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«The handle holds various files of this Leiden University dissertation. Author: Vonk, Freek Jacobus Title: Snake ...»

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The handle http://hdl.handle.net/1887/19952 holds various files of this Leiden University


Author: Vonk, Freek Jacobus

Title: Snake evolution and prospecting of snake venom

Date: 2012-09-06

Snake Evolution and

Prospecting of Snake Venom

Freek Jacobus Vonk

To my family and (snake) friends, with special thanks to my parents…..

Freek Jacobus Vonk

Snake Evolution and Prospecting of Snake Venom

Dissertation Leiden University

Cover photo: a wild king cobra (Ophiophagus hannah), Indonesia (Java). By Freek Vonk Printed by: Wöhrmann Print Services, Zutphen.

Copyright © 2012 by Freek J. Vonk, Leiden, The Netherlands. All rights reserved.

Snake Evolution and Prospecting of Snake Venom Proefschrift ter verkrijging van de graad van Doctor aan de Universiteit Leiden, op gezag van de Rector Magnificus Prof. Mr. P.F. van der Heijden, volgens besluit van het College voor Promoties te verdedigen op donderdag 6 september 2012 klokke 10:00 uur door Freek Jacobus Vonk Geboren te Dordrecht, Nederland 24 februari, 1983.

Promotiecommissie Promotor Prof. Dr. Michael K. Richardson Overige leden Prof. Dr. Carel J. ten Cate Prof. Dr. Herman P. Spaink Dr. Bart Vervust (University of Antwerp) Prof. Dr. Rob E. Poelman (Leiden University Medical Centre) Prof. Dr. Peter G.L. Klinkhamer The work described in this thesis was supported by a Toptalent grant from the Netherlands Organization for Scientific Research under grant number 021.002.034 and funding from the Naturalis Biodiversity Center (Leiden).


Chapter 1 Introduction 1

Chapter 2 Snake Venom: from Fieldwork to the 3 Clinic.

Chapter 3 Evolutionary Origin and Development 25 of Snake Fangs Chapter 4 Axial Patterning in Snakes and 39 Caecilians: Evidence for an Alternative Interpretation of the Hox Code.

Chapter 5 Massive Evolutionary Expansion of 60 Venom Genes in the King Cobra Chapter 6 An Efficient Analytical Platform for 78 On-line Microfluidic Profiling of Neurotoxic Snake Venoms Towards Nicotinic Receptor Like Affinity

Chapter 7 Summary of Thesis and Discussion 112

Samenvatting van T

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Snakes are limbless predators that have evolved two different ways of incapacitating prey: suffocation and envenomation. The evolution of these life-history strategies lie in close relation with the evolution of body-elongation and limblessness. Taken together, these adaptations underlie the massive radiation of snakes which occurred after the K-T boundary and extinction of the dinosaurs, presumably because multiple niches became available and many of their predators got extinct. In this thesis, I examine the evolution of these adaptations. A general question to address would be: what underlies these adaptations in snakes and how do they correspond with their ecology? In addition to these evolutionary questions, I also provide a summary in chapter one on the current status of using snake venom toxins in drug discovery and design, because the evolution of venom is closely related to the evolution of a serpentine body form and fangs, but also to the question of “why” snake venom is so interesting for the pharmacological prospector. The high potency and specifity of snake venom toxins is generated by millions of years of evolution and strong selective pressures, so it is my believe that the different chapters in my thesis are tightly bound together. It is believed that it was Aristotle who first suggested – on the basis of symptoms observed in snakebite victims – that snakes may also be used to cure certain diseases (it was not yet known that venom was responsible for the observed effects). And in 1835 venom was already (although unsuccessfully) used to treat rabies. This field of “bioprospecting” has accelerated especially in the last decade, mainly because the development of many new sequencing and screening techniques. In chapter two, we go into the development and evolution of the different snake venomconducting fangs. This has been a matter of great controversy among scientists for the last century and many different hypotheses have been proposed. This was particularly because the two groups of snakes with fangs in the front of their upper jaw (Elapidae and Viperidae), have only relatively recently in the 90s been shown not to form a monophyletic group, hinting towards an independent origin and evolution of front fangs. By using modern techniques such as in situ hybridization of snake embryos, and careful histological analysis we were able to reconstruct the evolutionary history and make a solid hypothesis for

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elongation and deregionalization in snakes, since that presumably has also been a major driver of venom evolution by providing the ability for snakes to coil up and act as coiled spring that can strike and envenomate prey at a distance. Since Hox determine the basic structure of an embryo we have performed many in situ hybridisations in snake embryos and carefully compared the expression profiles of different Hox genes. For the fourth chapter of this thesis we sequenced a draft genome of the largest venomous snake in the world, the king cobra (Ophiophagus hannah) and annotated many of its venom genes. We wanted to find out whether we could find evidence of gene duplication and / or gene modification of physiological genes for use in the venom gland. The birth-and-death model of gene evolution is the canonical framework for venom evolution, but was never actually thoroughly studies due to lack of genomic resources. Our genomic sequences let us study the king cobra venom genes in relation to its ‘normal’ physiological genes. In the fifth chapter, we have set out to develop a new efficient and rapid method of analysing snake venom mixtures and performing a biological assay at the same time. We used the acetylcholine binding protein (AChBP) as biological target, because acetylcholine receptors are also often involved in neurological disorders, such as Alzheimer’s and Myasthenia gravis. In the last chapter of this thesis, I discuss this work and try to put it into broader perspective. The field of evolutionary genomics, molecular evolution as well as bioprospecting is huge and broad. However, I strongly believe that working on the edge of different scientific disciplines will provide you with a better understanding of nature and allows you to answer more questions.

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Vonk FJ, Jackson K, Doley R, Madaras F, Mirtschin PJ and N Vidal.

This chapter has been published as 'cover article' in BioEssays 33, 269-79 (2011).

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Snake venoms are recognized here as a grossly under-explored resource in pharmacological prospecting. Discoveries in snake systematics demonstrate that former taxonomic bias in research has led to the neglect of thousands of species of potential medical use. Recent discoveries reveal an unexpectedly vast degree of variation in venom composition among snakes, from different species down to litter mates. The molecular mechanisms underlying this diversity are only beginning to be understood. However, the enormous potential that this resource represents for pharmacological prospecting is clear. New high-throughput screening systems offer greatly increased speed and efficiency in identifying and extracting therapeutically useful molecules. At the same time a global biodiversity crisis is threatening the very snake populations on which hopes for new venom-derived medications depend. Biomedical researchers, pharmacologists, clinicians, herpetologists and conservation biologists must combine their efforts if the full potential of snake venom derived medications is to be realized.

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Snakes are represented on earth today by some 3150 species 1. Of these the vast majority (ca. 2700 species, see Fig. 1) represent a single massive diversification event which occurred after the K-T boundary and extinction of the dinosaurs. This large and relatively recent group is known as Caenophidia or “advanced snakes” and characterized by the possession of a venom-delivery system or components of such a system 2. Snakes traditionally considered venomous are the 600 or so species with tubular front fangs, muscularized venom glands, and a bite significantly dangerous to humans (Viperidae, Elapidae and Atractaspidinae) - including well-known examples as the cobras, seasnakes, vipers and rattlesnakes. The remaining caenophidians were traditionally classified as “Colubridae”, meaning snakes with a venom gland whose venom poses no danger to humans, and who lack the fangs at the front of the mouth for injecting it. The “Colubridae” has been shown to be paraphyletic, and most of its subfamilies have recently been elevated to a familial rank in order to reflect their evolutionary distinctiveness (Fig. 1) 1,3.

“Colubrid” snakes have been largely neglected in venom research, because of the sole fact that bites have not been perceived as of medical importance, except few species as the African boomslang (Dispholidus typus) and twigsnakes (Thelotornis spp.) and the Asian yamakagashi (Rhabdophis tigrinus). However, during the last few years, there has been a trend towards studying the venoms of these harmless venomous snakes to, primarily, increase our understanding of venom evolution. It became evident that these harmless snakes do secrete a strong acting venom with powerful toxins, although in a significantly lower amount and without an efficient injection mechanism 2,4. This influenced the ongoing quest for venom molecules that may be useful in fighting human disease - a snake need not be dangerous to humans for its venom to still have a profound effect upon the human body. This field of biodiscovery is now rapidly emerging, especially due to the development of modern high-throughput screening assays that allow rapid identification of potential therapeutic agents.

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number of currently known species of each family and the number of red listed species. Families in grey used to make up the old traditional “Colubridae”. Phylogeny based on 1,5.

Snake venom contains a mixture of powerful proteins and peptides that have evolved to be targeted to receptors, ion channels or enzymes 6, in addition to some carbohydrates, nucleosides, lipids, and metal ions, whose functions are not all known 7,8. They interact with a wide variety of mammalian proteins and can disrupt the central and peripheral nervous systems, the blood coagulation cascade, the cardiovascular and neuromuscular systems, and homeostasis in general. These venom proteins act with great precision – different toxins recognize different subtypes of certain receptors with only subtle differences – and are very biologically active. The precision and power with which they work lies right at the centre of why they form such a valuable resource to biochemists, biomedical researchers, evolutionary biologists and others.

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components. In addition, some fundamental biological processes have been revealed by using toxins as probes to study cells and their receptors. Here we review the state of this field and emphasize that the emerging field of high-throughput screening assays applied to a wide range of unstudied venoms, has the potential to provide a solid basis for the discovery of new lead compounds for new drugs.

Forces driving evolutionary divergence in venom composition: diet, phylogeny, biogeography and ontogeny Only in relatively recent years has the remarkable variability of venom composition at the genus, species, subspecies, population and even individual levels been fully appreciated, and the underlying ecological forces and mechanisms of mutation at the molecular level started to be identified and understood.

Variation in venom composition has far-reaching implications. For the treatment of snakebite, for example, the importance of using pooled venom (mixtures of venom from several individuals of the same species representing different ages and geographical origins) has been emphasized in the production of antivenom to – in some cases - produce a serum that will be maximally effective against a bite from any snake of that species 9. However, it should be noted that many antivenoms provide excellent – sometimes even better – cross reactions against antigens not even included in the original mixture, a phenomenon which is not yet well understood.

Since different species of prey differ in their physiological reaction to venom molecules, it follows that venom composition is linked to diet and varies from the species level all the way to individuals. In saw-scaled vipers (Echis), venom from those species that feed on arthropods was highly toxic to scorpions 10. By contrast, scorpions were unaffected by venom from those species that feed on mammals 10. Phylogenetic analysis revealed repeated instances of co-evolution of venom composition with prey preference. Other examples of variation in venom composition, at the individual to the species level, correlated with diet have been found in coral snakes (Micrurus) 11, rattlesnakes (Crotalus and

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arietans) 18, and others.

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