* Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore; Australian Venom Research Unit, Department of Pharmacology, School of Medicine, University of Melbourne, Parkville, Victoria, Australia;
Population and Evolutionary Genetics Unit, Museum Victoria, G.P.O. Box 666E, Melbourne, Australia; and
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Medical College of Virginia
Correspondence: E-mail: dbskinim{at}nus.edu.sg.
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Abstract |
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Key Words: gene duplication sea snake venom gland phospholipase A2 evolution of toxins
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Introduction |
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Previously, we found that the three-finger (3FTx) toxins duplicated and diverged early during the evolutionary history of elapids to form a broad superfamily (Fry et al. 2003a, 2003b, 2003c; Fry and Wuster 2004). However, this superfamily continues to diversify, as shown by our finding of taxon-specific gene clusters. These evolutionary patterns are similar to what has been observed in multigene families involved in the adaptive immune response (e.g., immunoglobulins and major histocompatibility complex genes; Nei, Gu, and Sitnikova 1997). It is believed that gene duplication and subsequent divergence contributes to an organism's ability to react to a wide range of foreign antigens. In an analogous manner, snake toxins must react with diverse molecular targets in their prey. Thus, a birth-and-death mode of evolution generates a suite of toxins in order to allow predatory snakes to adapt to a variety of different prey species.
The sea snakes and sea kraits are unique in the snake world in possessing remarkably streamlined venom (Fry et al. 2003b), despite these two lineages independently colonizing the ocean (fig. 1). The venoms of sea snakes are relatively simple because of their specialized diet, consisting of a single class of vertebrates (fish). Such a streamlining of venoms is reflected by the remarkable level of cross-reactivity of sea snake antivenom (Chetty et al. 2004). An extreme divergence in venom evolution can extend even to the secondary loss of toxins (or venom). This has happened independently two times in the sea snake lineages, with Emydocephalus annulatus (Turtle-Head Sea Snake) and Aipysurus eydouxii (Marbled Sea Snake) both evolving to become obligate fish egg eaters (Glodek and Voris 1982; H. K. Voris and H. H. Voris 1983) with an accompanying loss of fangs and greatly atrophied venom glands (McCarthy 1987; Gopalakrishnakone and Kochva 1990). The toxicity of A. eydouxii venom is 40100 times lower than that of other sea snakes including the other six species in the same genus (Tu 1973, 1974). Similarly, the Australian terrestrial elapid Brachyurophis (shovel-nosed snakes) specializes in feeding upon reptile eggs (Scanlon and Shine 1988). The switch over to exclusive egg-feeding habit effectively removes the selection pressure to retain potentially toxic venom. Therefore, any change in toxin sequence (and hence bioactivity) would be a neutral mutation in these snakes as the toxins are not at all being used for prey capture.
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The purpose of this study was to examine the molecular evolution of the type IA PLA2 toxins present in A. eydouxii venom and to correlate any divergences relative to other snake venoms and to changes in the diet.
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Materials and Methods |
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Screening of cDNA Library
Miniscale plasmid isolations from 200 randomly selected colonies were carried out using QIAprep® spin miniprep kit (Qiagen). Purified plasmids were submitted to restriction enzyme analysis using SfiI. Clones containing cDNA inserts (196 clones; 98% efficiency) were subjected to DNA sequencing.
DNA Sequencing and Computer Analysis
Positive clones were subjected to sequencing using the M13 forward and reverse primers. The sequencing reaction was carried out using BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, Calif.), with an automated DNA sequencer (Model 3100A, Applied Biosystems). All DNA sequencing reactions were repeated twice, and all the nucleotide sequences were checked using the Chromas (DNA sequence analysis software, Technelysium Pty Ltd., Tewantin Qld, Australia). The cDNA sequences were compared to those in the GenBank databases using BLAST network services at National Center for Biotechnology Information. Nucleic acid and amino acid sequence homologies were obtained using Vector NTI program (Informax, Frederick, Md.).
Reverse transcription-PCR and TA Cloning
To amplify all possible cDNAs encoding PLA2, two rounds of reverse transcription (RT)-PCR was conducted. Fresh total RNA isolated from A. eydouxii venom gland was reverse-transcribed to cDNAs using PowerScript Reverse Transcriptase (Clontech). Gene-specific upstream primer P1 (5'-ATGTATCCTGCTCACCTTCTGGTC-3') was designed according to the conserved signal peptide sequences, and the downstream primer P2 (5'-CCTTGCGCTGAAGCCTCTCAAATA-3') was the sequences followed by the stop codon. PCR was carried out on a Thermal Cycler (Eppendorf Mastercycler, Westbury, N.Y.). The samples were subjected for 5 min to 95°C followed by 30 cycles of 1 min at 94°C, 20 s at 52°C, and 30 s at 72°C. The run was terminated by a 5 min elongation step at 72°C. High-fidelity polymerase of AdvantageTM 2 (Clontech) was used during PCR amplification to eliminate the PCR mismatching. RT-PCR products were analyzed on 1% agarose gels and purified using QIAquick® gel extraction kit (Qiagen). Then, the purified products of 470 base pairs were inserted into pGEM®-T Easy vector (Promega, Madison, Wis.) and transformed into E. coli DH5
. One hundred positive clones were sequenced, and the plasmid isolation, DNA sequencing, and computer analysis of these clones were performed as described above.
Phylogenetic Analysis
To minimize confusion, all protein sequences are referred to by their Swiss-Prot accession numbers (http://www.expasy.org/cgi-bin/sprot-search-ful) except for the toxins presented here, which are referred to by their GenBank accession numbers (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide&itool=toolbar). Sequences were aligned using the program CLUSTAL-X (Thompson et al. 1997), followed by visual inspection for errors. The A. eydouxii PLA2 sequences and representatives of the full PLA2 toxin diversity were analyzed using Bayesian inference implemented on MrBayes, version 3.0b4 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). The method uses Markov-chain Monte Carlo methods to generate posterior probabilities for each clade represented in the tree. The analysis was performed by running a minimum of 1 x 106 generations in four chains and saving every 100th tree. The log-likelihood score of each saved tree was plotted against the number of generations to establish the point at which the log-likelihood scores of the analysis reached their asymptote, and the posterior probabilities for clades were established by constructing a majority rule consensus tree for all trees generated after the completion of the burn-in phase. Alignments can be obtained by e-mailing Bryan G. Fry (bgf{at}unimelb.edu.au).
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Results |
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Phylogenetic analysis revealed that the A. eydouxii PLA2 toxins are all orthologs of each other relative to all other sequences (fig. 3). The relative distances among A. eydouxii PLA2 enzymes were also much shorter than those shown in toxins from a single species in the terrestrial or other marine snakes.
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Discussion |
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The results of this study show that A. eydouxii is unique among the snakes studied to date in that the venom is evolving at a much lower rate than comparative terrestrial snakes and even other marine snakes. While few species have been comprehensively studied and thus the sequences available do not fully represent the full diversity of toxins, a large number of terrestrial and marine snake PLA2 toxin sequences are available in the databases (>100). Phylogenetic analysis of all the available marine snake toxins and representative terrestrial snake toxins is revealing. The toxins from the terrestrial snakes and all the other marine species (true sea snake as well as sea krait) were paraphyletic for a particular species that had multiple toxins sequenced, and species-level paralogs were widespread (fig. 3). In contrast, all the A. eydouxii sequences formed a monophyletic group and were species-level orthologs. In addition, the A. eydouxii sequences formed a very tight clade in comparison to toxins from other marine species or the terrestrial snake. Consistent with previous mass spectrometry results of sea snake venoms (Fry et al. 2003b), a single PLA2 transcript was in much greater quantities than the others. Clone AY561154 was the most abundant PLA2, being present in six times the amounts of the others (table 1). However, this transcript was neither the most basal nor the most recently derived sequence.
While several gene duplication events are evident in the A. eydouxii sequences, the highly conserved sequences and low distances separating the toxins reveal an extremely low rate of diversification. Similarly, while the mutations occur with the unique toxin gene bias toward positions 1 and 2 of the codons in A. eydouxii PLA2 enzymes, there are very few mutations relative to PLA2 enzymes from other marine or terrestrial snakes.
As there is no longer a positive selection pressure acting upon the A. eydouxii venom for prey capture, mutations have accumulated that would have otherwise had a deleterious effect. The few mutations present have occurred in key structural and functional residues, resulting in toxins likely to have greatly diminished usefulness in prey capture. In PLA2 enzymes, several residues that are essential for the enzymatic activity are highly conserved. These residues are involved in the catalytic network (His48, Asp49, Tyr52, Tyr73, and Asp99), the hydrophobic region around the enzymatic site contributing to the substrate binding (Leu2, Phe5, Tyr22, Cys29, Cys45, Ala102, Ala103, and Phe106), and in the catalytically important Ca2+ binding (Tyr28, Gly30, Gly32, and Asp49) (Scott 1997). Substitution of these residues often leads to loss of catalytic properties. For example, in a subgroup of group II PLA2 enzymes, Asp49 is replaced by Lys49 (Maraganore and Heinrikson 1986) or Ser49 (Chijiwa et al. 2003). Asp49 is the most important residue involved in coordinating the Ca2+ ion that plays a crucial role in catalysis. Lys49-PLA2 enzymes lack the ability to bind Ca2+, and hence, they exhibit very low or no catalytic activity (Maraganore and Heinrikson 1986; de Oliveira et al. 2001).
In the present study, a very interesting finding is that the site of active site His48 in AY561155 is replaced with Arg48 (fig. 2). The proposed catalytic mechanism of PLA2 depends on the crucial role of His48; it polarizes and attracts a proton from a positionally conserved water molecule, which then participates in the formation of a tetrahedral intermediate. Upon collapse of the intermediate, hydrolysis products are released and three water molecules move into the active site (Scott et al. 1990). The substitution of His48 in AY561155 by Arg48 most likely disrupts the catalytic network, leading to significant, if not complete, reduction in the catalytic activity. This is the first natural substitution of active site His48 in PLA2 enzymes.
Snake venom PLA2 enzymes possess a common scaffold with four -helices and one ß-sheet, which are connected by five loops (Ohno et al. 1998). This scaffold is held together by six to seven highly conserved disulfide bonds (Dufton and Hider 1983). Interestingly, in one of the sequences presented here, AY561164, Cys44 is replaced by a Tyr44 and Tyr53 is replaced by Cys53 (fig. 2). In AY561156 PLA2, Cys125 is replaced by Tyr125, resulting in odd number of Cys residues. It would be interesting to see how these substitutions affect the conserved disulfide bonds and folding as well as the catalytic properties of these proteins.
In general, most of the amino acid substitutions in the A. eydouxii sequences (22 out of 30) are found in the surface loops; 4, 4, 11, 1, and 2 amino acid changes occurred in loop 1 through 5, respectively (fig. 2). Six and two amino acid changes occurred in helices 3 and 4, respectively. This corroborates our earlier finding that the higher rate of accelerated mutation occurs in the surface residues than in the buried residues in snake venom PLA2 enzymes (Kini and Chan 1999). Such natural substitution in the surface residues contributes to modifying the molecular surface to afford distinct and novel targeting to cells or tissues. In snake venom PLA2 enzymes, only one amino acid change has resulted in significant difference in their properties. For example, two Lys49-PLA2 called BP I and BP II from P. flavoviridis venom show only one amino acid replacement in loop 3 (Asp/Asn67) but exhibit considerably different potencies in enzymatic activity and in contraction of the isolated muscle tissue (Shimohigashi et al. 1995, 1996; Ohno et al. 1998). Thus, while the mutational rate is greatly slowed down in A. eydouxii toxins, it is likely that some of the PLA2 enzymes from A. eydouxii may have distinct physiological functions if they are indeed active.
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Conclusion |
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Supplementary Materials |
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Acknowledgements |
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Footnotes |
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