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Life Sciences - Biochemistry & Biophysics | Glycosyltransferases - Methods and Protocols


Methods and Protocols

Series: Methods in Molecular Biology, Vol. 1022

Brockhausen, Inka (Ed.)

2013, XVII, 425 p. 110 illus., 40 illus. in color.

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  • Includes cutting-edge methods and protocols
  • Provides step-by-step detail essential for reproducible results
  • Contains key notes and implementation advice from the experts

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 Protocols contains a wide range of studies, methods and protocols 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 protocols, and notes on troubleshooting and avoiding known pitfalls.


Authoritative and easily accessible, Glycosyltransferases: Methods and Protocols 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.

Content Level » Professional/practitioner

Keywords » bacterial glycosyltransferases - fluorescent microscopy - glycan antibodies - glycans - glycosyltransferases

Related subjects » Analytical Chemistry - Biochemistry & Biophysics - Organic Chemistry

Table of contents 

1. Glycan Microarray Screening Assay for Glycosyltransferase Specificities

Wenjie Peng, Corwin M. Nycholat, and Nahid Razi            


2. A Fluorescence-Based Assay for Core 1 3Galactosyltransferase (T-synthase) Activity                        

Tongzhong Ju and Richard D. Cummings 


3.  Structural and Biochemical Analysis of a Bacterial Glycosyltransferase

Fan Zhu, Ren Wu, Hua Zhang, and Hui Wu


4. Study of the Biological Functions of Mucin Type Core 3 O-glycans

Seung Ho Lee and Minoru Fukuda                                                   


5. Generation of Anti-Sulfated Glycan Antibodies using Sulfotransferase-deficient Mice

Hiroto Kawashima


6. Fluorescent Microscopy as a Tool to Elucidate Dysfunction and Mislocalization of Golgi Glycosyltransferases in COG Complex Depleted Mammalian Cells

Rose A. Willett, Irina D. Pokrovskaya, and Vladimir V. Lupashin


7. A Practical Approach to Reconstruct Evolutionary History of Animal Sialyltransferases and Gain Insights into the Sequence-Function Relationships of Golgi-Glycosyltransferases

Daniel Petit, Roxana Elin Teppa, Jean-Michel Petit, and Anne Harduin-Lepers 


8. Fluorescent Lectin Staining of Drosophila Embryos and Tissues to Detect the Spatial Distribution of Glycans during Development

E. Tian, Liping Zhang, and Kelly G. Ten Hagen


9. Photoaffinity Labeling of Protein O-Mannosyltransferases of the PMT1/PMT2 Subfamily

Martin Loibl and Sabine Strahl 


10. Enzymatic Analysis of the Protein O-glycosyltransferase, Rumi, Acting toward Epidermal Growth Factor-like (EGF) Repeats

Hideyuki Takeuchi and Robert S. Haltiwanger 


11. Enzymatic Characterization of Recombinant Enzymes of O-GlcNAc Cycling

Eun Ju Kim and John Hanover




12. Antibodies and Activity Measurements for the Detection of O-GlcNAc Transferase, and Assay of its Substrate, UDP-GlcNAc

Tony Lefebvre, Ludivine Drougat, Stéphanie Olivier-van Stichelen, Jean-Claude Michalski, and Anne-Sophie Vercoutter-Edouart


13. In vitro Glycosylation Assay for Bacterial Oligosaccharyltransferases

Matias A. Musumeci, Maria V. Ielmini, and Mario F. Feldman


14. In vitro UDP-sugar:Undecaprenyl-Phosphate Sugar-1-Phosphate Transferase Assay and Product Detection by Thin Layer Chromatography

Kinnari B. Patel and Miguel A. Valvano


15. In vitro O-Antigen Ligase Assay

Xiang Ruan and Miguel A. Valvano


16. Functional Identification of Bacterial Glucosyltransferase WbdN

Yin Gao, Anna Vinnikova, and Inka Brockhausen


17. Methods for the Pasteurella Glycosaminoglycan Synthases: Enzymes that Polymerize Hyaluronan, Chondroitin, or Heparosan Chains

Paul DeAngelis


18. Methods for Measuring Class I Membrane-bound Hyaluronan Synthase Activity

Paul H. Weigel, Amy J. Padgett-McCue, and Bruce A. Baggenstoss


19.  Creation and Characterization of Glycosyltransferase Mutants of Trypanosoma brucei

Luis Izquierdo, M. Lucia S. Güther, and Michael A.J. Ferguson


20. Mannose-6-Phosphate: A Regulator of LLO Destruction

Ningguo Gao and Mark A. Lehrman


21. N-Acetylglucosaminyltransferase (GnT) Assays using Fluorescent Oligosaccharide Acceptor Substrates: GnT-III, IV, V, and IX (GnT-Vb)

Shinji Takamatsu, Hiroaki Korekane, Kazuaki Ohtsubo, Suguru Oguri, Jong Yi Park, Akio Matsumoto, and Naoyuki Taniguchi


22. A Method for Determination of UDP-GlcNAc: GlcNAcβ1-6(GlcNAcβ1-2)Manα1-R [GlcNAc to Man] β1-4N-acetylglucosaminyltransferase VI Activity

Tomohiko Taguchi and Naoyuki Taniguchi


23. In vitro Assays of Orphan Glycosyltransferases and their Application to Identify Notch Xylosyltransferases

Maya K. Sethi, Falk F. R. Buettner, Angel Ashikov, and Hans Bakker


24. In vitro Folding of -1,4Galactosyltransferase and Polypeptide--N-acetylgalactosaminyltransferase from the Inclusion Bodies

Boopathy Ramakrishnan and Pradman K. Qasba


25. An Assay for 1,6-Fucosyltransferase (FUT8) Activity Based on the HPLC Separation of a Reaction Product with Fluorescence Detection

Hideyuki Ihara, Hiroki Tsukamoto, Naoyuki Taniguchi, and Yoshitaka Ikeda


26. Immunodetection of Glycosyltransferases in Gastrointestinal Tissues

Joana Gomes and Celso A. Reis


27.  Glycosyltransferases in Chemo-Enzymatic Synthesis of Oligosaccharides

Boris Tefsen and Irma van Die


28.  Tumor Targeting by a Carbohydrate Ligand-Mimicking Peptide

Shingo Hatakeyama, Toshiaki K. Shibata, Yuki Tobisawa, Chikara Ohyama, Kazuhiro Sugihara, and Michiko N. Fukuda


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

Catharina Steentoft, Eric Paul Bennett, and Henrik Clausen


30.  Generation of Monoclonal Antibodies to Native Active Human Glycosyltransferases

Malene Bech Vester-Christensen, Eric Paul Bennett, Henrik Clausen, and Ulla Mandel

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