Series Editors
......Page 1
Copyright Page......Page 2
Preface......Page 11
Methods in Enzymology......Page 12
Abstract......Page 40
Overview of Proteoglycan Structure, Nomenclature, and Function......Page 41
Growth Factor Signaling......Page 42
Autonomous Signaling......Page 43
Proteoglycan Downregulation: Endocytosis and Ectodomain Shedding......Page 46
Cell Adhesion......Page 47
Migration......Page 51
Immunoblotting for heparan sulfate proteoglycans......Page 54
Proteoglycan chimera oligomerization......Page 56
Qualitative and quantitative endocytosis assays......Page 58
Rac1 pull-down assay......Page 61
Multicellular migration assay......Page 63
Two-chambered migration assay (adhesion and migration)......Page 64
Acknowledgments......Page 65
References......Page 66
Dual Roles of Drosophila Glypican Dally-Like in Wingless/Wnt Signaling and Distribution......Page 69
Introduction......Page 70
Generation of Dally and Dlp Null Alleles......Page 71
Loss-of-function experiments show that dally and dlp are required for the proper distribution of extracellular Wg.........Page 72
The effects of overexpressing Dally and Dlp on extracellular Wg distribution......Page 74
Dlp Core Protein Determines Its Biphasic Activity in Wg Morphogen Signaling......Page 75
Generation of Dlp core protein transgenic line......Page 76
Immunofluorescent staining of imaginal discs......Page 77
Dlp Core Protein Can Interact with Wg Independent of Its GAG Chains......Page 78
Cell-binding experiment......Page 79
Immunostaining of cultured S2 cells......Page 80
Co-immunoprecipitaton of Dlp or Dlp core protein with Wg-GFP......Page 81
Co-immunoprecipitation......Page 82
Acknowledgments......Page 84
References......Page 85
Use of a Phage Display Antibody to Measure the Enzymatic Activity of the Sulfs......Page 87
Overview......Page 88
Method......Page 90
Materials......Page 91
Method......Page 93
Materials......Page 95
Method......Page 97
References......Page 98
Glycomics Profiling of Heparan Sulfate Structure and Activity......Page 101
HS structure and biosynthesis......Page 102
Heparan sulfates: A family of multifunctional cell regulators......Page 103
Decoding HS structure-function: Toward glycomics approaches......Page 104
RIP method for isolation of proteoglycans......Page 105
Profiling disaccharide composition......Page 106
Fluorescence HPLC analysis of HS disaccharide composition using BODIPY label......Page 108
Profiling HS oligosaccharides......Page 109
Liquid chromatography electrospray mass spectrometry......Page 110
Software tools for HS oligosaccharide characterization......Page 112
Method for HS oligosaccharide library production......Page 113
FGF signaling assay screening using BaF3 cells expressing FGFR1c......Page 115
References......Page 117
Microbe-Associated MolecularPatterns in Innate Immunity:Extraction and Chemical Analysisof Gram-Negative BacterialLipopolysaccharides......Page 122
Overview......Page 124
LPS and LOS Extraction Procedures......Page 126
Methods......Page 127
Hot phenol/water extraction (Westphal and Jann, 1965)......Page 128
Enzymatic hydrolysis......Page 129
Method......Page 130
Methods......Page 131
Tsai and Frasch (1982) protocol......Page 132
Method......Page 133
Monosaccharide determination: Acetylated methyl glycosides......Page 134
Method......Page 135
Monosaccharide absolute configuration (octyl glycosides, see Notes and tips: Leontein et al., 1978)......Page 136
Methods......Page 137
Determination of monosaccharides branching points (methylation analysis): protocol for neutral (Ciucanu and Kerek, 1.........Page 138
Methods......Page 140
Total fatty acid composition by methanolysis......Page 142
Methods......Page 145
Methods......Page 146
References......Page 147
Structural and Functional Analysis of Glycosphingolipids of Schistosoma mansoni......Page 149
Overview......Page 150
Isolation and Purification of S. mansoni Glycosphingolipids......Page 151
Structural Characterization of S. mansoni Glycosphingolipids......Page 153
High-performance thin-layer chromatography......Page 154
Mass spectrometric analysis of glycosphingolipids......Page 155
Analysis of the ceramide moiety......Page 156
Survey of S. mansoni glycosphingolipid structures......Page 158
Overlay technique for glycosphingolipids and immunostaining......Page 160
Analysis of glycan antigens of glycosphingolipids by ELISA......Page 161
Dendritic cells in the immune system......Page 162
Preparation of human monocyte-derived immature dendritic cells......Page 163
Adhesion of immature dendritic cells and K562 cells, expressing recombinant CLRs, to glycosphingolipids......Page 164
Modulation of dendritic cell function by S. mansoni glycosphingolipids......Page 166
References......Page 168
Biotoxicity Assays for Fruiting Body Lectins and Other Cytoplasmic Proteins......Page 173
Overview......Page 174
Expression of Fruiting Body Lectins in E. coli......Page 175
Toxicity Test Toward the Insect A. aegypti......Page 177
Toxicity Test Toward the Amoeba A. castellanii......Page 179
References......Page 180
Carbohydrate Signaling by C-Type Lectin DC-SIGN Affects NF-kappaB Activity......Page 183
Overview......Page 184
Dendritic Cell Stimulation with LPS and ManLAM......Page 185
RNA Interference in Dendritic Cells......Page 187
NF-kappaB Activation in DCs......Page 188
Phosphorylation and Acetylation of NF-kappaB by DC-SIGN Signaling......Page 189
Activity of Acetyltransferases in DC......Page 192
Transcription Regulation by Acetylation of p65......Page 193
Concluding Remarks......Page 194
Acknowledgments......Page 195
References......Page 196
Engineered Carbohydrate-Recognition Domains forGlycoproteomic Analysis of CellSurface Glycosylation and Ligandsfor Glycan-Binding Receptors......Page 197
Overview......Page 198
Engineering Glycan-Binding Specificity......Page 199
Protocol 1: Characterization of engineered CRDs using glycan arrays......Page 200
CRDs as Probes for Detection of Glycans on Blots......Page 203
Protocol 3: Generation of affinity columns......Page 204
Protocol 4: Cell fractionation on affinity columns......Page 205
Protocol 5: Gel analysis of isolated glycoproteins and identification using in-gel trypsin digestion followed by mass spe......Page 207
Protocol 7: Mass spectrometry of tryptic peptides......Page 208
Glycomics......Page 209
References......Page 210
Mannose 6-Phosphate Receptor Homology Domain-Containing Lectins in Mammalian Endoplasmic Reticulum-Associated Degradation......Page 212
Overview......Page 213
Quality Control of Newly Synthesized Glycoproteins......Page 214
Primary Structures of Yos9p, OS-9, and XTP3-B......Page 215
OS-9 and XTP3-B form a Complex with Membrane-Embedded Ubiquitin Ligase......Page 218
Method for immunoprecipitation followed by Western blotting......Page 219
Method for FAC analysis......Page 221
The Effect of OS-9 and XTP3-B on ERAD in Mammals......Page 222
Method for pulse-chase analysis......Page 223
Concluding Remarks......Page 224
References......Page 225
Multiple Functional Targets of theImmunoregulatory Activity ofGalectin-1: Control of Immune CellTrafficking, Dendritic CellPhysiology, and T-Cell Fate......Page 229
General Introduction......Page 230
Conceptual framework......Page 232
Lymphocyte isolation......Page 236
Chemotaxis Assay......Page 237
Isolation and culture of primary HUVECs......Page 238
In vitro flow chamber assay......Page 239
Experimental model of mouse peritonitis: Studying the effect of galectin-1 on PMN trafficking......Page 242
Summary of the peritonitis model in the mouse......Page 243
Microvascular beds......Page 244
Cremaster preparation......Page 245
Conceptual framework......Page 246
Preparation of recombinant galectin-1......Page 250
Differentiation and maturation of mouse DCs......Page 251
Receptor segregation......Page 253
Human allogeneic mixed leukocyte reaction......Page 254
Mouse allogeneic MLR......Page 255
Tumor protection assays......Page 256
Induction and assessment of EAE......Page 257
Conceptual framework......Page 259
Polarization of Th1, Th2, and Th17 cells......Page 262
Polarization of human T helper cells......Page 263
Glycophenotype analysis......Page 264
Cell death assays......Page 265
In vivo assays to study the role of galectin-1 in T helper cell fate......Page 266
Final Remarks and Future Directions......Page 267
References......Page 269
Manipulating Cell Surface Glycoproteins by Targeting N-Glycan-Galectin Interactions......Page 275
Galectins and Their N-Glycan Ligands......Page 276
Regulation of Glycoprotein Concentration at the Cell Surface by the Galectin-Glycoprotein Lattice......Page 278
Genetic and Metabolic Regulation of the Galectin-Glycoprotein Lattice......Page 279
Overview of Methods to Measure and Modulate the Galectin-Glycoprotein Lattice......Page 280
Co-immunoprecipitation of galectins with glycoprotein partners......Page 281
Flow cytometry to measure N-glycan branching and glycoprotein surface levels......Page 282
Mass spectroscopy of N-glycans......Page 283
Quantitative real-time PCR and mRNA half-life measurements of Golgi enzymes......Page 284
Gene-targeted deletions of Golgi enzymes......Page 285
N-X-S/T site mutation......Page 286
Competitive inhibition of galectin binding......Page 287
Overexpression of Golgi branching enzymes......Page 288
Metabolic supplementation of the hexosamine pathway......Page 289
TCR signaling......Page 290
T cell activation and proliferation......Page 291
References......Page 292
Galectin-1 and HIV-1 Infection......Page 297
Human immunodeficiency virus......Page 298
Structures of galectins......Page 300
Release/secretion of galectins......Page 301
General precautions for HIV-1-related studies (biosafety level for HIV-1 research; Byers et al., 2004)......Page 302
HIV-1 production in HEK 293T cells......Page 303
p24 ELISA......Page 304
Purification of HIV-1 with Optiprep......Page 305
AT-2 inactivation of HIV-1......Page 306
Peripheral blood mononuclear cells......Page 307
Cell activation......Page 308
Purification......Page 309
SDS-PAGE......Page 310
S-Carboxyamidomethylation of galectin-1......Page 311
Sugar antagonists to inhibit galectin's lectin activity......Page 313
Dominant-negative form of galectins......Page 314
Virus attachment assays......Page 315
Indirect evaluation of HIV-1 binding (luciferase activity)......Page 316
HIV-1 infection assay......Page 317
References......Page 318
The Glycomics of Glycan Glucuronylation in Drosophila melanogaster......Page 325
Introduction......Page 326
Extraction of proteins and lipids......Page 328
Permethylation......Page 330
Glycan analysis by NSI-MSn and automated data acquisition......Page 331
Release of O-linked oligosaccharides by beta-elimination......Page 333
Iatrobead chromatography for purification of total GSL......Page 338
DEAE Sephadex chromatography for fractionation of acidic and zwitterionic GSLs......Page 339
Analysis of intact GSLs by NSI-MSn data-dependent acquisition......Page 340
Discussion......Page 342
References......Page 345
Glycosyltransferases and Transporters that Contribute to Proteoglycan Synthesis in Drosophila: Identification and Functional Analyses Using the Heritable and Inducible RNAi System......Page 350
Glycosaminoglycan structures......Page 351
Biosynthesis of Drosophila GAGs: Glycosyltransferases, sulfotransferases, and sugar-nucleotide transporters.........Page 352
Core proteins of proteoglycans......Page 358
Function of proteoglycans......Page 359
Cloning and expression of Drosophila glycosyltransferases......Page 363
Assay of glycosyltransferase activity......Page 364
Cloning and expression of Drosophila sugar-nucleotide transporters including PAPS transporters......Page 365
Preparation of subcellular fractions of yeast and assay of sugar-nucleotide transport activity......Page 366
Establishment of RNAi flies......Page 367
Functional analyses of RNAi flies......Page 370
References......Page 373
O-GlcNAc Modification of the Extracellular Domain of Notch Receptors......Page 379
Overview......Page 380
Preparation of Recombinant EGF Domains by Yeast Expression System......Page 382
Purification of EGF20......Page 383
Mass Spectrometry......Page 384
Reduction, S-carbamidomethylation, and trypsin digestion......Page 385
Immunoblotting with CTD110.6 antibody......Page 387
beta4GalT-1 assay......Page 389
Hexosaminidase Treatment......Page 391
Deglycosylation of glycoproteins......Page 392
Conclusions and Future Directions......Page 393
References......Page 394
Regulation of Notch Signaling Via O-Glucosylation: Insights from Drosophila Studies......Page 398
Overview......Page 399
Genetic Identification and Characterization of rumi......Page 400
Drosophila Strains......Page 402
Methods......Page 403
Methods......Page 404
Methods......Page 405
Methods......Page 407
Generation of a Protein-Null Allele of rumi Via P-Element Excision......Page 408
Experimental Evidence that Rumi is a Protein O-Glucosyltransferase......Page 409
Methods......Page 412
Materials......Page 413
Methods......Page 414
Materials......Page 415
Methods......Page 416
References......Page 418
O-Fucosylation of Thrombospondin Type 1 Repeats......Page 422
Overview......Page 423
Glycosylation Site Mapping by Mass Spectrometry......Page 428
Sample preparation......Page 429
LC-MS of the digested sample......Page 430
References......Page 434
Use of Glycan Microarrays to Explore Specificity of Glycan-Binding Proteins......Page 438
Overview......Page 439
The Printed Glycan Microarray from the Consortium for Functional Glycomics (CFG)......Page 440
Analysis of GBPs on the CFG Glycan Microarray......Page 442
Defining a Glycan Motif Using Concentration-Dependent Binding of GBPs to the Printed Glycan Microarray......Page 443
The Concentration-Dependent Binding of Sambucus nigra Agglutinin to the Printed Glycan Microarray and a Method for Ranking.........Page 444
Using Microarrays to Identify a Glycan-Binding Motif for SNA......Page 446
Specificities of Human Galectin-8 and its Carbohydrate Recognition Domains (CRDs)......Page 452
Challenges in Identifying Physiological Ligands for GBPs......Page 460
Conclusion and Future Directions......Page 461
References......Page 463
Functional Roles of the Bisecting GlcNAc in Integrin-Mediated Cell Adhesion......Page 466
Overview......Page 467
Manipulation of GnT-III and GnT-V Genes in Cancer Cells......Page 469
Assays for Cell Spreading and Migration......Page 470
Construction of Various Integrin ฮฑ5beta1 Mutants by the Mutagenesis of Potential N-Glycos ......Page 472
N-Glycans Differentially Regulate Integrin-Mediated Cell Adhesion and Migration......Page 473
Identification of Important N-Glycosylation Sites for Functional Expression of ฮฑ5beta1 Int ......Page 475
Site-4 Is a Crucial N-Glycosylation Site on the ฮฑ5 Subunit for GnT-III Regulation ......Page 476
References......Page 477
Lectin-Based Glycoproteomic Techniques for the Enrichment and Identification of Potential Biomarkers......Page 481
Introduction......Page 482
Lectin Blotting and Separation of Total Glycoproteins in a Sample Using a Lectin(s) Immobilized on Paramagnetic Beads.........Page 485
Separation of Glycoproteins Using a Lectin Immobilized on Paramagnetic Beads Prior to Mass Spectrometry-Based Shotgun Prote......Page 487
Extraction of easily solubilized glycoproteins from tissues and separation on immobilized lectin(s) prior to shotgun prot......Page 488
Extraction of tightly membrane-associated glycoproteins from tissues and separation on immobilized lectin(s) prior to sho......Page 490
MS/MS Data Analysis and Confirmation of Glycoprotein Glycosylation Differences When Results from Two Tissue Sources are Com......Page 491
Conclusions......Page 493
References......Page 494
High-Throughput RNAi Screening for N-Glycosylation Dependent Loci in Caenorhabditis elegans......Page 497
Overview......Page 498
N-glycosylation in C. elegans......Page 499
C. elegans Strains, Culturing and RNAi Methods......Page 500
Tunicamycin treatment varies among strains......Page 502
Establishment of tunicamycin-hypersensitive genes......Page 503
Genome-wide RNAi Screen......Page 506
Discussion......Page 509
References......Page 512
The Acidic Environment of the Golgi Is Critical for Glycosylation and Transport......Page 514
Introduction......Page 515
Reporter Proteins for Transport Monitoring......Page 518
Establishment of Parent Cells for Screening Transport Mutant Cells......Page 519
Application of Transport Assay......Page 520
Measurement of Golgi pH......Page 523
Concluding Remarks......Page 525
References......Page 526
Enzymatic Synthesis of Lacto-N-Difucohexaose I Which Binds to Helicobacter pylori......Page 530
Overview......Page 531
Synthesis of Lacto-N-triose II Using beta-1,3-N-Acetylglucosaminyltransferase......Page 532
Materials......Page 533
Synthesis of Lacto-N-tetraose by Transglycosylation Using beta-1,3-Galactosidase......Page 534
Methods......Page 536
Methods......Page 537
Methods......Page 538
Methods......Page 539
Methods......Page 540
References......Page 541
The Drosophila 7-Pass Transmembrane Glycoprotein BOSS and Metabolic Regulation......Page 544
The fat body......Page 545
The brain......Page 546
Yeast G-protein-coupled receptor (GPCR) for glucose sensing
......Page 547
BOSS: Drosophila Orphan Membrane Receptor......Page 549
Sugar and Lipid Metabolism Is Impaired in boss Null Mutants......Page 551
Boss Mutant Flies Are Sensitive to Starvation
......Page 552
Biochemical Techniques for Detecting TAG......Page 554
Materials......Page 555
References......Page 556
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