Rui Li Phd Thesis for Hard Bound
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Glycation of Extracellular Matrix: A Solid-State NMR Study Rui Li Robinson College and Department of Chemistry University of Cambridge September 2019 This thesis is submitted for the degree of Doctor of Philosophy. Declaration This thesis is the result of my own work and includes nothing which is the outcome of work done in collaboration except as specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution. It does not exceed the prescribed word limit for the Degree Committee. Rui Li September 2019 Glycation of Extracellular Matrix: A Solid-State NMR Study Rui Li The main aim of this thesis is to investigate the glycation products, especially glycation intermediate products, generated in the glycation reaction model in vitro extracellular matrix (ECM) and ribose-5-phosphate using solid-state nuclear magnetic resonance (SSNMR) spectroscopy. The background and motivations of this project are outlined in chapter 1. Chapter 2 reviews the current understanding of the composition and organisation of the ECM, the structure of collagen and the critical roles collagen plays in the ECM. Chapter 3 introduces the glycation reaction and summarises the glycation products identified to date, with a discussion of the most widely-used characterisation techniques and in vitro model systems for studying the glycation reaction and glycation products. Chapter 4 covers the basic theory of SSNMR spectroscopy, which is the major analytical technique used in this thesis, and the principles behind the SSNMR experiments used in this project. Above three chapters provide the essential background knowledge for studying the glycation reaction chemistry and the glycation products in in vitro collagenous ECM model using SSNMR spectroscopy. Protocols for preparing, glycating and characterising in vitro ECM samples as well as all relevant experimental details are included in chapter 5, which is followed by a discussion in chapter 6 on the development of a method to extract collagen from in vitro ECM. In chapters 7 and 8, glycation products identified in different in vitro glycation models using SSNMR are outlined, where chapter 7 focuses on glycation products derived from the glycation reaction between non-isotope-enriched collagenous materials and 13C-enriched glycating agents, while chapter 8 concentrates on the effects of glycation on lysine and arginine residues in collagenous samples using 13C, 15N-enriched collagenous materials and 13C-enriched glycating agents. Chapter 9 discusses the development of a protocol to extract cellular metabolites, specifically carbonyl metabolites, and estimate their glycation potential, in order to understand the contribution that cell necrosis may have on glycation in patients. Conclusion and future work are outlined in chapter 10. This page is dedicated to my parents for their unreserved and unconditional love and support. “父兮生我,母兮鞠我。 拊我畜我,长我育我。 我复我,出入腹我。” Acknowledgements Nothing can really be accomplished solely on one’s own – my PhD work is no exception. So many people have helped me in different ways during this difficult yet rewarding journey. Foremost, I would like to thank all the members of the Duer group, past and present: Dave, Ying, Veronica, Yang, Rakesh, Ieva, Ulya, Anna, Adrian and Thomas for their training, support, generous assistance and good company throughout my time in Cambridge. It would not be possible to forget all the laughter we had in our cosy office 251, all the botanical garden trips we wandered, all the pastries and bread we shared from that lovely French bakery and homemade goodies from Adrian and Thomas (we are so well fed!). You guys are such lovely PhD buddies! Particularly, I would like to thank Prof. Melinda Duer for giving me the opportunity to join the group, for being such a supportive and enthusiastic supervisor during my time here in Cambridge and for giving me the encouragement and freedom to pursue my ideas. I could never ask for more than what I have got from a PhD supervisor. Special thanks go to Dr. Wing Ying Chow for her guidance through the exploration of the exciting DNP- SSNMR world, for answering all my stupid questions patiently and for recommending places to go and restaurants to try during my visit in Berlin. Heartfelt thanks go to Dr. Peter Grice, Mr. Duncan Howe, Mr Andrew Mason for training me on solution-state NMR spectrometers and running a smooth NMR service in the department. Thank Dr. Karin Muller for all her help with electron microscopy and sharing her wisdom during our sessions. I will miss your cooking very much! I would like to acknowledge all the funding bodies for their financial support throughout my PhD: Cambridge Trust and China Scholarship Council for my PhD scholarship, without which I could never have the opportunity to pursue a PhD in Cambridge; iNEXT for financing the trips to Berlin and the access to DNP-SSNMR and 700 MHz SSNMR spectrometers; Robinson College and the Department of Chemistry for their financial contribution towards my academic conference expenditures. Last but definitely not the least, I thank all my family and friends near or far for loving me, supporting me and keeping me company at my best and my worst, without whom I could never have made it this far. Especially Jo Xinyu, you have lit up my life in Cambridge, and I feel tremendously lucky to have you. List of Abbreviations Amino Acid 3-letter 1-letter Arginine Arg R Cysteine Cys C Glycine Gly G Histidine His H Hydroxylysine Hyl - Hydroxyproline Hyp O Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Tryptophan Trp W Tyrosine Tyr Y Acronym Definition 3-DG 3-Deoxyglucosone 3DG-H 3-Deoxyglucosone Hydroimidazolone AAA Amino Acid Analysis AEC 3-Amino-9-Ethylcarbazole AGEs Advanced Glycation Endproducts BSA Bovine Serum Albumin C7 Sevenfold Symmetric Phase Shift Scheme CSA Chemical Shift Anisotropy CEL Ne-(carboxylethyl)lysine CL Cell Lysate CMA N-(carboxymethyl)arginine CMC S-(carboxylmethyl)cysteine CMhL Ne-(carboxylmethyl)hydroxylysine CML Ne-(carboxylmethyl)lysine COL Collagenous CP Cross Polarisation DARR Dipolar-Assisted Rotational Resonance DCP Double Cross Polarisation DMEM Dulbecco’s Modified Eagle Medium DNA Deoxyribose Nucleic Acid ECM Extracellular Matrix ELN Elastin F6P Fructose-6-Phosphate FN Fibronectin Fru Fructose FSOBs Foetal Sheep Osteoblasts FTL Freeze-Thaw Lysis Fuc Fucose G6P Glucose-6-Phosphate GA Glyceraldehyde GAG Glycosaminoglycan G-H Glyoxal hydroimidazolone Glc Glucose GOLD Glyoxal Lysine Dimer HETCOR Heteronuclear Correlation HSA Human Serum Albumin LNL Liquid Nitrogen Lysis KSPG Keratan Sulfate Proteoglycan MAS Magic Angle Spinning MG-H MethylGlyoxal-derived 5-Hydro-5-imidazolone MODIC MethylglOxal Dimer Imidazole Crosslink MOLD MethylglOxal Lysine Dimer MS Mass Spectroscopy MW Molecular Weight NC Non-Collagenous NMR Nuclear Magnetic Resonance ns Number of Scans PC7 See POST-C7 PDSD Proton-Driven Spin Diffusion PG Proteoglycan PLL Poly-L-Lysine POST-C7 Permutationally Offset Stabilised C7 PTMs Post-Translational Modifications R5P Ribose-5-Phosphate Rib Ribose S/N Signal to Noise SEM Scanning Electron Microscopy SPC-5 Supercycled POST-C5 SQ-DQ Single Quantum-Double Quantum SSNMR Solid-State Nuclear Magnetic Resonance TEDOR Transferred Echo Double Resonance TEM Transmission Electron Microscopy THP Tetrahydropyrimidine VSMCs Vascular Smooth Muscle Cells Contents 1 INTRODUCTION .......................................................................................................................... 1 2 COLLAGEN AND THE EXTRACELLULAR MATRIX (ECM) ............................................ 3 2.1 COLLAGEN .................................................................................................................................... 4 2.1.1 THE COLLAGEN FAMILY ............................................................................................................. 4 2.1.2 COLLAGEN MOLECULES ............................................................................................................. 6 2.1.3 COLLAGEN FIBRILS AND HIGHER-ORDER STRUCTURES IN THE ECM ....................................... 9 2.1.4 ROLES OF COLLAGEN ................................................................................................................ 11 2.2 THE ECM, COLLAGEN AND DISEASES ...................................................................................... 11 2.3 NON-COLLAGENOUS ECM PROTEINS ...................................................................................... 12 2.4 SUMMARY ................................................................................................................................... 15 3 THE GLYCATION REACTION ................................................................................................ 16 3.1 GLYCATION AND ADVANCED GLYCATION ENDPRODUCTS (AGES) ....................................... 16 3.2 CONSEQUENCES OF GLYCATION ............................................................................................... 19 3.2.1 CONSEQUENCES OF COLLAGEN GLYCATION ...........................................................................