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Solutions to the Glycosylation Problem for Low- and High-Throughput Structural Glycoproteomics

  • Simon J. DavisEmail author
  • Max Crispin
Chapter

Abstract

N- and O-glycosylation profoundly affect the biological properties of glycoproteins, principally by influencing their structures and cellular trafficking, and by forming the recognition sites of carbohydrate-binding ligands. For crystallographers interested in studying the protein component of glycoproteins, the two most important aspects of glycosylation are (1) that it is often essential for the correct folding of a given protein and for ensuring its solubility, which generally necessitates expression of the molecule in eukaryotic cells, and (2) that there are now procedures for the efficient post-folding removal of N-linked glycans from glycoproteins and for minimizing the effects of O-glycosylation, which will generally benefit crystallogenesis. We provide an overview of how glycans influence glycoprotein folding and then identify the sources of structural heterogeneity at the heart of the ‘glycosylation problem’. We then discuss the options available to structural biologists for circumventing the problems associated with protein N- and O-glycosylation. Our emphasis is on methods for producing glycoproteins with homogeneous and/or removable N-glycosylation in mammalian cells that can be implemented in both very high yield, stable expression systems and in a high throughput format based on transient expression protocols. We also consider whether deglycosylation reduces protein stability and end by emphasizing the importance of using rigorous stereochemical and biosynthetic data when building glycosylation into partial or complete electron density.

Keywords

Glycosylation Endoglycosidases Mammalian expression systems Structural biology High throughput 

Abbreviations

CHO

Chinese hamster ovary

CNX

calnexin

CRT

calreticulin

Endo

endoglycosidase

ER

endoplasmic reticulum

GalNAc

α-N-acetylgalactosamine

GlcNAc

N-acetylglucosamine

GnT I

β1−2 N-acetylglucosamine transferase I

HEK

human embryonic kidney

IL

interleukin

IgSF

immunoglobulin superfamily

MALDI-TOF MS

matrix-assisted laser desorption/ionization-time of flight mass spectrometry

NB-DNJ

N-butyldeoxynojirimycin

PDB

Protein Data Bank

PNGase F

peptide-N-glycosidase F

s

soluble

S2

Schneider 2

SG

structural genomics

STP

serine-, threonine- and proline-rich

TCR

T-cell receptor

UGGT

UDP-glucose glycoprotein:glucosyltransferase

Notes

Acknowledgements

The authors wish to thank Veronica Chang, Radu Aricescu, Ray Owens, Jo Nettleship, Yvonne Jones, David Stuart, Neil Barclay, Chris Scanlan, Pauline Rudd, David Harvey, Mark Wormald, Tom Bowden and Raymond Dwek for many helpful discussions. We are particularly grateful to Ed Evans for his intellectual input and for his preparation of Fig. 6.9. This work was funded by the Wellcome Trust, the United Kingdom Medical Research Council and the Oxford Glycobiology Institute Endowment.

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.Nuffield Department of Clinical Medicine and MRC Human Immunology Unit, Weatherall Institute of Molecular MedicineUniversity of Oxford, John Radcliffe HospitalOxfordUK
  2. 2.Department of Biochemistry, Oxford Glycobiology InstituteUniversity of OxfordOxfordUK

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