UDP-glucose: glycoprotein glucosyltransferase 1 (UGGT1) substrate characterization by Nathan Masashi Doner A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto © Copyright by Nathan Masashi Doner 2018 UDP-glucose: glycoprotein glucosyltransferase 1 (UGGT1) substrate characterization Nathan Doner Master of Science Department of Molecular Genetics University of Toronto 2018 Abstract UDP-glucose: glycoprotein glucosyltransferase 1 (UGGT1) plays a quality control role in the ER by recognizing misfolded glycoproteins and glucosylating their Man9 N-glycans. To study the substrate specificity of UGGT1 and to produce an acceptor substrate for co-crystallization with UGGT1, I have purified Man9-glycosylated forms of bovine RiboB, RiboS, and S-protein. I have also produced variants of these proteins with the single N-glycan at Asn34 re-located to other sites. Using circular dichroism, I have established that RiboB is more thermostable than S-protein and RiboS. Accordingly, S-protein and RiboS are readily glucosylated and bound by human UGGT1. N34Q/Y76N The Man9 form of the S-protein variant bound to UGGT1 with a KD of 76 μM. This substrate is a candidate for co-crystallization with UGGT1. The methods described here can also be used to study other UGGT1 substrates and will facilitate work aimed at understanding how UGGT1 recognizes its misfolded glycoprotein substrates. ii Acknowledgements I would like to thank my supervisor, Dr. James Rini, for being an integral part of my learning process throughout my research. The countless discussions that we had about science, both pertaining to my research, and about other unanswered biological questions, were always stimulating and have contributed to my growth as a scientist. I would also like to thank Dr. Zhijie Li, who I view as an important technical mentor. He has helped me learn to be critical of methodology and how to be validate and be confident with the results of my own assays. Discussions with him have also stimulated my interest in many fields, both biological and non-biological. Additionally, the members of the Rini lab (especially Dr. Alan Wong, Aidan Tomlinson, Kristina Han, Dongxia Zhou, and Malathy Satkunarajah) and the members of the MaRS 16th floor have been very helpful and fun along the way and contributed positively to my experiences at the University of Toronto. Of course, I would like to thank my family and friends for being there for me outside of the lab, particularly my mother and Aislinn Sandre. iii Table of Contents Acknowledgements ........................................................................................................................ iii Table of Contents ........................................................................................................................... iv List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix List of Abbreviations ..................................................................................................................... xi Introduction .................................................................................................................................1 1.1 The secretory pathway .........................................................................................................1 1.1.1 Signal peptide...........................................................................................................1 1.1.2 N-linked glycan biosynthesis and transfer ...............................................................1 1.1.3 Calnexin cycle ..........................................................................................................4 1.1.4 Golgi apparatus ........................................................................................................5 1.1.5 ER-associated degradation (ERAD) ........................................................................6 1.2 UDP-glucose: glycoprotein glucosyltransferase (UGGT) ...................................................7 1.2.1 UGGT discovery ......................................................................................................7 1.2.2 UGGT structure .......................................................................................................8 1.2.3 UGGT in vitro glucosylation .................................................................................11 1.2.4 UGGT2 ..................................................................................................................14 1.2.5 UGGT in vivo glucosylation ..................................................................................14 1.2.6 Binding partner Sep15 (SELENOF) ......................................................................16 1.3 Rationale of the thesis ........................................................................................................16 Materials and Methods ..............................................................................................................17 2.1 Vector construction ............................................................................................................17 2.2 Cell culture .........................................................................................................................18 iv 2.2.1 Transfection ...........................................................................................................18 2.2.2 Protein production ..................................................................................................19 2.3 Protein purification and chromatography ..........................................................................19 2.3.1 UGGT1 ..................................................................................................................19 2.3.2 RiboB .....................................................................................................................19 2.4 RiboS and S-protein preparation and chromatography ......................................................20 2.5 Ribonuclease activity assay ...............................................................................................20 2.6 UGGT1 MALDI-TOF MS-based activity assay................................................................21 2.7 Circular Dichroism.............................................................................................................22 2.8 BIAcore surface plasmon resonance ..................................................................................22 Results .......................................................................................................................................23 3.1 Protein Expression .............................................................................................................23 3.1.1 UGGT1 expression ................................................................................................23 3.1.2 RiboB vectors and expression ................................................................................23 3.2 Protein Purification ............................................................................................................25 3.2.1 UGGT1 purification ...............................................................................................25 3.2.2 RiboB, RiboS and S-protein purification ...............................................................28 3.3 Substrate properties ............................................................................................................31 3.3.1 Circular Dichroism.................................................................................................31 3.3.2 Ribonuclease activity .............................................................................................34 3.4 UGGT1 activity .................................................................................................................35 3.4.1 MALDI-TOF-based activity assay ........................................................................35 3.4.2 Alternative activity assays .....................................................................................38 3.4.3 Effect of temperature .............................................................................................40 3.4.4 S-protein glycosylation mutant forms ....................................................................40 3.4.5 Estimation of KM and kcat .......................................................................................41 v 3.4.6 Effect of metal ions ................................................................................................45 3.5 UGGT1 substrate binding ..................................................................................................45 3.5.1 RiboB, RiboS, and S-protein .................................................................................45 3.5.2 KD estimation .........................................................................................................46 3.5.3 Glycan involvement in binding ..............................................................................48 Discussion and Conclusions ......................................................................................................50