Glycobiology Protocols

Glycobiology involves studies of complex carbohydrates and posttrans- tional modifications of proteins, and has become an important interdiscip- nary field encompassing chemistry, biochemistry, biology, physiology, and pathology. Although initial research was directed toward elucidation of the different carbohydrate structures and the enzymes synthesizing them, the field has now moved toward identifying the functions of carbohydrates. The pro- cols described in Glycobiology Protocols form a solid basis for investigations of glycan functions in health and disease. The cloning of many of the genes participating in glycosylation processes has helped to enhance our knowledge of how glycosylation is controlled, but has also added another dimension of complexity to the great heterogeneous variety of the structures of the oligos- charides of glycoproteins, proteoglycans, and glycolipids. A family of similar enzyme proteins exists for each glycosylation step. Glycosyltransferases are extremely specific for both the nucleotide sugar donor and the acceptor s- strate, but many other factors control sugar transfer, including the locali- tion and topology of enzymes, cofactors, possible chaperone proteins, and the availability of sugar acceptor substrates. The analysis of the intracellular organization of glycosylation and of the factors controlling the activities of the participating enzymes in the cell are important areas that need more research efforts. Another challenge for future research is to understand the glycodynamics of a cell, that is, how the cell responds to stimuli leading to biological and pathological changes in terms of alterations in glycosylation, and how this affects the biology of the cell.

Includes supplementary material: sn.pub/extras

Inhalt
Application of Fluorophore-Assisted Carbohydrate Electrophoresis for the Study of the Dolichol Pyrophosphate-Linked Oligosaccharides Pathway in Cell Cultures and Animal Tissues.- Partial Purification of Mannosylphosphorylundecaprenol Synthase From Micrococcus luteus.- Assay of Dolichyl-Phospho-Mannose Synthase Reconstituted in a Lipid Matrix.- O-Mannosylation in Mammalian Cells.- Methods for Analysis of Unusual Forms of O-Glycosylation.- Structural Analysis of Naturally Occurring Sialic Acids.- Nonradioactive Trans-Sialidase Screening Assay.- Structural Determination of O-Glycans by Tandem Mass Spectrometry.- Detailed Structural Analysis of N-Glycans Released From Glycoproteins in SDS-PAGE Gel Bands Using HPLC Combined With Exoglycosidase Array Digestions.- Molecular Modeling of Glycosyltransferases.- ?-Galactoside ?2,6-Sialyltransferase and the Sialyl ?2,6-Galactosyl-Linkage in Tissues and Cell Lines.- Visualizing Intracellular Distribution and Activity of Core2 ?(1,6)N-Acetylglucosaminyltransferase-I in Living Cells.- Gene Expression Analysis of Glycosylation-Related Genes by Real-Time Polymerase Chain Reaction.- Analysis of the Glycodynamics of Primary Osteoblasts and Bone Cancer Cells.- Micromethods for the Characterization of Lipid A-Core and O-Antigen Lipopolysaccharide.- Assay for a Galactosyltransferase Involved in the Assembly of the O7-Antigen Repeat Unit of Escherichia coli.- High-Throughput Quantitation of Metabolically Labeled Anionic Glycoconjugates by Scintillation Proximity Assay Utilizing Binding to Cationic Dyes.- Quantitative Analysis of Mucins in Mucosal Secretions Using Indirect Enzyme-Linked Immunosorbent Assay.- Molecular Dissection of the Mouse Zona Pellucida.- Soluble Adamantyl Glycosphingolipid Analogs as Probes of GlycosphingolipidFunction.- Diagnosis of Krabbe Disease by Use of a Natural Substrate.- In Vitro Assays of the Functions of Calnexin and Calreticulin, Lectin Chaperones of the Endoplasmic Reticulum.- Quantitative Measurement of Selectin-Ligand Interactions.- Binding and Inhibition Assays for Siglecs.