Beta cardiotoxin a novel protein isolated from the venom of ophiophagus hannah (king cobra) showing beta blocker activity

β-CARDIOTOXIN: A NOVEL PROTEIN ISOLATED FROM THE VENOM OF OPHIOPHAGUS HANNAH (KING COBRA) SHOWING BETA-BLOCKER ACTIVITY NANDHAKISHORE RAJAGOPALAN (M. Sc. (Life Sciences)) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT THE NATIONAL UNIVERSITY OF SINGAPORE DEPARTMENT OF BIOLOGICAL SCIENCES, FACULTY OF SCIENCE, NATIONAL UNIVERSITY OF SINGAPORE JANUARY, 2008 Wxw|vtàxw àÉ Åç ytÅ|Äç tÇw àxtv{xÜá ii iii ACKNOWLEDGEMENTS I would like to thank my supervisor Professor R Manjunatha Kini for his constant encouragement throughout my stay in Singapore. He provided me an opportunity to work in his lab as a visiting student from January to December 2003. He encouraged me to join the graduate program at NUS, which, I consider as a turning point in my life. What I really like about his management is the freedom he has given me in thinking and designing my work. This has molded me as an independent researcher over the past few years. Next, I would like to thank my co-supervisor Associate Professor Prakash Kumar. He has been a pillar of support throughout my time in Singapore. His useful suggestions during the manuscript preparation are not only reflected in the published article but will also influence the way I write in future. I would like to thank the graduate program run by the National University of Singapore for their financial support. I would also like to thank the Biomedical Research Council (BMRC) for their grant which funded the research. All this work would not have been possible without the support of our able collaborators. I would like to thank Associate Professor Peter Wong for helping me out whenever I had some experiments to be done at the Department of Pharmacology. I would also like to thank Dr. Zhu Yi Zhun for opening the doors of his lab for my work. I would like to thank the staff and students from the Department of Pharmacology for their technical support. Thanks to Assistant Professor Bian Jinsong, Dr. Wong Zong Jing, Mrs. Ting Wee Lee, Ms. Pei Ling, Ms. Kay Lee and Ms. Ning Li. I would also like to thank Assistant Professor Jayaraman Shivaraman and Ms. Sunita for their kind support for the structural studies. I would like to thank all the teachers who made a difference in my life. I would like to thank Mrs. Radha, Mrs. Meera, Mrs. Vijayalakshmi, Mrs. Saraswathi iv Chandrasekaran, Dr. Akbar Sha, Dr. M. Krishnan, Dr. A. S. Rao and Dr. Chellam Balasundaram. I would like to express my gratitude to my seniors Dr. Selvanayagam Nirthanan (Niru) and Dr. Rajamani Lakshminarayanan (Lakshmi) for their valuable suggestions and constant motivation. I would like to thank all my lab mates for making my stay fun and entertaining. Thanks to Dr. Pung Yuh Fen, for guidance during my stint as a trainee, Dr. Yajnavalka Banerjee, for providing great support and guidance, Dr. Md Abu Reza, Dr. Dileep Gangadharan, Dr. Syed Rehana, Dr. Joanna Pawlak, Dr. Kang Tse Siang, Rocky, Shifali and Dr. Raghurama Hegde for all the help they have done. I would like to thank Cho Yeow and Shi Yang for organizing great lab parties and Dr. Robin Doley for managing the complicated finances for these parties. I would like to thank Amrita and Girish for keeping me highly entertained during the writing of the thesis. I would also like to thank the “Kids Army” (a.k.a the undergrads) members Sin Min, Ee Xuan, Ming Zhi, Bee Har and Maulana for spreading their infectious enthusiasm. Last but not the least I would like to thank Ms. Tay Bee Ling for maintaining the lab finances and making sure we get things on time. I am grateful to my family for their support. Thanks to my father Mr. Rajagopalan and my mother Mrs. Lalitha Rajagopalan, my brothers Mr. Prasanna and Mr. Vasanth and my cousins Ms. Aarthi Ravichandran and Ms. Madhulika Ravichandran. Nandhakishore Rajagopalan January, 2008 v TABLE OF CONTENTS Page Dedication ii Acknowledgements iv Table of contents vi Summary xii Research collaborations xiv Acknowledgement of copyright xv Photo courtesy xvi List of figures xvii List of tables xx Abbreviations xxi CHAPTER ONE: INTRODUCTION 1.1 VENOMOUS SNAKES 1.1.1 Classification of venomous snakes 1.1.2 Ophiophagus hannah SNAKE VENOMS 10 1.2.1 Classification of venom proteins 10 1.2.1.1 Enzymatic proteins 10 1.2.1.2 Non-enzymatic proteins 13 1.2 1.3 THE THREE-FINGER TOXIN FAMILY 14 1.3.1 Neurotoxins 16 1.3.2 Non-conventional toxins 19 1.3.3 Muscarinic toxins 22 1.3.4 Fasciculins 23 1.3.5 Cardiotoxins 26 1.3.6 Calciseptine and FS2 toxin 29 1.3.7 Dendroaspin or mambin 31 1.3.8 Hannalgesin 33 1.3.9 3FTXs from Colubridae snakes 33 vi 1.3.10 3FTXs from Viperidae snakes 36 1.3.11 3FTXs as dual functional molecules 36 1.3.12 The 3FTX fold in non-venom proteins 37 1.3.13 The three-finger fold: ‘leprechauns ’ of molecular interactions 1.4 1.5 37 THE G PROTEIN-COUPLED RECEPTORS 44 1.4.1 General organization of GPCRs 45 1.4.2 The β-adrenergic signaling pathway 50 1.4.2.1 β1-AR signaling 52 1.4.2.2 β2-AR signaling 53 1.4.2.3 β3-AR signaling 56 1.4.3 Structures of β2-adrenergic receptor 56 AIM AND SCOPE OF THE THESIS 65 CHAPTER TWO: IDENTIFICATION AND ISOLATION OF βCARDIOTOXIN 67 2.1 INTRODUCTION 68 2.2 MATERIALS AND METHODS 70 2.2.1 Reagents and kits 70 2.2.2 O. hannah venom glands 70 2.2.3 Isolation of total RNA 70 2.2.4 Construction of cDNA library using 5’-RACE ready cDNA 71 2.2.5 TA-cloning 71 2.2.6 Preparation of E. coli DH5α competent cells 72 2.2.7 Heat-shock transformation 72 2.2.8 Isolation of plasmid DNA 73 2.2.9 Verification of clones using restriction digestion 74 2.2.10 DNA sequencing and analysis 74 2.2.11 O. hannah venom 74 2.2.12 Chemicals and columns 75 2.2.13 Isolation of novel proteins 75 vii 2.3 2.2.14 Molecular mass determination 75 2.2.15 N-terminal sequencing 76 2.2.16 Circular dichroism (CD) spectra 76 RESULTS 78 2.3.1 Construction of O. hannah venom gland cDNA library 78 2.4 2.3.2 Identification of novel proteins 78 2.3.3 Isolation and characterization of novel proteins 83 2.3.4 CD spectra of novel proteins 86 DISCUSSION 90 2.4.1 Identification and isolation of novel proteins from O. hannah venom 90 2.4.2 Identification of unique secondary structural conformation of β-cardiotoxin 2.5 CONCLUSIONS 93 96 CHAPTER THREE: MECHANISM OF ACTION OF β-CARDIOTOXIN 97 3.1 INTRODUCTION 98 3.2 MATERIALS AND METHODS 101 3.2.1 Animals 101 3.2.2 Methods of protein administration 101 3.2.3 In vivo toxicity study 102 3.2.4 Anticoagulant activity 102 3.3 3.2.4.1 Prothrombin time 102 3.2.4.2 Recalcification time 102 3.2.5 Hemolytic assay 103 3.2.6 ECG monitoring and heart rate determination 103 3.2.7 Isolated perfused heart 104 3.2.8 Measurement of isovolumetric cardiac performance 105 3.2.9 Competitive binding assay 105 RESULTS 107 3.3.1 In vivo toxicity study 107 viii 3.4 3.5 3.3.2 Anticoagulant activity 107 3.3.3 Hemolytic activity 108 3.3.4 Cardiac effects of β-cardiotoxin 108 3.3.5 Isolated perfused heart studies 112 3.3.6 Competitive binding assays 112 DISCUSSION 117 3.4.1 β-Cardiotoxin belongs to a new class of 3FTXs 118 CONCLUSIONS 121 CHAPTER FOUR: CHARACTERIZATION OF A MOLTEN GLOBULE INTERMEDIATE OF β-CARDIOTOXIN 122 4.1 INTRODUCTION 123 4.2 MATERIALS AND METHODS 124 4.2.1 Proteins and reagents 124 4.2.2 Thermal denaturation studies using CD spectroscopy 124 4.2.3 Chemical denaturation studies using CD spectroscopy 125 4.2.4 Effect of pH on secondary structure 125 4.2.5 Combined effects of pH and temperature on β-cardiotoxin structure 4.3 125 RESULTS 126 4.3.1 Thermal denaturation studies 126 4.3.2 Chemical denaturation studies 130 4.3.3 Effect of pH on secondary structure 130 4.3.4 Combined effects of pH and temperature on β-cardiotoxin structure 4.4 DISCUSSION 133 137 4.4.1 Identification of a unique α-helical ‘molten globule’ intermediate 4.5 CONCLUSIONS 137 142 CHAPTER FIVE: STRUCTURAL CHARACTERIZATION AND STRUCTURE-FUNCTION RELATIONSHIPS OF ix β-CARDIOTOXIN 143 5.1 INTRODUCTION 144 5.2 MATERIALS AND METHODS 145 5.2.1 Proteins and reagents 145 5.2.2 Crystallization of β-cardiotoxin 145 5.2.3 Data collection 146 5.2.4 Peptide synthesis 146 5.2.5 Purification of synthetic peptides 146 5.2.6 Mass determination 147 5.2.7 Air oxidation of peptides 147 5.2.8 Competitive binding assays 147 RESULTS 148 5.3.1 Crystallization of β-cardiotoxin 148 5.3.2 Synthesis and purification of peptides 148 5.3.3 Competitive binding assays 153 DISCUSSION 157 5.4.1 Crystallization of β-cardiotoxin 157 5.4.2 Structure-function relationships of β-cardiotoxin 157 CONCLUSIONS 160 5.3 5.4 5.5 CHAPTER SIX: CONCLUSIONS AND FUTURE PERSPECTIVES 161 6.1 GENERAL CONCLUSIONS 162 6.2 FUTURE PERSPECTIVES 165 6.2.1 3-Dimensional structure of β-cardiotoxin 165 6.2.2 Site-directed mutagenesis studies to determine the functional site 165 6.2.3 Characterization of the α-helical ‘molten globule’ intermediate 166 6.2.4 Nature of antagonism of β-cardiotoxin 166 6.2.5 Developing shorter bioactive peptides 166 6.2.6 Characterization of other novel proteins identified from the cDNA library 167 x Figure 5. Effects of ␤-cardiotoxin on cardiac function. Charts showing changes in ECG recorded before (above) and 10 after (below) the administration of control (A; 0.9% NaCl), mg/kg CM18 (B), and mg/kg ␤-cardiotoxin (C). Horizontal arrows of equal lengths highlight the changes in ECG patterns. D) Change in heart rate (BPM) 10 after the administration of control (0.9% NaCl dotted bar), CM18 (1 mg/kg gray bar), and ␤-cardiotoxin (1 mg/kg black bar). Each data set represents mean Ϯ sd (nϭ3). E) Dose-dependent reduction of heart rate 10 after the administration of ␤-cardiotoxin. Each data point represents mean Ϯ sd (nϭ3). indicated by an increase in the distance between successive QRS complexes on administration of ␤-cardiotoxin, suggesting a negative chronotropic effect that might cause bradycardia (Fig. 5C). Thus, a conventional CTX increases the heart rate, while ␤-cardiotoxin decreases the heart rate (Fig. 5D). This decrease in the heart rate induced by ␤-cardiotoxin is dose dependent (Fig. 5E). Thus, ␤-cardiotoxin induces negative chronotropism in the heart rate in rats unlike conventional CTXs. Isolated perfused heart studies The direct effects of ␤-cardiotoxin on cardiac tissue was determined using the Langendorff isolated perfused rat hearts. There were no changes in any of the cardiac parameters in the control group (Fig. 6A), whereas, ␤-cardiotoxin induced a negative chronotropic effect (Fig. 6B). ␤-Cardiotoxin at ␮M caused a marked reduction in the heart rate (Fig. 6C) without any significant change in the contractility as indicated by the LVDEP (Fig. 6D). Thus, the decrease in the heart rate induced by ␤-cardiotoxin in rats is most probably due to its direct action on the cardiac muscles. Interaction of ␤-cardiotoxin with human ␤-adrenergic receptors (␤-ARs) ␤-ARs are expressed abundantly in cardiomyocytes, and the adrenergic signaling cascade is responsible for the control of heart rate (31). Therefore, we hypothesized that the change in heart rate observed in anesthetized rats and isolated perfused rat hearts could be due to interaction of ␤-cardiotoxin with ␤-ARs, and hence we performed radioligand binding assays. At first, the nonspecific binding of radioligand (Ϫ)-[3H]CGP12177 to the receptor preparations was defined using ␮M (S)-(Ϫ)-propranolol hydrochloride. The radioligand showed only - 3% non-specific binding to ␤1-AR preparation and 1–2% nonspecific binding to ␤2-AR Figure 6. Effects of ␤-cardiotoxin on Langendorff perfused hearts. Charts showing changes in heart rate (HR) recorded before (above) and 10 after (below) the treatment with control (A; KH solution) and ␮M ␤-cardiotoxin (B) dissolved in KH solution. C) Changes in HR 10 after treatment with control (KH solution) shown by gray bar and ␮M ␤-cardiotoxin shown by black bar. D) Changes in LVDEP (mmHg) 10 after treatment with control (KH solution) shown by gray bar and ␮M ␤-cardiotoxin shown by black bar. Ϯ sd is indicated at top of box (nϭ3). FUNCTIONAL CHARACTERIZATION OF ␤-CARDIOTOXIN 3691 preparation. (S)-(Ϫ)-propranolol hydrochloride was also used as a positive control in the study, and IC50 values for the displacement of bound radioligand to ␤1-AR and ␤2-AR were determined as 3.5 and 0.5 nM, respectively (Fig. 7). The Ki of (S)-(Ϫ)-propranolol hydrochloride to ␤1-AR and ␤2-AR were calculated as 1.9 nM and 0.2 nM, respectively. These values are in good agreement with the Ki values given by the manufacturer (2.6 and 0.2 nM for ␤1-AR and ␤2-A, respectively; see Supplemental Fig. S3). ␤-Cardiotoxin showed a dose-dependent displacement of the radioligand (Ϫ)[3H]CGP-12177. IC50 values for the inhibition of ligand binding to ␤1-AR and ␤2-AR were determined as 10 and ␮M, respectively (Fig. 7A, B). The Ki for ␤-cardiotoxin binding to ␤1 and ␤2 ARs were calculated as 5.3 and 2.3 ␮M, respectively. Thus, ␤-cardiotoxin induces a nega- tive chronotropic effect on the heart rate by binding to ␤1-AR in cardiomyocytes. Its interaction with ␤2-AR in the bronchi may induce some respiratory symptoms because of bronco-constriction. This exogenous protein interacts with ␤-ARs and hence was named as ␤-cardiotoxin. DISCUSSION Cardiovascular diseases (CVDs) are widespread and are a major health issue in the developed nations. For example, a recent study revealed that ϳ71.3 million American adults suffer from one or more forms of CVD (32). CVD causes nearly one in every three deaths and was the number one killer in the USA as it was the cause for 37.3% deaths in 2003. The economic impact of CVD is huge, and it is estimated that the combined direct and indirect costs of treating CVDs in 2006 would be $403.1 billion (32). Beta-blockers, which are antagonists of ␤-ARs, are the drugs of choice in the treatment of CVDs. Most patients (93%) with myocardial infarction are given beta-blockers on arrival at hospitals. They are also prescribed after discharge as a chronic therapy (32). In addition, beta-blockers are used in the treatment of many pathological conditions afflicting the heart and vasculature, such as ventricular arrhythmias, heart failure, digitalis intoxication, and fetal tachycardia. Owing to the presence of ␤-ARs in various noncardiac tissues, beta-blockers have also been used to treat conditions like, migraine, essential tremor, situational anxiety, alcohol withdrawal, hyperparathyroidism, glaucoma, portal hypertension, and gastrointestinal bleeding (33). Most of the beta-blockers in clinical use currently are small molecules belonging to the aryloxypropanolamine class (34). Although they are widely used, physicians have encountered several problems associated with the currently available beta-blockers. All beta-blockers are available as racemic mixtures of Land D-enantiomers, where only the L-enantiomer exerts beta-blockade while the D-enantiomer, apart from being inert, may have adverse side effects. For example, in a clinical trial using the D-enantiomer of the widely used beta-blocker sotalol, it was reported that the mortality increased by 65% compared to placebo (35). Many beta-blockers exhibit varying levels of lipophilicity and so have the ability to cross membrane barriers and reach the central nervous system, causing adverse effects like hallucinations and insomnia (34). Some beta-blockers are shown to increase insulin resistance and raise the risk of diabetes (36). Thus, new betablockers with high specificity and low side effects are being actively pursued. Isolation of a novel protein from O. hannah venom Figure 7. Interaction of ␤-cardiotoxin with ␤-ARs. Displacement of radiolabeled ligand (Ϫ)-[3H]CGP-12177 by (Ϫ)propranolol (Open symbols) and ␤-cardiotoxin (solid symbols). Interaction with cloned human ␤1-AR (A) and ␤2-AR (B). Each data point represents mean Ϯ sd of replicates. 3692 Vol. 21 November 2007 On screening, the cDNA library from the venom gland tissue of O. hannah, we identified five new 3FTXs. One of these 3FTXs showed only ϳ55% sequence identity with conventional CTXs isolated from Naja sp. (Fig. The FASEB Journal RAJAGOPALAN ET AL. 1B). Subsequently, two other reports (37, 38) have described the sequencing of the same toxin and also some closely related isoforms from cDNA libraries (Fig. 1A and Supplemental Fig. S2B), which shows the usefulness of this approach for identification of novel low abundant proteins that have eluded detection by conventional approaches. Here we have described a twostep chromatographic approach used for the isolation of this novel protein (Fig. 2). Identification of unique secondary structural conformation of ␤-cardiotoxin CTXs from different cobra venoms have been classified into two distinct structural subclasses based on their CD spectra (39). Almost all CTXs studied so far fall clearly into either of the two classes. The ␤-cardiotoxin spectrum on the other hand, neither shows the positive maximum at 220 –225 nm shown by all group CTXs, nor has the intense maximum at 190 –195 nm like all group CTXs (Fig. 3). The secondary structural conformation of ␤-cardiotoxin is unique compared to both group CTX nanixain and group CTX CM18 (Fig. 3), and hence, it differs from both classes of CTXs. Thus, ␤-cardiotoxin has unique secondary structural elements compared to all other CTXs. Conventional CTXs have a few well-conserved residues apart from the eight Cys residues, which are thought to play an important role in maintaining structural integrity of the three-finger fold. Among them are Tyr 22 and Tyr 51, which have been shown by chemical modification to play vital structural and functional (especially Tyr 22) roles in CTXs (40). Replacement of Tyr 22 led to changes in interaction of ␤-sheet regions between loops I and II and replacement of Tyr 51 led to structural perturbation in the globular core region of the molecule, the overall effect being a destabilized structural core and highly perturbed dynamics in the 3-stranded ␤-sheet region (41). Unlike conventional CTXs, in ␤-cardiotoxin there are Val residues in both positions 23 and 53 (homologous to positions 22 and 51 of CTXs; Fig. 1B). These changes could possibly explain the observed unique secondary structural features of ␤-cardiotoxin. It would be interesting to see the effects of these two replacements on the tertiary structure of the protein. functionally distinct from conventional CTXs. ␤-Cardiotoxin was nonlethal up to a dose of 10 mg/kg, in contrast to CTXs, which are highly lethal proteins with LD50 values in the range of to mg/kg (1). Unlike CTXs, which show potent hemolytic activity (43– 45), ␤-cardiotoxin failed to show hemolytic activity on washed human erythrocytes (Fig. 4B). Further, unlike CTXs that cause an increase in heart rate when injected into anesthetized rats (Fig. 5B, D; ref. 46), ␤-cardiotoxin caused a dose-dependent decrease in heart rate indicated by the prolongation of successive QRS complexes in the ECG recordings (Fig. 5. C–E). We also found that it can act directly on the cardiac tissue causing a marked reduction in heart rate (negative chronotropism) in Langendorff preparations of perfused rat hearts without affecting the contractility (inotropism) (Fig. 6B–D). Many beta-blockers like esmolol exert a direct inhibitory effect on membrane Ca2ϩ channels apart from acting as beta-blocking agents. This inhibition of membrane currents would lead to pronounced negative inotropism leading to adverse complications like severe reduction of blood pressure (47). Peptides engineered from ␤-cardiotoxin would be useful therapeutic prototypes as they lack intrinsic negative inotropism like conventional beta-blockers. We have shown that the above mentioned pharmacological effects are due to its direct binding to ␤1- and ␤2-ARs. The Ki values indicate that it has higher affinity for ␤2-AR compared to ␤1-AR (5.3 and 2.3 ␮M for ␤1- and ␤2-ARs, respectively). The small molecule beta-blockers in current clinical use have strong affinities to the two receptors (in nanomolar ranges) compared to ␤-cardiotoxin that binds at low micromolar ranges. This may be caused by steric hindrance due to the larger molecular size of the protein compared to the small molecule drugs, and the affinities and specificity could be increased further by protein engineering approaches like mutagenesis and peptide minimization. ␤-Cardiotoxin belongs to a new class of 3FTXs with a unique molecular target in the prey. This finding further broadens the array of molecular targets identified for various 3FTXs. The ␤-AR blocking ␤-cardiotoxin along with the ␣1-AR blocking conopeptide from cone snail venom (28) form a novel class of exogenous peptide adrenergic-blocking agents that may have immense applications in developing novel research tools and therapeutic agents. ␤-Cardiotoxin belongs to a new class of 3FTXs 3FTXs constitute ϳ50% of the weight of most elapid and hydrophid venoms and are the leading cause of death and morbidity as they are highly lethal (42). As mentioned in the Introduction, despite the similarity in overall protein fold, they target different receptors, ion channels, or proteins to exhibit various pharmacological effects. As shown here, ␤-cardiotoxin exhibits unique biological effects compared to any of the 3FTXs known. Although its amino acid sequence shows similarity to CTXs (ϳ55% identity), it is structurally and FUNCTIONAL CHARACTERIZATION OF ␤-CARDIOTOXIN CONCLUSIONS In summary, we have described the identification and isolation of ␤-cardiotoxin, which is the first member of a new class of 3FTXs. Although it shows sequence homology to conventional CTXs, it has unique structural and functional features. Functionally, it directly acts on cardiac tissue causing bradycardia. These effects are mediated through its interaction with ␤-ARs. This protein could serve as prototype for rational design of highly specific and effective beta-blocking peptides 3693 having reduced side effects. Thus, this is the first report of an exogenous protein beta-blocker. We thank R. Lakshminarayanan (Department of Chemistry, National University of Singapore, Singapore) for helpful discussions. We thank Y. Banerjee and M. Dugar (Department of Biological Sciences, National University of Singapore, Singapore) for kindly providing nanixain. We also thank M. Ohta (Kobe Pharmaceutical University, Kobe, Japan) for kindly providing CM18. We also thank M. Radhakisan (Department of Biological Sciences, Nationalo University of Singapore, Singapore) for helping in the preparation of publication quality figures. This work was supported by a grant (R154 – 000-172–305) from Biomedical Research Council, Agency for Science, Technology and Research, Singapore. We also thank B. G. Fry (University of Melbourne, Melbourne, Australia) for providing the O. hannah venom glands. We thank H. Pei Ying, N. Li, T. W. Lee, and W. Z. Jing (Department of Pharmacy, National University of Singapore, Singapore) for invaluable technical help and support in all the pharmacological experiments. 17. 18. 19. 20. 21. 22. 23. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 3694 Hider, R. C., Karlsson, E., and Namiranian, S. 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Accepted for publication May 17, 2007. 3695 Gene 371 (2006) 246 – 256 www.elsevier.com/locate/gene Ohanin, a novel protein from king cobra venom: Its cDNA and genomic organization Yuh Fen Pung a , Sanjeed Vijaya Kumar a , Nandhakishore Rajagopalan a , Bryan G. Fry a,b , Prakash P. Kumar a,c,⁎, R. Manjunatha Kini a,d,⁎ b a Department of Biological Sciences, Faculty of Science, National University of Singapore, 117543 Singapore Australian Venom Research Unit, Level 8, School of Medicine, University of Melbourne, Parkville, Victoria 3010, Australia c Temasek Life Sciences Laboratory, National University of Singapore, 117604 Singapore d Department of Biochemistry, Virginia Commonwealth University Medical Center, Richmond VA 23298-0614, USA Received September 2005; received in revised form December 2005; accepted December 2005 Available online February 2006 Received by J.A. Engler Abstract Ohanin, from king cobra venom, is a novel protein which induces hypolocomotion and hyperalgesia in mice [Pung, Y.F., Wong, P.T.H., Kumar, P.P., Hodgson W.C., Kini, R.M., 2005. Ohanin, a novel protein from king cobra venom induces hypolocomotion and hyperalgesia in mice. J. Biol. Chem. 280, 13137–13147]. It is weakly similar to PRY-SPRY domains (B30.2-like domain). Here we report the complete cDNA and genomic organization of ohanin. Interestingly, cDNA sequence does not show significant sequence similarity to any known sequences, including those of B30.2-like domain-containing proteins. Its full-length cDNA sequence of 1558 bp encodes for prepro-ohanin with a propeptide segment at the Cterminal. Ohanin is the first member of a new subfamily of proteins containing B30.2-like domain with short N-terminal segment. We named this subfamily as vespryns. There are two mRNA subtypes differing in their 5′-untranslated regions. Southern hybridization study shows that ohanin is encoded by a single gene. Its genomic sequence is 7086 bp with five exons and four introns, and the two types of mRNAs are generated by alternative splicing of exon 2. Our results indicate that ohanin and vespryns may have evolved from the same ancestral gene as B30.2 domain. © 2006 Elsevier B.V. All rights reserved. Keywords: B30.2-like domain; PRY-SPRY domains; Vespryns; Alternative splicing 1. Introduction Snake venom is a cocktail of pharmacologically active peptides and proteins. It is rich in enzymes, such as phospholipases A2, metalloproteinases, serine proteinases, L-amino Abbreviations: SPRY domain, domain with unknown function in Ryanodine receptors and Dictyostelium discoideum; B30.2 domain, Domain which was named after the B30.2 exon maps within the Human Class I Histocompatibility complex. ⁎ Corresponding authors. Kini is to be contacted at Protein Science Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, 14 Science Drive 4, Singapore 117543. Tel.: +65 6874 5235; fax: +65 6779 2486. Kumar, Plant Morphogenesis Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, 14 Science Drive 4, Singapore 117543. Tel.: +65 6874 2879; fax: +65 6779 2486. E-mail addresses: dbskumar@nus.edu.sg (P.P. Kumar), dbskinim@nus.edu.sg (R.M. Kini). 0378-1119/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2005.12.002 acid oxidases, phosphodiesterases, acetylcholinesterases and nucleases (Karlsson, 1979; Torres et al., 2003). It also contains non-enzymatic proteins, such as three-finger toxins, serine proteinase inhibitors, helveprins/CRISPs, C-type lectin related proteins and waprins (Mochca-Morales et al., 1990; Kini, 2002; Torres et al., 2003; Yamazaki et al., 2003). However, snake venom proteins are still far from being completely cataloged and there is room to isolate and characterize novel snake venom proteins both structurally and functionally. One such example is the recently isolated venom protein, ohanin, from king cobra (Ophiophagus hannah) (Pung et al., 2005). It is the first member of a new family of snake venom proteins. Unlike other snake venom proteins, which are rich in Cys residues and disulfide bonds, ohanin has a single Cys residue. Thus ohanin is unique. It shows homology (49% similarity and 38% identity) to consensus sequence of PRY-SPRY domains. Ohanin lowers the locomotor activity and induces Y.F. Pung et al. / Gene 371 (2006) 246–256 hyperalgesia in mice (Pung et al., 2005). Both these biological activities are mediated through its effect on the central nervous system. We proposed that ohanin could serve both offensive and defensive roles by slowing down the locomotor activity and inducing pain in both preys and predators (Pung et al., 2005). Protein sequencing of ohanin showed that ohanin has a PRY domain followed by a partial SPRY domain. To further characterize this novel protein, we have cloned and sequenced its cDNA. In this paper, we show that ohanin is synthesized as a precursor protein and the prepro-protein has the complete SPRY domain with the presence of the C-terminal propeptide segment. The maturation of ohanin appears to occur by a cleavage at the dibasic Arg–Arg site. The results also show the presence of two ohanin mRNA subtypes differing in their 5′-untranslated regions. Analysis of genomic DNA sequence indicates that alternative splicing in ohanin gene leads to these two subtypes. Data presented here show that ohanin belongs to a new subfamily of proteins which contain the B30.2-like domain. We have named the protein subfamily as vespryns. This is the first report on the cDNA and genomic organization of this novel protein family. 2. Materials and methods 2.1. Materials King cobra venom glands and liver tissue were frozen in liquid nitrogen immediately after dissection and kept in − 80 °C until used. All the reagents and kits used for molecular biology study were as follows: oligonucleotides (1st Base Pte Ltd, Singapore), Platinum Taq polymerase, dNTP mix, λHindIII and Kb plus ladder (GIBCO BRL®, Carlsbad, CA), restriction endonucleases (New England Biolabs®, Beverly, MA), pGEMT-easy vector (Promega, Madison, WI), RNeasy® Mini kit, QIAGEN® OneStep RT-PCR kit, DNeasy® Tissue kit, QIAprep® Miniprep kit and QIAEX II Gel Extraction kit (Qiagen GmbH, Hilden, Germany), SMART™ RACE cDNA amplification kit, Universal GenomeWalker™ kit and BD Advantage™ Polymerase Mix (Clontech Laboratories Inc., Palo Alto, CA), PCR DIG probe synthesis kit, positively charged nylon membrane, DIG™ Easy Hyb buffer, anti-DIG alkaline phosphatase antibody and chemiluminescent substrate CDP-Star™ (Roche Diagnostics GmbH, Deutschland, Germany), ABI PRISM® BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (Version 3.0) (PE-Applied Biosystems, Foster City, CA) and Luria Bertani broth and agar (Q.BIOgene, Irvine, CA). 2.2. Isolation of total RNA Total RNA was isolated from king cobra venom gland using the RNeasy® Mini kit. For each extraction, 30 mg venom gland tissue was first pulverized in liquid nitrogen and further homogenized for 20 to 30 s using a Heidolph DIAX600 homogenizer (Schwabach, Germany) in the presence of 600 μl Buffer RLT. The integrity of the RNA extracted was examined by denaturing agarose gel electrophoresis. 247 2.3. Reverse transcription-polymerase chain reaction (RT-PCR) To generate gene-specific sequence, RT-PCR was performed using the QIAGEN® OneStep RT-PCR kit. In brief, RT-PCR mixture contained μl of QIAGEN OneStep RT-PCR Enzyme Mix and the final concentration of 250 ng total RNA as template, 0.4 mM dNTP mix and 0.6 μM degenerate primers in a total volume of 50 μl. The degenerate primers used were: RT1 (sense primer) 5′-GGNAAYTGGCARAARGCNGA-3′ and RT2 (antisense primer) 5′-CCACCANARNCCYTTYTGCCA-3′. The reverse-transcription and amplification conditions were: reverse-transcription at 50 °C/30 min; initial PCR activation step at 95 °C/15 min; immediately followed by 30 cycles of 3-step thermal cycling profile of denaturation at 94 °C/1 min, annealing at 50 °C/1 min, extension at 72 °C/2 and a final extension at 72 °C/10 min. The PCR products were fractionated by 1.5% agarose gel electrophoresis. The most intense bands were excised and purified using QIAEX II Gel Extraction kit before ligation to pGEMT-easy vector. Eight clones were sequenced with T7 (sense) 5′-GTAATACGACT CACTATAGGGC-3′ and SP6 (antisense) 5′-TATTTAGGTGA CACTATAG-3′ primers using the dideoxy chain termination method (Sanger et al., 1977) on an automated ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). ABI PRISM® BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (Version 3.0) was used to carry out the cycle sequencing reaction. Data were analyzed using the Sequencing Analysis 3.7 (Sample Manager) software (Applied Biosystems, Foster City, CA). 2.4. Construction of 5′- and 3′-RACE cDNA libraries The 5′- and 3′-RACE cDNA libraries were constructed using the SMART™ RACE cDNA amplification kit according to the manufacturer's protocol. 2.5. Isolation and sequencing of cDNA clones The 5′- and 3′-RACE-Ready cDNA libraries were constructed using SMART™ RACE kit For cDNA amplification, the 5′-RACE reaction mix consisted of 2.5 μl 5′-RACE-ReadycDNA, μl Universal Primer Mix (UPM) containing Long primer 5′-CTAATACGACTCACTATAGGGCAAGCAGTGG TATCAACGCAGAGT-3′ and Short primer 5′-CTAATAC GACTCACTATAGGGC-3′; and the final concentration of 1.5 U Platinum Taq polymerase, 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP mix and 0.2 μM GSP2 (antisense primer) 5′-CTTCCCAGCTAACCCAACAGCCCATTCCC-3′ in a total volume of 50 μl. The 3-step thermal cycling profile was as follows: cycle of hot start at 94 °C/1 min; 30 cycles of denaturation at 94 °C/30 s, annealing at 67 °C/30 s, extension at 72 °C/2 and followed by a final extension of 72 °C/10 min. The 3′-RACE reaction mix, which yielded the full-length cDNA, consisted of 2.5 μl 3′-RACE-Ready-cDNA, μl UPM and the final concentration of 1.5 U Platinum Taq polymerase, 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP mix and 0.2 μM 248 Y.F. Pung et al. / Gene 371 (2006) 246–256 GSP1 (sense primer) 5′-GATCATTTGATCCAGAGAAGACA CAGTCTC-3′ in a total volume of 50 μl. The 3-step thermal cycling profile was as follows: cycle of hot start at 94 °C/1 min; 30 cycles of denaturation at 94 °C/30 s, annealing at 68 °C/30 s, extension at 72 °C/3 min, followed by a final extension of 72 °C/ 10 min. The PCR products were fractionated by 1.5% agarose gel electrophoresis. The most intense bands for both the 5′- and 3′-RACE amplifications were excised, purified and ligated into pGEMT-easy vector. At least 16 clones from both the 5′- and 3′RACE libraries were fully sequenced using T7, SP6 and internal primers. 2.6. Isolation of genomic DNA Genomic DNA was isolated from king cobra liver tissue using the DNeasy® Tissue kit. For each extraction, 25 mg liver tissue was pulverized in liquid nitrogen using a mortar and pestle pre-cooled at − 80 °C. The integrity of the genomic DNA extracted was examined by 0.8% agarose gel electrophoresis. 2.7. Southern blot hybridization The digoxigenin (DIG)-labeled double-stranded DNA probes were prepared using the PCR DIG Probe Synthesis kit. The PCR reaction mix contained 0.5 μl ohanin cDNA in pGEMT-easy vector as template and a final concentration of 1.3 U Enzymes mix, 0.2 mM PCR DIG Labeling mix, 0.2 mM dNTP mix, 1× PCR buffer and 0.2 μM primers in a total volume of 25 μl. The gene-specific primers used were: P11 (sense) 5′-GCTGATGTGACGTTTGACTCAAACACA-3′ and P12 (antisense) 5′-AAGCCACCAGAGGCCCTTTTGCCA-3′. The 3-step thermal cycling involved a hot start at 95 °C/2 followed by 30 cycles of 95 °C/30 s, 63 °C/30 s, 72 °C/40 s and a final extension of 72 °C/7 min. The PCR product was purified and the concentration was determined by A260. Southern hybridization was performed according to the DIG System User's Guide (Roche Diagnostics GmbH, Deutschland, Germany). For each lane, 10 μg of genomic DNA was used. Detection of the signal was performed using the substrate CDP-Star™. 2.8. Construction of GenomeWalker libraries The GenomeWalker libraries were constructed using the Universal GenomeWalker kit according to the manufacturer's protocol. The libraries were made with 2.5 μg of genomic DNA restricted by DraI, EcoRV, PvuII and StuI, respectively. 2.9. Isolation and sequencing of genomic clones Genomic organization of ohanin was studied using both the gDNA PCR and ‘genome walking’ approaches. As a first step, gDNA PCR was performed using gene-specific primers designed from the coding region of ohanin cDNA. The primers used were as follows: gDNAsigpep (sense) 5′-ATGCTCC TGTTCACACTATGCTTT-3′ and gDNAstop (antisense) 5′-C CCTGTTTTAATGAAGAATTGTAACCTCTTA-3. The PCR reaction mix contained 0.5 μl gDNA as template and a final concentration of 1.5 U Platinum Taq polymerase, 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP mix and 0.2 μM primers in a total volume of 25 μl. The 3-step thermal cycling involved a hot start at 94 °C/2 followed by 30 cycles of 94 °C/1 min, 60 °C/45 s, 72 °C/3 and a final extension of 72 °C/10 min. The PCR products were fractionated on a 1.5% agarose gel and the band of interest was excised, purified and ligated into pGEMT-easy vector. Sixteen clones carrying the inserts were sequenced. Secondly, gDNA amplification was further performed to obtain the genomic sequence which corresponds to the 3′-UTR of the cDNA. The gene-specific primers used were: gDNA3UTR1 (sense) 5′-CTATATAGGGGCACGTGTTT CACTC-3′ and gDNA3UTR2 (antisense) 5′-TACTAACAGT GAGACTTTATTAGTAG-3′. The PCR reaction mix contained 0.5 μl gDNA as template and a final concentration of 1.5 U Platinum Taq polymerase, 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP mix and 0.2 μM primers in a total volume of 25 μl. The 3-step thermal cycling involved a hot start at 94 °C/1 followed by 30 cycles of 94 °C/1 min, 60 °C/30 s, 72 °C/3 and a final extension of 72 °C/10 min. The amplified fragment was purified, cloned and sequenced. Finally, the genomic sequence that corresponds to the 5′UTR of the cDNA was obtained from ‘genome walking’ and two additional gDNA PCR amplifications. The first ‘genome walk’ involved two sets of primers: adaptor primer (AP1-sense) 5′GTAATACGACTCACTATAGGGC-3′ provided in the kit and a 25-mer gene-specific primer gDNA5UTR1 (antisense) 5′-CTTT CTGCCAATTCCCAGGAGGTGA-3′; and the nested PCR adaptor primer (AP2-sense) 5′-ACTATAGGGC ACGCGT GGT-3′ provided in the kit and a 27-mer gene-specific primer gDNA5UTRnes2 (antisense) 5′-AGCCA GAGCCTTTCCAC CATTTTCCTG-3′. Primary and nested PCRs were performed as recommended by the BD GenomeWalker™ Kit user's manual with the following modifications. The 25.0 μl reaction mixture consisted of 0.5 μl of DNA template (either from each library or from primary PCR products), 1× PCR buffer, 0.2 mM dNTPs, 0.2 μM appropriate adaptor primers, 0.2 μM of appropriate gene-specific primers, 1× BD Advantage™ polymerase mix. The thermal cycling profile used was as follows: cycles of 94 °C/2 s, 72 °C/3 min; 32 cycles of 94 °C/2 s, 67 °C/4 followed by a final extension of 67 °C/7 min. The PCR products were purified, cloned and sequenced. A second gDNA PCR amplification was performed using the gene-specific primers 1-gDNA5UTR (sense) 5′-CGGA TCCTGCACAATAGTTTTATCTCC-3′ and 2-gDNA 5UTR (antisense) 5′-GACATGACCATTACAAACTCTAGC TTC-3′. The PCR reaction mix contained 0.5 μl gDNA as template and a final concentration of 1.5 U Platinum Taq polymerase, 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP mix and 0.2 μM primers in a total volume of 25 μl. The 3-step thermal cycling involved a hot start at 94 °C/2 followed by 30 cycles of 94 °C/20 s, 62 °C/30 s, 72 °C/3 and a final extension of 72 °C/10 min. The amplified fragment was purified, cloned and sequenced. The final gDNA PCR amplification was performed using the gene-specific primers 9-gDNA5UTR (sense) 5′-GATCATTT GATCCAGAGAAGACACAGT-3′ and 6-gDNA5UTR Y.F. Pung et al. / Gene 371 (2006) 246–256 (antisense) 5′-CAAACAAATGGTCATAAGCTGAGGTC TAC-3′. The PCR reaction mix contained 0.5 μl gDNA as template and a final concentration of 1× BD Advantage™ polymerase mix, 1× PCR buffer, 0.2 mM dNTP mix and 0.2 μM primers in a total volume of 25 μl. The 2-step thermal cycling involved a hot start at 95 °C/1 followed by 30 cycles of 95 °C/15 s, 67 °C/4 min, and a final extension of 67 °C/4 min. The amplified fragment was purified, cloned and sequenced. 3. Results 3.1. Cloning and sequencing of ohanin cDNA The amount of total RNA obtained from the king cobra venom gland was low (∼4 μg per 30 mg fresh weight), but the quality of RNA was relatively good (data not shown). We used a 249 combination of RT-PCR and RACE techniques to obtain the fulllength cDNA of ohanin. To isolate gene-specific sequences, RTPCR was first performed using the total RNA as template. Degenerate primers, RT1 and RT2, were designed based on ohanin's amino acid sequence (Pung et al., 2005). Analysis of the amplified fragment (∼300 bp) revealed that all eight randomly selected clones encoded for ohanin (data not shown). Two bands of approximately 550 and 600 bp were obtained from the 5′-RACE amplification in an attempt to clone the 5′coding region and 5′-UTR (Fig. 1A). Sequence analysis of 16 independent clones derived from the bands demonstrated the existence of two cDNA subtypes. We further designed a sense primer, GSP1, from the beginning of the 5′-UTR sequence for the 3′-RACE amplification. GSP1 and UPM yielded the full-length cDNA of ohanin (Fig. 1B). Sixteen individual clones were fully sequenced. Of these randomly selected cDNA clones, Fig. 1. Cloning and sequencing of ohanin cDNA. (A) 5′-RACE amplification. Partial coding region of ohanin together with its 5′-UTR was obtained from the 5′RACE amplification using GSP2 and UPM. (B) 3′-RACE amplification. The 3′-RACE amplification which yielded the full-length cDNA of 1558 bp exclusive of poly-A tail was obtained using GSP1 and UPM. (C) Nucleotide sequence and deduced amino acid sequence (gb: AY351433). Nucleotide sequence is presented in the 5′- to 3′-orientation. Deduced amino acid sequence by reverse-translation from the putative open reading frame is shown: the three ATGs are indicated in bold; the putative signal peptide is underlined; dibasic processing site is boxed and propeptide segment is marked in italics. The stop codon is marked by an asterisk and the polyadenylation signal, AATAAA, is underlined twice. The missing stretch of nucleotides in type II cDNA is shaded black (See text for details). 250 Y.F. Pung et al. / Gene 371 (2006) 246–256 Fig. 2. B30.2-like domain-containing proteins. (A) Sequence alignment of B30.2-like domains. The domains are from Pro-ohanin (gb: AY351433), Thaicobrin (sp: P82885), RFP (Ring finger protein, gb: NM172016), BTN (Butyrophilin, gb: NP038511), PRY-SPRY domains (conserved sequences of PRY-SPRY domains were provided by CDD data base), Beta subunit of SNTX (β-Stonustoxin, gb: Q91453), Alpha subunit of SNTX (α-Stonustoxin, gb: Q98989), Enterophilin (gb: AF126833) and SPRY domain-containing SOCS box protein (Suppressors of cytokine signaling: NP660116). Identical and conserved residues are shaded black and grey. The Arg–Arg dibasic cleavage site of pro-ohanin is indicated in bold and propeptide segment is marked in italics. Three conserved LDP, WEVE and LDYE motifs as described by Henry et al. (13, 14) are boxed. Gaps (–) were introduced for optimal alignment and maximum homology for the sequences. The arrows indicate the boundary of PRY and SPRY domains. The numbers in parentheses represent the percentage of similarity between the B30.2-like domain of pro-ohanin with other B30.2-like domain-containing proteins. The substitutions among the following groups of amino acid residues are considered as conserved changes: Y, F, and W; S and T; V, L, I and M; H, R and K; D, E, N and Q; A and G. (B) Schematic representation of proteins possessing B30.2-like domain. B30.2-like domains are shaded black and the unidentified domains are dotted. Y.F. Pung et al. / Gene 371 (2006) 246–256 two clones were of type I and 14 clones were of type II. Our results demonstrate that type I cDNA has a longer 5′-UTR region (236 bp), whereas type II differed by a 76-bp deletion from position 54 to 129 (Fig. 1C; segment shaded black). Except for the missing segment in the 5′-UTR of type II cDNAs, no other sequence differences were observed between the two types of ohanin cDNAs. The full-length cDNA (type I with 1558 bp) of ohanin (gb: AY351433) and its deduced amino acid sequence are shown in Fig. 1C. The cDNA (types I and II) encodes for a putative open reading frame (ORF) of 190 amino acid residues beginning with a start codon (ATG) at position 236 (type I numbering); ending with a termination codon (TAA) at position 808. The polyadenylation signal, AATAAA, is located 14 nucleotides upstream of the poly-A tail (Proudfoot and Brownlee, 1976). The ORF is larger than the ohanin protein sequence we reported previously (Pung et al., 2005). It has a signal peptide of 20 amino acid residues, followed by 107 amino acids of mature ohanin and a 63 amino acid-long propeptide segment at the C-terminal (Fig. 1C). Thus ohanin is synthesized as a preproprotein in the venom gland. We have named the precursor of ohanin as pro-ohanin. The cleavage at the dibasic Arg–Arg site probably results in the removal of C-terminal propeptide segment leading to mature ohanin (Fig. 1C). 3.2. Amino acid alignment of B30.2-like domains of pro-ohanin and other proteins Comparison of the full-length cDNA sequence using the nucleotide BLAST algorithm (http://www.ncbi.nlm.nih.gov/ BLAST/) (Altschul et al., 1997) did not display any significant sequence similarity with other nucleotide sequences deposited in the GenBank data base. This was surprising as the protein sequence showed weak similarity to B30.2-like domains (Pung et al., 2005). A Conserved Protein Domain Data base (CDD) (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (Marchler-Bauer et al., 2003) search, however, revealed that the deduced pro-ohanin sequence (from residue to 170) shares an overall identity of 15% to 36% and similarity of 22% to 51% with PRY-SPRY domains (B30.2-like domains). Fig. 2A shows the alignment of B30.2-like domains of pro-ohanin with other B30.2-like domain-containing proteins. The B30.2 domain, which maps within the B30.2 exon of the Human Class I Histocompatibility complex, is a conserved domain of 160 to 170 amino acid residues (Vernet et al., 1993; Henry et al., 1997a). One main characteristic of B30.2-like domains is the existence of three highly conserved motifs, LDP, WEVE, and LDYE (Henry et al., 1997b, 1998). It should be noted that mature ohanin has the first two motifs, whereas LDYE motif is found within the propeptide segment (Fig. 2A). This conserved domain, usually located at the Cterminal, is associated with N-terminal domains of different protein families (Fig. 2B). These protein families include RBCC proteins (RING-finger, B-box and Coiled-coil domain proteins) (Vernet et al., 1993; Orimo et al., 2000; Meyer et al., 2003), butyrophilin-related proteins (Ogg et al., 1996; Rhodes et al., 2001), as well as α- and β-subunits of stonustoxin 251 (Ghadessy et al., 1996) as shown in Fig. 2B. In addition, SOCS box proteins (Suppressors Of Cytokine Signaling proteins) (Hilton et al., 1998; Yao et al., 2005) and enterophilin-related proteins (Gassama-Diagne et al., 2001) also contain the B30.2-like domain (Fig. 2B). The deduced pro-ohanin sequence, from residue to 170, shows weak similarity to the complete B30.2-like domain proteins, suggesting that pro-ohanin may have been evolved from the same ancestral gene as the B30.2 domain. Unlike all other subfamilies, ohanin/pro-ohanin has an extremely short Nterminal segment of residues. In addition to ohanin, two other proteins, namely Thaicobrin (sp: P82885) and an ohaninlike protein (Naja naja atra) (Li et al., 2004) from cobra venoms, have been identified so far in this subfamily. We named this new family of snake venom proteins as vespryns (Venom PRY-SPRY domain-containing proteins). 3.3. Southern blot hybridization As mentioned above, there are two mRNA subtypes for ohanin. It was of interest to determine whether these two mRNAs are products of two independent genes or derived through alternative splicing. As a first step, we performed the genomic Southern hybridization experiment. King cobra genomic DNA was digested with EcoRI, HindIII, BamHI and NdeI, separately. These genomic DNA digests were hybridized with a 297-bp DIG-labeled probe designed from nucleotide 319 to 616 of its cDNA (Fig. 1C). We observed a single band in all four digests (Fig. 3), suggesting that ohanin is encoded by a single gene in the king cobra genome. 3.4. Cloning and sequencing of ohanin gene To determine the genomic organization of ohanin gene, genomic DNA PCR and ‘genome walking’ approaches were used. Ohanin cDNA sequence (Fig. 1C) was used to map the exon– intron boundaries (Fig. 4). In the first amplification, gDNAsigpep and gDNAstop were used to amplify its coding region. The resultant fragment was ∼1.9 kb (Fig. 4A). Our attempts to PCR amplify the 3′-UTR region of the genomic DNA yielded another ∼750 bp band as shown in Fig. 4A. Fig. 3. Genomic Southern blot of ohanin. Genomic DNA of king cobra (10 μg each lane) was digested with EcoRI, HindIII, BamHI or NdeI enzymes. Southern hybridization shows the presence of one single band in all four digests. Thus ohanin is encoded by a single gene in the king cobra genome. The migration position of λHindIII marker is indicated. 252 Y.F. Pung et al. / Gene 371 (2006) 246–256 We tried to amplify the 5′-UTR region from the genomic DNA using primers designed from the transcription start site to the signal peptide region. However, no band was obtained after several attempts. This led us to suspect that our primers may have been interrupted by the presence of intron(s) or the thermal cycling profile used was still not optimal. Hence, genome walker libraries were constructed. As shown in Fig. 4A, the ‘genome walk’ was performed using antisense primers, gDNA5UTR1 Y.F. Pung et al. / Gene 371 (2006) 246–256 253 Fig. (continued). and gDNA5UTRnes2 with adaptor primers (AP1 and AP2) from the kit. The resultant ∼1.65 kb fragment was fully sequenced. We obtained another ∼1.8 kb further upstream by a gDNA PCR performed using primers 1-gDNA5UTR and 2-gDNA 5UTR designed from the 5′-region of the cDNA and the previously obtained ∼1.65 kb genomic DNA fragment (Fig. 4A). With the optimized thermal cycling profile, we further generated another fragment of ∼1.4 kb corresponding to the transcription start site region of the cDNA using primers 9-gDNA5UTR and 6-gDNA5UTR (Fig. 4A). Thus, we have obtained a total of 7086 bp of the gene sequence, spanning from 5′-UTR to 3′-UTR regions of ohanin cDNA (Fig. 4A and B). Sequences flanking the splice junctions were determined for all the exons and introns of ohanin (Table 1). The donor and acceptor splice sites of the exon–intron boundaries conform to the rule that intron begins with GT and ends with AG (Breathnach and Chambon, 1981). Ohanin gene contains five exons and four introns. Out of five exons identified, the coding region of ohanin is made up from two exons. Exons 1, and encode mainly the 5′-UTR region. Interestingly, exon is spliced out in one of the mRNA subtypes (Figs. 1C and 4B and C). Exon comprises of the remaining 5′-UTR region (11 bp), signal peptide and the first eight amino acid residues of ohanin. Exon encodes for ohanin spanning from residues to 107, the propeptide segment as well as the sequence corresponding to the 3′-UTR (Fig. 4B and C). 4. Discussion Ohanin is a novel protein isolated from king cobra venom (Pung et al., 2005). It induces hypolocomotion and hyperalgesia in mice via both intraperitoneal and intracerebroventricular injection. Both the pharmacological actions are probably mediated through its effect on the central nervous system (Pung et al., 2005). This is a unique toxin which shows similarity to PRY-SPRY domains. cDNA cloning and sequencing show that ohanin is synthesized as a prepro-protein in the venom gland (Fig. 1). Further, there are two types of mRNA encoding ohanin; they differ in their 5-UTR. Interestingly, ohanin cDNA sequence shows no homology to other nucleotide sequences deposited in the GenBank data base including those of the B30.2-like domaincontaining proteins. However, the deduced protein sequence shares weak similarity to the PRY-SPRY domains (Fig. 2). Unlike other subfamily members of B30.2 domain-containing proteins, ohanin has a relatively short N-terminal extension (Fig. 2B). This new subfamily of venom proteins, vespryns, consists of ohanin (sp: P83234), Thaicobrin (sp: P82885) and an ohanin-like protein from N. naja atra (Li et al., 2004). Fig. 4. Gene structure of ohanin. (A) Strategy used for cloning and sequencing of ohanin gene. The fragments obtained from gDNA PCR amplification are indicated by solid arrows; whereas the fragment from ‘genome walking’ is indicated by dashed arrow. Step 1: The region corresponding to the coding region of ohanin was amplified using the primers gDNAsigpep and gDNAstop. Step 2: The 3′-UTR was further amplified using the primers gDNA3UTR1 and gDNA3UTR2. Step 3: As for 5′-UTR, primer pairs used for ‘genome walking’ were gDNA5UTR1 and AP1 for primary amplification; gDNA5UTRnes2 and AP2 for secondary amplification. Steps and 5: Primer pairs, 1-gDNA5UTR and 2-gDNA5UTR, as well as 9-gDNA5UTR and 6-gDNA5UTR, were used to obtain the remaining 5′-UTR region from the genomic DNA. (B) Ohanin gene sequence. Using both the genomic DNA PCR and ‘genome walking’ strategies, the full-length genomic sequence of 7086 bp was obtained. Exon–intron boundaries were determined based on cDNA and genomic sequences. Exons are shaded grey and indicated by upper case letters while introns are indicated by lower case letters. The missing exon in type II cDNA is shaded black. The three ATGs are indicated in bold; the putative signal peptide is underlined; dibasic processing site is boxed; propeptide segment is marked in italics, the stop codon is indicated by an asterisk and the polyadenylation signal, AATAAA, is underlined twice. (C) Genomic organization of ohanin. Ohanin gene comprises of five exons and four introns. Exons to have the sizes of 53, 76, 95, 96 and 1238 bp, respectively. The introns are 1160, 1743, 1292 to 1333 bp, respectively. In the case of alternative splicing, the whole exon is excluded producing a shorter transcript of 1482 bp. The complete cDNA was named type I, while the shorter cDNA corresponding to the alternative splicing (missing exon 2) was named type II cDNA. 254 Y.F. Pung et al. / Gene 371 (2006) 246–256 Table The exon–intron boundaries of ohanin gene Exon Size (bp) Intron Size (bp) 5′-donor splice site 53 76 95 96 1238 1160 1743 1292 1333 …AGAAGgtaag… …TAAAGgtaga… …CACAGgtaaa… …GAAAGgtaag… 3′-acceptor splice site …tatagACTCC… …tgtagGAAGC… …tttagGAGTC… …tgcagCTGAT… 4.1. Analysis of ohanin cDNA It should be noted that in addition to the start codon (AUG) at position 236, two other in-frame putative start codons are found further upstream in the same ORF at positions 146 and 152 (Fig. 1C). In general, the AUG closest to the 5′-end of an mRNA is the start signal for protein synthesis (Kozak, 1981, 1984). The context surrounding AUG is also crucial for the determination of the translation start site (Kozak, 1989). Based on the survey of 699 vertebrate mRNAs, GCC(A/G)CCaugG was identified as the most consensus sequence for eukaryotic translation start site (Kozak, 1987a,b). Kozak (1989) also highlighted the importance of purine at position − and G at position +4. The absence of purine at position − (usually an A) will impair the translation initiation more profoundly than any other nucleotides in the consensus sequence as it will lead to leaky scanning. In the absence of purine at position − 3, however, G at the position + is essential for efficient translation (Kozak, 1989). A careful analysis at the three possible start codons revealed that none of them have the consensus sequence (Kozak, 1987a,b, 1989). Only the first AUG at position 146 fits well with the (− 3, + 4) rule with the presence of G at both positions. However, no suitable signal peptide required for its secretion into the venom can be identified with the first two AUG sites. Hence we propose that the third AUG at position 236 is the start codon for ohanin, although there is a possibility that the other two AUGs can be utilized as the start codon (see below). Signal peptide is a hallmark of all secreted proteins. Since ohanin is secreted in the snake venom, it is expected to have a suitable signal peptide. Signal peptides generally have three common structurally and functionally distinct building blocks; a short, positively charged N-terminal (n-region) for its penetrability; a central hydrophobic region (h-region) that generally extends across the lipid head-groups of membranes and also helps in positioning the more polar C-terminal (c-region) in an exposed and extended conformation to be accessible for signal peptidase cleavage (Von Heijne, 1986, 1998). Based on the statistical analysis of 161 nonhomologous signal peptide sequences from the collection of 450 eukaryotic sequences, Von Heijne (1986, 1998) has suggested the (−3, −1) rule. According to this rule, the residue in the −3 position must not be aromatic (Phe, His, Tyr, Trp), charged (Asp, Glu, Lys, Arg) or large and polar (Asn, Gln). In addition, the residue in −1 position must be small (Ala, Ser, Gly, Cys, Thr or Gln). Recently determined crystal structure of E. coli signal peptidase (Paetzel et al., 1998) further confirms the importance of three building blocks and (−3, −1) rule of signal peptide as proposed by Von Heijne (1986, 1998). With the three potential translation start sites, signal peptides with 50, 48 and 20 amino acid residues are possible. The signal peptide prediction software (http://www.cbs.dtu.dk/services/ SignalP/) (Bendtsen et al., 2004) indicates that the cleavage site for prepro-ohanin is between Ala and Ser with all three AUGs as starting codons. Amino terminal sequencing by Edman degradation of ohanin (Pung et al., 2005) supports this predicted cleavage site. However, only the third AUG results in a 20residue signal peptide, whereas the other two will generate unusually long signal peptides of 50- and 48-residue, respectively. Further the 20-residue signal peptide has the highest probability of 0.532 compared to 0.311 and 0.257 for 50- and 48residue signal peptides. Since all toxin genes sequenced have 16to 27-residue signal peptides (Smith, 1990), we propose that the third AUG acts as the start codon for ohanin. The putative 20residue signal peptide has all the distinctive structural features; a basic n-region, a central hydrophobic h-region and a more polar c-region. In addition, Ala residues at position − and − also conform to Von Heijne's (− 3, − 1) rule. 4.2. Analysis of ohanin gene Despite the presence of two types of mRNAs, there is only a single gene which encodes ohanin as indicated by Southern hybridization (Fig. 3). This gene has five exons and four introns. Genomic DNA sequence analysis indicates that the heterogeneity at the 5′-UTR of the two types of mRNAs are due to the occurrence of alternative splicing from a single gene (Fig. 4B). About half of all mammalian genes are estimated to have more than one splice forms (Mironov et al., 1999; Brett et al., 2000; Croft et al., 2000; Kan et al., 2001; Okazaki et al., 2002). So far five major forms of alternative splicing have been identified. They consist of exon-skipping, alternative 3′ splicesite, alternative 5′ splice-site, mutually exclusive exons and intron retention (Ast, 2004). Analysis of ohanin genomic DNA indicates that the splicing pattern belongs to exon-skipping as the missing segment in type II cDNA is encoded solely by exon (Fig. 4). The implication of alternative splicing of ohanin gene at the 5′-UTR is not clear at this time. However, alternative splicing at the 5′-UTR is probably a mechanism that allows for the use of several differently regulated promoters for the same gene as demonstrated by most of the splicing events in mammalian genes (Mironov et al., 1999). In this study, we have also shown that a single intron is inserted right before residue of ohanin, leaving the last exon coding for PRY-SPRY domains (B30.2-like domain) intact (Fig. 4B and C). Interestingly, at the nucleotide level, this ‘B30.2 exon’ does not show sequence similarity to either coding or 3′-UTR regions. Similar genomic organization among ohanin and other B30.2-like domain-containing proteins indicates these proteins may have evolved from the same ancestral gene. 4.3. Molecular evolution of ohanin/pro-ohanin It is interesting to note that all the snake venom proteins reported so far are highly similar to non-venomous body proteins. For example, sarafotoxins are similar to endothelins, Y.F. Pung et al. / Gene 371 (2006) 246–256 Cobra venom factors are similar to complement C3, and threefinger toxins are similar to proteins such as CD59. Thus, it has been postulated that snake toxins arise from recruitment events of genes from various body protein families during evolution (Fry, 2005). Under positive selection pressure, B30.2 domain may have been selected to duplicate with MHC class I gene. Subsequently, this domain has been recruited in the venom gland. The basis for the multiple and independent recruitment events of certain protein families for the use as toxins are unclear. However, it is hypothesized that the chosen protein families are likely to be favored in the snake's adaptive evolution, particularly for its feeding habits. These protein families must also be beneficial or ‘economical’ for use as stable molecular scaffolds to incorporate various functional motifs on their surface to exert different pharmacological actions. 4.4. Functional implication(s) of B30.2-like domain The B30.2-like domains are the only common domains found in transmembrane proteins (BTNs), intracellular proteins (RFPs, SSA/Ro) and secreted proteins (ohanin/pro-ohanin, Thaicobrin, α- and β-subunits SNTX) (Fig. 2B) (Henry et al., 1997b, 1998). However, the biological implication(s) of this Cterminal domain in the diverse functionally and structurally unrelated protein families is not known. Recently, TRIM5α, originally identified from rhesus monkey, was found to confer resistance to retroviral infection (HIV type 1) (Sawyer et al., 2005). Functional studies using chimeric TRIM5α have further shown that a 13 amino acid residue ‘patch’ in the SPRY domain of TRIM5α is responsible for the inhibitory effect (Sawyer et al., 2005). Mature ohanin with an 8-amino acid residue Nterminal segment, followed by PRY and partial SPRY domains, induces hypolocomotion and hyperalgesia in mice (Pung et al., 2005). 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[...]... of thermal imaging of warm-blooded prey at night Elapidae is a diverse family of venomous snakes found in Americas, Asia, Africa and Australia (Figure 1.2 D) This family includes cobras, kraits, mambas, coral snakes and a large diversity of Australian elapids They possess short, fixed fangs in the front of the mouth Hydrophidae, consisting of sea snakes, are considered as a sub family of elapidae by... 1.1.2 Ophiophagus hannah This thesis deals with proteins isolated from the venom of Ophiophagus hannah (King cobra) This snake belongs to the family elapidae and it is the longest venomous snake in the world, growing to a maximum length of 18 feet (Figure 1.3) They also have a relatively long lifespan of up to 25 years (Veto et al., 2007) King cobra is distributed in parts of Southeast Asia, South China... delivery apparatus like front fangs evolved in elapidae (cobras and kraits), hydrophiidae (sea snakes), viperidae (vipers) and crotalidae (rattle snakes); among which, the viperidae and crotalidae have the most sophisticated venom delivery apparatus with retractable fangs (Figure 1.1) Presently, there are about 3200 species of snakes and approximately 1300 of them are venomous (Hider et al., 1991) Of the. .. from snake venoms have been isolated and characterized by employing more sophisticated techniques from proteomics and genomics We have constructed a cDNA library from the venom gland mRNA of Ophiophagus hannah (king cobra) and identified five novel proteins From the Cys numbers and pattern we concluded that all these new proteins belong to the three-finger toxin family of snake venom proteins We have... Calliophis, and Maticora species (coral snakes), and most venomous snakes of Australia Short, fixed fangs; venom injected by succession of chewing movements Indopacific region: Pelamis platurus (pelagic sea snake), Laticauda colubrina (colubrine sea krait or yellow-lipped sea krait), Laticauda semifasciata (Erabu black-banded sea krait) Fangs similar to those of elapidae; highly neurotoxic venom; rarely... all families of snake venoms Cyeteine-rich secretory proteins (CRISPs) or helothermine-related venom proteins are proteins with 16 conserved Cys residues They are known to modulate the activity of various ion channels Sarafotoxins Isolated from the venom of Atractaspis engaddensis These isopeptides are structurally and functionally related to mammalian endothelins They are potent vacoconstrictors acting... have venomous species of snakes (Table 1.1) (Harris, 1991) Colubridae is the largest family of snakes (Figure 1.2 A) They dominate most parts of the world except Australasia Most snakes in this family are harmless except a few like the African Boomslang (Dispholidus typus) Viperidae (true vipers) are the most widespread snakes found throughout Europe, Africa, Asia and Americas, but absent in Australia... Unlike mammalian PLA2, snake PLA2 enzymes induce various pharmacological effects like pre- and postsynaptic neurotoxicity, myotoxicity, cardiotoxicity, hemolytic activity, anticoagulant activity, antiplatelet activity, hypotension, hemorrhage and edema L-amino acid oxidase (LAO) Found widely in elapidae, viperidae and crotalidae venoms Flavo enzymes catalyzing the conversion of L-amino acid substrates... to α-keto acids LAOs from snake venoms also induce pharmacological effects like induction or inhibition of platelet aggregation, association with mammalian epithelial cells and induction of apoptosis and anti bacterial activity Serine Proteinases Found widely in elapidae and viperidae venoms Snake venom serine proteinases, in addition to their contribution to the digestion of prey, affect various physiological... They are heavier and bulkier compared to other families and have a distinct triangular head Their venom delivery apparatus is very advanced and they possess retractable fangs Crotalidae is sometimes considered as a sub family of viperidae Crotalids are similar to true vipers, except for the presence of heat sensing pits between the nostril and the eye (Figure 1.2 C) This gives them the ability of thermal . support. Thanks to my father Mr. Rajagopalan and my mother Mrs. Lalitha Rajagopalan, my brothers Mr. Prasanna and Mr. Vasanth and my cousins Ms. Aarthi Ravichandran and Ms. Madhulika Ravichandran β -CARDIOTOXIN: A NOVEL PROTEIN ISOLATED FROM THE VENOM OF OPHIOPHAGUS HANNAH (KING COBRA) SHOWING BETA- BLOCKER ACTIVITY NANDHAKISHORE RAJAGOPALAN (M. Sc. (Life Sciences)) A THESIS SUBMITTED. great support and guidance, Dr. Md Abu Reza, Dr. Dileep Gangadharan, Dr. Syed Rehana, Dr. Joanna Pawlak, Dr. Kang Tse Siang, Rocky, Shifali and Dr. Raghurama Hegde for all the help they have