University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2014-09-12 Towards a Biochemical Reconstitution of Nepenthes Pitcher Fluid for the Treatment of Celiac Disease Yang, Menglin Yang, M. (2014). Towards a Biochemical Reconstitution of Nepenthes Pitcher Fluid for the Treatment of Celiac Disease (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/28475 http://hdl.handle.net/11023/1740 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Towards a Biochemical Reconstitution of Nepenthes Pitcher Fluid for the Treatment of Celiac Disease by Menglin Yang A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOCHEMISTRY AND MOLECULAR BIOLOGY CALGARY, ALBERTA SEPTEMBER, 2014 © Menglin Yang 2014 Abstract Celiac disease (CD) is an autoimmune disorder that is triggered by the incomplete digestion of gliadins in dietary gluten due to the abundance of P and Q residues in their protein sequence(s). This thesis provides an initial assessment of the proteolytic activity of Nepenthes plant extracts, which is attributed to the aspartic proteases, nepenthesin I and II, potential as an oral protease therapeutic for CD. To this end, nepenthesin I and II were produced recombinantly and characterized. The recombinant nepenthesins were able to reconstitute the proteolytic activity of Nepenthes extracts except for cleavage after P, which was attributed to a previously unidentified protease. Nevertheless, the Nepenthes extracts and recombinant nepenthesin I/II were assessed for their capacity to detoxify gliadins. Although the recombinant nepenthesins alone did not appear sufficient, the Nepenthes plant extracts appeared to efficiently detoxify gliadin, which supports the proposed formulations potential as an effective oral therapeutic for CD. ii Preface Portions of this thesis resulted in the following peer-reviewed or in preparation publications: 1) Rey, M., Yang, M., Burns, K.M., Yu, Y., Lees-Miller, S.P., and Schriemer, D.C. (2013). Nepenthesin from monkey cups for hydrogen/deuterium exchange mass spectrometry. Molecular and Cellular Proteomics, 12(2), 464-472 2) Yang, M., Hoeppner, M., Rey, M., Man, P. and Schriemer, D.C. (2014). Recombinant nepenthesin II for hydrogen/deuterium exchange mass spectrometry. In preparation. iii Acknowledgements First and foremost, I would like to thank my supervisor, Dr. David Schriemer, for giving me the opportunity to work on this project and for his immense support throughout my graduate studies. For their various experimental contributions to this project, I would like to thank Drs. Martial Rey, Laurent Brechenmacher and Kelvin Ma, as well as, Ronghua Yu. Thank you to my committee members, Drs. Hans Vogel and Tony Schryvers, for their advice and guidance throughout my graduate studies. Thank you to all of the members of the Schriemer laboratory for providing a great work environment. Thank you to my mother and father, Lanlan and Shanning, for their support in my career decisions. Finally, thank you to Patricia Lan for your unwavering support in all of my decisions. iv Dedication To Patti. v Table of Contents Abstract ........................................................................................................................................... ii Preface ............................................................................................................................................ iii Acknowledgements ........................................................................................................................ iv Dedication ....................................................................................................................................... v Table of Contents ........................................................................................................................... vi List of Tables ................................................................................................................................. ix List of Figures and Illustrations ...................................................................................................... x List of Symbols, Abbreviations, and Nomenclature .................................................................... xiii CHAPTER ONE: INTRODUCTION ............................................................................................. 1 1.1 Celiac Disease: A General Overview .................................................................................. 1 1.2 Epidemiology ....................................................................................................................... 1 1.3 Basis for Disease Development ........................................................................................... 3 1.4 Pathogenic Mechanism ........................................................................................................ 3 1.5 Disease Symptoms and Outcomes ....................................................................................... 7 1.6 Treatments for CD- The Gluten-Free Diet ........................................................................... 8 1.7 Proposed Therapeutics for CD ............................................................................................. 9 1.8 Oral Proteases: A Promising Treatment for CD ................................................................ 10 1.8.1 Oral Proteases: AlV003 ............................................................................................. 11 1.8.2 Oral Proteases: STAN1 .............................................................................................. 12 1.8.3 Oral Proteases: AN-PEP ............................................................................................ 13 1.8.4 Oral Proteases: Limitations and Future Directions .................................................... 13 1.9 A New Oral Protease Candidate for the Treatment of CD- Nepenthesin .......................... 14 1.10 Research Hypothesis and Objectives ............................................................................... 18 CHAPTER TWO: IDENTIFICATION OF THE PROTEOLYTIC COMPONENTS OF NEPENTHES PITCHER FLUID .................................................................................................. 19 2.1 Introduction ........................................................................................................................ 19 2.2 Experimental Procedures ................................................................................................... 20 2.2.1 Chemicals ................................................................................................................... 20 2.2.2 Horticulture of Nepenthes Plants ............................................................................... 20 2.2.3 Preparation of Nepenthes fluid for Proteome Studies ................................................ 22 2.2.4 Proteome Mass Spectrometry and Data Analysis ...................................................... 22 2.2.5 Visualization of the Nepenthes fluid Proteome over Time ........................................ 23 2.2.6 In-gel Processing ........................................................................................................ 23 2.2.7 Activity Assays .......................................................................................................... 24 2.2.8 Pepstatin A Purification ............................................................................................. 24 2.2.9 Determination of Cleavage Specificities ........................................................................ 25 2.3 Results and Discussion ...................................................................................................... 26 2.3.1 In-solution Proteome Analyses of Nepenthes Pitcher Secretions .............................. 26 2.3.2 Activity and Cleavage Preferences of Nepenthes Pitcher Fluid ................................ 34 2.3.3 Pepstatin A Purified Nepenthes Pitcher Fluid ............................................................ 39 2.4 Conclusions ........................................................................................................................ 41 2.5 Contributions to the Chapter .............................................................................................. 41 vi CHAPTER THREE: RECOMBINANT RECONSTITUTION OF THE PROTEOLYTIC ACTIVITY OF NEPENTHES PITCHER FLUID ........................................................................ 42 3.1 Introduction ........................................................................................................................ 42 3.2 Experimental Procedures ................................................................................................... 43 3.2.1. Chemicals .................................................................................................................. 43 3.2.2. Plasmid Preparation .................................................................................................