Functional Glycomics (eBook)

Front Cover 1
Methods in Enzymology: Functional Glycomics 4
Copyright Page 5
Contents 6
Contributors 14
Preface 20
Methods in Enzymology 22
Section one: Stem Cell 50
Chapter One: beta1,4-Galactosyltransferase V: A Growth Regulator in Glioma 52
1. Overview 53
2. Experimental 54
3. Results 57
4. Conclusion and Future Direction 67
Acknowledgments 71
References 71
Chapter Two: Roles of Polysialic Acid in Migration and Differentiation of Neural Stem Cells 74
1. Overview 75
Acknowledgments 83
References 83
Chapter Three: Structural and Functional Analysis of Chondroitin Sulfate Proteoglycans in the Neural Stem Cell Niche 86
1. Overview 88
2. Immunohistochemistry of Embryonic and Postnatal Mouse Brain Sections 91
3. Method to Stain Embryonic Sections for NSPC Markers 92
4. Analysis of the Adult Neurogenic Niche and SVZ-Derived Cells by Immunocytochemistry 93
5. Immunocytochemistry of Acutely Dissociated Cells 95
6. Isolation of NSPCs by Immunopanning or by Immunoisolation Using Paramagnetic Beads (EasySep) 96
7. Neurosphere Cultures and Various Methods for Their Analysis 98
8. Disaccharide Analysis of CS/DS Chains from Embryonic Brain and Conditioned Neurosphere Culture Media and Effect of Sodium C 104
9. Analysis of NSPC-Proliferation In Vitro and In Vivo 105
10. Analysis of CSPG Functions in NSPCs Using Chondroitinase ABC Treatment in Culture 105
11. Analysis of Chondroitin Sulfate Functions in the Neural Stem Cell Niche 107
12. RT-PCR and Semiquantitative Analysis of the Synthetic Machinery for Glycosaminoglycans 108
13. In Situ Hybridization of Sulfotransferases in Tissue and Neurosphere Sections 112
14. Microscopy 115
15. Conclusion and Outlook 115
Acknowledgments 115
References 115
Chapter Four: Transcript Analysis of Stem Cells 122
1. Introduction 123
2. qRT-PCR as a Tool for Determining Transcript Analysis for Glycan-Related Gene Expression 124
3. Examples of the Applications of qRT-PCR Transcript Analysis to Investigate Changes in Glycan-Related Gene Expression in Ste 132
Acknowledgments 137
References 137
Chapter Five: Directing Stem Cell Trafficking via GPS 142
1. Overview 143
2. Rationale for GPS 144
3. Guiding Principles and Method for GPS 146
4. Method of a(1,3)-Fucosylation of Cell Surface Using FTVI 148
5. Detection of E-Selectin Ligand Expression Following GPS 149
6. Testing for E-Selectin Ligand Activity Using E-Selectin-Ig Chimera (Three-Step Method for Flow Cytometry) 150
7. Summary 151
Acknowledgments 152
References 152
Chapter Six: Functional Assays for the Molecular Chaperone Cosmc 156
1. Overview 157
2. T-Synthase and Cosmc 159
3. T-Synthase Activity Assay 162
4. Assay Activity for Function of Cosmc 165
5. Conclusion and Future Directions 168
Acknowledgments 169
References 169
Chapter Seven: Core 3-Derived O-Glycans Are Essential for Intestinal Mucus Barrier Function 172
1. Introduction 173
2. Generation of C3GnT-/- Mice 175
3. Disruption of the C3GnT Gene Eliminates Core 3-Derived O-Glycans and Exposes the Tn Antigen in Murine Colon 177
4. Deficiency of C3GnT Results in Reduced Muc2 Expression in Colon and Impaired Mucosal Integrity 180
5. C3GnT-/- Mice are Highly Susceptible to Dextran Sodium Sulfate-Induced Colitis 182
6. Conclusion and Future Direction 187
Acknowledgments 189
References 189
Chapter Eight: Core3 Glycan as Tumor Suppressor 192
1. Overview 193
2. Generating Core3 Glycan Expressing Cell Lines 194
3. Detection Methods of Core3 Expression 195
4. Migration Assay Using Attractant 196
5. Determination of Major Integrin Using Functional Blocking Antibodies 196
6. Tumor Formation Assay 198
7. Western Blotting and Lectin Blotting 199
8. Heterodimerization Assay 200
9. FAK Signaling 201
References 202
Chapter Nine: Characterization of Mice with Targeted Deletion of the Gene Encoding Core 2 beta1,6-N-Acetylglucosaminyltransferas 204
1. Introduction 205
2. Generation and Genotyping of C2GnT2 KO Mice 206
3. Mice Lacking C2GnT2 Have Reduced Core 2 and no Core 4 Enzyme Activity and Altered Glycosylation 208
4. Phenotyping C2GnT2 KO Mice 210
5. Conclusions and Future Directions 217
Acknowledgments 219
References 219
Chapter Ten: Analyzing Physiological Function of Polypeptide GalNAcT-1-Deficient Mice in Humoral Immunity 222
1. Overview 223
2. Assay for In Vitro B Lymphocyte Activation 223
3. Assay for In Vivo Antibody Production 224
4. Immunohistochemical Staining of Frozen Sections 226
5. Apoptosis Detection Using Antibody Against Caspase 3 Active Form 228
6. Apoptosis Detection by TUNEL System 231
Acknowledgment 232
References 232
Chapter Eleven: beta3GnT2 (B3GNT2), a Major Polylactosamine Synthase: Analysis of B3gnt2-Deficient Mice 234
1. Overview 235
2. Glycogenes for Polylactosamine Synthesis (i.e., beta1,3-N-Acetylglucosaminyltransferase Genes) 235
3. N-Glycan Polylactosamine is Greatly Reduced in B3gnt2-/- Mice 236
4. Phenotype of B3gnt2-/- Lymphocytes Lacking Polylactosamine on N-Glycans 242
5. Protocols 246
Acknowledgments 251
References 251
Chapter Twelve: Targeted Genetic Inactivation of N-Acetylglucosaminyltransferase-IVa Impairs Insulin Secretion from Pancreatic be 254
1. Overview 255
2. Engineering GnT-IVa-Deficient Mice 256
3. GnT-IV Enzymology 258
4. Glucose and Insulin Homeostasis 259
5. Immunohistochemical Analysis 260
6. Islet Cell Preparation and Culture 263
7. Pulse-Chase Labeling 264
8. Cell Surface Half-Life Time of GLUT2 265
9. GLUT2 Glycan Analysis by Lectin Blot 266
10. Cell-Surface Protein Cross-Linking 267
Acknowledgment 269
References 269
Chapter Thirteen: The Ashwell-Morell Receptor 272
1. Overview 273
2. Endogenous Ligands of the AMR 276
3. Hepatocytes in Molecular Clearance Mechanisms of the Liver 277
4. Genotyping HL-1- or HL-2-Deficient Mice 278
5. Hematology and Coagulation Analyses Methods (Partly Adapted from Ellies et al., 2002 Grewal et al., 2008
Acknowledgments 285
References 285
Chapter Fourteen: Roles of GlcNAc-6-O-Sulfotransferases in Lymphoid and Nonlymphoid Tissues 292
1. Overview 293
2. Establishment of a Mouse Colon Adherent Cell Line and Cell Culture 295
3. Treatment of CAdC1 Cells with SCFAs 296
4. RT-PCR 296
5. Histology and Immunostaining 297
6. Preparation of Colonic-Mucin-Enriched Fraction 298
7. Carbohydrate Structural Analysis 299
8. Induction of Colitis by Dextran Sulfate Sodium 303
Acknowledgments 304
References 304
Chapter Fifteen: Core O-Glycans Required for Lymphocyte Homing: Gene Knockout Mice of Core 1 beta1,3-N-Acetylglucosaminyltransfera 306
1. Introduction 307
2. Lymphocyte Homing Assay 309
3. Staining of Lymph Nodes by L- and E-Selectin-IgM Chimeric Proteins after Glycosidase Treatment 312
4. Probing of L-Selectin Ligands on Membrane Filters with L- and E-selectin-IgM Chimeric Proteins 314
5. Conclusions 316
Acknowledgments 318
References 318
Chapter Sixteen: Immunohistochemical Analysis of Carbohydrate Antigens in Chronic Inflammatory Gastrointestinal Diseases 320
1. Overview 321
2. Immunohistochemical Analysis Using Conventional Immunostaining 323
3. Immunohistochemical Analysis Using Multiple Immunostaining 328
4. Immunohistochemical Analysis Using L-SelectinIgM Chimera Binding 332
Acknowledgments 336
References 336
Chapter Seventeen: Genetic Defects in Muscular Dystrophy 340
1. Overview 341
2. Mouse Models of Muscular Dystrophy 343
3. Approach to Phenotype Analysis in Mouse Muscular Dystrophy Models 355
4. Summary 361
Acknowledgments 361
References 361
Chapter Eighteen: POMT1 is Essential for Protein O-Mannosylation in Mammals 372
1. Overview 373
2. Experimental 374
3. Discussion/Conclusions 386
Acknowledgments 388
References 389
Chapter Nineteen: POMGnT1, POMT1, and POMT2 Mutations in Congenital Muscular Dystrophies 392
1. Overview 393
2. Methods 394
3. Procedures for Enzymatic Activity and Mutation Search 397
Acknowledgment 399
References 399
Chapter Twenty: Cellular and Molecular Characterization of Abnormal Brain Development in Protein O-Mannose N-Acetylglucosaminyltr 402
1. Overview 403
2. Analysis of a-DG Glycosylation and Laminin Binding by Western Blot 404
3. Histological Analysis of POMGnT1 Knockout Brain 405
4. Lamination Defects in the Neocortex of POMGnT1 Knockout Mice 406
5. Analysis of the Pial Basement Membrane by Laminin Immunostaining 408
6. Analysis of the Pial Basement Membrane by Transmission Electron Microscopy 409
7. Analysis of the Glia Limitans by GFAP Immunofluorescence Staining 411
Acknowledgments 413
References 414
Chapter Twenty-One: Investigating the Functions of LARGE: Lessons from Mutant Mice 416
1. Overview 417
2. Human LARGE and Relevance to Disease 418
3. Identification of Mice Carrying Mutations in Large 419
4. Loss of Functional Large Protein Results in Hypoglycosylation of a-Dystroglycan 422
5. Phenotypes of Mice with Mutations in Large 423
6. Expression of LARGE Genes 427
7. Does Large Encode a Functional Glycosyltransferase? 428
8. Largemyd Mice as a Model for Therapeutic Approaches to Dystroglycanopathy 430
Acknowledgments 430
References 430
Chapter Twenty - Two: A Tumor Suppressor Function of Laminin-Binding a-Dystroglycan 436
1. Background 437
2. Methods 438
Acknowledgment 444
References 444
Chapter Twenty - Three: Tumor Formation Assays 446
1. Overview 447
2. Animal Care and Protocol Approval 448
3. Disinfection of Mice 448
4. Analgesia and Anesthesia in Mice 448
5. IP Injection 450
6. IV Injection into the Tail Vein 450
7. IV Tumor Formation Assay Using Immune-Deficiency Mice 452
8. SC Inoculation 454
9. FP Inoculation 456
10. Testicular Inoculation 457
11. Prostate Inoculation 457
Acknowledgment 460
References 460
Author Index 462
Subject Index 488
Color Plates 498