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Solutions to the glycosylation problem for low- and high-throughput structural glycoproteomics

Solutions to the glycosylation problem for low- and high-throughput structural glycoproteomics
Solutions to the glycosylation problem for low- and high-throughput structural glycoproteomics

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.

Endoglycosidases, Glycosylation, High throughput, Mammalian expression systems, Structural biology
127-158
Springer
Davis, Simon J.
77c9e91c-a2c6-498a-9128-bf164a8da8de
Crispin, Max
cd980957-0943-4b89-b2b2-710f01f33bc9
Owens, Raymond
Nettleship, Joanne
Davis, Simon J.
77c9e91c-a2c6-498a-9128-bf164a8da8de
Crispin, Max
cd980957-0943-4b89-b2b2-710f01f33bc9
Owens, Raymond
Nettleship, Joanne

Davis, Simon J. and Crispin, Max (2010) Solutions to the glycosylation problem for low- and high-throughput structural glycoproteomics. In, Owens, Raymond and Nettleship, Joanne (eds.) Functional and Structural Proteomics of Glycoproteins. Dordrecht. Springer, pp. 127-158. (doi:10.1007/978-90-481-9355-4_6).

Record type: Book Section

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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More information

e-pub ahead of print date: 12 November 2010
Published date: 2010
Keywords: Endoglycosidases, Glycosylation, High throughput, Mammalian expression systems, Structural biology

Identifiers

Local EPrints ID: 414541
URI: http://eprints.soton.ac.uk/id/eprint/414541
PURE UUID: 739e333e-bcea-4dde-9af8-c9deccf4f959
ORCID for Max Crispin: ORCID iD orcid.org/0000-0002-1072-2694

Catalogue record

Date deposited: 03 Oct 2017 16:31
Last modified: 06 Jun 2024 01:59

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Contributors

Author: Simon J. Davis
Author: Max Crispin ORCID iD
Editor: Raymond Owens
Editor: Joanne Nettleship

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