National Natural Toxins Research Center (NNTRC)

VIPER DAYThursday, April 20, 2017

Texas A&M University-Kingsville®

NNTRC Research Team

Dr. Elda E. SánchezExecutive Director

Tuchakorn LertwanakarnPh.D. Student from Chulalongkorn University in Bangkok, Thailand

Mark HockmullerSerpenatriumCurator

Ilse DiegoGraduate Student

Ifey IbikUndergraduate

Dr. Montamas SuntravatAssistant Research Professor

Jessenia MarquezGraduate Student

Sungmin YooGraduate Student

Thomas HelmkeUndergraduate Student

Nora Diaz De Leon Administrative Associate III

Former NNTRC Student & Keynote Speaker

Dr. Maria Perez-Johnson Dr. Maria Perez-Johnson was born in Kingsville, and graduated from Bishop High School. She attended Texas A&M University-Kingsville, and graduated from (TAMUK) with a major in Biology and a minor in Chemistry. She joined the Ronald E. McNair Scholars program as an undergraduate and conducted Research at the National Natural Toxins Research Center under the guidance of Drs. John C Perez and Elda Sánchez. Her research involved isolating proteins from snake venom for potential biomedical applications. She also did a summer

internship at Texas A&M University-College Station where she isolated proteins from fungi to develop isolate proteins to aid in other biomedical applications. She graduated Magna Cum Laude in 1997 and following graduation began her studies at the University of Health Science Center, Texas College of Osteopathic Medicine in Fort Worth, Texas. She completed her Pediatric residency at Texas A&M University, Driscoll Children’s Hospital. She is board certified in Pediatrics and has practiced Pediatric Emergency Medicine at Driscoll Children’s Hospital, Edinburg Children’s Hospital, Children’s Hospital of San Antonio, and Dayton Children’s Hospital in Ohio. She has served at the Pediatric Department Chair at Driscoll Children’s Hospital and has served on numerous committees at various institutions to focus on the health of the children of South Texas.

Viper Day Made Possible By:

The Office of the PresidentDr. Steven H. Tallant

The Office of the ProvostDr. Heidi Anderson

The College of Arts & SciencesDr. Dolores Guerrero,

Dean

Former Distinguished Alumni Dr. Roger Campos, D.D.S. Campos Family Dental PC

San Antionio, TX

The Office of Student AccessDr. Mary Gonzalez,

Associate VP

Sahiti MallelaGraduate Student

Juan SalinasAnimal Room Technician

Paige RamosUndergradute Animal

Room Technician

Interested in Texas A&M-Kingsville?Visit: www.tamuk.edu

Interested in science or scientific fields of study? Visit the Science Departments online:

www.tamuk.edu/nntrc www.tamuk.edu/chemistry

www.tamuk.edu/biology

National Natural Toxins Research CenterTexas A&M University-Kingsville975 West Avenue B, MSC 224Kingsville,Texas 78363-8208

Victoria ParraUndergraduate Student

Chesney MiglGraduate Student

Iheoma NnadiUndergraduate Student

AGENDA8:30 A.M - 9:30 A.M.

Registration, Ballroom A & Poster Viewing

9:30 A.M - 9:45 A.M. Mistress of Ceremony

Dr. Elda E. Sanchez, DirectorNational Natural Toxins Research Center

Viper Day Welcome Dean’s Office - Dr. Dolores Guerrero

9:45 A.M - 10:15 A.M. Introduction of the Speaker Keynote Presentation

Maria Perez-Johnson, DOAward Presentation

10:15 A.M - 10:30 A.M. Break &

Poster Reviews

10:30 A.M - 12 P.M. Viper Day Trivia Game

Presentation of Scholarships

12 P.M - 1 P.M. Lunch (free), Ballroom B

1 P.M - 3 P.M. Tours of the Serpentarium and the National Natural Toxins

Research Center

ABSTRACTSThe Design Of An X-Aptamer Against Snake Venom Disintegrins

Ilse Diego1,2, Montamas Suntravat1, Sara E. Lucena1, Curtis Lam3, Nancy Ward3, Mark Shumbera3, David Gorenstein3, Peter Davies4, and Elda E. Sánchez1&2 1National Natural Toxins Research Center, Texas A&M University-Kingsville, Kingsville, TX 2Department of Chemistry, Texas A&M University-Kingsville, Kingsville, Texas USA 3AM Biotechnologies, LLC, Houston, TX 4Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX

The use of aptamers rather than antibody-based antitoxins can result in the development of a new venom antidote, useable under severe conditions and suitable for immediate administration in the field without risk of serious side effects. Envenomation by the family Viperidae is characterized by hemorrhage, local necrosis, edema, and systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity. Snake venom metalloproteinases (SVMPs) and disintegrins are the important factors responsible for hemorrhage and also interfere the hemostasis pathway. Metalloproteinases from snake venoms are classified into three major classes (P-I to P-III) based on their structural domains. The disintegrin domain is a part of the P-II and P-III SVMPs, which have been reported to possess more diverse biological activities than the P-I class of SVMPs. The aim of this project is to design an X-aptamer against a disintegrin from the venom of Crotalus atrox and determine its neutralizing abilities on both disintegrins and SVMPs. Reverse-Phase C18 High Performance Liquid Chromatography was used to purify and isolate disintegrins from this venom. Fractions 6-9 showed protein bands at about 8 kDa, which were identified as disintegrins using SDS-PAGE and N-terminal sequencing. Disintegrins will be further biotinylated in vitro for the X-aptamer selection. X-aptamers can open new avenues not only for treating envenomations but also in identifying novel cellular targets of venom toxins providing the foundation for designing innovative therapeutics for the treatment of various illnesses.

Cytotoxicity Of A Beta-Cardiotoxin Isolated From King Cobra Venom (Ophiophagus hannah) On Three Different Mammalian Cell Types Tuchakorn Lertwankarn1,

Montamas Suntravat2, Kittipong Tachampa1, Elda E. Sánchez2&3 1Department of Physiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand 2National Natural Toxins Research Center, Texas A&M University-Kingsville, Kingsville, TX 3Department of Chemistry, Texas A&M University-Kingsville, MSC 161, Kingsville, TX

Beta-cardiotoxin (ß-CTX), the novel three-finger toxin from king cobra venom (Ophiophagus haanah), has been discovered since 2007. It is known to decrease the heart rate without changing cardiac contractile stage in the isolated perfuse heart rat, which indicates that it possesses beta-blocker activity. Therefore, it could have potential for cardiovascular drug development and design. The cytotoxicity testing against different mammalian cell types will provide useful insight into the toxicity of a novel beta-blocker agent. The objective of this study was to isolate ß-CTX and to characterize cytotoxicity on mouse skeletal muscle cells (C2C12), rat smooth muscle cells (A7r5), and human umbilical vein endothelial cells (HUVEC). We have isolated ß-CTX from the crude venom of the king cobra using reverse phase and cation exchange chromatography. For the first step purification with reverse phase chromatography, fractions 25-28 contained neurotoxins with molecular weights about 8 kDa and ß-CTX with a molecular weight of 7 kDa as determined by SDS-PAGE and N-terminal sequencing. Fractions 25-28 were pooled for further purification of ß-CTX using cation exchange chromatography. Purified ß-CTX will be tested for cytotoxicity on C2C12, A7r5, and HUVEC cell lines using the MTT assay. ß-CTX may have utility in the development of therapeutic tools in the treatment of cardiovascular diseases.

The Isolation And Characterization Of Snake Venom Cysteine-Rich Secretory Proteins (svCRiSP) From The Venoms Of The Southern Pacific Rattlesnake And King Cobra SnakesVictoria Parra1, Tuchakorn Lertwankarn1, Sara Lucena1, Montamas Suntravat1, Elda E. Sanchez1&2 1National Natural Toxins Research Center, Texas A&M University-Kingsville, Kingsville, TX 2Department of Chemistry, Texas A&M University-Kingsville, MSC 161, Kingsville, TX

Snake venom can contain hundreds of different molecules that work together to subdue prey; however, when isolated into single molecules, they can be found to have therapeutic benefits. There are many snake venom molecules that have been characterized and biological functions known. One molecule in particular, the snake venom cysteine-rich secretory protein (svCRiSP), has mystified many in the field of toxinology. Although biological svCRiSP’s have been recognized for many years as abundant components in the venom of numerous species of snakes, little is known of the role that these toxins play in the pathophysiology of snakebite. It has been established that several svCRiSPs inhibit ion channel activities, including possible effects on smooth muscle contraction, and at least two svCRiSPs, (ES-CRiSP and natrin) affect cell-signaling pathways in vascular endothelial cells. It is the hypothesis, based on the limited information available in the literature for Ophanin, and preliminary results obtained with Hellerin, a newly discovered rattlesnake CRiSP, that svCRiSPs play an important role in the pathophysiology of snakebite by initiating a pro-inflammatory response in tissues at the site of the snakebite. The goal of this project is to isolate Hellerin from the venom of the Southern Pacific Rattlesnake, and Ophanin from the venom of the King Cobra snake to determine their role in some important functions of smooth muscle cells, and vascular physiology, with special emphasis in vascular permeability.

A Novel Recombinant Waprin Cloned From The Venom Gland Of The Venezuelan Mapanare (Bothrops colombiensis) With Anticoagulant Activity Thomas J. Helmke1,

Sungmin Yoon1, Montamas Suntravat1, Elda E. Sánchez1&2, Alexis Rodriguez-Acosta3 1National Natural Toxins Research Center, Department of Chemistry, Texas A&M University-Kingsville, Kingsville, TX 2Department of Chemistry, Texas A&M University-Kingsville, MSC 161, Kingsville, TX 3Laboratorio de Inmunoquímica y Ultraestructura, Instituto Anatómico de la Universidad Central de Venezuela, Caracas 1041, Venezuela.

Waprin is a small protein containing 50 amino acid residues and belongs to the Whey acidic protein (WAP) family. The WAP domain is found in mammalian proteins such as SLPI (antileukoprotease) and Elafin (an elastase-specific inhibitor of human skin), which are highly divergent regarding a broad range of biological functions involving the innate immune system and inhibiting various cellular proteins. The role of waprin in snake venom is unknown. Only one waprin from Oxyuranus microlepidotus venom has been examined, and it was shown to act as an antimicrobial agent. The aim of this study was to produce waprin cloned from the venom gland cDNA library of Bothrops colombiensis and to characterize it regarding its biological activity. Waprin was amplified by PCR, cloned into a pET-32a vector, and expressed in Escherichia coli BL21. Affinity purified waprin was cleaved from the fusion tags by enterokinase cleavage. Recombinant waprin was further purified by two-step chromatography consisting of HiTrapTM Benzamidine FF column, followed by Talon Metal Affinity column. The activated clotting time (ACT), clot rate (CR), and platelet function (PF) were used to determine the effect of waprin on hemostasis using the Sonoclot assay. A significant increase in the ACT and a significant decrease in the CR were observed after the addition of waprin to human whole blood. No change in PF was observed; therefore, according to these results, waprin is demonstrating anticoagulant activity.

A Novel Method Of Transcriptome Acquisition For cDNA Library Construction From The Venom Gland Of Crotalus atrox Chesney E. Migl1,

Chesney E. Migl1, Montamas Suntravat1, Clayton Hilton2, Elda E. Sánchez1&3 1National Natural Toxins Research Center, Texas A&M University-Kingsville, Kingsville, TX, 2Dick and Mary Lewis Kleberg College of Agriculture, Natural Resources and Human Sciences, Veterinary Tech Program, Texas A&M University-Kingsville, Kingsville, TX 3Department of Chemistry, Texas A&M University-Kingsville, MSC 161, Kingsville, TX

Investigation into snake venom is a rapidly growing field of research, with enormous application for medical treatment, new pharmaceuticals, and insights into complicated biochemical processes. One of the vital tools of this research are collections of tissue-specific transcriptomes, called cDNA libraries. Collecting venom tissue has traditionally involved sacrificing the snake and harvesting the entire venom gland. While this yields an abundance of tissue to sample, the resulting loss of the specimen limits potential downstream experimentation, particularly ontological and longitudinal comparisons. This study examines an alternative method to create functional cDNA libraries without sacrificing valuable or difficult to obtain animals. The first stage involved taking a biopsy sample during survival surgery on a Western Diamondback Rattlesnakes (Crotalus atrox) to determine biopsy tolerance, sample feasibility, and its impact on subsequent venom production for the animal. For the second stage we will analyze the quality and quantity of the RNA retrieved and attempt to create a functional cDNA as a demonstration of the viability of the alternative biopsy method.

Expression, Purification And Characterization Of The New Recombinant Crotamines From The Venom Gland Of Crotalus oreganus helleri: Their Role On

Antimicrobial ActivitySungmin Yoon1; Montamas Suntravat1, Elda E. Sánchez1&2 1National Natural Toxins Research Center, Texas A&M University-Kingsville, Kingsville, TX2Department of Chemistry, Texas A&M University-Kingsville, MSC 161, Kingsville, TX

Crotamine, a highly basic and non-enzymatic toxin, is the major toxic component found in the venom of the South American rattlesnake, Crotalus durissus terrificus. Based on the three-dimensional structure of crotamine, its cysteine-pairing pattern and folding structure are very similar to those of the other small toxins identified in different rattlesnake venoms and the antimicrobial peptide human ß-defensins. Native, recombinant, and synthesized crotamine (YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG) has been found to contain antimicrobial activity against a wide variety of bacteria and fungi. In the present work, we aim to express recombinant propeptide (start codon methionine) and mature (start codon tyrosine) crotamine-like isoforms, helleramines, cloned from the venom gland cDNA library of Crotalus oreganus helleri and to characterize their antimicrobial activity against Gram-negative and Gram-positive bacteria. Propeptide and mature helleramines 5J11 and 6O01 cloned into two different vectors, pET32a and pGEX-4T-1 (5J11-pGEX-4T-1, 6O01-pGEX-4T-1, 5J11-pET32a, and 6O01-pET32a), were expressed in Escherichia coli BL21. The result of expression from pGEX-4T-1 vector is an N-terminus Glutathione S-transferase (GST)-tagged fusion protein, which was purified using Glutathione Sepharose 4B resins. For the pET32a vector, recombinant polyhistidine-tagged proteins were purified using TALON® cobalt-based immobilized Metal Affinity Chromatography (IMAC). Purified tag-cleaved recombinant helleramines will be further investigated for their antimicrobial activities. The production of recombinant helleramines with biological activity may provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development.

CONT’D

Molecular Basis For The Inhibitory Effect Of Two Novel C-Type Lectins From The Southern Pacific Rattlesnake On Platelet Aggregation

Montamas Suntravat1, Amanda Cruz1, Esteban Cuevas1, Chairat Atphaisit1, Thomas Helmke1, Sara Lucena1, Elda E Sánchez1

1National Natural Toxins Research Center, Texas A&M University-Kingsville, Kingsville, TX

The C-type lectin toxins (CTLs) are important components of the hemorrhagic venoms of pit vipers. Many venom CTLs target components of the platelet membrane thrombotic machinery such as the platelet glycoprotein Ib-IX (GPIb-IX) protein complex and its interactions with Von Willebrand factor (VWF). The goal of this study is to investigate the molecular basis for the anti-thrombotic activity of a specific CTL toxin, Hellericetin. We have isolated two Hellericetins from the crude venom of the Southern Pacific Rattlesnake (Crotalus oreganus helleri) using cation exchange and reverse phase chromatography. Hellericetin 1 is either a homodimer or a heterodimer of two closely related subunits, and Hellericetin 2 is a heterodimeric CTL. Both Hellericetins have strong inhibitory activity on ristocetin-induced platelet aggregation with IC50s of 138 nM and 125 nM, respectively, but not by ADP and collagen, suggesting that the Hellericetins might act on GPIb and/or VWF receptors. We will use an enzyme-linked immunosorbent assay (ELISA) to measure the direct binding of the Hellericetins to VWF and the effect of Hellericetins on VWF binding to the ligand-binding domain of GPIba knowledge will contribute to the development of new classes of drugs for the treatment of athero-thrombotic diseases.

Detection of Venom L-Amino Acid Oxidases Antigens Via A Lateral Flow Colloidal Gold Immunoassay Test Strip

Sahiti Mallela1, Dr. Montamas Suntravat1, Dr. Sara E. Lucena1, Dr. Alexis Rodriguez-Acosta3, Dr. Elda E. Sánchez1&2 1National Natural Toxins Research Center, Department of Chemistry, Texas A&M University-Kingsville, Kingsville, TX 2Department of Chemistry, Texas A&M University-Kingsville, MSC 161, Kingsville, TX 3Laboratorio de Inmunoquímica y Ultraestructura, Instituto Anatómico de la Universidad Central de Venezuela, Caracas 1041, Venezuela.

Snakebites are considered the most serious envenomation regarding morbidity and mortality. This is owing to the huge quantity of venom provided by snakebites as well as the copious classes of toxin molecules present in snake venom. An immunochromatographic strip was developed for the detection of venom L-amino acid oxidases (LAAOs) in the blood of patients that have been bitten by venomous snakes. This test will allow us to determine the severity of a snakebite by observing the intensity of the reaction of LAAOs with antibodies against LAAOs. The test strip is composed of a sample pad, a conjugate pad, an absorbent pad, and a test membrane. The strip contains a colloidal gold-tagged antibody (anti-LAAO) made in rabbits against a main snake toxin, LAAO, which is conjugated onto the pad. Non-colloidal gold-tagged anti-LAAO and goat anti-rabbit antibody are also blotted on the nitrocellulose membrane for the test and control lines, respectively. Samples to be tested for the presence of LAAOs are added to the sample pad. As the serum migrates chromatographically it carries the gold-tagged antibody with it. If LAAOs are present, a specific antibody/LAAO/colored antibody complex will form on the test band area of the membrane. The absence of the test band indicates no LAAOs are present. The method control will always have a positive colored line since the gold-tagged antibody will continue to migrate and bind to the goat anti-rabbit antibody. If the control line does not appear, the test is rendered invalid. A total of 19 individual venom samples consisting of 7 species (Crotalus atrox, C. adamanteus, C. scutulatus scutulatus, Agkistrodon piscivorus, Sistrurus catenatus edwardsii, Micrurus fulvius and Micrurus tener tener) were used to test the strip. Venoms from C. adamanteus and C. atrox had a much quicker response at 5 min, while the C. scutulatus scutulatus venoms showed up in 15 min. Venoms from Micrurus ssp were used as negative controls since they are known to have very little to no LAAOs. This test strip is rapid (5 min) and easy to perform with no requirement of professional skills, reagents or equipment. The immunochromatographic strip will allow medical personnel to evaluate the severity of a snakebite in individual patients to assess treatment with antivenom.

Purification And Characterization Of Cysteine Rich-Secretory Proteins (CRISPs) From The Venom Of The Southern Pacific Rattlesnake (Crotalus Oreganus Helleri): Their Role

On Blood And Lymphatic Endothelial Cell PermeabilityJessinia Marquez1, Victoria Parra1, Montamas Suntravat1, Elda E. Sánchez1&2 1National Natural Toxins Research Center, Texas A&M University-Kingsville, Kingsville, USA 2Department of Chemistry, Texas A&M University-Kingsville, MSC 161, Kingsville, TX 78363, USA.

Cysteine-rich Secretory Proteins (CRISPs) have long been recognized as ubiquitous components of many snake venoms, however, no clear explanation has been provided for the role they play in venoms. Some CRISPs have been shown to inhibit ion channel activities and have major effects on cell signaling pathways in vascular endothelial cells. We speculate that CRISPs, via combined effects on cell signaling pathways and ion channel activities, disrupt normal interstitial fluid dynamics adjacent to the snakebite, accelerating the transfer of the macromolecular toxins in the venom into the lymphatic circulation, which plays a critical role in venom absorption and distribution into the systemic circulation. The rapid delivery of these toxins into the circulation contributes to the acute effects of envenomation and the rapid incapacitation and death of the snake’s prey. The goal of our study is to characterize the cellular and molecular basis for the effects of Hellerin, a newly identified CRISP isolated from the venom of the Southern Pacific rattlesnake, on the function of blood and lymphatic endothelial cells. Knowledge gained from these studies will contribute to a new level of understanding of the pathophysiology of snakebite and the development of new therapies for the treatment of this devastating disease.personnel to evaluate the severity of a snakebite in individual patients to assess treatment with antivenom.