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Generating heparan sulfate saccharide libraries for glycomics applications

Nature Protocols volume 5, pages 821–833 (2010)Cite this article

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Abstract

Natural and semi-synthetic heparan sulfate (HS) saccharide libraries are a valuable resource for investigating HS structure–function relationships, enabling high-throughput glycomics studies. Owing to the difficulty of chemical or in vitro enzymatic synthesis of HS saccharides, the structural diversity displayed in saccharides from tissue or cell sources cannot be readily accessed. In contrast, saccharide libraries can be generated by partial digestion of tissue-derived HS polysaccharide chains and chromatographic fractionation of the resulting saccharide mixtures. Fractionation is initially on the basis of hydrodynamic volume, using size exclusion chromatography. Further fractionation, on the basis of charge using strong anion exchange, can subsequently be applied. Desalting and sample concentration follows each fractionation step. Chromatographic fractions are generated that contain purified, or partially purified, saccharides. Here we describe a comprehensive protocol for generation of structurally diverse natural saccharide libraries from HS variants that is fast (∼3 weeks) and reproducible.

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Figure 1
Figure 2: A flow chart summarizing production of semi-purified natural HS saccharide libraries.
Figure 3: Analysis of heparinase digestion time points using PAGE.
Figure 4: Analysis of heparinase digestion time points using SEC.
Figure 5: SEC fractionation of heparinase partial digests of HS variants.
Figure 6: Fraction pooling and PAGE analysis for SEC fractionation of heparinase partial digests of HS variants.
Figure 7: SAX-HPLC of SEC peak fraction pools from HS partial digests.

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Acknowledgements

We were funded by grants 6E/16799 and B65/8/02649 from the Biotechnology & Biological Sciences Research Council (to J.E.T. and A.K.P.), the Engineering & Physical Sciences Research Council (Basic Technology Grant GR/S79268/01 and Basic Technology Translation Grant EP/G037604 to J.E.T. and A.K.P.), Wellcome Trust grant 082502 (to E.A.Y. and J.E.T.) and a Medical Research Council Senior Research Fellowship (to J.E.T.). We thank Mark Skidmore, Scott Guimond and Rebecca Miller, for their useful discussions.

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  1. Andrew K Powell

    Present address: Current address: School of Pharmacy & Biomolecular Sciences, Liverpool John Moores University, Liverpool, UK.,

Authors and Affiliations

  1. Centre for Glycobiology, School of Biological Sciences, University of Liverpool, Liverpool, UK

    Andrew K Powell, Yassir A Ahmed, Edwin A Yates & Jeremy E Turnbull

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Contributions

A.K.P. was involved in all aspects (particularly establishing and applying the protocol) and wrote the manuscript. Y.A.A. applied the methodologies toward saccharide preparation from diverse HS sources and assisted in preparing the manuscript. E.A.Y. generated the chemically modified heparin polysaccharides and gave advice. J.E.T. obtained funding, initially established the SEC and SAX methodology in the laboratory, edited the manuscript and provided overall supervision.

Corresponding author

Correspondence to Jeremy E Turnbull.

Supplementary information

Supplementary Figure 1. Troubleshooting a failed heparinase digestion by increasing the amount of enzyme.

Selectively 2, 6-desulfated porcine mucosal heparin was digested with (a) 2.5 mU/mg (2.5 mU/ml) and (b) 5 mU/mg (5 mU/ml) heparinase II. Aliquots were removed at sequential time points, heated to 100 °C to terminate the digestion, and analysed by PAGE, alongside a pool of size-estimated SEC fractions (containing ∼ 4-16 mers) from bovine lung heparin (Heparin stds). PAGE gels were stained with Azure A and digital images recorded using a DVC camera operated with DVC view software version 2.2.8. (PDF 681 kb)

Supplementary Figure 2. Troubleshooting/optimization of heparinase digestion using a multi-step process.

Selectively 2-O desulfated porcine mucosal heparin was analysed, using a Superdex™ peptide 7.5/300 small scale SEC column, run using an AKTApurifier™ 10 at a flow rate of 0.5 ml / min in 0.5 M ammonium hydrogen carbonate, whilst monitoring the elution profile by absorbance at λabs =232nm, (a) before digestion, following digestion with 1 mU/mg (25 mU/ml) (b) heparinase III or (c) heparinase II for 24 hrs, (d) 5 mU/mg (125 mU/ml) heparinase II for 8 hrs and (e) 1 mU/mg (25 mU/ml) each of heparinases I, II and III in combination for 8 hrs. (f) Superdex™ 16/60 medium scale SEC analysis of 2-O desulfated heparin pooled digestion aliquots (equivalent), removed and heated to 100 °C, to terminate the digestion, at 12 and 23 hours during digestion of 250 mg with a combination of 1 mU/mg (25 mU/ml) each of heparinase I, II and III. Data was recorded using the Unicorn 5.0 software and exported into SigmaPlot 11. (PDF 145 kb)

Supplementary Figure 3. Desalting of HS partial digest SEC fraction pools.

Porcine mucosal HS (a) 2 mer (b) 4mer and (c) 6, 8 and 10mer fraction pools from Superdex™ 30 SEC were desalted using two HiPrep™ 26/10 Desalting columns in series, run at flow rate of 5 ml / min in deionised water, whilst monitoring the elution profile by absorbance at λabs =232nm (—) and conductance (—). Data was recorded using the Unicorn 5.0 software and exported into SigmaPlot 11. Fractions across the first eluting peak, containing the HS saccharide, (—) were pooled to obtain minimum contamination with salt (ammonium hydrogen carbonate; —) whose elution position was confirmed by the peak with high conductance. (PDF 98 kb)

Supplementary Table 1. Monitoring heparinase III digestion of an HS variant by absorbance at λabs =232nm.

Porcine mucosal HS (413 mg) was digested with 0.1 mU/mg (5 mU/ml) of heparinase III for 2hrs, a further 10 mU heparinase III was added at 2 hurs and then again at 24 hrs, before heating the sample to 100 °C at 26 hrs to terminate digestion. (PDF 68 kb)

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Powell, A., Ahmed, Y., Yates, E. et al. Generating heparan sulfate saccharide libraries for glycomics applications. Nat Protoc 5, 821–833 (2010). https://doi.org/10.1038/nprot.2010.17

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