Design and Characterization of Galectin-3 Fusion Proteins and Novel Multivalent Galectin-3 Ligands Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Master of Science (M.Sc.) Biotechnologie Sophia Böcker aus Eisenhüttenstadt Berichter: Universitätsprofessor Dr. rer. nat. Lothar Elling Universitätsprofessor Dr. rer. nat. Wilhelm Jahnen-Dechent Tag der mündlichen Prüfung: 24.04.2018 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek verfügbar. Acknowledgment My first thanks go to Univ.-Prof. Dr. rer. nat. Lothar Elling for his supervision, for letting me work on this interesting topic and dive into the world of galectins and sugars. Thank you for being a member of the laboratory for biomaterials. I also gratefully thank Univ.-Prof. Dr. rer. nat. Wilhelm Jahnen-Dechent for kindly agreeing to be my second reviewer. Furthermore, I thank Univ.-Prof. Dr. rer. nat. Marc Spehr to be my third examiner. I enjoyed all the inspiring collaborations and want to thank: Dr. rer. nat. Sebastian Lowins, each experiment with you was a lot of fun; Anne Rix, you enabled cell experiments with my proteins, thank you especially for performing FACS-analysis with HUVECs. A special thanks to Prof. Dr. rer. nat. Andreas Walther and Prof. Dr. rer. nat. Martin Möller (DWI Leibniz Institute for Interactive Materials, Aachen) for the opportunity to perform measurements using their SPR device. Of course, I have to thank for the financial support. Thanks for being a scholarship holder of the Research Training Group “GRK 1035: Biointerface – Detection and Control of Interface- induced Biomolecular and Cellular Functions” by DFG. Other thanks go to the excellence initiative of the German federal and state governments through ERS@RWTH Aachen University. I thank all the students I supervised for their participation in my topic and for giving me the chance to practice leadership. I learned a lot of new perceptions. I want to cordially thank all present and former members of the laboratory for biomaterials, especially Anna Eisele, Bastian Lange, Dr. rer. nat. Christiane Kupper, Dennis Hirtz, Dominic Laaf, Manja Henze, Dr. rer. nat. Ruben R. Rosencrantz and Thomas Fischöder. Thank you for the nice working atmosphere. An extra thanks to Manja for not breaking-down the contact and all the motivating talks. The final thanks are the biggest thanks and go to my family and to the very best, Ruben. Without you this thesis would never have been finished. Thank you for your help, support and motivation. Publications derived from this work S. Böcker, L. Elling, Biotinylated N-Acetyllactosamine and N,N-Diacetyllactosamine based Oligosaccharides as Novel Ligands for Human Galectin-3. Bioengineering 2017, 4(2), 31. S. Böcker, L. Elling, Binding Characteristics of Galectin-3 Fusion Proteins. Glycobiology 2017, 27, 457-468. S. Böcker, D. Laaf, L. Elling, Galectin binding to neo-glycoproteins: LacDiNAc conjugated BSA as ligand for human galectin-3. Biomolecules 2015, 5, 1671-1696. A. Šimonová, C. E. Kupper, S. Böcker, A. Müller, K. Hofbauerová, H. Pelantová, L. Elling, V. Křen, P. Bojarová, Chemo-enzymatic synthesis of LacdiNAc dimers of varying length as novel galectin ligands. Journal of Molecular Catalysis B: Enzymatic 2014, 101, 47-55. C. E. Kupper, S. Böcker, H. L. Liu, C. Adamzyk, J. van de Kamp, T. Recker, B. Lethaus, W. Jahnen-Dechent, S. Neuss, G. Müller-Newen, L. Elling, Fluorescent SNAP-tag galectin fusion proteins as novel tools in glycobiology. Current Pharmaceutical Design 2013, 19, 5457-5467. Presentations S. Böcker, D. Laaf, L. Elling (2015), lecture/poster: “LacDiNAc Conjugated BSA: a Neo- glycoprotein as Multivalent and Selective Ligand for Galectin-3”, COST Action CM1102 MultiGlycoNano Spring Training School, Bangor, UK S. Böcker (2013), lecture: “Binding Studies of Truncated Galectin-3“, 24th Joint Glycobiology Meeting, Halle-Wittenberg, Germany S. Böcker, C. E. Kupper, L. Elling (2013), lecture/poster: “Fluorescent SNAP-Tag Galectin-3 Fusion Protein as a Novel Tool in Glycobiology”, 10th Carbohydrate Bioengineering Meeting, Prague, Czech Republic S. Böcker, C. E. Kupper, L. Elling (2012), poster: “Fluorescent SNAP-Tag Galectin-3 Fusion Protein as a Novel Tool in Glycobiology”, 23rd Joint Glycobiology Meeting, Wageningen, The Netherlands Table of contents Abbreviations .......................................................................................................................................... I Summary .............................................................................................................................................. III Zusammenfassung ............................................................................................................................... IV 1. Introduction ................................................................................................................................... 1 1.1 Carbohydrates – The sweet world ................................................................................................. 1 1.2 Galectins – Sweet and sour effects .............................................................................................. 10 1.3 Design of galectin-3 ligands – Turning sweet into affine ........................................................... 17 1.4 The aim ....................................................................................................................................... 26 1.4.1 Design, cloning, expression, and characterization of truncated galectin-3 ......................... 26 1.4.2 Design of novel multivalent neo-glycoproteins as promising ligands for galectin-3 .......... 26 1.5 References ................................................................................................................................... 28 2. Binding characteristics of galectin-3 fusion proteins – Influence of truncation and fusion . 39 Abstract ............................................................................................................................................... 39 2.1 Introduction ................................................................................................................................. 40 2.2 Materials and methods ................................................................................................................ 41 2.2.1 Cloning of galectin constructs ............................................................................................. 41 2.2.2 Expression and purification ................................................................................................. 42 2.2.3 SDS-PAGE and western blot .............................................................................................. 43 2.2.4 Size exclusion chromatography ........................................................................................... 43 2.2.5 Immobilization of recombinant galectin to Sepharose beads .............................................. 43 2.2.6 Self-association/crosslinking assays of galectin-3 .............................................................. 44 2.2.7 Galectin binding assay on asialofetuin ................................................................................ 45 2.2.8 Inhibition of galectin binding with (Di-)LacNAc-linker-tBoc ............................................ 45 2.2.9 Surface plasmon resonance spectroscopy ........................................................................... 45 2.2.10 Flow cytometry with human umbilical vein endothelial cells ............................................. 46 2.3 Results and discussion ................................................................................................................ 47 2.3.1 Production and characterization of full-length and truncated galectin-3 fusion constructs 47 2.3.2 Self-association/crosslinking potential of galectin-3 fusion proteins .................................. 49 2.3.3 Binding of galectin-3 fusion proteins to asialofetuin in a solid-phase assay ....................... 52 2.3.4 Surface plasmon resonance spectroscopy of galectin-3 fusion proteins on immobilized asialofetuin .......................................................................................................................... 57 2.3.5 Binding of galectins to human umbilical vein endothelial cells .......................................... 61 2.4 Conclusion .................................................................................................................................. 62 2.5 Contributions ............................................................................................................................... 63 2.6 References ................................................................................................................................... 63 3. Neo-glycoproteins as novel ligands for human galectin-3 –Albumin as carrier for multivalent presentation of non-biotinylated and 6-biotinylated tetrasaccharides to gain high- affinity ligands ..................................................................................................................................... 67 Abstract ............................................................................................................................................... 67 3.1 Introduction ................................................................................................................................