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Glycan Remodeling with Processing Inhibitors and Lectin-Resistant Eukaryotic Cells

  • Veronica T. Chang
  • Robert A. Spooner
  • Max Crispin
  • Simon J. Davis
Part of the Methods in Molecular Biology book series (MIMB, volume 1321)

Abstract

Some of the most important and interesting molecules in metazoan biology are glycoproteins. The importance of the carbohydrate component of these structures is often revealed by the disease phenotypes that manifest when the biosynthesis of particular glycoforms is disrupted. On the other hand, the presence of large amounts of carbohydrate can often hinder the structural and functional analysis of glycoproteins. There are often good reasons, therefore, for wanting to engineer and predefine the N-glycans present on glycoproteins, e.g., in order to characterize the functions of the glycans or facilitate their subsequent removal. Here, we describe in detail two distinct ways in which to usefully interfere with oligosaccharide processing, one involving the use of specific processing inhibitors, and the other the selection of cell lines mutated at gene loci that control oligosaccharide processing, using cytotoxic lectins. Both approaches have the capacity for controlled, radical alteration of oligosaccharide processing in eukaryotic cells used for heterologous protein expression, and have great utility in the structural analysis of glycoproteins.

Key words

Mammalian expression N-glycan processing Kifunensine Lectin selection Protein crystallization 

Notes

Acknowledgements

This work was supported by the European Commission as SPINE (contract QLG2-CT-2002-00988) under the Integrated Programme “Quality of Life and Management of Living Resources,” the Wellcome Trust, Cancer Research UK, the Biotechnology and Biological Sciences Research Council, the Medical Research Council, and the Glycobiology Institute Endowment.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Veronica T. Chang
    • 1
  • Robert A. Spooner
    • 2
  • Max Crispin
    • 3
  • Simon J. Davis
    • 1
  1. 1.Radcliffe Department of Medicine and MRC Human Immunology Unit, John Radcliffe HospitalUniversity of OxfordOxfordUK
  2. 2.Life SciencesUniversity of WarwickCoventryUK
  3. 3.Department of Biochemistry, Oxford Glycobiology InstituteUniversity of OxfordOxfordUK

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