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

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Functional and Structural Proteomics of Glycoproteins

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.

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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

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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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  1. Nuffield Department of Clinical Medicine and MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, UK

    Simon J. Davis

  2. Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK

    Max Crispin

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Correspondence to Simon J. Davis .

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  1. Oxford Protein Production Facility, Roosevelt DR, Oxford, OX3 7BN, United Kingdom

    Raymond Owens

  2. Oxford Protein Production Facility, Roosevelt Dr, Oxford, OX3 7BN, United Kingdom

    Joanne Nettleship

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Davis, S.J., Crispin, M. (2010). Solutions to the Glycosylation Problem for Low- and High-Throughput Structural Glycoproteomics. In: Owens, R., Nettleship, J. (eds) Functional and Structural Proteomics of Glycoproteins. Springer, Dordrecht. https://doi.org/10.1007/978-90-481-9355-4_6

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