Chemokines, Part B (eBook)

Front Cover 1
Methods in Enzymology: Chemokines, Part B 4
Copyright Page 5
Contents 6
Contributors 12
Preface 16
Methods in Enzymology 18
Chapter 1: Chapter One Isolation, Identification, and Production of Posttranslationally Modified Chemokines 48
1. Introduction 49
2. Isolation and Stimulation of Peripheral Blood Mononuclear Cells (PBMC) 50
3. Concentration of Isolated Proteins 51
4. Affinity Chromatography 52
5. Specific Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) 52
6. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 53
7. Ion Exchange Chromatography 54
8. Reverse-Phase High-Pressure Liquid Chromatography (RP-HPLC) 55
9. Ion Trap Mass Spectrometry 55
10. Edman Degradation 56
11. Total Protein Quantification Methods 57
12. Illustration: Isolation and Identification of Natural Posttranslationally Modified CXCL8 Isoforms 58
13. Solid-Phase Peptide Synthesis 59
14. Citrullination of Chemokines 66
15. Identification of Enzymes Generating the Natural Posttranslationally Modified Chemokines, as Exemplified by Aminopeptidase N(A 68
16. Comparison of the Heparin-Binding Properties of Chemokine Isoforms 69
Acknowledgments 70
References 70
Chapter 2: Chapter Two Homo- and Hetero-Oligomerization of Chemokines 76
1. Introduction 77
2. Methods to Detect and Quantify Oligomerization 80
3. Methods for Collecting Residue-Specific Information on Chemokine Oligomers 88
4. Conclusions 92
Acknowledgments 92
References 92
Chapter 3: Chapter Three Lymphotactin Structural Dynamics 96
1. Introduction 97
2. Production of Biologically Active Lymphotactin 99
3. Ltn Folds into Two Unrelated Native State Structures 101
4. Kinetics of Interconversion in the Ltn Conformational Equilibrium 102
5. Engineering Conformationally Restricted Lymphotactin Variants 105
6. Functional Analysis of Distinct Ltn Native State Conformations 106
7. GAG Binding Residues are Linked to the Ltn Conformational Equilibrium 110
8. Conclusions 112
References 113
Chapter 4: Chapter Four Interactions of Chemokines with Glycosaminoglycans 116
1. Introduction 117
2. Methods to Detect, Quantify, and Characterize Chemokine: Gag Interactions 125
3. Biophysical Methods 132
4. Summary 142
Acknowledgments 143
References 143
Chapter 5: Chapter Five Multiple Approaches to the Study of Chemokine Receptor Homo- and Heterodimerization 150
1. Introduction 151
2. Biochemical Techniques to Measure Chemokine Receptor Oligomerization 152
3. Resonance Energy Transfer (RET) Techniques 156
4. Sequential BRET-FRET (SRET) Technology 163
5. Conclusion 164
Acknowledgments 164
References 165
Chapter 6: Chapter Six Plasmon Resonance Methods in Membrane Protein Biology: Applications to GPCR Signaling 168
1. Introduction 169
2. Plasmon Spectroscopy 170
3. Sensor Construction and Sample Deposition 174
4. Lipid Bilayer Deposition 176
5. Spectral Data Analysis 177
6. Membrane Protein Insertion 181
7. Ligand and G-Protein Binding by GPCRs 183
8. Conclusions 186
. Conflicts of Interests 189
Acknowledgments 189
References 189
Chapter 7: Chapter 7 Tyrosine Sulfation of HIV-1 Coreceptors and Other Chemokine Receptors 192
1. Introduction 193
2. Production and Use of Tyrosine-Sulfated Peptides Derived from Chemokine Receptors 200
3. Study of Chemokine-Receptor Sulfation on the Plasma Membrane 205
4. Bacterial Expression of Tyrosine-Sulfated Peptides and Proteins 206
5. Protocols 208
6. Conclusions 212
References 213
Chapter 8: Chapter 8 Activation Mechanisms of Chemokine Receptors 216
1. Introduction 217
2. Current Models for 7TM Receptor Activation 220
3. Constitutive Activity of 7TM Receptors 221
4. Activation of Chemokine Receptors by Small-Molecule Agonists 222
5. Experimental Procedures 228
6. IP3 Assay in Transiently Transfected COS-7 Cells 230
References 231
Chapter 9: Chapter 9 The Duffy Antigen Receptor for Chemokines 236
1. Introduction 237
2. Methods for the Study of DARC as a Malarial Receptor 239
3. Methods for the Study of DARC as a Chemokine Sink 243
4. Isolation of Erythrocytes and Measurement of Chemokines 244
5. DARC as a Chemokine Transcytosis Receptor 245
6. The Assay of Chemokine Transcytosis by DARC 246
7. Conclusions 247
References 248
Chapter 10: Chapter Ten Hetero-Oligomerization of Chemokine Receptors 252
1. Introduction 252
2. Coimmunoprecipitation 253
3. Resonance Energy Transfer Techniques 257
4. Developing Techniques 266
References 267
Chapter 11: Chapter 11 Subsecond Analyses of G-Protein Coupled-Receptor 272
1. Introduction 273
2. Analysis of GPCR Function by Flow Cytometry 274
3. Small-Volume Rapid Mix Device Flow Cytometry 276
4. General Listing of Materials 277
5. Optimizing For Analysis of Molecular Assemblies by Flow Cytometry: Receptor Affinity 279
6. Modular Molecular Assemblies of GPCR Ternary Complexes on Beads 279
7. Preparation of G-Protein Coated Beads (Ga.betagamma Beads) 281
8. Analysis of Modular Dissassembly of LRG Modules 284
9. Summary and Outlook 290
Acknowledgments 290
References 290
Chapter 12: Chapter 12 The Use of Receptor Homology Modeling to Facilitate the Design of Selective Chemokine Receptor Antagonists 294
1. Introduction 295
2. Receptor Expansion of Rhodopsin Models as an Initial Approach 305
3. The Use of beta2-Adrenergic Receptor Structure as an Alternative Template 312
4. Conclusions 319
References 321
Chapter 13: Chapter 13 Characterizing Proteolytic Processing of Chemokines by Mass Spectrometry, Biochemistry, Neo-Epitope Antibodies and Functional Assays 326
1. Introduction 327
2. In vitro Processing and Characterization of Proteolysis 328
3. In Vivo Functional Characterization of Proteolysis 348
4. Summary 349
Acknowledgments 349
References 350
Chapter 14: Real-Time in Vitro Assays for Studying the Role of Chemokines in Lymphocyte 356
1. Introduction 357
2. Methods for Investigation of Lymphocyte Crawling and Transendothelial Migration (TEM) Under Shear Flow 359
Acknowledgments 375
References 375
Chapter 15: A Microfluidics-Based Method for Analyzing Leukocyte Migration to Chemoattractant Gradients 378
1. Introduction 379
2. Preparation of Microfluidic Devices 381
3. Generation of Chemoattractant Gradients 383
4. Preparation of Cells 384
5. Experimental Setup 385
6. Data Analysis 388
7. Conclusion 390
Acknowledgments 391
References 391
Chapter 16: Two-Photon Microscopy and Multidimensional Analysis of Cell Dynamics 394
1. Introduction 395
2. 2P Microscope Systems 397
3. Fluorescent Reporters 403
4. Imaging Preparations 405
5. Image Acquisition 410
6. Multidimensional Analysis 413
7. Presentation of 2P Microscopy Images 418
References 420
Chapter 17: Zymosan-Induced Peritonitis as a Simple Experimental System for the Study of Inflammation 424
1. Introduction 425
2. Materials 429
3. Methods 430
4. Expected Results 433
5. Other Considerations 437
Acknowledgments 439
References 439
Chapter 18: A Chemokine-Mediated in vivo T-cell Recruitment Assay 442
1. Introduction 443
2. In Vitro Activation of T Lymphocytes 444
3. In Vivo Chemokine-Mediated Recruitment 449
4. Use of In Vivo Recruitment Assay for Chemokine Studies 452
5. Conclusion 456
Acknowledgments 456
References 457
Author Index 458
Subject Index 478
Color Plates 486