Mapping the Conformational Dynamics of E-selectin upon Interaction with its Ligands Thesis by Fajr Aleisa In Partial Fulfillment of the Requirements For the Degree of Master of Science King Abdullah University of Science and Technology Thuwal, Kingdom of Saudi Arabia 05/15/2013 2 EXAMINATION COMMITTEE APPROVALS FORM Committee Chairperson: Prof. Jasmeen Merzeban Committee Member: Prof. Christopher Gehring Committee Member: Dr. Samah Zeineb Gadhoum 3 © 05/15/2013 Fajr Aleisa All Rights Reserved 4 ABSTRACT Mapping the Conformational Dynamics of E-selectin upon Interaction with its Ligands Fajr Aleisa Selectins are key adhesion molecules responsible for initiating a multistep process that leads a cell out of the blood circulation and into a tissue or organ. The adhesion of cells (expressing ligands) to the endothelium (expressing the selectin i.e., E- selectin) occurs through spatio-temporally regulated interactions that are mediated by multiple intra- and inter-cellular components. The mechanism of cell adhesion is investigated primarily using ensemble-based experiments, which provides indirect information about how individual molecules work in such a complex system. Recent developments in single-molecule (SM) fluorescence detection allow for the visualization of individual molecules with an good spatio-temporal resolution (nanometer spatial resolution and millisecond time resolution). Furthermore, advanced SM fluorescence techniques such as Förster Resonance Energy Transfer (FRET) and super-resolution microscopy provide unique opportunities to obtain information about nanometer-scale conformational dynamics of proteins as well as nano-scale architectures of biological samples. Therefore, the state-of-the-art SM techniques are powerful tools for investigating complex biological system such as the mechanism of cell adhesion. In this project, several constructs of fluorescently labeled E-selectin will be used to study the conformational dynamics of E-selectin binding to its ligand(s) using SM-FRET and combination of SM-FRET and force microscopy. These studies will be beneficial to fully understand the mechanistic details of cell adhesion and migration of cells using the established model system of hematopoietic 5 stem cells (HSCs) adhesion to the selectin expressing endothelial cells (such as the E- selectin expressing endothelial cells in the bone marrow). 6 ACKNOWLEDGEMENTS I would like to sincerely thank my supervisor professor Jasmeen Merzaban for giving me the chance to be part of her marvelous scientific journey, for her continuous motivation, support, and guidance and for sharing her great knowledge. Also, my sincere gratitude to Dr. Kosuke Sakashita, my teacher, for his valuable knowledge, for teaching me, hearing my ideas, for his patience and for helping me to become more confident and express my thoughts and, to professor Samir Hamdan for his support, continuous help, valuable advices during this project and for allowing me to use equipment and materials from his lab generously. I would like to extend my appreciation to professor Satoshi Habuchi, Dr. Zeineb Gadhoum and professor Chrisopher Gehring for their guidance and support. My great appreciation goes to my all my lab mates and lab managers from Cell Migration and Signaling lab and DNA Replication lab for their continuous support, valuable discussions and good times. Thank you to my best friends for believing me, and supporting me, you are the best. Most importantly, my heartfelt love and gratitude goes to my parents: AbdulRahmaan and Noura for simply everything that can or cannot be listed, for being the two most amazing people in my life, to my life friends, siblings: Basma, Taghreed, Rasha, Ibrahim, Mohammad, Hanouf and Khalid for being the best at all in everything all the time, and to the two people that are always sweet in my life, my niece and nephew, my best friends: Sara and AbdulRahmaan and above all, I thank Allah. Lastly, thank you to everyone who supported me during my education and to anyone missed from this acknowledgment. 7 TABLE OF CONTENTS EXAMINATION COMMITTEE APPROVALS FORM 2 COPYRIGHT PAGE 3 ABSTRACT 4 ACKNOWLEDGEMENTS 6 TABLE OF CONTENTS 7 LIST OF ABBREVIATIONS 9 LIST OF SYMBOLS 14 LIST OF ILLUSTRATIONS 15 LIST OF TABLES 17 1. INTRODUCTION 18 1.1 Multi-step Paradigm of Cellular Recruitment 18 1.2 Selectins, their ligands, and their role in controlling cell migration 22 1.3 Tools to study biophysical properties of Adhesion molecules 28 2. MATERIALS AND METHODS 35 2.1 PCR reactions 35 2.2 DNA gel electrophoresis analysis and gel extraction 42 2.3 Ligation dependent cloning and Bacterial transformation 44 2.4 Virus preparation and Sf9 cells infection 50 2.5 CHO-K1 cells transfection 52 2.6 FACS analysis 54 8 2.7 Patch purification of the mE-Bio2 and mE-Bio3 recombinant proteins in 54 CHO-K1 2.8 Western blot analysis 55 3. RESULTS 58 3.1 Expression of recombinant mE-IgG protein in Rosetta gami bacterial cells 59 3.2 PCR reactions to generate various E-selectin chimeras 60 3.3 Preparation of the Insect and Mammalian cell Expression vectors 64 3.4 Bacmids preparation to express E-selectin constructs in Insect cells 68 3.5 Expression of mE-Bio3 and mE-Bio2 recombinant proteins in CHO-K1 75 mammalian cells 4. DISCUSSION 82 BIBLIOGRAPHY 85 APPENDICES 90 9 LIST OF ABBREVIATIONS BM Bone marrow bp Base pair CBD Complement binding domain CD24 Cluster of differentiation 24 CD34 Cluster of differentiation 34 CD62E Cluster of differentiation 62 Endothelia CHO-K1 Chinese hamster ovarian-K1 cells CR Consensus repeats Cys Cysteine D-MEM Dulbecco's modified eagle medium DNA Deoxyribonucleic acid dNTPs deoxyribonucleotide DTT Dithiothreitol E-selectin Endothelial selectin E.coli Escherichia coli EDTA Ethylenediaminetetraacetic acid EGF Endothelium growth factor ESL-1 E-selectin ligand-1 FBS Fetal bovine serum FL Full length 10 FRET Fluorescent Resonance Energy Transfer GlyCAM-1 Glycosylation-dependent cell adhesion molecule-1 GmR Gentamycin Resistance goi Gene of interest gp200 200 kDa glycoprotein GT Glycosyltransferase HEV High endothelial venules HPC Hematopoietic progenitor cells HRP Horse radish peroxidase HSC Hematopoietic stem cells IL-1 Interleukin 1 family IPTG Isopropyl β-D-1-thiogalactopyranoside kb Kilo base kDa Kilo Dalton kV Kilovolt L-selectin Lymphocytes selectin LDS Lithium dodecyl sulfate LEC-CAMS Lectin cell adhesion molecules LFA-1 Lymphocyte function-associated antigen 1 LMP Low melting point LPS Lipopolysaccharides M Molar 11 MAd-CAM-1 Mucosal vascular addressin cell adhesion molecule 1 mL Milliliter mM Millimolar MOI Multiplicity of infection ms Millisecond NEAA Non-essential amino acids nm Nanometer P-selectin Platelets selectin PLN Peripheral lymph nodes PNAd Peripheral node addressin PBS Phosphate buffered saline PBST Phosphate-buffered saline-Tween-20 PCR Polymerase chain reaction PEI Polyethyleneimine pfu Plaque-forming unit Pfu Pyrococcus furiosus PMSF Phenylmethylsulfonyl fluoride ProK Proteinase K PSGL-1 P-selectin glycoprotein ligand-1 PVDF Polyvinylidene fluoride r-mE-sel Recombinant mouse E-selectin RA Rheumatoid arthritis 12 rcf Relative centrifugal force RE Restriction enzyme RO Reverse osmosis rpm Revolution per minute RT Room temperature SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis Sf9 Spodoptera frugiperda-derived cell line SFM Serum free media SOC Super Optimal Broth with glucose SOP Splicing by overlapping PCR SP Secretion signal peptide TB Tris-Glycine buffer TBST Tris-buffered saline-Tween-20 TE Tris-EDTA TNF Tumor Necrosis Factor UV Ultraviolet X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside β-ME β-mercaptoethanol µg Microgram µL Microliter µm Micrometer 13 LIST OF SYMBOLS °C Degree Celsius Ca+2 Calcium De novo Process that starts from the beginning x g Gravity-related acceleration H2O Water in vitro In glass in vivo In life koff Dissociation rate NaCl Sodium chloride Ni Nickel 14 LIST OF ILLUSTRATIONS Figure 1.1: The multi-step paradigm of cell migration and homing 19 Figure 1.2: Selective homing of lymphocytes to PLN 20 Figure 1.3: Selectins domain structure 23 Figure 1.4: Crystal structure of E-selectin binding domain (lectin) in complex 25 with sLeX Figure 1.5: The bent and extended conformations of Selectins 30 Figure 1.6: Fluorescent labeling of selectin at two locations on the protein for 32 unraveling selectin dynamic behavior by FRET Figure 2.1: mE-IgG-Bio1 construction 38 Figure 2.2: mE-Bio2 construction 39 Figure 2.3: mE- Bio3 construction 41 Figure 2.4: LR reaction 46 Figure 2.5: Inoculation of candidate colonies and expansion of culture 49 Figure 3.1: Positions of disulfide bonds in lectin/EGF domains 58 Figure 3.2: Western blot of the full length (FL) r-mE-sel protein 59 Figure 3.3: E-selectin constructs 61 Figure 3.4: PCR construction of recombinant mE-selectin (r-mE-sel) 63 Figure 3.5: Genetic map of pDEST8 and pEF-DEST51 destination vectors 65 Figure 3.6: Colony PCR products for recombinant pDEST8 and pEF-DEST51 66 Figure 3.7: PCR products for recombinant pDEST8 and pEF-DEST51 67 Figure 3.8: DH10Bac transposition in the baculovirus bacmid, bMON14272 69 15 Figure 3.9: Colony PCR products for recombinant bacmids from DH10Bac 69 Figure 3.10: Colony PCR products for recombinant bacmids from Genehogs 70 Figure 3.11: Microscope images reveal that Sf9 insect cells were successfully 71 infected with pDEST8-mE-Bio2 and pDEST8-mE-Bio3 plasmids Figure 3.12: Flow Cytometric analysis of Sf9 cells 5 days post-infection with 73 E-selectin constructs Figure 3.13: Flow diagram outlining