Glycobiology (eBook)

Front Cover 1
Methods in Enzymology: Glycobiology 4
Copyright Page 5
Contents 6
Contributors 16
Preface 24
Methods in Enzymology 26
Section One: Proteoglycans and Sulfotransferases 54
Chapter One: Auxiliary and Autonomous Proteoglycan Signaling Networks 56
Abstract 56
1. Overview of Proteoglycan Structure, Nomenclature, and Function 57
2. Growth Factor Signaling 58
3. Integrin Interactions 59
4. Autonomous Signaling 59
5. Proteoglycan Downregulation: Endocytosis and Ectodomain Shedding 62
6. Cell Adhesion 63
7. Migration 67
8. Experimental Procedures 70
Acknowledgments 81
References 82
Chapter Two: Dual Roles of Drosophila Glypican Dally-Like in Wingless/Wnt Signaling and Distribution 86
Abstract 87
1. Introduction 87
2. Generation of Dally and Dlp Null Alleles 88
3. Examination of Dally and Dlp in Wg Signaling and Gradient Formation in the Wing Disc 89
4. Dlp Core Protein Determines Its Biphasic Activity in Wg Morphogen Signaling 92
5. Dlp Core Protein Can Interact with Wg Independent of Its GAG Chains 95
6. The Ratio of Dlp/Fz2 Determines the Biphasic Activity of Dlp or Dlp Core Protein in Wg Signaling 99
7. Conclusion 101
Acknowledgments 101
References 102
Chapter Three: Use of a Phage Display Antibody to Measure the Enzymatic Activity of the Sulfs 104
Abstract 104
1. Overview 105
2. ELISA for Sulf Activity Against the RB4CD12 Epitope in Immobilized Heparin/HS 107
3. Flow Cytometry-Based Sulf Assay Against the Cell Surface RB4CD12 Epitope 108
4. Ex Vivo Sulf Assay Against the RB4CD12 Epitope in Cryostat-Cut Sections 112
Acknowledgments 115
References 115
Chapter Four: Glycomics Profiling of Heparan Sulfate Structure and Activity 118
Abstract 118
1. Overview 119
2. Experimental 122
3. Conclusions and Future Perspectives 134
Acknowledgments 134
References 134
Section Two: Lectins, Immunity and Infection 140
Chapter Five: Microbe-Associated Molecular Patterns in Innate Immunity 142
Abstract 143
1. Overview 144
2. LPS and LOS Extraction Procedures 146
3. Purification of the Crude Extracts 149
4. SDS-PAGE 151
5. Carbohydrate Analysis: Monosaccharide Determination, Absolute Configuration, and Definition of Branching Points... 154
6. Fatty Acids Compositional Analysis (GC-MS) 162
References 167
Chapter Six: Structural and Functional Analysis of Glycosphingolipids of Schistosoma mansoni 170
Abstract 171
1. Overview 171
2. Isolation and Purification of S. mansoni Glycosphingolipids 172
3. Structural Characterization of S. mansoni Glycosphingolipids 174
4. Immunochemical Characterization of Glycan Antigens 181
5. Interaction of S. mansoni Glycosphingolipids with Dendritic Cell Receptors 183
6. Conclusions 189
References 189
Chapter Seven: Biotoxicity Assays for Fruiting Body Lectins and Other Cytoplasmic Proteins 194
Abstract 194
1. Overview 195
2. Expression of Fruiting Body Lectins in E. coli 196
3. Toxicity Test Toward the Insect A. aegypti 198
4. Toxicity Test Toward the Nematode C. elegans 200
5. Toxicity Test Toward the Amoeba A. castellanii 200
6. Statistics 201
Acknowledgments 201
References 201
Chapter Eight: Carbohydrate Signaling by C-Type Lectin DC-SIGN Affects NF-kappaB Activity 204
Abstract 204
1. Overview 205
2. Dendritic Cell Stimulation with LPS and ManLAM 206
3. RNA Interference in Dendritic Cells 208
4. NF-kappaB Activation in DCs 209
5. Phosphorylation and Acetylation of NF-kappaB by DC-SIGN Signaling 210
6. Activity of Acetyltransferases in DC 213
7. Transcription Regulation by Acetylation of p65 214
8. Concluding Remarks 215
Acknowledgments 216
References 217
Chapter Nine: Engineered Carbohydrate-Recognition Domains for Glycoproteomic Analysis of Cell Surface Glycosylation and Ligands 218
Abstract 219
1. Overview 219
2. Engineering Glycan-Binding Specificity 220
3. CRDs as Probes for Detection of Glycans on Blots 224
4. CRDs as Affinity Tools for Probing of Cell Surface Glycosylation 225
Acknowledgments 231
References 231
Chapter Ten: Mannose 6-Phosphate Receptor Homology Domain-Containing Lectins in Mammalian Endoplasmic Reticulum-Associated Deg 234
Abstract 234
1. Overview 235
2. Quality Control of Newly Synthesized Glycoproteins 236
3. Primary Structures of Yos9p, OS-9, and XTP3-B 237
4. OS-9 and XTP3-B form a Complex with Membrane-Embedded Ubiquitin Ligase 240
5. Sugar Recognition Specificity of OS-9 and XTP3-B 243
6. The Effect of OS-9 and XTP3-B on ERAD in Mammals 244
7. Concluding Remarks 246
Acknowledgments 247
References 247
Chapter Eleven: Multiple Functional Targets of the Immunoregulatory Activity of Galectin-1 252
Abstract 253
1. General Introduction 253
2. Regulation of Immune Cell Trafficking, Recruitment, and Chemotaxis 255
3. Galectin-Glycan Lattices in the Control of DC Physiology 269
4. Galectin-Glycan Lattices in the Control of T Helper Cell Fate 282
5. Final Remarks and Future Directions 290
Acknowledgments 292
References 292
Chapter Twelve: Manipulating Cell Surface Glycoproteins by Targeting N-Glycan-Galectin Interactions 298
Abstract 298
1. Overview 299
2. Galectins and Their N-Glycan Ligands 299
3. Regulation of Glycoprotein Concentration at the Cell Surface by the Galectin-Glycoprotein Lattice 301
4. T Cells and the Galectin-Glycoprotein Lattice 302
5. Genetic and Metabolic Regulation of the Galectin-Glycoprotein Lattice 302
6. Overview of Methods to Measure and Modulate the Galectin-Glycoprotein Lattice 303
References 315
Chapter Thirteen: Galectin-1 and HIV-1 Infection 320
Abstract 320
1. Overview 321
2. Experimental 325
References 341
Section Three: Drosophila 348
Chapter Fourteen: The Glycomics of Glycan Glucuronylation in Drosophila melanogaster 350
Abstract 350
1. Introduction 351
2. Experimental Procedures and Results 353
3. Discussion 367
Acknowledgments 370
References 370
Chapter Fifteen: Glycosyltransferases and Transporters that Contribute to Proteoglycan Synthesis in Drosophila 376
Abstract 377
1. Overview of Glycosaminoglycan Biosynthesis in Drosophila 377
2. Identification of Drosophila Glycosyltransferases and Sugar-Nucleotide Transporters that Contribute to Proteoglycan Synthes 389
3. Establishment and Functional Analysis of RNAi Flies for the Glycosyltransferases and Sugar-Nucleotide Transporters that Con 393
References 399
Section Four: NOTCH Signaling 406
Chapter Sixteen: O-GlcNAc Modification of the Extracellular Domain of Notch Receptors 408
Abstract 408
1. Overview 409
2. Preparation of Recombinant EGF Domains by Yeast Expression System 411
3. In Vitro O-GlcNAc Transferase Assay 413
4. Mass Spectrometry 413
5. Detection of O-beta-GlcNAc Modification Using Antibodies 416
6. Galactosyltransferase Labeling 418
7. Hexosaminidase Treatment 420
8. Conclusions and Future Directions 422
Acknowledgments 423
References 423
Chapter Seventeen: Regulation of Notch Signaling Via O-Glucosylation 428
Abstract 429
1. Overview 429
2. Genetic Identification and Characterization of rumi 430
3. Drosophila Strains 432
4. Drosophila Culture and Husbandry 433
5. A Genetic Screen to Identify New Notch Regulators 434
6. Mapping and Sequencing 435
7. Rescue Experiments 437
8. Generation of a Protein-Null Allele of rumi Via P-Element Excision 438
9. Experimental Evidence that Rumi is a Protein O-Glucosyltransferase 439
10. Enzyme Assay for Protein O-Glucosyltransferase Activity 442
11. Analysis of the Product Obtained from the Protein O-Glucosyltransferase Assay 443
12. Detection of O-Linked Glucose on Notch EGF Repeats by Mass Spectrometry 445
Acknowledgments 448
References 448
Section Five: New Development 452
Chapter Eighteen: O-Fucosylation of Thrombospondin Type 1 Repeats 454
Abstract 454
1. Overview 455
2. Glycosylation Site Mapping by Mass Spectrometry 460
Acknowledgments 466
References 466
Chapter Nineteen: Use of Glycan Microarrays to Explore Specificity of Glycan-Binding Proteins 470
Abstract 470
1. Overview 471
2. The Printed Glycan Microarray from the Consortium for Functional Glycomics (CFG) 472
3. Analysis of GBPs on the CFG Glycan Microarray 474
4. Defining a Glycan Motif Using Concentration-Dependent Binding of GBPs to the Printed Glycan Microarray 475
5. The Concentration-Dependent Binding of Sambucus nigra Agglutinin to the Printed Glycan Microarray and a Method for Ranking 476
6. Using Microarrays to Identify a Glycan-Binding Motif for SNA 478
7. Specificities of Human Galectin-8 and its Carbohydrate Recognition Domains (CRDs) 484
8. Challenges in Identifying Physiological Ligands for GBPs 492
9. Conclusion and Future Directions 493
Acknowledgments 495
References 495
Chapter Twenty: Functional Roles of the Bisecting GlcNAc in Integrin-Mediated Cell Adhesion 498
Abstract 498
1. Overview 499
2. Manipulation of GnT-III and GnT-V Genes in Cancer Cells 501
3. Assays for Cell Spreading and Migration 502
4. Construction of Various Integrin a5beta1 Mutants by the Mutagenesis of Potential N-Glycos 504
5. N-Glycans Differentially Regulate Integrin-Mediated Cell Adhesion and Migration 505
6. Identification of Important N-Glycosylation Sites for Functional Expression of a5beta1 Int 507
7. Site-4 Is a Crucial N-Glycosylation Site on the a5 Subunit for GnT-III Regulation 508
8. Future Perspectives 509
Acknowledgments 509
References 509
Chapter Twenty-One: Lectin-Based Glycoproteomic Techniques for the Enrichment and Identification of Potential Biomarkers 514
Abstract 514
1. Introduction 515
2. Lectin Blotting and Separation of Total Glycoproteins in a Sample Using a Lectin(s) Immobilized on Paramagnetic Beads... 518
3. Separation of Glycoproteins Using a Lectin Immobilized on Paramagnetic Beads Prior to Mass Spectrometry-Based Shotgun Prote 520
4. Glycomics Strategy for Identifying Potential Glycoprotein Cancer Markers 524
5. MS/MS Data Analysis and Confirmation of Glycoprotein Glycosylation Differences When Results from Two Tissue Sources are Com 524
6. Conclusions 526
Acknowledgments 527
References 527
Chapter Twenty - Two: High-Throughput RNAi Screening for N-Glycosylation Dependent Loci in Caenorhabditis elegans 530
Abstract 530
1. Overview 531
2. N-glycosylation in C. elegans 532
3. RNAi in C. elegans 533
4. C. elegans Strains, Culturing and RNAi Methods 533
5. Genome-wide RNAi Screen 539
6. Discussion 542
References 545
Chapter Twenty - Three: The Acidic Environment of the Golgi Is Critical for Glycosylation and Transport 548
Abstract 548
1. Introduction 549
2. Reporter Proteins for Transport Monitoring 552
3. Establishment of Parent Cells for Screening Transport Mutant Cells 553
4. Transport Assay of Reporter Proteins 554
5. Application of Transport Assay 554
6. Mutagenesis 557
7. Selection of Mutant Cells 557
8. Measurement of Golgi pH 557
9. Analysis of Glycosylation Using Lectin Staining 559
10. Concluding Remarks 559
Acknowledgment 560
References 560
Chapter Twenty - Four: Enzymatic Synthesis of Lacto-N-Difucohexaose I Which Binds to Helicobacter pylori 564
Abstract 565
1. Overview 565
2. Synthesis of Lacto-N-triose II Using beta-1,3-N-Acetylglucosaminyltransferase 566
3. Synthesis of Lacto-N-tetraose by Transglycosylation Using beta-1,3-Galactosidase 568
4. Preparation of Recombinants FUT1 and FUT3 571
5. Synthesis of Lacto-N-fucopentaose I and Lacto-N-difucohexaose I with the Aid of Fucosyltransferases 573
Acknowledgments 575
References 575
Chapter Twenty - Five: The Drosophila 7-Pass Transmembrane Glycoprotein BOSS and Metabolic Regulation 578
Abstract 578
1. Introduction 579
2. BOSS: Drosophila Orphan Membrane Receptor 583
3. BOSS Responds to Extracellular Glucose 585
4. Sugar and Lipid Metabolism Is Impaired in boss Null Mutants 585
5. boss Mutant Flies Are Sensitive to Starvation 586
6. Biochemical Techniques for Detecting TAG 588
References 590
Author Index 592
Subject Index 622
Color Plates 636