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Molecular & Cellular Proteomics 7:215-246, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Evolution of an Arsenal

Structural and Functional Diversification of the Venom System in the Advanced Snakes (Caenophidia)^*

Bryan G. Fry^,, Holger Scheib^¶,||, Louise van der Weerd^**, Bruce Young, Judith McNaughtan, S. F. Ryan Ramjan, Nicolas Vidal^¶¶, Robert E. Poelmann^** and Janette A. Norman^,||||

From the Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute and Department of Oral Medicine and Surgery, School of Dental Science, University of Melbourne, Parkville, Victoria 3010, Australia, ^¶ SBC Lab AG, Seebüelstrasse 26, 8185 Winkel, Switzerland, ^** Molecular Imaging Laboratories Leiden, MRI Facility, Departments of Anatomy and Radiology, 2300 RC, Leiden, The Netherlands, Department of Biology, Washburn University, Topeka, Kansas 66621, ^¶¶ UMR 7138, Département Systématique et Evolution, Muséum National d’Histoire Naturelle, CP 26, 57 rue Cuvier, 75005 Paris, France, and ^|||| Molecular Biology, Museum Victoria, G. P. O. Box 666, Melbourne, Victoria 3001, Australia

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Venom is a key innovation underlying the evolution of advanced snakes (Caenophidia). Despite this, very little is known about venom system structural diversification, toxin recruitment event timings, or toxin molecular evolution. A multidisciplinary approach was used to examine the diversification of the venom system and associated toxins across the full range of the 100 million-year-old advanced snake clade with a particular emphasis upon families that have not secondarily evolved a front-fanged venom system (80% of the 2500 species). Analysis of cDNA libraries revealed complex venom transcriptomes containing multiple toxin types including three finger toxins, cobra venom factor, cysteine-rich secretory protein, hyaluronidase, kallikrein, kunitz, lectin, matrix metalloprotease, phospholipase A₂, snake venom metalloprotease/a disintegrin and metalloprotease, and waprin. High levels of sequence diversity were observed, including mutations in structural and functional residues, changes in cysteine spacing, and major deletions/truncations. Morphological analysis comprising gross dissection, histology, and magnetic resonance imaging also demonstrated extensive modification of the venom system architecture in non-front-fanged snakes in contrast to the conserved structure of the venom system within the independently evolved front-fanged elapid or viperid snakes. Further, a reduction in the size and complexity of the venom system was observed in species in which constriction has been secondarily evolved as the preferred method of prey capture or dietary preference has switched from live prey to eggs or to slugs/snails. Investigation of the timing of toxin recruitment events across the entire advanced snake radiation indicates that the evolution of advanced venom systems in three front-fanged lineages is associated with recruitment of new toxin types or explosive diversification of existing toxin types. These results support the role of venom as a key evolutionary innovation in the diversification of advanced snakes and identify a potential role for non-front-fanged venom toxins as a rich source for lead compounds for drug design and development.

Previous studies indicate significant morphological variation in the venom gland (7–9) and dentition of snakes (10). The fang may or may not be enlarged and can range from a solid tooth with or without grooving to an enclosed canaliculate channel as present in elapids and viperids (10). Few comparative studies of the caenophidian venom system have been performed, although West (11, 12) and Sarkar (13) described several different structural and topological features. A distinction was attempted between the venom glands of the front-fanged and non-front-fanged snakes with the glands in most non-front-fanged snakes termed "Duvernoy’s glands" (7).

Similarly, very little has been revealed about the composition of venoms from advanced snake lineages other than the three clades with high pressure, hollow front-fang venom delivery systems (Atractaspis, elapids, and viperids). Despite accounting for the majority of the families (Fig. 1 and Ref. 6) and 1900 of the 2500 advanced snake species, the multiple non-front-fanged families have received scant attention. Only a few studies have been undertaken, and even fewer have sequenced or bioactivity-tested individual toxins (2, 14–18). Studies of the first full-length toxins from non-front-fanged snakes were revealing, particularly the isolation and characterization of a potently neurotoxic three-finger toxin (3FTx)¹ from the colubrid snake Coelognathus radiatus (2). This toxin type had long been considered the hallmark of elapid venoms and had been the subject of intense study (19). Follow-up molecular phylogenetic studies demonstrated the shared origin of 3FTx and other toxin types across the entire advanced snake radiation (3).

View larger version (49K): [in this window] [in a new window]   FIG. 1. Cladogram of evolutionary relationships of advanced snakes (1, 6, 58–61) showing relative timing of toxin recruitment events and derivations of the venom system. MRI images are shown for representatives. Acn, Acetylcholine esterase; LAO, L-amino oxidase; C3B, FAMC3B cytokine; CNP-BPP, c-type natriuretic peptide-bradykinin-potentiating peptide; GrTx, glycine-rich toxin; Hya, hyaluronidase; RAP, renin-like aspartic protease; VEGF, vascular endothelial growth factor.

It has been shown previously that snake venoms evolve via a process by which a gene encoding for a normal body protein, typically one involved in key regulatory processes or bioactivity, is duplicated, and the copy is selectively expressed in the venom gland (20). The newly created toxin type evolves via the birth-and-death model of protein evolution in which a toxin multigene family is created by further gene duplication events followed by the deletion of some copies and conversion of others to non-functional copies or pseudogenes (19).

In addition to gene duplication, mutation is an important process that generates a tremendous diversity of venom toxins within these multigene families. The newly created toxin multigene families preserve the molecular scaffold of the ancestral protein but modify key functional residues at the tips of loops to acquire a myriad of newly derived activities (19, 20). Other mutations can include the selective expression of a particular domain, such as the expression of the disintegrin domain from snake venom metalloprotease, ADAM-type (SVMP/ADAM) toxins in viperid venoms (21). These toxins have an unusual combination of precise specificity and extreme potency, characteristics that make them particularly amenable for use as investigational ligands or as leads for drug design and development (22–24).

In this study we used a multidisciplinary approach to (a) characterize the venom transcriptomes of representative caenophidian snakes, (b) determine the timing of toxin recruitment events and patterns of toxin diversification, and (c) characterize changes in the venom delivery architecture. From comparison with the more widely studied elapids and viperids, we determined whether there are significant structural or functional differences in the evolution of the venom system and their associated toxins in the poorly characterized non-front-fanged caenophidian snakes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Species Studied and Molecular Phylogeny of Advanced Snakes—
A total of 107 species representing each of the major lineages of advanced snakes were included in the study and examined using one or more of the following techniques: clone sequencing of cDNA libraries, gross dissection and examination of dentition, histology of venom glands and ducts, and magnetic resonance imaging (see "Appendix I" for details). In most cases different individuals were used as a source of material for each of these analyses.

Molecular Modeling of Toxins—
3D models for caenophidian toxins were generated based on the assumption that homologous proteins share similar 3D structures (25). In other words, the three-dimensional structure of a target protein can be modeled if its sequence is homologous to at least one template protein whose 3D structure has been determined experimentally by applying either x-ray or NMR techniques (26). In this work aligning the protein sequences of target and template(s) was carried out in SPDBV (27), and the initial alignments were refined manually. From these alignments 3D models were built directly in SPDBV applying the "Build Preliminary Model" option, which is disabled in the currently distributed public version of the software. Loops were built by scanning a database of known loop structures using the same software, and suitable specimens were selected after visual inspection. The enthalpy of the resulting models was minimized applying two times 200 steps of Steepest Descent minimization. Finally the quality of each model structure was assessed in iMolTalk (28), and a Van der Waals surface was calculated in MolMol (29). In MolMol electrostatic potentials were calculated applying the "simplecharge" command and mapped on the model structure surface. The families of venom proteins were analyzed by superimposing the structures in SPDBV (27), and conserved and variable structural regions were identified. In the molecular modeling of representative proteins, blue surface areas indicate positive charges, red surface areas indicate negative charges, and model pairs show sides of the protein rotated by 180°.²

Molecular Phylogeny of Toxin Sequences—
Molecular phylogenetic analyses of toxin transcripts were conducted using the translated amino acid sequences. Comparative sequences from other venomous reptiles and outgroups were obtained through BLAST searching (www.expasy.org/tools/blast/) using representative toxin sequences. To minimize confusion, all sequences obtained in this study are referred to by their GenBank^TM accession numbers (www.ncbi.nlm.nih.gov/sites/entrez?db=Nucleotide), and sequences from previous studies are referred to by their UniProt/Swiss-Prot accession numbers (www.expasy.org/cgi-bin/sprot-search-ful). Resultant sequence sets were aligned using the program ClustalX followed by visual inspection for errors. When presented as sequence alignments, the leader sequence is shown in lowercase, the prepro region is underlined, cysteines are highlighted in black, and functional residues are in bold. Datasets were analyzed using Bayesian inference implemented on MrBayes, version 3.0b4. 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.³

Gross Dissection and Analysis of Dentition—
Gross dissection was performed on freshly euthanized and formalin-fixed specimens to document the relative size and position of the venom gland and associated skeletal musculature. Features of the maxillary dentition were scored using data from a previous study (10) in which five morphological states were defined: 1) smooth surface and no enclosed venom canal, 2) no enclosed venom canal and surface with shallow furrow, 3) deep groove running the majority of the length of the tooth, 4) deep groove present but restricted to less than half the length of the tooth, and 5) enclosed venom canal.

Histological Analysis—
Histological sections were prepared from the intact head and the excised venom delivery system. Whole heads were removed, and a cut was made to the underside to allow fast penetration of the fixative (10% neutral buffered formalin). After a minimum of 2 days excess tissue was removed, and specimens were immersed in Kristensen’s decalcification solution and placed on a rotor for up to 3 weeks (depending on the size of the head). Before processing the heads were bisected longitudinally for cutting transversely, at 3 µm, in two separate blocks. The processing schedule was: 10% formalin, 2 h; absolute ethanol, 4 x 1 h; Histolene, 3 x 1 h; paraffin wax, 2 x 90 min. The sections were taken every 100 µm, and matching sections were stained with periodic acid-Schiff stain and Masson’s trichrome stain. In other specimens, the venom gland, venom duct, and the adjacent bony and muscular tissue were excised and placed in decalcifying solution (Cal-Ex, Fisher) for 72–168 h. Each sample was dehydrated and cleared through a progressive ethanol series and Cyto-Sol (Fisher) prior to embedding in Paraplast (Fisher). Serial sections were cut at 10–12 µm. All species were sectioned in the frontal plane; when available, the contralateral venom delivery system was sectioned either parasagittally or transversely. Sections were stained using a variant of Van Gieson’s stain, which provides clear distinction between connective tissue, muscle, and epithelium, or with hematoxylin and eosin.

Magnetic Resonance Imaging—
Magnetic resonance imaging (MRI) was used to examine the three-dimensional shape and internal anatomy of the venom glands. Formalin-ethanol-fixed heads were first submersed in Fomblin (Solvay Solexis) to prevent air artifacts. Depending on head size, imaging was performed on either 9.4-T (small/medium) or 17.6-T (large) vertical 89-mm-bore systems (Bruker BioSpin, Rheinstetten, Germany) with a Bruker Micro2.5 gradient system of 1 T/m and transmit/receive birdcage radiofrequency coil with diameter of 10–30 mm. Bruker ParaVision 3.0 software was used for image acquisition. Anatomical images were acquired using a 3D gradient echo sequence. The field of view and matrix were varied to fit the individual samples, resulting in voxel sizes between (40)³ mm³. Imaging parameters were: echo time = 8 ms; repetition time = 40 ms; flip angle, 20°; four to eight averages; total scan time between 3 and 9 h per sample, depending on size and resolution. Image segmentation of the glands was performed manually in Amira 4.1 (Mercury Computer Systems Inc.), and 3D surface renderings were generated for all species.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Isolation and Characterization of Venom Toxin Transcripts—
Analysis of venom gland cDNA from non-front-fanged snake libraries revealed the presence of transcripts with homology to previously characterized venom toxins from front-fanged snakes and venomous lizards (helodermatids and varanids) (Tables I and II). Transcripts sequenced were 3FTx (Figs. 2 and 3), C3/cobra venom factor (CVF) (Fig. 4), cysteine-rich secretory protein (CRISP) (Figs. 5 and 6), hyaluronidase (Fig. 7), kallikrein (Figs. 8 and 9), kunitz (Figs. 10 and 11), lectin (Figs. 12 and 13), matrix metalloprotease (MMP) (Fig. 14), phospholipase A₂ (PLA₂) Type IB (Fig. 15), SVMP/ADAM (Figs. 16, 17, and 18), and waprin (Fig. 19). Transcripts of five of these toxin types were also recovered from the cDNA libraries of the representative elapid Oxyuranus microlepidotus (3FTx, CRISP, kunitz, and waprin) and the viperid Causus rhombeatus (kallikrein).

View larger version (97K): [in this window] [in a new window]   FIG. 2. Sequence alignment of representative 3FTx. Species shown are E. polylepis (1, EU029668), P. mossambicus (2, EU029669), L. poecilogyrus (3, EU029670; 6, EU029672; 7, EU029673), T. dhara (4, EU029671), D. typus (5, EU029674; 9, EU036636; 17, EU029681; 19, EU029683), T. jacksonii (8, EU036635; 18, EU029682; 20, EU029684; 21, EU029685), T. biscutatus (10, EU029675; 12, EU029677; 13, EU029678), Leioheterodon madagascariensis (11, EU029676), T. dhara (14, EU029686; 15, EU029679; 16, EU029680), C. radiatus (22, P83490), Dendroaspis jamesonii (23, P25682), from B. multicinctus (24, Q9PW19; 25, Q9YGJ0), Bungarus candidus (26, P81783), and Naja sputatrix (27, Q9W7I3). Also included is the representative non-toxin peptide brain -neuropeptide (28, Q9WVC2) from Mus musculus.

View larger version (56K): [in this window] [in a new window]   FIG. 3. Bayesian molecular phylogeny of 3FTx. The outgroup is the non-toxic brain -neuropeptide (Q9WVC2) from M. musculus. The ribbon model of EU029668 E. polylepis shows β-strands in yellow.

View larger version (39K): [in this window] [in a new window]   FIG. 4. Molecular evolution of C3/CVF toxins. A, partial sequence alignment of the representative toxin forms shown from top to bottom: Q2XXR5 from L. madagascariensis, Q49HM6 from Austrelaps superbus, Q01833 from N. naja, and Q91132 from Naja kaouthia as well as the non-toxin forms P23667 from Xenopus laevis and Q90633 from Gallus gallus. B, Bayesian molecular phylogenetic analysis of representative toxin and non-toxin body forms. Outgroups are the non-toxin sequences P23667 X. laevis and Q90633 G. gallus.

View larger version (121K): [in this window] [in a new window]   FIG. 5. Sequence alignment of representative full-length (unless otherwise indicated) CRISP toxins. 1, Q2XXQ5 from D. typus; 2, partial sequence Q2XXP5 from T. dhara; 3, partial sequence Q2XXP4 from T. biscutatus; 4, partial sequence Q2XXP7 from P. olfersii; 5, partial sequence Q2XXQ0 from L. poecilogyrus; 6, Q2XXQ3 from E. polylepis; 7, partial sequence Q2XXQ1 from L. madagascariensis; 8, Q2XXP9 from O. microlepidotus; 9, Q3SB03 from Hoplocephalus stephensii; 10, Q3SB05 from P. textilis; 11, Q8UW11 from L. curtus; 12, Q8AVA3 from Pseudechis porphyriacus; 13, Q8AVA4 from Pseudechis australis; 14, Q8JI38 from Laticauda semifasciata; 15, Q8JI39 from Trimeresurus flavoviridis; 16, P79845 from P. mucrosquamatus; 17, Q8JGT9 from R. tigrinus; 18, Q2XXP1 from Varanus varius; 19, Q91055 from H. horridum; 20, the non-toxin representative Q91XA3 from M. musculus.

View larger version (54K): [in this window] [in a new window]   FIG. 6. A, Bayesian molecular phylogeny of CRISP toxins. Outgroups are non-toxin forms Q034O1 and Q91XA3 from M. musculus. B, ribbon view of Q2XXQ5 from D. typus. -Helices are in red, and β-strands are in yellow. Residues postulated to bind to ion channels (without blocking them) are in white (Glu-55, Tyr-65, Ser-76, and Tyr-136 in PR-1; Ser-195 and Arg-196 in CRD); Ser-195 and Arg-196 are colored orange because they are part of a putative ion channel blocking site. The EX2F motif is in magenta, i.e. Glu-197 and Phe-200. Channel blocking motif hypothesized in this study is shown: part I in pink containing Val-199 and Phe-200 (of the DVF, EX2F motifs) but also Asn-202 and Asn-220; part II in blue containing Gln-209, Ser-210, and Asn-211; part III in orange consisting of Ser-195, Arg-196, and Phe-226; and part IV in green consisting of the C-terminal tail Pro-228 and Asn-229.

View larger version (63K): [in this window] [in a new window]   FIG. 7. Hyaluronidase. A, partial sequence alignment of toxin sequences. 1, EU091710 from L. poecilogyrus; 2, A3QVP2; 5, A3QVP3; 13, A3QVP1; 14, A3QVP0; 15, A3QVN9, all from Bitis arietans; 3, A3QVN8; 4, A3QVN7; 6, A3QVP5; 9, A3QVN6, all from Echis pyramidum leakeyi; 7, A3QVN5; 8, A3QVN3; 10, A3QVN4, all from Cerastes cerastes; 11, A3QVN2 Echis ocellatus; 12, A3QVP4 from Echis carinatus sochureki. Also shown are the representative non-toxins: 16, Q12794 from Homo sapiens; 17, Q05A56 from M. musculus. B, Bayesian molecular phylogeny; the outgroup is Q9W6K5 from Xenopus laevis.

View larger version (115K): [in this window] [in a new window]   FIG. 8. Sequence alignment of the representative full-length (unless otherwise indicated) kallikrein toxins. 1, partial sequence Q2XXM2 from P. olfersii; 2, Q5MCS0 from L. curtus; 3, Q6T6S7 from Bitis gabonica; 4, P81824; 5, Q9PTU8, both from Bothrops jararaca; 6, P33589 from Lachesis muta; 7, Q2XXN0 from V. varius; 8, P43685 from H. horridum; 9, the non-toxin representative Q9POG3 from H. sapiens.

View larger version (31K): [in this window] [in a new window]   FIG. 9. Bayesian molecular phylogeny of kallikrein toxins. Outgroups are non-toxin forms Q9GOG3 and Q9UBX7 from H. sapiens.

View larger version (40K): [in this window] [in a new window]   FIG. 10. Sequence alignment of kunitz toxins with functional residues (protease-inhibiting reactive bond) shown in bold and the leader sequence shown in lowercase. 1, EU029687 from T. dhara; 2, EU029688 from P. olfersii; 3, P24541 from Eristocophis macmahonii; 4, P82966 from O. hannah; 5, P19859 from N. naja; 6, P00981 from Dendroaspis polylepis; 7, Q7LZE4 from O. scutellatus; 8, Q90WA0 from P. textilis; 9, P00989 from B. multicinctus; 10, the representative non-toxin P04815 from Bos taurus.

View larger version (41K): [in this window] [in a new window]   FIG. 11. A, Bayesian molecular phylogeny of kunitz toxins. The outgroup is the non-toxin P04815 from B. taurus. B, ribbon view of EU029687 from T. dhara. -Helices are in red, β-strands are in yellow, and the functional residue Ala-18 is shown in green.

View larger version (58K): [in this window] [in a new window]   FIG. 12. Sequence comparison of representative lectin toxins. The homomeric forms shown are: 1, EU029691; 2, EU029689; 3, EU091713, all from E. polylepis; 4, EU029699 from L. madagascariensis; 5, EU029697; 6, EU029702, both from L. poecilogyrus; 7, EU029700 from P. olfersii; 8, EU029696 from T. jacksonii; 9, Q90WI6 from B. multicinctus; 10, Micrurus corallinus sequence published but not database-curated (62); 11, Q6TRS6 from Bothrops jararacussu; and 12, Q6T7B7 from B. gabonica. Representative heterodimeric forms (- and β-chains, respectively) shown are: 13, Q8JIV6; and 14, Q8JIV7A, both from D. acutus. Also included is the representative non-toxin form: 15, P83300 from Anser anser.

View larger version (43K): [in this window] [in a new window]   FIG. 13. Bayesian molecular phylogeny of lectin toxins. The outgroup is the non-toxin P83300 from A. anser. *, M. corallinus sequence published but not database-curated (62).

View larger version (61K): [in this window] [in a new window]   FIG. 14. Matrix metalloprotease. A, partial sequence alignment of EU091709 from L. poecilogyrus (1), Q9DE15 from G. gallus (2), A2VCV4 from X. laevis (3), Q98TC6 from Cyprinus carpio (4), P50757 from Oryctolagus cuniculus (5), and Q9TUL8 from Equus caballus (6). B, Bayesian molecular phylogeny of lectin toxins. The outgroup is the non-toxin P52176 from B. taurus.

View larger version (45K): [in this window] [in a new window]   FIG. 15. PLA2 (Type IB) analyses. A, sequence alignment of representative toxins. 1, EU029703 from T. biscutatus; 2, P80966 from O. hannah; 3, Q9PSN5 from Notechis scutatus; 4, Q9W7J3 from P. textilis; 5, the non-toxin representative Q8JFB2 from L. semifasciata pancreas. B, Bayesian molecular phylogeny; the outgroup is Q8JFB2 from L. semifasciata liver. The ribbon model of TRI002F09 from T. biscutatus shows the conserved catalytic residues (63). -Helices are in red, and β-strands are in yellow.

View larger version (92K): [in this window] [in a new window]   FIG. 16. N-terminal sequence alignment of SVMP/ADAM toxins. The partial sequences from non-front-fanged advanced snakes are: 1, EU029705 from P. olfersii; and 2, EU029707 from L. poecilogyrus. The full-length unique truncated forms are: 3, EU029708; 4, EU029713; 5, EU029717; and 6, EU029727, all from P. mossambicus. The representative viperid venom sequences are: 7, O42138 from Agkistrodon contortrix laticinctus; and 8, Q4VM08 from Macrovipera lebetina. 9, the representative elapid sequence Q8JGN1 from Naja mossambica. 10, the representative atractaspidid venom form Q9PT48 from A. engaddensis. The representative non-toxin forms are: 11, Q9UKQ2 (ADAM28 type); and 12, Q9H2U9 (ADAM7 type) from H. sapiens.

View larger version (74K): [in this window] [in a new window]   FIG. 17. C-terminal sequence alignment of SVMP/ADAM toxins. The partial sequences from non-front-fanged snakes are: 1, EU029734; 2, EU029736; 3, EU029737, all from D. typus; 4, EU029735 from T. jacksonii; 5, EU029738; 6, EU029739, both from L. madagascariensis; and 7, EU036637 from T. dhara. The typical viperid venom forms are: 8, O42138 from A. contortrix laticinctus; and 9, Q4VM08 from M. lebetina. 10, the representative truncated disintegrin form Q6T6T2 from B. gabonica. 11, the representative elapid sequence Q8JGN1 from N. mossambica. 12, the representative atractaspidid venom form Q9PT48 from A. engaddensis. The representative non-toxin forms are: 13, Q9UKQ2 (ADAM28 representative); and 14, Q9H2U9 (ADAM7 representative) from H. sapiens.

View larger version (53K): [in this window] [in a new window]   FIG. 18. Bayesian molecular phylogeny of SVMP/ADAM toxins. The outgroup is the non-toxin ADAM28 sequence Q9UKQ2 from H. sapiens. *, a published but uncurated sequence from Micropechis ikaheka (64).

View larger version (38K): [in this window] [in a new window]   FIG. 19. A, sequence comparison of representative waprin toxins from non-front-fanged advanced snakes (1, EU029745 from E. polylepis; 2, EU029744 from L. poecilogyrus; 3, EU029743; 4, EU029746; 5, EU029742, all from P. olfersii; 6, EU029741 from R. tigrinus; 7, EU029740 from T. jacksonii) and from front-fanged advanced snakes (8, P60589 from Naja nigricollis; 9, P83952 from O. microlepidotus) and of the representative non-toxin form (10, P19957 from H. sapiens). B, Bayesian molecular phylogeny of waprin toxins. The outgroup is the non-toxin P83300 from A. anser. The ribbon model of EU029740 from T. jacksonii shows β-strands in yellow.

Reflective of sequence variation of proteins in the CRISP family, its members have been found to interact with different target proteins, i.e. cyclic nucleotide-gated ion channels as well as L-type Ca²⁺ and BK_Ca K⁺ channels (30–32). Binding to the respective channels is speculated to be a general property of CRISP and was attributed to a predominantly hydrophobic cavity involving residues from both domain PR-1 and cysteine-rich domain (CRD), whereas specific channel block supposedly is taking place with residues of CRD (30–35). Six amino acids were identified for the calcium channel blocker Triflin (Protein Data Bank code 1WVR (30)) to bind to ion channels (numbering according to Triflin): Glu-44, Tyr-54, Ser-65, Tyr-125 (all four in PR-1), Thr-184, and Arg-185 (both in CRD). An additional four amino acids were conserved and postulated to affect ion channel function of this neurotoxin. They are all located in CRD: Phe-189, Leu-195, Tyr-205, and Phe-215. However, this work reveals that none of these 10 positions is conserved throughout the CRISP toxin family. Even more so our in silico studies could not confirm suggested functional motifs, namely EX₂F and DVF, at least as a general principle of CRISP. Differences in CRISP function of toxic venom proteins and the non-toxic representative from mouse are subtle and likely to be found in CRD, i.e. Leu-220 and Leu-230 (numbering according to mouse; Fig. 5). The promiscuous susceptibility of CRISP toward different ion channels is reflected in their high degree of sequence variation. In the absence of obvious sequence and structure differences between toxic and non-toxic specimens (Figs. 5 and 6) it will be necessary to acquire further data on the function of the CRISP family members to identify the functionally discriminating amino acids.

The hyaluronidase transcript sequenced from Liophis poecilogyrus showed significant sequence similarity to those sequenced from viperid venoms (36), and the snake sequences all formed a clade (Fig. 7). Kallikrein transcripts isolated from Philodryas olfersii retained residues essential for the potent hypotensive action mediated by the liberation of bradykinin from kininogen (Fig. 8).

Kunitz-type toxins belong to the superfamily of bovine pancreatic trypsin-like inhibitors. Although they share the same overall 3D fold, their antitrypsin activity may vary. It has been long known that the residue in the so-called P1 site of the trypsin inhibitor is positively charged (arginine, lysine, or histidine), whereas this residue in a chymotrypsin inhibitor usually is large and hydrophobic (leucine, phenylalanine, tyrosine, or asparagine) (37). From the multiple sequence alignment presented in Fig. 10 it can be concluded that kunitz-type toxins from P. olfersii, Ophiophagus hannah, Naja naja, and Bungarus multicinctus are likely to inhibit chymotrypsin, whereas the others putatively are trypsin inhibitors. Our 3D modeling efforts showed that the kunitz toxins vary, i.e. in the length of their C- but also their N-terminal tails (Figs. 10 and 11). The N-terminal tail is held in place by formation of two disulfide bonds involving the first and sixth as well as the second and fourth cysteines. The inhibitory residues are solvent-exposed and reside in a long surface loop immediately after the second cysteine, which indicates that the position of these residues is rather conserved among kunitz toxins (Figs. 10 and 11). Therefore, changes in polarity and charge are expected to affect the physicochemical properties of this region.

Within the lectin toxins, variation of a key tripeptide motif has been shown to have significant impact upon functionality (38) with EPN conferring mannose binding ability, whereas QPD confers galactose binding. In this study, both were obtained as well as new variants of this motif containing EAP, QAP, and LTD (Figs. 12 and 13). The EPN motif appears to be basal, whereas the QPD motif is an early emerging variant, and the other variants evolved subsequently at different times during the evolution of the animals themselves. The viper venom-specific heterodimeric forms have lost this motif entirely.

A novel MMP toxin had been reported previously from the venom of the lethal natricid snake Rhabdophis tigrinus (39). However, only a very small fragment was obtained so the MMP-subtype relationship remained unclear. In this study, a full-length MMP was obtained from the L. poecilogyrus library and was shown to be closest to MMP2 (Fig. 14) rather than showing the MMP9 relationship hypothesized in the previous R. tigrinus study.

A Type IB PLA₂ transcript was isolated from Trimorphodon biscutatus that retained not only the ancestral pancreatic loop but also the catalytic diad residues histidine and aspartate as well as two conserved tyrosine residues that together with structural water form the catalytic center (Fig. 15). This toxin is most likely responsible for conferring the presynaptic neurotoxicity observed for this venom (40). The electrostatic surfaces of PLA₂ IB clearly indicate the overall similarity of the five toxins studied both in terms of size and shape as well as with regard to the physicochemical properties. It can be concluded that all PLA₂ IB toxin specimens exhibit very similar neurotoxic function.

The SVMP/ADAM sequences were shown to be of the PIII type, consistent with previously reported fragments from Dispholidus typus (41). Numerous transcripts were also recovered with significant variations including changes in the number and spacing of cysteine residues and large scale deletions. Caenophidian 3FTx, lectin, MMP, SVMP, and waprin transcripts were all characterized by changes in ancestral cysteines in addition to the evolution of new cysteines (Figs. 2, 12, 16, 17, and 19). In contrast, transcripts of the caenophidian C3/CVF, CRISP, hyaluronidase, kallikrein, kunitz, and PLA₂ Type IB toxins preserved the ancestral cysteine numbers and spacing (Figs. 4, 5, 8, 10, and 15). Large deletions were detected in transcripts of the lectin and SVMP/ADAM toxins. Multiple transcripts of a deleted form of the lectin toxin were isolated from Enhydris polylepis (Fig. 12) in which a large stretch of residues including an ancestral cysteine is deleted, leaving a free cysteine, potentially facilitating dimerization. Another lectin toxin version from the E. polylepis cDNA (again for which multiple transcripts were obtained) had a significant change in sequence to the second half of the protein as a consequence of a frameshift mutation. The resulting new transcript contains overall four cysteine bonds and therefore may fold into a stable, bioactive molecule. A major truncation was detected in transcripts of the SVMP/ADAM toxins isolated from Psammophis mossambicus (Fig. 16). These transcripts consisted solely of the propeptide region normally post-translationally cleaved from the functional enzymatic region, and some isoforms had evolved new cysteines within this domain.

Structural Variation of the Venom System—
The combination of MRI, histological analysis, gross dissection, and examination of dentition revealed extensive variations in the relative size of the venom gland and lumen and variation in the course of the venom duct (Figs. 1, 20, and 21 and see "Appendix II"). Large venom glands, similar in size to those of some elapids, were found in representatives from each of the different non-front-fanged families, such as the colubrid snake Telescopus dhara, the homalopsid snake Cerberus rynchops, the psammophiine snake P. mossambicus, and the dipsadid snake Helicops leopardinus. The lethal colubrid snake D. typus had the most robust glands of all non-front-fanged snakes studied. In contrast, the venom glands of two colubrid snakes (Pituophis guttatus and Dasypeltis scabra) were found to be greatly atrophied as was that of the pareatid Pareas carinatus. The course of the venom ducts ranged from nearly craniad in the elapid and viperid to varying degrees of medial or craniomedial in the non-front-fanged species.

View larger version (63K): [in this window] [in a new window]   FIG. 20. Transverse histology of Masson’s trichrome-stained sections showing the relative size of venom glands (proportional to head size) for H. leopardinus (A), D. typus (B), Homalopsis buccata (C), P. guttatus (D), and D. scabra (E). VG, venom gland; SG, supralabial gland.

View larger version (180K): [in this window] [in a new window]   FIG. 21. Histology using modified Van Gieson’s stain. A, frontal section through Ahaetulla nasuta showing the structural differences between the supralabial gland and venom gland. B, transverse section showing a proximal to distal transition in the epithelial lining of the venom duct of Psammophis schokari. C, frontal section showing the relatively small venom vestibule of D. punctatus located adjacent to the fang. D, frontal section through R. tigrinus showing the venom vestibule located immediately adjacent to the venom gland. E, frontal section through the venom duct of T. capensis showing the concentric rings of surrounding connective tissue. F, transverse section through the venom duct of Coluber constrictor showing the venom duct opening to the oral cavity. G, frontal section through Psammodynastes pulverulentus showing the direct course of the venom duct from the venom gland to the fang sheath. H, frontal section through the fang sheath and diverticulum of Malpolon monspessulanus. I, frontal section through Oligodon smithii. showing the accessory venom duct extending away from the fang sheath. D, venom duct; F, fang; O, oral cavity; S, fang sheath; SG, supralabial gland; VG, venom gland; V, venom vestibule.

The greatest level of morphological variation was found in the topographical relationship between the venom duct and the fang. In some non-front-fanged taxa (e.g. Heterodon) the venom duct opens directly into the oral cavity rather than to the lumen of the fang sheath and the surface of the fang. In other cases the venom duct courses directly to the fang sheath with no venom vestibule located at the juncture between these two structures as it is in all front-fanged taxa. Most commonly in non-front-fanged snakes the venom duct opened both into the fang sheath and directly to the roof of the mouth immediately adjacent to the fang sheath; this dual opening was achieved by the venom duct, or in some cases the associated venom vestibule, paralleling the long axis of the fang sheath forming a prominent diverticulum. In a few non-front-fanged taxa there was a secondary venom duct that arose from the distal portion of the fang sheath and extended peripherally to contact the roof of the mouth. Combining information for the different structural features revealed the non-front-fanged snakes to possess a highly variable venom system architecture with all combinations of epithelial lining, venom vestibule, and venom duct topography identified (see "Appendix II").

The maxillary dentition was also revealed to be extremely variable among the non-front-fanged snakes, ranging from solid smooth fangs (type 1) to grooved fangs of varying depth and length (types 2–4) (Table III). Tooth size was also variable with significant enlargement occurring on multiple independent occasions. Particularly enlarged teeth were observed in representatives from all families, such as Dispholidus and Oligodon in Colubridae, Tomodon and Waglerophis in Dipsadidae, Macropisthodon in Natricidae, Malpolon and Rhamphiophis in Psammophiinae, and Homalopsis in Homalopsidae. In contrast, Atractaspis plus all elapids and viperids possessed type 5 dentition with an enclosed venom canal, and relative tooth size was also much less variable.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the sequencing surveys undertaken in this study, transcripts of 3FTx, CRISP, and SVMP/ADAM were the most phylogenetically widespread dominantly secreted toxin types, occurring in at least seven of the 10 non-front-fanged snakes examined. Other phylogenetically widespread dominant toxin transcripts were lectin and waprin. Identification of 3FTx transcripts as a common component of caenophidian venom is consistent with previous studies showing active expression of potent 3FTx in a range of non-front-fanged snakes (2, 14, 40). Similarly for the other toxin types, the congruence with previous LC/MS data on the same species provides additional evidence of active expression of bioactive proteins in the venom glands of non-front-fanged snakes (14). Multiple transcripts of the majority of toxins were recovered from individual cDNA libraries; this pattern is consistent with accelerated diversification in toxin multigene families as observed in elapids and viperids (19). Numerous transcripts were recovered with significant variations including changes in key functional residues, changes in the number and spacing of cysteine residues, and large scale deletions. These modifications represent potential neofunctionalization (evolution of novel bioactivities). These results support previous suggestions that the venom system is a basal characteristic of the advanced snakes (3, 4) and also previous crude venom studies (14, 16, 17). This has important implications for understanding the evolution and ecology of the advanced snake radiation and identifies venoms from non-front-fanged snakes as an important bioresource.

Despite their relative presence, these toxin types in different lineages should not be considered the only classes present in a particular venom. This caveat is due to the limited sequencing that detected only the major toxin transcripts for each species. Other types may be present at lower expression levels. It is likely that more detailed exploration of their venoms will reveal the presence of additional toxin types. This is demonstrated by this study recovering only 3FTx, CRISP, factor X, kunitz, and Type IB PLA₂ from O. microlepidotus but not other toxin types previously sequenced from this species such as factor V, natriuretic peptides, or nerve growth factor.

Toxin Structure-Function Relationships—
Structure-function relationships of a number of the toxin types sequenced in this study have been well characterized in venoms from snakes (Atractaspis, elapids, or viperids) or helodermatid lizards (Table II) (20). This includes information on the key amino acid residues necessary for conferring toxicity (e.g. CRISP proteins (30–35)), the role of cysteine spacing in determining folding structure and toxicity, and a demonstrated role for novel mutations such as large scale deletions in conferring new bioactivities.

CRISP toxin transcripts were present in the majority of the venom glands examined. Previous analysis of CRISP toxin sequences from the venom of the natricid snake R. tigrinus showed that it lacked the EX₂F motif thought to be responsible for the smooth muscle paralytic effect as well as the KX₆KR motif hypothesized to be essential for the inhibition of cyclic nucleotide-gated calcium channels. Subsequent bioactivity testing confirmed that R. tigrinus CRISP toxin indeed lacked these activities (31, 32, 42, 43), but it was unclear whether this was representative of CRISP toxins from the venoms of other non-front-fanged snakes. In this study, forms containing the EX₂F motif were present in some venoms (Figs. 5 and 6). Translation and expression of these encoded proteins would thus likely induce smooth muscle paralysis. It is notable that CRISP toxins are particularly rich in the venoms of species that are reptilian feeders (14) such as T. dhara and T. biscutatus. It may be that the toxic hypothermic effect is useful in slowing down the movement of exothermic prey. Studies on this aspect may shed significant light into the evolutionary pressures driving the molecular diversification of this toxin type.

Cation-permeable channels (sodium-, potassium-, and calcium-selective channels as well as cyclic nucleotide-gated channels, which are unselective for monovalent Na⁺ and K⁺ but also allow transfer of Ca²⁺ cations) often contain more than one binding site for channel blockers, one generally within the transmembrane bundle to bind e.g. anesthetics and one or more at their extracellular side that interact with toxins. A most effective channel block occurs when the ion permeation pore is directly occluded by a toxin from the extracellular side. This pore in many cases is bevelled, narrowing toward a selectivity filter (i.e. in potassium, calcium, and sodium channels). To attract positively charged ions this pore contains both acidic and aromatic residues. Therefore, extracellular channel blockers often contain positive charges, i.e. guanidinium groups as in arginine, which are flanked by H-bond donors, like in conotoxins and dendrotoxins. Due to their size, venom proteins can affect channel gating only from the extracellular side. We postulate that to occlude the channel pore these proteins require a 3D structure motif that (i) is accessible to interact with an ion channel, (ii) consists of one (ideally positively) charged residue to interact with the charged amino acids inside the ion permeation pore, (iii) exhibits one or more H-bond donor(s) in vicinity to the charged residue, and (iv) possibly contains an aromatic amino acid to form either - or cation- interactions with the channel pore.

Structural studies revealed that CRISP is a two-domain protein of conserved fold consisting of a pathogenesis-related PR-1 and a CRD, which are linked together via a flexible hinge (30, 44). Although significantly smaller than PR-1, the CRD spans the C-terminal six of a total of 16 conserved cysteines. The resulting three disulfide bonds stabilize the CRD fold and leave only marginal room for movement in this domain. It is widely assumed that hydrophobic residues of a concave cleft including both the PR-1 and the cysteine-rich domain would bind the CRISP to the ion channel (30), whereas actual channel block is hypothesized to involve amino acids of CRD only. The fact that CRISP toxins block a variety of ion channels may indicate that different residues located at different positions of CRD are responsible for occluding the extracellular entry to the channel. This hypothesis is supported by the observation that CRD can move relative to the PR-1 domain, which may even lead to exposure of residues that in the experimental x-ray structures (Protein Data Bank codes 1RC9 (44) and 1XX5 (31)) are found at the interface to PR-1. Yet the amino acids actually blocking ion channels remained unidentified to date and were mainly postulated from multiple sequence alignments. To our understanding, the cysteine-rich domain contains three sites, which may putatively block ion channels: (i) the loop between β11 and 7 spanning eight residues between the first and second cysteine in CRD, (ii) the loop connecting 7 and 8 containing up to six amino acids, and (iii) the C-terminal tail following the sixth conserved CRD cysteine.

A hypothesized functional EX₂F motif (34, 35) was found three to six residues C-terminal to the first CRD cysteine. In this study we could not confirm this motif to be conserved and thus functional throughout the CRISP family. However, the widespread taxonomical presence is suggestive that if this motif is not basal it is at least early emerging. Whereas the aromatic residue was conserved (either phenylalanine or tyrosine), the glutamate occurred in only five of 16 sequences. At the same position five asparagines, one glutamine, and two lysines were found, but interestingly no aspartate was found. In contrast, the C-terminal neighbor to glutamate in position +3 of the first CRD cysteine is highly conserved exhibiting only aspartate (nine occurrences) and asparagine (11 occurrences). This position corresponds to the first residue in the DVF motif that has been identified to selectively interact with L-type calcium channels (35). It ranges from position 4 to 6 after the first CRD cysteine. This motif was first identified in -conotoxin TxVII (45) and was later found in the venom protein ablomin (35). Our structural studies showed that the side chain of the aspartate/asparagine in DVF was oriented toward the core of CRD forming several H-bonds with the third and most C-terminal a-helix 9. The second and third residues of this motif are located at the surface of CRD with their side chains pointing toward the solvent. Yet valine was found only three times in this study, whereas in 14 instances this residue was charged (four times lysine and 10 times glutamate and aspartate). The third residue of this motif was large and hydrophobic and, with the exception of the sequence Q3SB05 from Pseudonaja textilis, aromatic. We hypothesize that these two residues may dock to ion channels because they satisfy, in most cases, the four criteria postulated earlier: (i) spatial accessibility, (ii) a charged amino acid, (iii) nearby H-bond donors, and (iv) an aromatic residue in spatial proximity to the charged amino acid. Exceptions to criterion ii are several hydrophobic or polar residues found in D. typus, L. poecilogyrus, E. polylepis, Lapemis curtus, Protobothrops mucrosquamatus, and Heloderma horridum. Interestingly potential H-bond donors are available in positions –1 and –2 of the fourth CRD cysteine (Fig. 5).

Alternatively, interactions with L-type calcium channels may involve one of the following motifs. (i) The loop tip between helices 7 and 8 may be involved. Here the first residue of the loop is either polar (Gln or Asn) or charged (Lys or Glu) followed by a mostly polar (Asn, Ser, or Thr) and an either polar (Ser, Asn, Thr, or His) or charged amino acid (Lys or Asp) just before the third conserved cysteine. This region contains charged residues in many toxins and is structurally exposed. However, it lacks an aromatic amino acid in its vicinity. (ii) The residues in position +1 or +2 of the first and in +1 of the fifth conserved CRD cysteine may be involved. The former was postulated to be involved in cyclic nucleotide-gated channel block and often contains a basic amino acid (see below and Fig. 5); the latter is predominantly phenylalanine. This substructure is also accessible and is held in place by a nearby disulfide bond. (iii) Positions +1, +2, and +3 after the sixth CRD cysteine may be involved. This part of the C terminus is mostly positively charged and polar. It is relatively flexible and accessible and may therefore interact with L-type calcium channels.

Morita and co-workers (32, 33, 35) proposed one or two residues immediately C-terminal of the first conserved CRD cysteine to be critical for interaction with cyclic nucleotide-gated ion channels. Their work revealed that the C-terminal domains of pseudechetoxin and pseudecin, which are very similar to CRD, were identical but for two residues. The KR motif in pseudechetoxin made this protein more susceptible to cyclic nucleotide-gated block than the NY of pseudecin. In this work, seven of 19 specimens had a positively charged residue in either position +1 or +2 of the first cysteine (Fig. 5). For these specimens, we therefore postulate some activity to block cyclic nucleotide-gated channels.

Modification of structural residues was also evident in numerous caenophidian venom toxin transcripts. It has been shown that changes in the spacing of ancestral cysteines, the occurrence of newly evolved cysteines, and modifications in structural residues flanking the cysteines can alter the three-dimensional structure of the molecule and thus the residues contributing to the surface chemistry with consequences for bioactivity (20). Such modifications can produce toxin variants that have specificities or potencies differing radically from previously characterized forms, including the potential emergence of neofunctionalizations.

Most of the 3FTxs sequenced in this study retained the ancestral 10-cysteine arrangement (19) (Fig. 2) as had been shown previously for the -neurotoxic 3FTx from the venom of the colubrid C. radiatus (2). However, the conservation of the ancestral cysteines does not preclude bioactivities other than -neurotoxicity as typified by a form from the venom of the colubrid snake Boiga dendrophila that retained the ancestral cysteines yet was only weakly -neurotoxic but had a newly derived presynaptic mode of neurotoxicity (16). As the sequences obtained in this study were even more divergent, including the evolution of alternate cysteines, it is quite likely that a multiplicity of novel activities has been derived.

In addition to variations in functional residues and cysteines, mutants were also sequenced with frameshifts, truncations, and putative exon deletions. Novel transcripts of the SVMP/ADAM toxin type were isolated from P. mossambicus that were comprised solely of the propeptide domain (Fig. 16). Although forms of SVMP/ADAM have been identified previously that selectively express a particular domain (e.g. comprised solely of the disintegrin domain (19)) this is the first time that the prepro domain has been discovered as being selectively expressed as the sole domain. The putative exon deletion in the lectin toxin variant from E. polylepis not only removed a large stretch of residues but also one of the ancestral cysteines, leaving a free cysteine, potentially facilitating dimerization (Fig. 12). Similarly the putative frameshift in the lectin toxin variant from E. polylepis produced transcripts with even numbers of cysteines, and therefore these sequences may be able to fold to produce stable frameworks (Fig. 12). Thus, genomic mutations in both the caenophidian SVMP/ADAM and lectin toxins have the potential to produce functional proteins with significantly altered bioactivities.

Venom toxins from non-front-fanged snakes may also prove useful for investigations of the structure-function relationships of the normal body proteins from which they were derived (20). In some cases, much more is known about the toxic forms than the ancestral body homologues. For example, the normal body forms of the CRISP proteins are poorly characterized with virtually nothing known of their bioactivities. Even the activities of forms that may play important roles, such as those with high expression levels in specific tissue or organs, remain unknown. The basal activity of the CRISP venom protein is likely to be relaxation of peripheral smooth muscle, such as helothermine from H. horridum (46), and may indicate a tissue-specific role for the ancestral normal body protein. The kallikrein toxins are an example of where an ancestral body action (liberation of bradykinin from kininogen) is preserved, but some toxin isoforms have derived additional activities (e.g. cleavage of fibrinogen). Similarly the two independently recruited types of PLA₂ proteins in snake venoms have derived activities that the normal body form lacks (e.g. antiplatelet toxicity, myotoxicity, and neurotoxicity). Consistent with this, the toxic forms contain a positively charged hotspot on the surface that is lacking in the ancestral form from the pancreas (Fig. 15). The venom proteins therefore represent exquisite natural "knock-out" studies of tremendous usefulness in elucidating the structure-function relationships of the non-toxin, body homologues of physiological importance. The relative assignment of conserved/variable functional residues should not be considered definitive as not all sites/proteins are well characterized, and other transcripts/toxin types may contain functional residues that confer ancestral or novel bioactivities, especially for the toxin types not listed above.

Timing of Toxin Recruitment Events—
The phylogenetic distribution of venom toxins in the Toxicofera (venomous squamates) provides insights into the timing of toxin recruitment events (Fig. 1). The majority of snake venom toxins (14 of 27) are either shared with the two other Toxicofera lineages (Iguania and Anguimorpha) (7 of 27) or occur near the base of the Caenophidia snake clade (7 of 27). The independent evolutions of advanced front-fang architectures in Atractaspis and Viperidae are linked with recruitments of new toxin types (Fig. 1). In the viperids, two new toxin types, c-type natriuretic peptide-bradykinin-potentiating peptide and PLA₂ (Type IIA), are closely linked to the evolution of advanced venom delivery systems and ambush feeding, whereas four other toxin types were evolved later on (renin-like aspartic protease, cytokine FAM3B, glycine-rich toxin, and waglerin). The evolution of advanced venom architecture in viperids also coincides with significant molecular diversification of two existing toxin types (SVMP/ADAM and kallikrein). The evolution of sarafotoxins in Atractaspis is also closely timed with the evolution of advanced front fangs, but whether as a cause or an effect (occurring before or after) the evolution of hollow fangs remains to be elucidated. In the elapids, the only two additional toxin types currently known do not coincide with development of a high pressure front-fanged venom system with the hole-punching, short stubby fangs useful for penetrating tough reptile scales. Instead the evolution of new venom systems appears to also be linked to the explosive diversification of existing toxin types: 3FTx and PLA₂ (Type IB) toxins in the elapids with numerous new bioactivities developed for each toxin type. The only documented newly evolved toxin types are found within the rapidly radiated Australian elapid snakes. One evolved at the base of this clade (factor X), and one evolved in the common ancestor of Pseudonaja and Oxyuranus (factor V).

The very long, derived glands of C. rhombeatus do not appear to be linked to the evolution of new toxin types either with the dominant toxin types being CRISP, kallikrein, Type IIA PLA₂, and SVMP/ADAM (including RGD disintegrins). Within Atractaspis the venom differences between long and short glanded species is unclear. It is also unknown whether the long glands of some Calliophis species are linked to significant changes in venom composition.

The early recruitment of a LYNX/SLUR-like gene to form the 3FTx toxin multigene family was one of the most significant developments in snake venom evolution, priming the Caenophidia for extensive diversification. Indeed, venoms from non-front-fanged species have been shown to be just as toxic as well characterized extremely potent elapid venoms (40). A unique nuance of this potent and fast acting toxin type is that, unlike most other toxins, the lethality is almost the same whether injected subcutaneously, intramuscularly, or even intravenously (19), and therefore delivery just under the skin is as efficient as straight into the bloodstream. The highest expression levels of this toxin type in the non-front-fanged snake families are in the gracile, fast moving forms that have only slightly or moderately enlarged rear maxillary teeth and predate upon fast moving, soft, thin skinned, non-dangerous prey items such as geckos and frogs (14). In contrast, the long fanged D. typus is an arboreal snake that includes birds as a major prey item, and the venom is rich in forms of the SVMP/ADAM toxins that are potently prothrombin-activating. The fish-eating aquatic homalopsid snakes, which also have large maxillary dentition, favor hemotoxic lectin toxins as well as ADAM toxins. The elapid snakes, which puncture tough reptile skin with their short, strong hollow front fangs, also typically express 3FTx transcripts as the dominant mRNA species. However, they favor the derived forms that lack the second and third ancestral cysteines.

Within the elapids additional toxin types were evolved later on but also with tremendous impact, such as factor X at the base of the Australian elapid clade and factor V in the common ancestor of Pseudonaja/Oxyuranus. The recruitment of factor X and factor V in the Australian clade of elapids would have also tremendously aided prey capture with only a small amount of these very active enzymes needed to cause severe disruption of blood chemistry. However, the activity of factor X toxins is rate-limited by the requirement for cofactors, particularly the essential need to form a 1:1 complex with factor V (47). Thus, the recruitment of factor V into the venom of the Oxyuranus/Pseudonaja clade resulted in a virtually complete, perfect toxin complex, greatly increasing the relative toxicity of the venoms and thus significantly aiding in the prey capturing ability. Subsequent to the recruitment of factor V, the Oxyuranus/Pseudonaja common ancestor split into two very different forms despite the two genera remaining very closely genetically related.

The Oxyuranus species are very large snakes with extremely long fangs and high venom yields. Unlike most Australian snakes, which often feed on reptiles, these snakes feed exclusively on mammalian prey including large rats and bandicoots, dangerous prey items capable of an adept defense potentially causing serious injury to the snake predators. The snakes successfully feed on such dangerous prey by minimizing prey contact time through the use of a snap-release form of striking along with a tendency for multiple strikes, thus overwhelming the prey items with copious amounts of the extraordinarily potent venom delivered deep into the tissues several times. A limitation of this form of prey capture is that the snakes need to be very warm to successfully use this energetically costly form of prey capture. Consequently their habitat is limited only to the tropical north of Australia and in New Guinea (Oxyuranus scutellatus) and the baking heat of the channel country in the Australian outback (O. microlepidotus).

In contrast, the Pseudonaja species have taken a very different evolutionary path, taking advantage of possessing such toxic venom to greatly minimize the amount of biological energy expended in venom production. Pseudonaja species are smaller snakes with very short fangs and much smaller venom yields, utilizing constriction to hold non-dangerous prey items (such as reptiles) in place while envenomating. As these snakes do not expend as much energy in prey capture, they do not have to be as warmed up as the Oxyuranus species. These snakes consequently have taken advantage of their extremely toxic venom to become one of the most adaptable and successful snake types of the entire Australian continent.

Another conspicuous finding of this study was the resolution of the lectin toxins into a single clade with homomeric forms not reciprocally monophyletic in relation to the heterodimeric form found exclusively in viperid venoms (Fig. 13). The homomeric and the heterodimeric lectin forms had been considered previously to be the result of two separate toxin recruitment events (3) or the result of a single recruitment event (18). However, reconstructing the evolutionary history had been hampered by the scarcity of homomeric sequences. In this study, we sequenced numerous lectin toxin transcripts from four additional non-front-fanged snake families. This allowed for a more robust reconstruction of the molecular evolutionary history and thus the confirmation that all forms of the snake venom lectin toxins are the result of a single toxin recruitment event (Fig. 1) and that the viperid venom heterodimeric forms arose from within the homomeric basal form (Fig. 13). This study also allowed for the inference that the EPN mannose-binding motif is basal whereas the QPD galactose-binding variant was an early emerging derivation that predated the viperid radiation.

Derivations of the Venom System—
Taub (7) described extensive morphological variation within the venom gland (his "Duvernoy’s" gland) of non-front-fanged snakes. The present study demonstrates that similar morphological variation exists in the venom duct as well as in the topographic relationship between the venom duct and the fang sheath (see "Appendix I"). These morphological variations do not assort along established evolutionary lines, nor do they correlate well with the attributes of the venoms detailed in this study. This is in contrast to the front-fanged Elapidae and Viperidae families, which each have a well defined, characteristic venom apparatus at the familial level with some minor variations (Fig. 1), such as the size of venom glands and connections of the compressor and other head muscles. The same is true for the front-fanged genus Atractaspis but not for the non-front-fanged snakes now included in the Atractaspidinae subfamily, which were shown in this study to be quite variable.

The mechanics of venom injection in front-fanged snakes relies on pressurization of the venom delivery system (48–51). The colubrid snake D. typus is the only species with well characterized rudimentary compressor musculature (52), although there is evidence that this has been convergently evolved in the lamprophiine snake Mehelya capensis (52) and in the non-front-fanged atractaspidine Brachyophis revoili (53). The general absence of these features from the venom delivery system of non-front-fanged snakes suggests that a substantially different mechanism(s) is present within this group. The limited skeletal muscle contact with the venom gland in non-front-fanged snakes (54), the relatively weak wall of the venom duct, and the presence of a venom vestibule would all oppose the pressurization of the venom delivery system (but see Jansen and Foehring (55)). The multiple topographic relationships between the venom duct and the fang, including taxa in which the venom duct opens to the roof of the mouth rather than the fang sheath, clearly indicate that deformation of the fang sheath need not be an important part of venom delivery in non-front-fanged snakes.

Interestingly the colubrid family also contained a clade in which the venom gland was greatly atrophied, the American "rat snake" clade of colubrid snakes, as typified by P. guttatus (Figs. 1 and 20). The American rat snakes have secondarily evolved a new form of prey capture (powerful constriction) and prey preference (rodents). Subsequently the gland has become greatly atrophied. Similarly the African D. scabra, which feeds exclusively on bird eggs, also has greatly atrophied venom glands (Fig. 20). This is paralleled in the sea snake Aipysurus eydouxii that, subsequent to switching from feeding on fish to feeding exclusively on fish eggs, has greatly atrophied venom glands (Fig. 1) and almost entirely lost fangs accompanied by significant accumulation of deleterious mutations to the toxins still transcribed (56, 57). Similarly the lizard egg-eating Brachyurophis species of Australian terrestrial elapids also have significant reduction of the venom system. Finally the other few lineages displaying an absence or reduction of the glands are malacophagous (Asian pareatids and some American dipsadids) (1).

Changes in gland size were not restricted to atrophying but also included spectacular lengthening. In each of the front-fanged clades, tremendous elongation has occurred independently: Atractaspis engaddensis, Atractaspis microlepidota, Atractaspis micropholis, and Atractaspis scortecci in the Atractaspinae, Calliophis intestinalis and Calliophis bivirgata in the Elapidae, and Causus resimus and C. rhombeatus in the Viperidae. The elongations extend to a quarter of the body length. The biological advantage gained by these elongations remains to be elucidated. In at least the case of Causus the change in gland length was not accompanied by a significant shift in venom profile or recruitment of new toxin types. Instead, having typical viperid venom composition with the major toxin types sequenced (SVMP, kallikrein, and PLA₂ (Type IIA)) in this study being consistent with molecular weights obtained in a previous LC/MS study by us (14). It remains to be investigated whether there are significant venom compositional differences between long and short glanded forms within the Atractaspis and Calliophis genera.

Snakes of Potential Medical Concern—
This study has important implications in regard to human medicine by providing new information regarding snake venom composition and thus potential clinical effects of envenomation. It also highlights the potential danger of snakes regarded as of trivial concern based solely upon the lack of severe bites, but in some cases the rarity of reports is due to the rarity of the snake in captivity or encounters in the wild.

This study has shown that the venom transcriptome of Thrasops jacksonii is very similar to the extremely potent venom of the very closely related (and proven lethal) D. typus, particularly the abundance of PIII-type SVMP/ADAM toxins. The lethality of D. typus venom is the result of high expression levels of a potent prothrombin-activating form of the SVMP/ADAM toxin type. In the clinical pathology, the prothrombin activation causes consumptive coagulopathy, resulting in the disappearance of fibrinogen from the blood and the elevated presence of d-dimer products with the ultimate net result of incoagulable blood. Predation by T. jacksonii on live mammalian prey results in rapid death in a manner identical to that produced by D. typus: profound disruption of hemostasis manifested by rapid and copious bleeding from the mouth and nostrils.⁴ Clearly potent venom toxins are actively expressed in this species. We advise exotic animal keepers to treat this species with the utmost caution until studies of the action of T. jacksonii venom upon the hemostatic system are undertaken, including the relative neutralization by D. typus antivenom. In at least this case, absence of evidence should not be taken as evidence of absence.

Conclusion—
Collectively these findings strongly support that there has been extensive evolutionary modification of the venom system in the advanced snake radiation (Caenophidia). All variables appear to change independently ranging from the biochemical variation and specialization of the venoms to the dentition and glandular morphology. The long evolutionary history of the advanced snake radiation (100 million years) has resulted in extensive diversification of the venom system and shows how little we really know about venom macroevolution and the potential role in the ecology/evolution of the animals themselves. Furthermore the toxin types identified here should not be regarded as the only toxins present in those venoms. Additional experimental efforts will certainly reveal new isoforms of these toxins, and some classes will almost certainly be confirmed as evolving earlier than currently appreciated. Further entirely new toxin types may yet be discovered in any one of the poorly studied snake lineages. Our major findings are of a multidisciplinary nature with uses ranging from evolutionary biology to protein evolution and highlight the underutilization of snake venoms as bioresources in drug design and development.

	ACKNOWLEDGMENTS

	FOOTNOTES

 
Received, March 2, 2007, and in revised form, September 10, 2007.

Published, MCP Papers in Press, September 17, 2007, DOI 10.1074/mcp.M700094-MCP200

¹ The abbreviations used are: 3FTx, three-finger toxin; CRISP, cysteine-rich secretory protein; SVMP, snake venom metalloprotease; ADAM, a disintegrin and metalloprotease; BLAST, Basic Local Alignment Search Tool; 3D, three-dimensional; SPDBV, Swiss-PdbViewer; MRI, magnetic resonance imaging; T, tesla; CVF, cobra venom factor; MMP, matrix metalloprotease; PLA₂, phospholipase A₂; CRD, cysteine-rich domain.

² Homology model coordinates can be obtained from H. Scheib. E-mail: holger{at}moltalk.org.

³ Sequence alignments can be obtained from B. G. Fry. E-mail: bgf{at}unimelb.edu.au.

⁴ B. G. Fry, personal observations.

^* This work was supported by grants (to B. G. F.) from the Australian Academy of Science, Australian French Association for Science and Technology, Australia and Pacific Science Foundation, CASS Foundation, Ian Potter Foundation, International Human Frontiers Science Program Organisation, Netherlands Organisation for Scientific Research, and University of Melbourne (Faculty of Medicine and Department of Biochemistry) and a Department of Innovation, Industry and Regional Development Victoria Fellowship; by Australian Research Council Grant DP0451663 (to B. G. F. and J. A. N.); and by an Australian Government Department of Education, Science and Training/EGIDE International Science Linkages grant (to B. G. F., J. A. N., and N. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TMEBI Data Bank with accession number(s) EU029668–EU029765, EU036635–EU036637, and EU091709–EU091713.

^|| Present address: Nestlé Research Center, Vers-chez-les-Blanc, P. O. Box 44, 1000 Lausanne, Switzerland.

To whom correspondence should be addressed. E-mail: bgf{at}unimelb.edu.au

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