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Fasciola Gigantica

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Fasciola Gigantica MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF TYPE 1 CYSTATIN STEFIN-2 OF THE LIVER FLUKE FASCIOLA GIGANTICA BY MISS SINEE SIRICOON A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE DOCTOR OF PHILOSOPHY (BIOMEDICAL SCIENCES) GRADUATE PROGRAM IN BIOMEDICAL SCIENCES FACULTY OF ALLIED HEALTH SCIENCES THAMMASAT UNIVERSITY ACADEMIC YEAR 2015 COPYRIGHT OF THAMMASAT UNIVERSITY MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF TYPE 1 CYSTATIN STEFIN-2 OF THE LIVER FLUKE FASCIOLA GIGANTICA BY MISS SINEE SIRICOON A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE DOCTOR OF PHILOSOPHY (BIOMEDICAL SCIENCES) GRADUATE PROGRAM IN BIOMEDICAL SCIENCES FACULTY OF ALLIED HEALTH SCIENCES THAMMASAT UNIVERSITY ACADEMIC YEAR 2015 COPYRIGHT OF THAMMASAT UNIVERSITY (1) Dissertation Title Molecular and biochemical characterization of type 1 cystatin stefin-2 of the liver fluke Fasciola gigantica Author Miss Sinee Siricoon Degree Doctor of Philosophy (Biomedical Sciences) Department/Faculty/University Graduate Program in Biomedical Sciences Faculty of Allied Health Sciences Thammasat University Dissertation Advisor Associate Professor Hans Rudi Grams, Dr. rer. nat. Dissertation Co-Advisor Professor Vithoon Viyanant, Ph.D. Dissertation Co-Advisor Professor Peter Smooker, Ph.D. Dissertation Co-Advisor Assistant Professor Suksiri Vichasri Grams, Dr. rer. nat. Dissertation Co-Advisor Assistant Professor Amornrat Geadkaew, Ph.D. Academic Years 2015 ABSTRACT Cysteine proteases including cathepsin B and cathepsin L are involved in physiological and biological processes in all living organisms and imbalanced activity of these proteases may cause several diseases. They are also important antigens in the trematode genus Fasciola and have vital roles in parasite survival including protection, infection and nutrition of the liver fluke. The biological roles and application of parasite cathepsins, e.g. in chemotherapy and as vaccines have been studied but data concerning their regulation is still incomplete. Especially, the cysteine protease inhibitors of the cystatin superfamily have not been investigated in depth in trematodes. This research was conducted to characterize the molecular (2) and biochemical properties of a type 1 cystatin (Stefin-2) in the liver fluke Fasciola gigantica. In the present study, a cDNA encoding type 1 cystatin of the parasite, FgStefin-2, was isolated from total RNA of adult F. gigantica by RT-PCR. A FgStefin-2- specific probe detected gene and transcript in Southern and Northern analyses of genomic DNA and total RNA of the fluke. FgStefin-2, which unusually for type 1 cystatins carries a signal peptide, was expressed as active recombinant protein in Escherichia coli (rFgStefin-2). Purified rFgStefin-2 was used for cysteine protease inhibition assays, functional analysis and polyclonal antibody production. The polyclonal antibody was used to study the distribution of native FgStefin-2 in 2- and 4-week-old juveniles and adult F. gigantica and to detect rFgStefin-2 and native FgStefin-2 in crude worm (CW) extract and excretory/secretory (ES) products of adult F. gigantica in immunoblots. Immunohistochemical analysis showed that FgStefin-2 is located in several tissues of the parasite including the prostate gland in adults and gut epithelium in all stages. Immunoblots demonstrated that the polyclonal antibody reacted with rFgStefin-2, CW extract and ES product of the adult parasite, but did not cross-react with rFgMDCd10, rFgStefin-1 and CW extracts of other trematodes. The inhibitory properties of rFgStefin-2 against bovine cathepsin B, bovine cathepsin L and the proteolytic activity of ES products, CW extracts from metacercariae and adult parasite were characterized by using fluorogenic substrates and by zymography using gelatin as substrate. The recombinant protein exhibited inhibitory activity against cysteine proteases (cathepsins B and L) and the proteolytic activity of ES products, CW extracts from metacercariae and adult parasites. This protein was found to be active over a wide pH range and heat stable. Preliminary study on immunomodulatory properties of the protein showed that it may interfere with an immune response mechanism that is involved in polyclonal T-cell proliferation. Furthermore, the novel cathepsin B5 of F. gigantica, FgCB5, was expressed and purified in a yeast-based eukaryotic expression system. Immunoblots showed that procathepsin B5 was detected in CW extract and in minor amounts in ES product. Analyses of recombinant cathepsin B5 revealed that it was active at acidic pH, able to effectively digest several of the host substrates and also exhibited (3) substantial exopeptidase activity. Inhibitory activities of rFgStefin-1, rFgStefin-2 and human cystatin C against rFgCB5 demonstrated nanomolar inhibition constants. This experiment was used to confirm that rFgStefin-2 has been evolutionary adapted to block cathepsin B. In summary, FgStefin-2 may play important roles, for example in the protection of the minute newly excysted juvenile from autoproteolysis and in the regulation of cysteine protease activity in the reproductive system. This knowledge may help to evaluate application of the protein as diagnosis tool, drug target and vaccine candidate against fascioliasis. Keywords: cystatin, cysteine protease, protease inhibitor, Fasciola gigantica (4) ACKNOWLEDGEMENTS This Ph.D. dissertation was completed with the support and collaboration of several people and institutes. First, I would like to express my deepest gratitude to my major advisor, Assoc. Prof. Dr. Hans Rudi Grams, for his constant support, encouragement, kindness, solving technical problems and invaluable advice throughout my study. Without his virtue, this dissertation might not have been possible. Beside my advisor, I would like to thanks my co-advisor, Professor Dr. Vithoon Viyanant, Thailand Center of Excellence in Drug Discovery and Development (TCEDDD), Thammasat University who always encouraged and suggested me about this study. Furthermore, I am also deeply grateful to Assist. Prof. Dr. Suksiri Vichasri Grams for her guidance valuable advice, suggestions and comments. Equally, I am very thankful to Assist. Prof. Dr. Amornrat Geadkaew for her kindness and suggestion in my laboratory work. My special thanks and deep appreciation are bestowed on Prof. Dr. Peter Smooker, who provided the excellent opportunity to conduct research in his laboratory at RMIT University, Australia for his kind advice, intensive supervision, invaluable suggestions and encouragement. I would like to express my deep thankfulness to Assoc. Prof. Dr. Smarn Tesana, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand for his kind acceptance to be the chair committee of my dissertation defense committee and his kindness, comments and corrections of my thesis. My dissertation would have not been accomplished without the financial support of the Thailand Research Fund through a Royal Golden Jubilee scholar under the supervision of Assoc. Prof. Dr. Hans Rudi Grams and the support from Thammasat University through a graduate student grant. Many thanks to all of my friends, seniors, juniors, instructors and staff members of the Graduate Program in Biomedical Sciences, Faculty of Allied Health (5) Sciences, Thammasat University for unconditional support, their friendship and helpfulness. Finally, I would like to express my deepest thankfulness to my beloved family, my father, my mother and all of my best friends for their strong encouragement, understanding, never-ending support and who stayed with me at all times. I would not have achieved my goal without them. Sinee Siricoon (6) TABLE OF CONTENTS Page ABSTRACT (1) ACKNOWLEDGEMENTS (4) TABLE OF CONTENTS (6) LIST OF TABLES (17) LIST OF FIGURES (18) LIST OF ABBREVIATIONS (24) CHAPTER 1 INTRODUCTION 1 CHAPTER 2 OBJECTIVES 4 CHAPTER 3 REVIEW OF LITERATURE 5 3.1 Fascioliasis 5 3.1.1 Definition and classification of Fasciola species 5 3.1.2 Geographic distribution and epidemiology of Fasciola spp. 5 3.1.3 The Morphology and life cycle of Fasciola spp. 10 3.1.4 Parasite physiology in the mammalian host 17 3.1.4.1 Gut 17 3.1.4.2 Reproductive System 19 3.1.5 Pathogenesis 22 (7) TABLE OF CONTENTS (Cont.) Page 3.1.6 Diagnosis of fascioliasis 23 3.1.7 Treatment of fascioliasis 25 3.1.8 Control and prevention of fascioliasis 26 3.1.9 Vaccine against animal fascioliasis 29 3.2 Proteases 29 3.2.1 Definition and Classification of proteases 29 3.2.2 Cysteine proteases and substrate specificity 34 3.2.3 Physiological roles of cysteine proteases 42 3.2.4 Mammalian cathepsins 42 3.2.4.1 Cathepsin L 43 3.2.4.2 Cathepsin B 43 3.2.5 Fasciola papain-like cysteine proteases 47 3.2.5.1 Cathepsin L-like proteases of Fasciola 49 3.2.5.2 Cathepsin B-like proteases of Fasciola 54 3.3 Cystatins and the mechanism of their interaction with cysteine 58 proteases 3.3.1 Cysteine protease inhibitors of mammalian and pathogenic 65 organisms 3.3.1.1 Ixodes scapularis (blood feeding tick) 65 3.3.1.2 Schistosoma spp. 65 3.3.1.3 Fasciola hepatica 66 3.3.1.4 Parasitic nematodes 67 3.3.1.5 Homo sapiens 69 3.4 Immunomodulation by nematode cystatins 76 3.4.1 Interference with antigen presentation and T cell responses 76 3.4.2 Modulation of the cytokine production 79 (8) TABLE OF CONTENTS (Cont.) Page 3.4.3 Effects on the inducible nitric oxide production 79 CHAPTER 4 MATERIALS and METHODS 81 4.1 Molecular cloning and sequence analysis of FgStefin-2 81 4.1.1 Molecular cloning of FgStefin-2 cDNA 81 4.1.1.1 PCR amplification of FgStefin-2 cDNA 81 4.1.1.2 Agarose gel electrophoresis 82 4.1.1.3 Isolation of PCR product from agarose gel by 83 using QIAquick® Gel Extraction Kit 4.1.2 Molecular cloning of the PCR-amplified FgStefin-2 cDNA 84 and transformation of the bacterial host cells with the recombinant plasmid 4.1.2.1 Ligation of FgStefin-2 PCR product into the pGEM® - T Easy vector 84 4.1.2.2 Preparation and storage of chemical competent E.
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