ISOLATION AND CHARACTERIZATION OF CONOTOXINS FROM THE VENOM OF CONUS PLANORBIS AND CONUS FERRUGINEUS by Adriana Pak A Thesis Submitted to the Faculty of Charles E. Schmidt College of Science in Partial Fulfillment of the Requirements for the degree of Master of Science Florida Atlantic University Boca Raton, Florida ISOLATION AND CHARACTERIZATION OF CONOTOXINS FROM THE VENOM OF C. PLANORBIS AND C. FERRUGINEUS by AdrianaPak This thesis was prepared under the direction of the candidate's thesis advisor, Dr. Frank Mari, Department of Chemistry and Biochemistry, and has been approved the members of her supelVisory committee. It was submitted to the faculty of the Charles E. Schmidt College of Science and was accepted in partial fulfillment of the requirements for the degree of Master of Science. SUPERVISORY COMMITTEE: Fran , --,....,...,·hesis Advisor ~vr/~-- L~ Deguo Du, Ph.D Jer e E. Haky, Ph.D. Chair, Department of emistry and Biochemistry us ell Ivy, Ph.D. Interim Dean, The Charles E. Schmidt College of Science ~~r2?r--~ , P~~.JDate 11 ACKNOWLEDGEMENTS I would like to thank Dr. Frank Mari for all his guidance, time, and the opportunity to work with him. I would also like to thank Dr. Lyndon West and Dr. Deguo Du for their guidance and for serving on my committee. Furthermore, I would like to thank Dr. Katarzyna Pisarewicz for friendship and guiding me in the beginning. Thank you also Dr. Jose Riveira-Ortiz, Dr. Vered Marks, Dr. Herminsul Cano and Alberto Padilla, for being great friends and lab mates. Thank you also to Pam Mongkhonsri, Tanya T. Kelley for friendship and helping me several challenge tasks. I also want to thank Dr. Carolina Moller for guiding me and giving me a good example of being mom and scientist at same time. Thank to all members of Dr. Mari’s lab for a nice and warm environment in the lab. Finally I would like to thank my husband Pedro, my daughters Alexis and Michelle, my parents and my brother for all love and patience during this course of my life. And most important to God, my strength and my Savior. iii ABSTRACT Author: Adriana Pak Title: Isolation and Characterization of Conotoxins from the Venom of Conus planorbis and Conus ferrugineus Institution: Florida Atlantic University Thesis Advisor: Dr. Frank Mari Degree: Master’s of Science Year: 2014 The venom of marine gastropods belonging to the genus Conus has yielded numerous structurally and functionally diverse peptidic components. The increase variety of bioactive peptides identified in cone snail venoms is the product of the variety of molecular adaptations taken by Conus species in evolving neuroactive molecules to suit their diverse biological purposes. Toxins from cone snails are classified into two major groups. One group consists of disulfide-rich peptides commonly termed conotoxins; the second group comprises peptides with only one disulfide bond or none. In this work, we present the discovery and characterization from the marine snails C. planorbis and C. ferrugineus. Both species are commonly found in the Indo-Pacific iv region and are very similar and is not distinguishable by size and shape of the shell. Novel P and T-Supefamiles were found in both species along with small linear peptides with have a high frequency of tyrosine residues. Each chapter contains a detailed look at the discovery process for the isolation and characterization of C. planorbis and C. ferrugineus. At discussion part, we also compared the peptides isolated in this work with other peptides from the literature. v ISOLATION AND CHARACTERIZATION OF CONOTOXINS FROM THE VENOM OF CONUS PLANORBIS AND CONUS FERRUGINEUS LIST OF FIGURES .........................................................................................................viii LIST OF TABLES............................................................................................................. xi CHAPTER I - INTRODUCTION ...................................................................................... 1 1.1 Conus planorbis and Conus ferrugineus .................................................................. 5 1.2 Post Translational Modifications of Peptides ........................................................... 6 1.3 Biology of Conus species.......................................................................................... 7 CHAPTER II - MATERIAL AND METHODS............................................................... 10 2.1 Crude Venom Extraction ........................................................................................ 10 2.2 Purification and Isolation of a Peptide.................................................................... 11 2.2.1 Size-Exclusion Superdex Peptide HR 10/30 High Performance Liquid Chromatography (SE-HPLC).................................................................................... 11 2.2.2 Size-Exclusion High Liquid Chromatography (SE-HPLC)............................. 11 2.2.3 Analytical Reverse Phase HPLC ..................................................................... 12 2.3 Mass Spectrometry.................................................................................................. 12 2.3.1 AB QSTAR XL MS/MS System ..................................................................... 12 2.3.2 Voyager-DE STR............................................................................................. 12 vi 2.4 Nuclear Magnetic Resonance ................................................................................. 13 2.5 Reduction and Alkylation of Cysteyl Residues ...................................................... 14 2.6 Peptide Sequencing................................................................................................. 14 2.7 Nomenclature.......................................................................................................... 15 CHAPTER III - RESULTS............................................................................................... 16 3.1 Isolation and characterization of the venom of C. ferrugineus............................... 16 3.1.1 Purification of fraction Fer_B03g.................................................................... 16 3.1.2 Purification of fraction Fer_B05p................................................................... 21 3.1.3 Purification of fraction Fer_D04ij ................................................................... 24 3.1.4 Purification of fraction Fer_D05r .................................................................... 34 3.1.5 Purification of fraction Fer_F06k .................................................................... 37 3.1.6 Purification of fraction Fer_F07i ..................................................................... 42 3.2 Isolation and characterization of the venom of C. planorbis.................................. 45 3.2.1 Purification of Pla_A06j .................................................................................. 45 3.2.2 Purification of Pla_A06k ................................................................................. 46 3.2.3 Purification of Pla_A06l .................................................................................. 46 3.2.4 Purification of fraction Pla_A04j..................................................................... 55 3.2.5 Purification of fraction Pla_A04u.................................................................... 59 3.2.6 Purification of fraction Pla_B04f..................................................................... 62 3.2.7 Purification of fraction Pla_B05g .................................................................... 66 CHAPTER IV - DISCUSSION AND CONCLUSION.................................................... 72 REFERENCES ................................................................................................................. 77 vii LIST OF FIGURES Figure 1. 1: C. planorbis (left) and C. ferrugineus (right).................................................. 5 Figure 1. 2: Conus ferrugineus and Conus planorbis range map ....................................... 6 Figure 3. 1: Size Exclusion chromatogram of the venom from C. ferrugineus (Fer_B) in Superdex-Peptide Column ................................................................................ 17 Figure 3. 2: Analytical RP-HPLC chromatogram of Fer_B 03 ........................................ 18 Figure 3. 4: MALDI-TOF MS of reduced and alkylated of Fer_B03g ............................ 20 Figure 3. 5: Analytical RP-HPLC chromatogram of Fer_B 05p ...................................... 22 Figure 3. 6: TOF MS Spectrum of Fer_B05p................................................................... 23 Figure 3. 7: Size Exclusion Chromatogram for C. ferrugineus (Fer_D) .......................... 26 Figure 3. 8: Analytical RP-HPLC chromatogram of Fer_D 04........................................ 27 Figure 3. 9: Purification of peak i on Analytical RP-HPLC chromatogram of Fer_D04 . 28 Figure 3. 10: MALDI TOF MS Spectrum of Fer_D04ij before and after reduction and alkylation............................................................................................................... 30 Figure 3. 11: 1D NMR Spectrum of Fer_D04ij................................................................ 32 Figure 3. 12: 2D NMR TOCSY Spectrum of Fer_D04ij.................................................. 33 Figure 3. 13: Analytical RP-HPLC chromatogram of Fer_D........................................... 34 Figure 3. 14: QSTAR TOF MS Spectrum of native Fer_D05.........................................