Ccl27/Ccl2 Ccl28

UNIVERSITY OF CALIFORNIA, SAN DIEGO Structural and Functional Analysis of the Chemokine CCL27 and the Expression and Purification of Silent Chemokine Receptors D6 and DARC A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry and Biochemistry by Ariane L. Jansma Committee in charge: Professor Tracy M. Handel, Chair Professor Patricia Jennings, Co-Chair Professor William Gerwick Professor Susan Taylor Asst. Professor Faik A. Tezcan 2009 This Dissertation of Ariane L. Jansma is approved, and it is acceptable in quality and form for publication on microfilm and electronically: ________________________________________________________ ________________________________________________________ ________________________________________________________ ________________________________________________________ Co-Chair ________________________________________________________ Chair iv TABLE OF CONTENTS Signature Page ................................................................................................................ iii Table of Contents............................................................................................................. iv List of Figures.................................................................................................................... x List of Tables...................................................................................................................xiii List of Abbreviations........................................................................................................xiv Acknowledgements.........................................................................................................xvi Curriculum Vitae.............................................................................................................xvii Abstract of the Dissertation ............................................................................................. xx INTRODUCTION...............................................................................................................1 CHAPTER 1......................................................................................................................7 Introduction to Chemokines ..............................................................................................7 1.1 Chemokines and Chemokine Receptors................................................................7 1.2 Chemokine Oligomerization and GAG Interaction ...............................................12 1.3 Chemokines and Disease ....................................................................................17 1.4 Biological Diversity of Chemokines ......................................................................19 1.5 Acknowledgements..............................................................................................23 CHAPTER 2....................................................................................................................24 Optimization of the Expression and Purification of CCL27 and CCL28 ..........................24 2.1 Introduction to CCL27 and CCL28 .......................................................................24 2.2 Results .................................................................................................................26 2.2.1: Protein expression and purification .............................................................26 2.2.2: Protein expression of CCL28, ubiquitin fusion ............................................26 2.2.3: Initial purification of CCL28 .........................................................................28 iv 2.2.4: Ubiquitin cleavage and HPLC purification...................................................29 2.2.5: Preparation of MOPS media .......................................................................31 2.2.6: Confirmation of CCL27 and CCL28 function...............................................33 CHAPTER 3....................................................................................................................35 NMR Analysis of the Structure, Dynamic and Unique Oligomerization Properties of the Human Chemokine CCL27 .............................................................................................35 3.1 Summary..............................................................................................................35 3.2 Introduction ..........................................................................................................37 3.3 Results .................................................................................................................41 3.3.1: CCL27 oligomerizes, forming a tetramer at high concentrations ................41 3.3.2: NMR assignment and structure determination of the monomeric form.......51 3.3.3: Characterization of internal dynamics .........................................................55 3.3.4: Attempts to characterize the CCL27 dimer interface ..................................56 3.3.5: Analysis of mutants of CCL27 by PFG diffusion NMR ................................67 3.3.6: The interaction with glycosaminoglycans induces oligomerization .............70 3.4 Discussion............................................................................................................75 3.5 Conclusions..........................................................................................................79 3.6 Materials and Methods.........................................................................................80 3.6.1: Protein expression and purification .............................................................80 3.6.2: NMR spectroscopy......................................................................................80 3.6.3: Heparin binding assay.................................................................................87 3.6.4: Solubility assays..........................................................................................88 3.6.5: Chemical cross-linking ................................................................................88 3.7 Acknowledgements..............................................................................................89 CHAPTER 4....................................................................................................................90 v Characterization of the Interaction of CCL27 with Glycosaminoglycans and with the Human CCR10 Chemokine Receptor .............................................................................90 4.1 Summary..............................................................................................................90 4.2 Introduction ..........................................................................................................92 4.3 Results .................................................................................................................96 4.3.1: Comparison of CCL27 and CCL28 .............................................................96 4.3.2: Addition of an amino-terminal Phenylalanine to CCL27..............................97 4.3.3: Amino-terminal modifications of CCL27 produce partial agonists.............100 4.3.4: The chemical nature of the N-terminus is not critical for signaling............102 4.3.5: Deletion of N-terminal residues results in antagonist................................103 4.3.6: C-terminal truncation of CCL27 results in a partial agonist.......................106 4.3.7: CCL27 interactions with Heparin tetrasaccharide .....................................109 4.3.8: Residue specific identification of the Heparin binding sites for CCL27 .....113 4.3.9: Analysis of transendothelial migration.......................................................116 4.4 Discussion..........................................................................................................118 4.5 Conclusion .........................................................................................................123 4.6 Materials and Methods.......................................................................................124 4.6.1: Expression and purification of WT and mutant CCL27 and CCL28 ..........124 4.6.2: Cell culture ................................................................................................124 4.6.3: Chemotaxis assays ...................................................................................124 4.6.4: Calcium flux and desensitization assays...................................................125 4.6.5: In vitro Heparin binding assays .................................................................125 4.6.6: NMR analysis, 15N-1H HSQC and chemical shift perturbation ..................126 4.7 Acknowledgements............................................................................................128 vi CHAPTER 5 ..................................................................................................................129 Preliminary Functional Analysis of CCL28 Suggests a Role for the C-terminal Region Independent of Leukocyte Migration .............................................................................129 5.1 Summary............................................................................................................129 5.2 Introduction ........................................................................................................130 5.3 Results ...............................................................................................................132

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