Variability in the Venom of Conus regius By Alejandro Uribe-Benninghoff A Thesis Submitted to the Faculty of the Charles E Schmidt College ofScience in Partial Fulfillment of the Requirements for the Degree of Master of Science Florida Atlantic University Boca Raton, FL December 1999 Variability in the Venom of Conus regius By Alejandro Uribe-Benninghoff This thesis was prepared under the direction of the candidate's thesis advisors, Dr. Jim Hartman, Department of Biological Sciences, and Dr. Frank Mari, Department of Chemistry & Biochemistry, and has been approved by the members of his supervisory 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. SUP ERVISORY COMMITTEE: Dr. Fran . Mari Thes i<:.. Co Advisor Dr~' a v idBi n n i ~~ --- - -- -- ---- - ---- ---- Ch~it;~partm~ e nt of Biological ~cienccs Vice Provost ii ACKNOWLEDGEMENTS Special gratitude to both Dr. Frank Mari and Dr. Jim Hartmann, thesis advisors and friends, for giving me the opportunity to come to Florida Atlantic University and make one of my lifelong dreams come true. I would also like to thank my friend and soul mate, Teresa Koledin, for giving me her help during endless hours in the lab during every step of the project, both above and below the water. Herminsul Cano for his invaluable guidance in HPLC procedures in cone snail venom analysis and give special thanks to all the members of the U.S.S. Conotoxin diving team. Their precious help took this project to "soaring" new depths. Acknowledgements for Cognetix Inc., Salt Lake City, for its help with the cDNAIPCR experiments. Finally and most importantly, I would like to thank my parents, Alejandro Uribe and Olga Maria Benninghoff, my aunt, Diana Benninghoff, my brother and my sisters, for their never-ending love and support throughout all of these years. Ill ABSTRACT Author: Alejandro Uribe-Benninghoff Title: Variability in the Venom of Conus regius Institution: Florida Atlantic University Thesis Co-Advisors: Dr. James X. Hartmann & Dr. Frank Mari Degree: Master of Science Year: 1999 The venom of two different geographical populations of Conus regius has been isolated and characterized. Comparisons between the chromatographic profiles of the venom of these two populations exhibited similarities and differences among the venom's constituents. MALO I-TOF and PCR analysis techniques ratified the differences present in the venom of both populations. It is postulated that these differences could reflect the rapid adaptive nature of cone snails in an actual stage of speciation. Molecular weights of the _venom's constituents were compared with those of patented conopeptides in the Swiss Protein Database. Results of this comparison indicated that a number of the peptides isolated for both of the populations of C. regius had the same molecular weight as other patented conopeptides. In combination with the PCR analysis of these conopeptides, it has been proposed that some of the venom constituents of the venom of C. regius could have pharmacological applications for vertebrate systems. IV TABLE OF CONTENTS LIST OF TABLES ............................................................................. vii LIST OF FIGURES ........................................................................... viii 1. INTRODUCTION .. ..................................................................... .. 1 1.1 General Background ....................................................................... 1 1.2 Gross Anatomy of the Venom Apparatus ......................................... .... .4 1.3 Cone Snail Venom ......................................................................... 8 1.4 Conopeptide Action .. ......................................................... ............ 13 1.5 Conopeptide Evolution .. ................. ................................................ 15 1.6 Conus Peptides and Phylogenetic specificity .......................................... 16 1. 7 Purpose and Scope of the Research ... .......................... ... .. ................... 19 1.8 Objectives .................................................................. ............... 20 2. MATERIALS AND METHODS ....................................................... 22 2.1 Venom Collection ............................ ............................................ 22 2.2 Venom Extraction ........................ ................................................. 22 2.3 Peptide Isolation ........................................................................... 24 2.4 Mass Spectrometry ........................................................................ 25 2.5 eDNA Analysis ........................................................................... 26 3. RESULTS AND DISCUSSION ......................................................... 27 3.1 Venom Characterization ................................................................. 27 3.2 eDNA Analysis ...........................................................................40 v 3.3 Possible Venom Constituents ........................................................... .42 - 4. CONCLUSIONS ........................................................................... 46 5. REFERENCES .............................................................................. 49 VI LIST OF TABLES Table 3.1a: MALDI-TOF results for the chromatographic profiles of the venom of Conus regius specimens belonging to the Florida Keys ............... 30 Table 3.1b: MALDI-TOF results for the chromatographic profiles of the venom of Conus regius specimens belonging to Curacao ......................... 31 Table 3.1c: Molecular weight similarities and retention volumes of peaks shared by the chromatographic profiles of C. regius for both geographical areas ........................................................................... 36 Table 3.1d: Molecular weight differences and retention volumes of peaks in the chromatographic profiles of C. regius for both geographical areas ............... 3 8 Table 3.3a: Molecular weights of venom components of C. regius compared to molecular weights ofthe Swiss Protein Database of patented conopeptides ..................................................................... .45 VII LIST OF FIGURES -. Figure 1.7a: Conus regius ................................................................... 21 Figure 1.7b: Conus regius citrinus ......................................................... 21 Figure 1. 7c: Conus regius shells showing "transitional stage" .......................... 21 Figure 2.1a: Collection sites for C. regius and C. regius citrinus ...................... 23 Figure 3.1a: Reversed-Phase HPLC chromatogram for the venom of a Conus regius specimen collected in the Florida Keys (Pickles Reef, Plantation Key) ............................................................... 28 Figure 3.1b: Reversed-Phase HPLC chromatogram for the venom of a Conus regius specimen collected in the island ofCuracao .......................... 29 Figure 3.1c: Chromatographic profiles for two Conus regius specimens (Florida Keys) showing presence of four "signature peaks" ............... 33 Figure 3.1d: Chromatographic profiles for two Conus regius specimens (Curacao) showing presence of four "signature peaks" .................... 34 Figure 3.1e: Chromatographic profiles of C. regius from the Florida Keys (top) and C. regius from Curacao (bottom) showing conserved areas (enclosed by boxes) ............................................ 37 VIII 1. INTRODUCTION 1.1 General Background The class Gastropoda contains the largest number of mollusk species. Within this class, the families Conidae, Turridae, and Terebridae constitute the suborder Toxoglossa. Members of the Toxoglossa suborder are characterized by the possession of a venom apparatus, of which the most venomous are the marine snails belonging to the genus Conus. A number of human fatalities and intoxications have resulted from their stings. 1 There are around 500 species in the family Conidae , all of them members of the single genus Conus, and with few exceptions these are confined to tropical and subtropical waters. 2 Only a few number of species extend outside the tropics and those that do usually show a high level of endemicity in such cool areas as South Africa. southern Australia, and southern Japan. There are approximately 150 species present in the Western Atlantic out of which 60 are endemic to the Florida coastline. Despite great variation among these snails in shell size and shape, they all have a rough conical shape. 3 The shells are distinguished for rich colors and varied patterns. The whorls are rolled upon themselves below a small, sharp apex; the narrow aperture is as long as the body whorl and usually notched near the suture.4 Color patterns in cones are very important but 1 variable features in their identification. Colors are usually the product of depositing metabolic waste products, although they are not fully understood. 5 -. Cone snails are predominantly nocturnal in habit, burrowing in sand, under coral or beneath rocks during the daytime. They become active at night, when feeding usually occurs. Generally, each cone snail species is a highly specialized venomous predator. Certain Conus will feed on only a single prey species. Various Conus species feed on fish (piscivorous), others feed on gastropod mollusks (molluscivorous), or polychaete worms as well as two smaller phyla (hemichordates and echiuroid