Glycosyltransferases Methods and Protocols 1st Edition by Inka Brockhausen ISBN 1627034641 9781627034647

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ISBN 10: 1627034641

ISBN 13: 9781627034647 

Author: Inka Brockhausen 

Glycosyltransferases (GTs) are essential for the biosynthesis of complex glycoconjugates and are powerful tools to study the functions of complex glycans in health, development and disease. Complex glycoconjugates, such as glycoproteins, proteoglycans and glycolipids, are assembled by GTs which synthesize specific linkages between sugars or sugars and protein. This is in contrast to the non-specific or less specific chemical glycation reactions, transglycosylation and reverse glycosylation reactions. Glycosyltransferases: Methods and Prools contains a wide range of studies, methods and prools which form a solid basis for investigations of the role and mechanisms, biology and pathology involving GTs. Written in the successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible prools, and notes on troubleshooting and avoiding known pitfalls.

Authoritative and easily accessible, Glycosyltransferases: Methods and Prools is a vital contribution to glycobiology and glycopathology, and to applications of these enzymes in biotechnology and drug development. It will prove invaluable to students, postdoctoral fellows, and senior scientists carrying on research of GTs that has been intensified over the last years.

Table of contents:

Part I: Glycosyltransferase Assays and Activity Measurements

1. Glycan Microarray Screening Assay for Glycosyltransferase Specificities
2. A Fluorescence-Based Assay for Core 1 β3Galactosyltransferase (T-synthase) Activity
3. Photoaffinity Labeling of Protein O-Mannosyltransferases of the PMT1/PMT2 Subfamily
4. Enzymatic Analysis of the Protein O-glycosyltransferase, Rumi, Acting toward Epidermal Growth Factor-like (EGF) Repeats
5. Enzymatic Characterization of Recombinant Enzymes of O-GlcNAc Cycling
6. Antibodies and Activity Measurements for the Detection of O-GlcNAc Transferase, and Assay of its Substrate, UDP-GlcNAc
7. In vitro Glycosylation Assay for Bacterial Oligosaccharyltransferases
8. In vitro UDP-sugar:Undecaprenyl-Phosphate Sugar-1-Phosphate Transferase Assay and Product Detection by Thin Layer Chromatography
9. In vitro O-Antigen Ligase Assay
10. Functional Identification of Bacterial Glucosyltransferase WbdN
11. Methods for the Pasteurella Glycosaminoglycan Synthases: Enzymes that Polymerize Hyaluronan, Chondroitin, or Heparosan Chains
12. Methods for Measuring Class I Membrane-bound Hyaluronan Synthase Activity
13. Creation and Characterization of Glycosyltransferase Mutants of Trypanosoma brucei
14. Mannose-6-Phosphate: A Regulator of LLO Destruction
15. N-Acetylglucosaminyltransferase (GnT) Assays using Fluorescent Oligosaccharide Acceptor Substrates: GnT-III, IV, V, and IX (GnT-Vb)
16. A Method for Determination of UDP-GlcNAc: GlcNAcβ1-6(GlcNAcβ1-2)Manα1-R [GlcNAc to Man] β1-4N-acetylglucosaminyltransferase VI Activity
17. In vitro Assays of Orphan Glycosyltransferases and their Application to Identify Notch Xylosyltransferases
18. In vitro Folding of β-1,4Galactosyltransferase and Polypeptide-α-N-acetylgalactosaminyltransferase from the Inclusion Bodies
19. An Assay for α1,6-Fucosyltransferase (FUT8) Activity Based on the HPLC Separation of a Reaction Product with Fluorescence Detection

Part II: Structural, Biochemical, and Cellular Studies of Glycosyltransferases
20. Structural and Biochemical Analysis of a Bacterial Glycosyltransferase
21. Study of the Biological Functions of Mucin Type Core 3 O-glycans
22. Fluorescent Microscopy as a Tool to Elucidate Dysfunction and Mislocalization of Golgi Glycosyltransferases in COG Complex Depleted Mammalian Cells
23. A Practical Approach to Reconstruct Evolutionary History of Animal Sialyltransferases and Gain Insights into the Sequence-Function Relationships of Golgi-Glycosyltransferases
24. Fluorescent Lectin Staining of Drosophila Embryos and Tissues to Detect the Spatial Distribution of Glycans during Development
25. Immunodetection of Glycosyltransferases in Gastrointestinal Tissues

Part III: Glycoengineering, Antibodies, and Applications
26. Generation of Anti-Sulfated Glycan Antibodies using Sulfotransferase-deficient Mice
27. Glycosyltransferases in Chemo-Enzymatic Synthesis of Oligosaccharides
28. Tumor Targeting by a Carbohydrate Ligand-Mimicking Peptide
29. Glycoengineering of Human Cell Lines Using Zinc Finger Nuclease Gene Targeting – SimpleCells with Homogeneous GalNAc O-glycosylation Allow Isolation of the O-glycoproteome by One-step Lectin Affinity Chromatography
30. Generation of Monoclonal Antibodies to Native Active Human Glycosyltransferases

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Tags: Inka Brockhausen, Glycosyltransferases, Methods, Protocols