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Application of Human Glycosyltransferases in N-Glycan Synthesis and Their Substrate Specificity Studies

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Application of Human Glycosyltransferases in N-Glycan Synthesis and Their Substrate Specificity Studies Georgia State University ScholarWorks @ Georgia State University Chemistry Dissertations Department of Chemistry 12-15-2016 Application of Human Glycosyltransferases in N-glycan Synthesis and Their Substrate Specificity Studies Angie Dayan Calderon Molina Georgia State University Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss Recommended Citation Calderon Molina, Angie Dayan, "Application of Human Glycosyltransferases in N-glycan Synthesis and Their Substrate Specificity Studies." Dissertation, Georgia State University, 2016. https://scholarworks.gsu.edu/chemistry_diss/128 This Dissertation is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. APPLICATION OF HUMAN GLYCOSYLTRANSFERASES IN N-GLYCAN SYNTHESIS AND THEIR SUBSTRATE SPECIFICITY STUDIES by ANGIE DAYAN CALDERON MOLINA Under the Direction of Peng G. Wang, PhD ABSTRACT Glycoscience is important in many areas such as human health, energy and material science. Glycans have been shown to be involved in the pathophysiology of almost every major disease. Additional glycan structure knowledge is required to help advance personal medicine, and pharmaceutical developments, among others. For glycoscience to advance there is a need for large quantities of well-defined glycans and have quick access to glycosyltransferases for manipulating glycan synthesis. Herein, we will cover our efforts on studying the substrate specificities of human glycosyltransferases such as FUT8 and Gn-T V, and their application on N-glycan synthesis. Complex asymmetric N-glycan isomer structures have been related to many diseases such as breast cancer, among others. Synthesis of complex asymmetric N-glycan isomer structures including: a1,6 core-fucosylated, and tri-antennary structures can be achieved by taking advantage of the high specificity of glycosyltransferases that can work as unique catalyst to generate well-defined glycan structures. INDEX WORDS: FUT8, fucosylation, N-glycan, glycosyltransferases, Gn-T I, Gn-T II, Gn-T V. APPLICATION OF HUMAN GLYCOSYLTRANSFERASES IN N-GLYCAN SYNTHESIS AND THEIR SUBSTRATE SPECIFICITY STUDIES by ANGIE DAYAN CALDERON MOLINA A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the College of Arts and Sciences Georgia State University 2016 Copyright by Angie Dayan Calderon Molina 2016 APPLICATION OF HUMAN GLYCOSYLTRANSFERASES IN N-GLYCAN SYNTHESIS AND THEIR SUBSTRATE SPECIFICITY STUDIES by ANGIE DAYAN CALDERON MOLINA Committee Chair: Peng George Wang Committee: Binghe Wang Jenny Yang Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University December 2016 iv DEDICATION I would like to dedicate this work to my family and friends who supported me throughout my time in graduate school. I would especially like to thank my parents, Fernando Calderon and Beatriz Molina for their unconditional support, encouragement and love. I would also like to thank Xuan Wang for his patience, love, and encouragement during all of this time. Finally, I want to dedicate this dissertation to my grandparents who would have been very proud of this accomplishment Jesus Molina and Aura Rodriguez. v ACKNOWLEDGEMENTS First and foremost, I would like to sincerely thank my advisor Dr. Peng George Wang. I am especially grateful for his guidance, encouragement and support throughout all these years of hard work and struggles. I would also like to thank Dr. Binghe Wang, who has also been supportive every step of the way and has seen me grow and mature since my undergraduate years in his research lab. I would also like to thank Dr. Jenny Yang, for all her support and knowledge. I want to thank Dr. Barbara Baumstark and Dr. Al Baumstark for all of their encouragement, understanding, support all throughout my academic career at GSU. I want to especially thank my collaborator Dr. Lei Li for all his guidance and knowledge, without his support this work could not have been done. Thanks to all of the Wang group lab members especially Yunpeng, Jun, Cheng, Jingyao, Wanyi, Zhigang, Yuxi, Xu, Hailiang, Xuan, Kristina, Shanshan, and all others for their help in my research. Last but not least I want to thank the Bio- Bus program, coordinators, all the fellows and volunteers who have encourage me and helped me become a better teacher. 1 TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................. v LIST OF TABLES ............................................................................................................ 5 LIST OF FIGURES .......................................................................................................... 6 1 INTRODUCTION .................................................................................................... 11 1.1 Purpose ............................................................................................................... 11 1.2 Introduction ....................................................................................................... 12 1.3 Glycosylation importance ................................................................................. 14 1.4 N-Glycosylation site ........................................................................................... 14 1.5 N-Glycosylation difference ............................................................................... 15 1.6 Protein glycosylation pathway ......................................................................... 16 1.7 Therapeutic N-linked glycoproteins ................................................................ 18 1.8 References: ......................................................................................................... 19 2 SUBSTRATE SPECIFICITY OF FUT8 AND CHEMOENZYMATIC SYNTHESIS OF CORE-FUCOSYLATED ASYMMETRIC N-GLYCANS ....................... 21 2.1 Abstract .............................................................................................................. 21 2.2 Introduction ....................................................................................................... 21 2.3 Results and Discussion ...................................................................................... 24 2.4 Conclusion .......................................................................................................... 30 2.5 Acknowledgements ............................................................................................ 30 2 2.6 Experimental ...................................................................................................... 31 2.6.1 Materials and enzymes ................................................................................. 31 2.6.2 Expression and purification of human FUT8 ............................................. 31 2.6.3 Substrate specificity study of FUT8 ............................................................. 33 2.6.4 General methods for glycan synthesis and purification .............................. 36 2.6.5 HPLC profiles and MS data of purified N-glycans ..................................... 37 2.6.6 ESI-MS and 1H NMR data of purified N604 .............................................. 53 2.6.7 NMR spectra and data of purified core-fucosylated N-glycans .................. 54 2.7 References .......................................................................................................... 60 3 FUT8: FROM BIOCHEMISTRY TO SYNTHESIS OF CORE- FUCOSYLATED N-GLYCANS. .............................................................................................. 62 3.1 Abstract .............................................................................................................. 62 3.2 Introduction ....................................................................................................... 63 3.3 FUT8 specificity studies .................................................................................... 65 3.4 Traditional versus chemo-enzymatic synthesis of core fucosylated N- glycans. 67 3.5 Conclusion .......................................................................................................... 74 3.6 Acknowledgements ............................................................................................ 75 3.7 References .......................................................................................................... 75 3 4 ENZYMATIC SEPARATION OF ASYMMETRIC BI- AND TRI- ANTENNARY N-GLYCAN ISOMERS USING FUT8 AND GN-TV ................................... 78 4.1 Abstract .............................................................................................................. 78 4.2 Introduction ....................................................................................................... 78 4.3 Results and Discussion ...................................................................................... 81 4.3.1 Generation of asymmetric biantennary and triantennary N-glycans ......... 82 4.3.2 Substrate Specificity for human Gn-T V ..................................................... 83 4.3.3 Enzymatic separation of N-glycan isomers ................................................. 84 4.4 Conclusion .........................................................................................................
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