respectively (Fig. 2). Therefore, all possible cDNAs encoding PLA2s were ampli®ed by PCR against cDNA library with primers, OG5 and OG6, from the 5 '- and 3'-UTRs common to both NnkPLA-I and NnkPLA-II and possibly to cDNA clones, if any, of CM-II and CM-III. The ampli®ed DNAs (D3) were employed for dot blot hybridization to search cDNAs encoding CM-II and CM-III. The probes utilized were 5'-
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CAACTGCTATGATGAAGCCGA-3 ' (OG7) which corresponds to residues 48± 55 of CM-II and CM-III and 5 '-CAACTGCTATAATGAAGCCGA-3 ' (OG8) which corresponds to residues 48±55 of NnkPLA-I and NnkPLA-II. One of the major dierences between NnkPLA-I and NnkPLA-II and CM-II and CM-III was noted at position 52: Asn for NnkPLA-I and NnkPLA-II and Asp for CM-II and CM-III which are brought about by one nucleotide substitution A to G. The oligonucleotide OG8 speci®c to NnkPLA-I and NnkPLA-II hybridized to D3, in addition to NnkPLA-I and NnkPLA-II, even at 608C (Fig. 3). Although the oligonucleotide OG7 for cDNA clones, if any, of CM-II and CM-III hybridized to D3 at 508C, it was washed out at 608C in any cases (Fig. 3). Hybridization of OG7 and OG8 was not observed for vector pTV118N employed as a control. These results revealed that cDNAs encoding NnkPLA-I and NnkPLA-II de®nitely exist in the cDNA library, but cDNAs corresponding to CM-II and CM-III are not present (Fig. 2). The presence of only two PLA2 isozymes in N. naja kaouthia venom is in
Fig. 3. Dot blot hybridization of NnkPLA-I, NnkPLA-II and PLA2 cDNAs (D3) ampli®ed by PCR from N. naja kaouthia (Malaysia) venom gland cDNA library (see Materials and methods section) with probe OG8 (5'-CAACTGCTATAATGAAGCCGA-3') speci®c for NnkPLA-I and NnkPLA-II (A) and with probe OG7 (5'-CAACTGCTATGATGAAGCCGA-3 ') speci®c for cDNAs, if any, of CM-II and CM-III (B). After hybridization at 488C for 12 h, the membranes were washed at 28C intervals from 50 to 608C. The hybridization pro®les at 50 and 608C are shown. a1, NnkPLA-I; a2, NnkPLA-II; b1, D3; and b2, vector pTV118N as a negative control.
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contrast to the fact that Crotalidae snake venoms contain more PLA2 isozymes (Ogawa et al., 1992,1995,1996; Nakashima et al., 1993,1995; Nobuhisa et al., 1996). In order to assess how many PLA2 isozyme genes exist in N. naja kaouthia genome, Southern blot analysis was conducted for its genomic DNAs extracted from its liver and digested with restriction enzymes EcoRI and BamHI. As shown in Fig. 4, only two bands were detected when the coding-region probe (nucleotides 103±349) which corresponds to the mature protein-coding region of NnkPLA-I was employed. A similar result was obtained for the full-length probe (nucleotides 1±640). Even when the 3 '-UTR probe (nucleotides 420±640) was used, the number of bands detected was not likely to be increased (Fig. 4). These results strongly suggested that N. naja kaouthia genome contains only two PLA2 isozyme genes. When the nucleotide sequences of NnkPLA-I and NnkPLA-II were compared, six nucleotide substitutions were noted, two in the signal peptide domain and four in the open reading frame. All the substitutions were found to occur at nonsynonymous site. Proÿ12, Pheÿ8, Asn15, Arg59, Asp81 and Asp83 in NnkPLA-I are replaced by Serÿ12, Serÿ8, Ser15, Gly59, Gly81 and Asn83, respectively, in NnkPLA-II (the positions with minus number indicate those in the signal peptide). The nucleotide sequences of NnkPLA-I and NnkPLA-II were compared with
Fig. 4. Southern blot analysis of Malaysian N. naja kaouthia genomic DNAs. Three probes, the codingregion probe (nucleotides 103±349), the 3'-UTR probe (nucleotides 420±640) and the full-length probe (nucleotides 1±640), were prepared from NnkPLA-I (A). Genomic DNAs extracted from the liver were digested with EcoRI and BamHI, respectively, electropheresed on 0.8% agarose gels, transferred onto Hybond-N+ nylon membranes and hybridized with the probes (B).
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those of cDNAs, BmPLA2, NsPLA2, LlPLA2 and AlPLA2, which encode group I snake venom gland PLA2s, BmPLA2 (Bungarus multicinctus ) (Danse et al., 1990), NsPLA2 (Notechis scutatus ) (Ducancel et al., 1988a), LlPLA2 (Laticauda laticauda ) (Guignery-Frelat et al., 1987) and AlPLA2 (Aipysurus laevis ) (Ducancel et al., 1988b), respectively (the nucleotide sequences of BmPLA2, LlPLA2 and NsPLA2 are in Fig. 1). It was found that the 5'- and 3 '-UTRs (86±87%) are more conserved than the protein-coding regions (74%), being in contrast to ordinary genes in which the noncoding regions are more variable than the protein-coding region (Kubo et al., 1987; Ohno et al., 1987; Matsuoka et al., 1988; Strathmann and Simon, 1990). Thus, the signi®cance of group I snake venom gland PLA2 isozymes in molecular evolution was evaluated by mathematical analysis of the nucleotide sequences of NnkPLA-I, NnkPLA-II, BmPLA2, NsPLA2, LlPLA2 and AlPLA2. The KN values for the 5 '- and 3 '-UTRs and the KS and KA values for the protein-coding region were computed for all the pairs of cDNAs. Table 1 shows the data for 11 pairs. The values between NnkPLA-I and NnkPLA-II was excluded because these have the highly homologous sequences. These genes appear to have diverged quite recently. The following two characteristics are prominent from the data. First, KN/KS values are smaller than 1, indicating that nucleotide substitutions have occurred in the protein-coding region more frequently than in the UTRs. Second, KA/KS values are close to 1, indicating that nucleotide substitutions in the protein-coding region tend to cause amino acid change. These facts indicate that group I snake venom gland PLA2s have evolved in an accelerating manner.
Table 1 The KS, KA, KN, KN/KS and KA/KS values for 11 pairs of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2 isozymesa Pair of cDNAs
KS
KA
KN
KN/KS
KA/KS
NnkPLA-I vs. BmPLA2 NnkPLA-I vs. NsPLA2 NnkPLA-I vs. LlPLA2 NnkPLA-I vs. AlPLA2 NnkPLA-II vs. BmPLA2 NnkPLA-II vs. NsPLA2 NnkPLA-II vs. LlPLA2 NnkPLA-II vs. AlPLA2 BmPLA2 vs. NsPLA2 BmPLA2 vs. LlPLA2 NsPLA2 vs. LlPLA2
0.268 0.355 0.463 0.364 0.275 0.356 0.466 0.369 0.391 0.413 0.216
0.277 0.346 0.428 0.378 0.274 0.345 0.408 0.377 0.346 0.415 0.241
0.162 0.152 0.166 0.150 0.162 0.152 0.166 0.150 0.089 0.149 0.077
0.604 0.432 0.356 0.411 0.588 0.426 0.356 0.406 0.227 0.361 0.356
1.032 0.974 0.925 1.040 0.996 0.970 0.876 1.022 0.885 1.005 1.245
a Other combinations other than a pair of NnkPLA-I and NnkPLA-II gave the similar values as listed in each column.
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4. Discussion Screening of Malaysian N. naja kaouthia venom gland cDNA library by probe D1, DNA fragment(s) prepared by RT-PCR for its venom gland mRNAs with primers OG1 and OG2, gave two PLA2 cDNAs, NnkPLA-I and NnkPLA-II. No additional clones were detected even when probe D2 containing the 3' moiety of the protein-coding region and the 3 '-UTR was employed. Similarly as this fact, Southern blot analysis (Fig. 4) implied that N. naja kaouthia genome codes for only two PLA2 isozymes. NnkPLA-I diers from NnkPLA-II in sequence only at four positions, 15, 59, 81 and 83 (Fig. 2). The sequences of NnkPLA-I and NnkPLA-II are similar to those of CM-II and CM-III, respectively. Asn-52, Gly-80, Asp-81 and Asp-83 of NnkPLA-I are replaced by Asp, Asn, Gly and Asn, respectively, in CM-II. On the other hand, NnkPLA-II diers from CM-III only at two positions. Asp-20 and Asn-52 of NnkPLA-II are substituted by Asn and Asp, respectively, in CM-III. cDNA cloning and dot blot hybridization (Fig. 3) showed that no clones corresponding to CM-II and CM-III exist in Malaysian N. naja kaouthia venom gland cDNA library. This reveals that PLA2s from Malaysian N. naja kaouthia venom are distinct from those of Thailand N. naja kaouthia venom. Such geographical variation of venom components within the same snake species has been known (Chippaux et al., 1991; Daltry et al., 1996; Tsai et al., 1996). For instance, according to Tsai et al. (1996), Russell's viper PLA2s can be separated into two types; one having Asn (Thailand, Taiwan, Burma and Pakistan) and the other Ser at the N-terminus (Southern India and Sri Lanka). Pair of CM-II and NnkPLA-I and that of CM-III and NnkPLA-II appear to be in orthologous relationship in evolution. All snake venom PLA2s whose crystal structures have been clari®ed possess a common scaold constructed from four a-helices and a b-sheet which are connected by ®ve loops (Fig. 2) (Ohno et al., 1998). The four amino acid substitutions between NnkPLA-I and NnkPLA-II are found only in the loop regions. Even in CM-II and CM-III, three of four substitutions occur in the loop regions. Diverged biological functions of proteins appear to be exerted or carried in the loop region. Loop 2 of PLA2s are known to construct a part of the Ca2+ binding site (Dijkstra et al., 1981,1983). Substitutions between NnkPLA-I and NnkPLA-II occur at loops 3 and 4 which are believed to construct a lipid-water interface recognition site. Although two Lys-49-PLA2s, called BPI and BPII, from T. ¯avoviridis venom show only one amino acid replacement (Asp/Asn) at position 67 in loop 3, they exhibited considerably dierent potencies in enzymatic activity and in contraction of the isolated muscle tissue (Shimohigashi et al., 1995,1996; Ohno et al., 1998). It is thus likely that NnkPLA-I and NnkPLA-II, or CM-II and CM-III, have distinct physiological functions due probably to dierent surface interactions. Analysis of the nucleotide sequences of NnkPLA-I, NnkPLA-II, BmPLA2, NsPLA2, L1PLA2 and A1PLA2, all of which encode group I snake venom gland PLA2s, showed that the protein-coding region except for the signal peptide-coding
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domain is more variable than the 5 '- and 3 '-UTRs (Fig. 1) and that nonsynonymous nucleotide substitutions are almost equal to synonymous nucleotide substitutions in the protein-coding region (Table 1). These features are the same as have been observed for genes encoding group II Viperidae snake venom gland PLA2 isozymes which are thought to have evolved in an accelerating manner (Nakashima et al., 1993,1995; John et al., 1994; Ogawa et al., 1995,1996; Kordis and Gubensek, 1996; Nobuhisa et al., 1996). This indicates that group I snake venom gland PLA2 isozymes have also evolved in an accelerating manner, possibly to gain new physiological functions. Thus, accelerated evolution is a common phenomenon for PLA2s in both Viperidae and Elapidae snake venom glands. It has recently been established that serine proteases of Crotalinae snake venom glands have evolved in an accelerating manner (Deshimaru et al., 1996). It may be said that accelerated evolution is universal for plural protein families in snake venom glands.
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