Identification, Characterization, and Utilization of Glycosyltransferases DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Nicholas Roman Pettit Graduate Program in Chemistry The Ohio State University 2011 Dissertation Committee: Dr. Peng George Wang, Advisor Dr. Zucai Suo Dr. Karin Musier-Forsyth Copyright by Nicholas Pettit 2011 3 ABSTRACT Among the many biomolecules found in nature, such as amino acids and DNA, carbohydrates are among the most important and abundant. The assembly of carbohydrates to build various oligosaccharides or glycans is of considerable interest in the glycobiology community, as understanding, controlling, and manipulating such assemblies can yield many interesting insights. This is because glycans are directly involved with many critical biological processes such as molecular recognition, cell-to- cell communication, protein folding and stability, as well as being involved many unique disease state phenotypes. The assembly of these glycans is commonly accomplished by a class of enzymes called glycosyltransferases, and it is these enzymes which have been exploited in vitro for the synthesis of attractive oligosaccharides. However, the identification, characterization, and subsequent exploitation of a glycosyltransferase is a very tedious and long process which stems from the root problem that knowing the primary function of a glycosyltransferase must be determined experimentally. This is because glycans are not the result of primary gene products, and identifying the function and specificity of a glycosyltransferase is not straight forward. In this thesis, the work has been focusing on developing a method by which function can be quickly assigned to a putative glycosyltransferase, as well as characterizing several bacterial ii glycosyltransferases, and lastly exploiting glycosyltransferase functions to perform various functions Chapter 1 provides a general introduction to the function of glycans, the function and biosynthesis of cell surface oligosaccharides, an overview of the methodologies used to determine glycosyltransferase functions, and lastly a review of chemical approaches to study and exploit oligosaccharide biosynthesis. Chapter 2 and 3 are primarily associated with characterization of bacterial glycosyltransferases and biosynthesis of O-antigen structures in two different strains of E. coli. Chapter 2 focuses on investigating the biosynthesis of the O-antigen repeating unit in E. coli O127 and E. coli O128; whereas Chapter 3 focuses on the detailed biochemical characterization of WbnI, a bacterial homologue of the important human blood group B galactosyltransferase. In Chapter 4, a high throughput technology was developed by which a putative glycosyltransferase can be quickly cloned, expressed, and relative substrate specificity determined. This work utilizes ligation independent cloning, in vitro protein expression, and SAMDI mass spectrometry along with a library of donor/acceptor substrates, by which enzymatic activity of putative glycosyltransferases is assessed. In Chapter 5, glycosyltransferases and sugar nucleotide biosynthesis are exploited both in vivo and in vitro for the synthesis of interesting and novel oligosaccharides. Finally, Chapter 6 summarizes the main results of all the studies included in this thesis, and also provides further directions for each project. iii DEDICATION Dedicated to my mother and father, Susan and Paul Pettit for all their love and support. iv ACKNOWLEDGMENTS My years at The Ohio State University have turned out to be the most intellectually challenging and important years in my life. I was challenged in ways I never anticipated by my advisor, my lab mates, my peers, and my teachers. For this, I feel that there are many people that deserve my gratitude. Let me start by first thanking my advisor, Professor Peng George Wang. His unmatched passion, love, and enthusiasm for science has been an inspiration for me, and will always be in my future endeavors. Through his constant challenges and encouragements I feel like I have matured as a scientist and as an individual. This dissertation would not have been possible without Dr. Wang‘s guidance and advice. I also need to acknowledgement my lab-mates, whom have played an integral part in my years at The Ohio State University. Firstly, I want to thank Dr. Weiqing Han for putting up with my constant bombardment of questions pertaining to my research. He was a terrific mentor for the development of my biochemical and genetic skills, and without his daily advice and conversations I would have certainly been lost. I also thank Dr. Guohui Zhao for allowing me to theorize with him about my projects, as he always made time in his day to aid me. I also owe gratitutde to several of the organic chemists in the lab for their advice, specifically TJ Styslinger and Cai Li. TJ was kind enough to v synthesize many compounds that I required for the progress of my research, as well as NMR and mass spectrometric analysis of the compounds that I synthesized. Cai Li played vital role in one of my projects, and his knowledge of synthetic chemistry and application of said chemistry was very useful in the progress of our collaboration. In addition to my current lab-mates, there are also several former lab-mates that deserve my gratitude. First and foremost, I want to thank my first mentor while in the Wang Lab, Dr. Wen Yi. He was responsible for the initial development of my biochemistry and molecular biology techniques, as well as teaching me the ins and outs of the Wang Lab. Dr. Lei Li generously taught me many useful tricks and strategies in molecular cloning, which have become essential techniques in my research. Dr. Robert Woodward has been extremely helpful in the proof reading and discussions pertaining to my publications. There are also many other lab-mates and friends from the past and present Wang Lab that I want to thank for their support and company. I want to also thank our collaborators, Dr. Milan Mrksich from The University of Chicago. His students, Lan Ban and Andreea Stuparu were helpful in obtaining and analyzing our data, as well as always being available should we have questions. I also want to thank my committee members, who generously offered their valuable time in aiding me in my dissertation and defense. Lastly, I need to thank my parents, friends, and family, whom have been critical for support and love during my time at Ohio State. I wouldn‘t be who I am today if it vi wasn‘t for these people. No words can express how thankful I am for all the support, love, and advice that these people have given me during the last four years. vii VITA November 21, 1984 ................................................. Born, Allison Park, PA 2007......................................................................... Bachelor of Science, Chemistry ................................................................................. Miami University, Oxford, OH 2007-2009 ............................................................... Graduate Teaching Assistant ................................................................................. The Ohio State University, ................................................................................. Columbus, OH 2007 – 2011............................................................. Research Assistant ................................................................................. The Ohio State University ................................................................................. Columbus, OH PUBLICATIONS Research Publications 1. Pettit, N.; Cai, L.; Han, W.; Liang, Z.; Guan, W.; Wang, P. G. American Journal of Biomedical Science 2011, 3, 107-115 2. Pettit, N.; Styslinger, T.; Mei, Z.; Han, W.; Zhao, G.; Wang, P.G. Biochemical and Biophysical Research Communications 2010, 402, 190-195 3. Han, W.; Li, L.; Pettit, N.; Yi, W.; Woodward, R.; Liu, X.; Guan, W.; Bhatt, V.; Song, J.; Wang, P.G. Methods Mol Biol. 2010, 600, 93-110 4. Liu, X.; Xia, C.; Lei, L.; Guan, W.; Pettit, N.; Zhang, H.; Chen, M.; Wang, P.G. Bioorganic & Medicinal Chemistry 2009, 17, 4910-4915 viii FIELDS OF STUDY Major Field: Chemistry ix TABLE OF CONTENTS Page Abstract ............................................................................................................. ii Dedication .........................................................................................................iv Acknowledgments............................................................................................. v Vita .................................................................................................................. viii List of Schemes ................................................................................................ xiv List of Tables ................................................................................................... xv List of Figures ................................................................................................. xvii List of Abbreviations .......................................................................................xxi Chapters 1. Introduction .......................................................................................... 1 1.1 Bacterial Cell Surface Oligosaccharides ......................................... 3 1.2 Polysaccharide Synthesis and Exploiting Nature to do Chemistry . 6 1.2.1 Traditional Chemical Synthesis