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Structural and Biochemical Characterization of UDP-Glucose: Glycoprotein Glucosyltransferase

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Structural and Biochemical Characterization of UDP-Glucose: Glycoprotein Glucosyltransferase Structural and Biochemical Characterization of UDP- glucose: Glycoprotein Glucosyltransferase by Xuyao Li A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Biochemistry University of Toronto © Copyright by Xuyao Li 2016 Structural and Biochemical Characterization of UDP-glucose: Glycoprotein Glucosyltransferase Xuyao Li Master of Science Department of Biochemistry University of Toronto 2016 Abstract Endoplasmic reticulum quality control is a crucial process in eukaryotic cells to ensure glycoprotein folding. It utilizes N-glycan modification as an indicator of glycoprotein folding status. Monoglucosylated glycoproteins are recognized by calnexin and calreticulin that recruit ERp57 to assist folding. The glycoproteins are released from calnexin and calreticulin upon removal of the glucose residue by glucosidase II. UDP-glucose: glycoprotein glucosyltransferase (UGGT) recognizes incompletely folded glycoprotein and adds a glucose residue to the glycoprotein. Addition of the glucose residue leads to re-association of the glycoprotein with calnexin and calreticulin for further folding. In this study, we focus on characterizing UGGT and its substrate interaction. We expressed Fabs against UGGT as a tool for crystallization. The binding of Fabs to UGGT was assayed by the surface plasmon resonance technique. We also assayed UGGT activity using a radioactive UDP-Glc-based assay. The results provided a basis for understanding UGGT and its substrate interaction. ii Acknowledgments It has been a great journey since I began the master program. I would like to thank everyone that helped me in this project. I would like to express my immense gratitude to my supervisor, Dr. James M. Rini. This thesis would not have been possible without his guidance and support. His inspiration has opened my eyes to the wonders of science. I would also like to acknowledge both members of my supervisory committee, Dr. David B. Williams and Dr. Gil Privé for their precious advice and input throughout the project. I would like to thank the Department of Biochemistry and all my colleagues in the Rini lab for their help and support. They have made such a friendly and peaceful environment for research and learning. I especially thank Dr. Sachdev Sidhu for providing the phage display Fab constructs, Dr. Cordula Enenkel for instructing me in yeast culture, Jesse Hackett for providing the original UGGT construct, Malathy Satkunarajah and Dongxia Zhou for their technical support, Alan Wong and Zhijie Li for their support and comments on the thesis, and Kristina Han for supporting me and providing the Her2 antibody expressed with kifunensine. Last but not least, I would like to thank my family and friends for always being there for me. iii Table of Contents Acknowledgments .......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................ vii List of Figures .............................................................................................................................. viii List of Abbreviations ..................................................................................................................... ix Chapter 1 Introduction .................................................................................................................... 1 1.1 Protein folding and quality control in the ER ..................................................................... 1 1.1.1 N-linked glycosylation ............................................................................................ 1 1.1.2 Glucose processing ................................................................................................. 3 1.1.3 Glucosidase II ......................................................................................................... 3 1.1.4 The calnexin/calreticulin cycle ............................................................................... 4 1.1.5 UDP-glucose glycoprotein glucosyltransferase ...................................................... 7 1.2 Phage display Fabs ............................................................................................................ 15 1.3 Thesis objectives ............................................................................................................... 16 Chapter 2 Materials and Methods ................................................................................................. 17 2.1 Chemicals, plasmids and cell culture ................................................................................ 17 2.2 Plasmid constructions ....................................................................................................... 17 2.3 Mammalian expression and purification of hUGGT1 ...................................................... 18 2.4 Bacterial expression and purification of Fabs ................................................................... 20 2.5 Mammalian expression and purification of Fabs and antibodies ...................................... 22 2.6 Papain digestion of antibodies .......................................................................................... 24 2.7 Fab UGGT affinity assay .................................................................................................. 24 2.8 UGGT glucosylation assay ............................................................................................... 24 iv Chapter 3 Results .......................................................................................................................... 25 3.1 Expression, purification and crystallization of UGGT ..................................................... 25 3.1.1 Expression of UGGT ............................................................................................ 25 3.1.2 Purification of UGGT ........................................................................................... 25 3.1.3 Proteolysis of UGGT ............................................................................................ 25 3.1.4 Crystallization of UGGT ....................................................................................... 26 3.2 Preparation of Fabs as a tool for crystallization ................................................................ 28 3.2.1 Bacterial expression and purification of Fabs ....................................................... 28 3.2.2 Mammalian expression and purification of antibodies ......................................... 31 3.2.3 Papain digestion of antibodies .............................................................................. 31 3.2.4 Mammalian expression and purification of Fabs .................................................. 31 3.2.5 Fab UGGT affinity assay ...................................................................................... 37 3.2.6 Co-crystallization of UGGT with Fabs ................................................................. 37 3.3 UGGT enzyme assay ........................................................................................................ 37 3.3.1 Preparation of UGGT acceptor substrates ............................................................ 37 3.3.2 UGGT glucosylation assay ................................................................................... 43 Chapter 4 Discussion .................................................................................................................... 45 4.1 Expression, purification and crystallization of UGGT ..................................................... 45 4.1.1 Mammalian expression of UGGT ......................................................................... 45 4.1.2 Proteolysis of UGGT ............................................................................................ 45 4.1.3 Crystallization of UGGT ....................................................................................... 46 4.2 Generation of Fabs ............................................................................................................ 46 4.2.1 Bacterial expression of Fabs ................................................................................. 47 4.2.2 Mammalian expression of Fabs/antibodies ........................................................... 47 v 4.2.3 Fab UGGT affinity assay ...................................................................................... 48 4.3 Design of RiboB constructs .............................................................................................. 49 4.4 Preparing M9 glycan containing protein ........................................................................... 50 4.5 UGGT glucosylation assay ............................................................................................... 51 Chapter 5 Future Directions .......................................................................................................... 53 5.1 Fragmentation of UGGT ................................................................................................... 53 5.2 Fabs as a tool for characterization .................................................................................... 53 5.3 Preparation of
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