In the past decade, there has been an explosion of progress in understanding the roles of carbohydrates in biological systems. This explosive progress was made with the efforts in determining the roles of carbohydrates in immunology, neurobiology and many other disciplines, examining each unique system and employing new technology. This volume represents the first of three in the Methods in Enzymology series, including Glycomics (vol. 416) and Functional Glycomics (vol. 417), dedicated to disseminating information on methods in determining the biological roles of carbohydrates. These books are designed to provide an introduction of new methods to a large variety of readers who would like to participate in and contribute to the advancement of glycobiology. The methods covered include structural analysis of carbohydrates, biological and chemical synthesis of carbohydrates, expression and determination of ligands for carbohydrate-binding proteins, gene expression profiling including micro array, and generation of gene knockout mice and their phenotype analyses.
Author(s): Minoru Fukuda
Series: Methods in Enzymology 415
Edition: 1
Publisher: Academic Press
Year: 2006
Language: English
Pages: 383
Front......Page 3
Table of Contents......Page 6
Contributors to Volume 415......Page 10
Preface......Page 14
Previous Volumes in Methods in Enzymology......Page 16
Section I. N-Glycan Processing......Page 40
Analysis of LLO Biosynthesis by Incorporation of Radiolabeled Precursors......Page 42
Approaches for Analysis of Non-Radioactive LLOs......Page 43
LLO Analysis by Fluorophore-Assisted Carbohydrate Electrophoresis......Page 45
Monosaccharide Profiling by FACE......Page 50
LLO and Monosaccharide-P-Dolichol Extraction......Page 51
G0-3M1-9Gn2-P-P-Dol and Free Glycan Analysis: Derivatization with ANTS or ANDS......Page 52
Partial Purification and Separation of Phosphosugars and Nucleotide Sugars......Page 53
Monosaccharide Profiling FACE......Page 54
LLO Extraction and Partial Purification......Page 55
Oligosaccharide Profiling by FACE......Page 57
References......Page 58
[2] Identification of N-Glycan-Binding Proteins for E3 Ubiquitin Ligases......Page 59
Overview......Page 60
Methods......Page 61
Methods......Page 62
Methods......Page 64
Methods......Page 65
Methods......Page 66
References......Page 68
Overview......Page 70
Overview of Class 1 (GH47) Expression and Purification......Page 74
Enzyme Expression and Purification......Page 75
alpha-Mannosidase Assays and Kinetic Analyses......Page 77
Glycan Binding Studies by SPR......Page 79
Summary......Page 81
References......Page 83
Introduction......Page 85
In Vitro PNGase Assay......Page 86
Glycoprotein Substrate for In Vivo PNGase Assay......Page 88
Cell Extracts and Western Blotting......Page 89
Cycloheximide-Decay Experiment......Page 90
Technical Considerations......Page 91
References......Page 92
Section II. Structural Analysis......Page 96
Overview......Page 98
Materials for Tissue Homogenization......Page 99
Method for Homogenization......Page 100
Method for Tryptic Digestion......Page 101
Method for Release of N-Glycans......Page 102
Method for Release of O-Glycans: Reductive Elimination......Page 103
Method for Desalting of O-Glycans......Page 104
Buffers for Enzymatic Digestions......Page 105
Method for Acid Hydrolysis......Page 106
Method for Permethylation......Page 107
MS Analyses......Page 108
Materials for MALDI Analysis......Page 109
Method for MALDI Analysis......Page 110
Method for Electrospray Ionization Mass Spectrometry (ES-MS)......Page 111
Method for the Preparation of Partially Methylated Alditol Acetates......Page 112
Automated Analysis of N-Glycan MALDI Fingerprints......Page 113
Materials......Page 114
Methods......Page 115
Creating MSD and MSA Files......Page 116
The PARC Mass Spectrum Viewer......Page 118
Menus......Page 122
References......Page 123
Overview......Page 126
Materials and Reagents......Page 127
PA-Oligosaccharide Preparation from Glycoprotein......Page 128
3-DM Using HPLC......Page 129
HPLC Analysis......Page 130
Materials and Methods......Page 133
Structural Assignment of MSn Spectral Matching......Page 136
MS Analysis......Page 137
References......Page 140
[7] Determination of Glycosylation Sites and Disulfide Bond Structures Using LC/ESI-MS/MS Analysis......Page 142
Overview......Page 143
Methods......Page 144
Identifying Cys-Containing Peptides and N-Linked Glycopeptides......Page 145
Identifying Disulfide-Bonded Pairs of Peptides......Page 146
Digestion with Trypsin, Chymotrypsin, or Endoproteinase Glu-C (Protocol 3)......Page 149
Complete Reduction with DTT and Alkylation with IAM (Protocol 5)......Page 150
References......Page 151
[8] Identification of O-GlcNAc Sites on Proteins......Page 152
Introduction......Page 153
Materials......Page 154
Procedure......Page 155
Materials......Page 156
Galactosyltransferase Labeling......Page 157
Procedure......Page 158
Removing O-GlcNAc by Hexosaminidase Digestion......Page 160
Procedure for Coupling the Sample the PVDF Disk for Manual Edman Degradation......Page 161
Site Mapping by beta-Elimination and Michael Addition with Dithiothreitol......Page 162
Materials......Page 163
Protein Digestion......Page 164
Mild BEMAD Treatment......Page 165
Thiol Affinity Chromatography......Page 166
Chemoenzymatic Enrichment of O-GlcNAc-Modified Proteins......Page 167
Procedure......Page 168
Future Directions......Page 169
References......Page 170
Section III. Carbohydrate Synthesis and Antibiotics......Page 174
Introduction......Page 176
Enzyme Production Materials......Page 177
General Procedure for the Bacterial Expression......Page 178
General Procedure for the Mammalian Expression System......Page 180
Enzyme Purification......Page 181
Dowex Resin and Size Exclusion Chromatography......Page 182
Preparative High-Performance Liquid Chromatography......Page 183
Notes......Page 184
Notes......Page 186
O-Glycans......Page 187
Globoside Glycans......Page 188
References......Page 189
Overview......Page 192
General Methods......Page 197
Conjugation to Tetanus Toxoid......Page 199
References......Page 201
Overview......Page 203
Culture of H. pylori with AGS Cells Stably Expressing alpha4GlcNAc-Capping Structure......Page 205
Expression of CD43 Expressing alpha4GlcNAc......Page 206
Preparation of Human Gastric Mucins......Page 208
Cell Motility and Morphology......Page 209
Detection of alpha-D-glucosyl Cholesterol by Mass Spectrometry......Page 210
In Vitro Assay of Cholesterol alpha-Glucosyltransferase from H. pylori......Page 211
Continuous Spectrophotometric Assay for Cholesterol alpha-Glucosyltransferases......Page 212
Future Perspective......Page 215
References......Page 216
Overview......Page 219
Tetracycline......Page 223
Spectinomycin......Page 224
Pactamycin......Page 226
Hygromycin......Page 228
Streptomycin......Page 230
Neamine-Containing Aminoglycosides and Apramycin......Page 232
Binding of Neamine-Containing Aminoglycosides to Mutant Ribosomes......Page 234
Acknowledgment......Page 237
References......Page 238
Overview......Page 241
Characterization of Vibrio cholerae Neuraminidase by Using a Mechanism-Based Fluorescent Labeling Reagent......Page 243
Synthesis of Suicide Substrate of Neuraminidase 1......Page 244
Inhibition and Specific Labeling of VCNA......Page 245
Purification of the Fluorescence-Labeled Peptide by Anti-Dansyl Antibody Column......Page 246
MALDI-TOF MS and MS/MS Finger Printing Analysis of the Labeled Peptide......Page 247
References......Page 250
Overview......Page 252
Experimental Design......Page 253
Identification of Localization and Catalytic Domains......Page 255
Localization Domain Plasmids......Page 256
Catalytic Domain Plasmids......Page 257
Flow Cytometry Analysis of Cell Surface Glycoproteins......Page 258
Day 3: Split Cells and Add Rapamycin......Page 259
Western Blot Analysis of Secreted Glycoproteins......Page 260
Variables Affecting Enzyme Activity......Page 261
Immunofluorescence Microscopy......Page 265
Applications and Future Directions......Page 266
References......Page 267
Introduction......Page 269
Materials and Methods......Page 272
Ac4ManNAz (1,3,4,6-tetra-O-acetyl-N-azidoacetyl-alpha,beta-D-mannosamine......Page 273
Ac4GalNAz (1,3,4,6-tetra-O-acetyl-N-azidoacetyl-alpha,beta-D-galactosamine......Page 274
Phosphine-PFP (2-diphenylphosphanyl-terephthalic acid 1-methyl ester 4-pentafluorophenyl ester)......Page 276
Metabolic Labeling of Glycans in Cell Culture......Page 277
Reaction of Azido Sugars with Labeling Reagents in Cell Culture and in Tissue Lysates......Page 278
Enrichment and Identification of Metabolically Labeled Glycans......Page 280
Discussion......Page 281
Detection of Azide-Labeled Glycans......Page 283
Enrichment and Identification of Azide-Labeled Glycans......Page 284
Further Reading......Page 287
Section IV. Carbohydrate Ligand Specificity......Page 290
Introduction......Page 292
General Considerations in Developing Activity-Based Probes......Page 293
Specific Case: Carbohydrate Processing Enzymes......Page 295
Case Study: Probing Exoglycosidases in Cell Lysates......Page 298
Step 2: Chemoselective Ligation Reaction......Page 302
Step 3: Gel-Base Profiling of the Proteome......Page 303
Experimental Procedures for Labeling E. coli Cell Lysates Using Probe 6......Page 304
References......Page 306
Introduction......Page 308
Oligosaccharide Microarrays as Tools in HIV Glycobiology......Page 309
Materials and Equipment......Page 310
Preparation of High-Mannose Microarrays......Page 311
Hybridization Experiments......Page 312
Microarrays of Synthetic Heparin Oligosaccharides for High-Throughput Screening of Heparin-Protein Interactions......Page 313
Preparation of Heparin Arrays......Page 316
Incubation with Heparin-Binding Proteins......Page 317
Microarrays of Aminoglycosides......Page 318
Materials and Equipment......Page 319
Preparation of Aminoglycoside-Bearing Microarrays......Page 320
Synthesis of RNA for Microarray Applications......Page 322
Hybridization of RNA to Aminoglycoside Microarrays......Page 323
Hybridization of Proteins to Aminoglycoside Microarrays......Page 324
Materials and Equipment......Page 325
Preparation of Carbohydrate Microarrays......Page 326
Hybridization of Bacteria with Carbohydrate Microarrays......Page 327
References......Page 328
[18] Identification of Ligand Specificities for Glycan-Binding Proteins Using Glycan Arrays......Page 331
Overview......Page 332
Construction of the CFG Streptavidin‐Biotin Glycan Array (Plate Glycan Array)......Page 333
Direct Binding Assays......Page 335
Analysis of Binding Specificity on the CFG Plate Array......Page 336
Construction of the CFG Covalent Printed Glycan Array (Printed Glycan Array)......Page 337
Direct Binding Assays......Page 338
Analyzing the Covalent Printed Array Results......Page 339
Assays of Cells and Organisms......Page 340
Fluorescence Reporters Used in Glycan Array Assays......Page 341
Example Data Output From the Plate and Printed Glycan Arrays......Page 345
References......Page 347
Introduction......Page 350
Principle of FAC......Page 351
Immobilization of Lectins......Page 353
Preparation of Miniature Column......Page 355
Operation of FAC......Page 356
Full Specificity Analysis Using More Than 100 Glycans......Page 358
Concentration Dependence Analysis......Page 361
Concluding Remarks......Page 363
References......Page 364
Overview......Page 365
Materials......Page 367
Step 2: Preparation of N-aminooxyacetyl-DHPE (AOPE)......Page 368
Materials......Page 369
Preparation of NGLs from Other Saccharides......Page 370
Materials......Page 371
Semi-Preparative HPTLC Purification Procedure......Page 372
Status of the Core Monosaccharide in AO-NGLs......Page 373
Preparation of Liposomes......Page 375
Procedures......Page 376
References......Page 378
Introduction......Page 380
Evanescent-Field Fluorescence-Assisted Microarray System......Page 381
Fabrication of Lectin Microarray......Page 382
Scanning and Data Analysis......Page 383
Validation of Specific Binding Between Oligosaccharides and Immobilized Lectins......Page 384
Real-Time Scanning of Binding Reactions......Page 385
Application of Glycoprotein Probes on the Lectin-Antibody Hybrid-Array Slide......Page 386
Concluding Remarks......Page 387
Acknowledgments......Page 388
References......Page 389
Author Index......Page 392
Subject Index......Page 414
