Skip to main content

Advertisement

SpringerLink
Log in

Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

O-sulfotransferases (OSTs) are critical enzymes in the cellular biosynthesis of the biologically and pharmacologically important heparan sulfate and heparin. Recently, these enzymes have been cloned and expressed in bacteria for application in the chemoenzymatic synthesis of glycosaminoglycan-based drugs. OST activity assays have largely relied on the use of radioisotopic methods using [35S] 3′-phosphoadenosine-5′-phosphosulfate and scintillation counting. Herein, we examine alternative assays that are more compatible with a biomanufacturing environment. A high throughput microtiter-based approach is reported that relies on a coupled bienzymic colorimetric assay for heparan sulfate and heparin OSTs acting on polysaccharide substrates using arylsulfotransferase-IV and p-nitrophenylsulfate as a sacrificial sulfogroup donor. A second liquid chromatography-mass spectrometric assay, for heparan sulfate and heparin OSTs acting on structurally defined oligosaccharide substrates, is also reported that provides additional information on the number and positions of the transferred sulfo groups within the product. Together, these assays allow quantitative and mechanistic information to be obtained on OSTs that act on heparan sulfate and heparin precursors.

Herapan sulfate O-sulfotranferase coupled enzyme colorimetric assay

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Similar content being viewed by others

Abbreviations

2-OST:

2-O sulfotransferase

3-OST-1:

3-O sulfotransferase isoform 1

6-OST-1:

6-O sulfotransferase isoform 1

6-OST-3:

6-O sulfotransferase isoform 3

Ac:

Acetyl

AST-IV:

Aryl-sulfotransferase

AT:

Antithrombin III

C5-epi:

C5 epimerase

CDSNS HP:

Completely de-O-sulfonated heparin

cGMP:

Current good manufacturing process

FTMS:

Fourier transform mass spectrometry

GAGs:

Glycosaminoglycans

GlcA:

Glucuronic acid

GlcN:

Glucosamine

HILIC:

Hydrophilic interaction liquid chromatography

IdoA:

Iduronic acid

LC-MS:

Liquid chromatography-mass spectrometry

NDST:

N-deacetylase N-sulfotransferase

PAP:

3′-adenosine 5′-phosphate

PAPS:

3′-adenosine 5′-phosphosulfate

PNP:

para-nitrophenol

PNPS:

para-nitrophenylsulfate

S:

Sulfo

References

  1. Esko JD, Kimata K, Lindahl U (2008) Proteoglycans and sulfated glycosaminoglycans. In: Varki A (ed) Essentials of glycobiology, 2nd edn, 16. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  2. Ly M, Laremore TN, Linhardt RJ (2010) Proteoglycomics: recent progress and future challenges. OMICS 14:389–399

    Article  CAS  Google Scholar 

  3. Capila I, Linhardt RJ (2002) Heparin–protein interactions. Angew Chem Int Edn 41:391–412

    Article  Google Scholar 

  4. Kreuger J, Spillman D, Li J-P, Lindahl U (2006) Interactions between heparan sulfate and proteins: the concept of specificity. J Cell Biol 174:323–327

    Article  CAS  Google Scholar 

  5. Gallagher JT (1989) The extended family of proteoglycans: social residents of the pericellular zone. Curr Opin Cell Biol 1:1201–1218

    Article  CAS  Google Scholar 

  6. Hardingham TE, Fosang AJ (1992) Proteoglycans: many forms and many functions. FASEB J 6:861–870

    CAS  Google Scholar 

  7. Bernfield M, Götte M, Park PW, Reizes O, Fitzgerald ML, Lincecum J, Zako M (1999) Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 68:729–777

    Article  CAS  Google Scholar 

  8. Garner OB, Yamaguchi Y, Esko JD, Videm V (2008) Small changes in lymphocyte development and activation in mice through tissue-specific alteration of heparan sulphate. Immunology 125:420–429

    Article  CAS  Google Scholar 

  9. Islam T, Butler M, Sikkander SA, Toida T, Linhardt RJ (2002) Further evidence that periodate cleavage of heparin occurs primarily through the antithrombin binding site. Carbohydr Res 337:2239–2243

    Article  CAS  Google Scholar 

  10. Jordan RE, Oosta GM, Gardner WT, Rosenberg RD (1980) The binding of low molecular weight heparin to hemostatic enzymes. J Biol Chem 255:10081–10090

    CAS  Google Scholar 

  11. Linhardt RJ (2003) Heparin: structure and activity. J Med Chem 46:2551–2554

    Article  CAS  Google Scholar 

  12. Zhang F, Yang B, Ly M, Solakyildirim K, Xiao Z, Wang Z, Beaudet JM, Torelli AY, Dordick JS, Linhardt RJ (2011) Structural characterization of heparins from different commercial sources. Anal Bioanal Chem 401:2793–2803

    Article  CAS  Google Scholar 

  13. Liu H, Zhang Z, Linhardt RJ (2009) Lessons learned from the contamination of heparin. Nat Prod Rep 26:313–321

    Article  CAS  Google Scholar 

  14. Wang Z, Zhang Z, McCallum SA, Linhardt RJ (2010) NMR quantification for monitoring heparosan K5 capsular polysaccharide production. Anal Biochem 398:275–277

    Article  CAS  Google Scholar 

  15. Wei Z, Swiedler SJ, Ishihara M, Orellana A, Hirschberg CB (1993) A single protein catalyzes both N-deacetylation and N-sulfation during the biosynthesis of heparan sulfate. Proc Natl Acad Sci U S A 90:3885–3888

    Article  CAS  Google Scholar 

  16. Wang Z, Yang B, Zhang Z, Ly M, Takieddin M, Mousa S, Liu J, Dordick JS, Linhardt RJ (2011) Control of the heparosan N-deacetylation leads to an improved bioengineered heparin. Appl Microbiol Biotech 91:91–99

    Article  CAS  Google Scholar 

  17. DeAngelis PL, Liu J, Linhardt RJ (2013) Chemoenzymatic synthesis of glycosaminoglycans: re-creating, re-modeling, and re-designing nature’s longest or most complex carbohydrate chains. Glycobiology 23:764–777

    Article  CAS  Google Scholar 

  18. Esko JD, Selleck SB (2002) Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu Rev Biochem 71:435–471

    Article  CAS  Google Scholar 

  19. Paul P, Suwan J, Liu J, Dordick JS, Linhardt RJ (2012) Recent advances in sulfotransferase enzyme activity assays. Anal Bioanal Chem 403(6):1491–1500

    Article  CAS  Google Scholar 

  20. Limtiaco JFK, Beni S, Jones CJ, Langeslay DJ, Larive CK (2011) NMR methods to monitor the enzymatic depolymerization of heparin. Anal Bioanal Chem 399:593–603

    Article  CAS  Google Scholar 

  21. Nilsson M, Khajeh M, Botana A, Bernstein MA, Morris GA (2009) Diffusion NMR and trilinear analysis in the study of reaction kinetics. Chem Commun 1252–1254

  22. Gamage N, Barnett A, Hempel N, Duggleby RG, Windmill KF, Martin JL, McManus ME (2006) Human sulfotransferases and their role in chemical metabolism. Toxicol Sci 90:5–22

    Article  CAS  Google Scholar 

  23. Wang Z, Dordick JS, Linhardt RJ (2011) E. coli K5 heparosan fermentation and improvement by genetic engineering. Bioengin Bugs 2:1–15

    Article  Google Scholar 

  24. Satishchandran C, Markham GD (1989) Adenosine-5′-phosphosulfate kinase from Escherichia coli K12. Purification, characterization, and identification of a phosphorylated enzyme intermediate. J Biol Chem 264:15012–15021

    CAS  Google Scholar 

  25. Karamohamed S, Milsson J, Nourizad K, Ronaghi M, Pettersson B, Nyrén P (1999) Production, purification, and luminometric analysis of recombinant Saccharomyces cerevisiae MET3 adenosine triphosphate sulfurylase expressed in Escherichia coli. Prot Expr Purif 15:381–388

    Article  CAS  Google Scholar 

  26. Xu Y, Pempe EH, Liu J (2012) Chemoenzymatic synthesis of heparin oligosaccharides with both anti-factor Xa and anti-factor IIa activities. J Biol Chem 287:29054–29061

    Article  CAS  Google Scholar 

  27. Duncan M, Liu M, Fox C, Liu J (2006) Characterization of the N-deacetylase domain from the heparan sulfate N-deacetylase/N-sulfotransferase 2. Biochem Biophys Res Commun 339:1232–1237

    Article  CAS  Google Scholar 

  28. Pervin A, Gallo C, Jandik KA, Han X-J, Linhardt RJ (1995) Preparation and structural characterization of large heparin-derived oligosaccharides. Glycobiology 5:83–95

    Article  CAS  Google Scholar 

  29. Hileman RE, Smith AE, Toida T, Linhardt RJ (1997) Preparation and structure of heparin lyase-derived heparan sulfate oligosaccharides. Glycobiology 7:231–239

    Article  CAS  Google Scholar 

  30. Liu R, Liu J (2011) Enzymatic placement of 6-O-sulfo groups in heparan sulfate. Biochemistry 50:4382–4391

    Article  CAS  Google Scholar 

  31. Shailubhai K, Singh RK, Schmuke JJ, Jacob GS (1996) An enzymatic procedure for the preparation and purification of 3′-phosphoadenosine 5′-phospho-[35S]sulfate ([35S]PAPS): applications in syntheses of 8-azido and 8-bromo derivatives of [35S]PAPS. Anal Biochem 243:165–170

    Article  CAS  Google Scholar 

  32. Burkart MD, Wong C–H (1999) A continuous assay for the spectrophotometric analysis of sulfotransferases using aryl sulfotransferase IV. Anal Biochem 274:131–137

    Article  CAS  Google Scholar 

  33. Burkart MD, Izumi M, Chapman E, Lin C–H, Wong C–H (2000) Regeneration of PAPS for the enzymatic synthesis of sulfated oligosaccharides. J Org Chem 65:5565–5574

    Article  CAS  Google Scholar 

  34. Pi N, Armstrong JI, Bertozzi CR, Leary JA (2002) Kinetic analysis of NodST sulfotransferase using an electrospray ionization mass spectrometry assay. Biochemistry 41:13283–13288

    Article  CAS  Google Scholar 

  35. Xiong J, Bhaskar U, Li G, Fu L, Li L, Zhang F, Dordick JS, Linhardt RJ (2013) Immobilized enzymes to convert N-sulfo, N-acetyl heparosan to a critical intermediate in the production of bioengineered heparin. J Biotechnol 167:241–247

    Article  CAS  Google Scholar 

  36. Xu D, Song D, Pederson LC, Liu J (2007) Mutational study of heparan sulfate 2-O-sulfotransferase and chondroitin sulfate 2-O-sulfotransferase. J Biol Chem 282:8356–8367

    Article  CAS  Google Scholar 

  37. Marshall AD, Darbyshire JF, Hunter AP, McPhie P, Jakoby WB (1997) Control of activity through oxidative modification at the conserved residue Cys66 of aryl sulfotransferase IV. J Biol Chem 272:9153–9160

    Article  CAS  Google Scholar 

  38. Habuchi H, Tanaka M, Habuchi O, Yoshida K, Suzuki H, Ban K, Kimata K (2000) The occurrence of three isoforms of heparan sulfate 6-O-sulfotransferase having different specificities for hexuronic acid adjacent to the targeted N-sulfoglucosamine. J Biol Chem 275:2859–2868

    Article  CAS  Google Scholar 

  39. Tran VM, Nguyen TKN, Raman K, Kuberan B (2011) Applications of isotopes in advancing structural and functional heparanomics. Anal Bioanal Chem 399:559–570

    Article  CAS  Google Scholar 

  40. Sheng J, Xu Y, Dulaney SB, Huang X, Liu J (2012) Uncovering biphasic catalytic mode of C5-epimerase in heparan sulfate biosynthesis. J Biol Chem 287:20996–21002

    Article  CAS  Google Scholar 

  41. Li K, Bethea HN, Liu J (2010) Using engineered 2-O-sulfotransferase to determine the activity of heparan sulfate C5-epimerase and its mutants. J Biol Chem 285:11106–11113

    Article  CAS  Google Scholar 

  42. Linhardt RJ, Wang HM, Loganathan D, Bae JH (1992) Search for the heparin antithrombin III-binding site precursor. J Biol Chem 267:2380–2387

    CAS  Google Scholar 

  43. Edavettal SC, Lee KA, Negishi M, Linhardt RJ, Liu J, Pederson LC (2004) Structural analysis of the sulfotransferase (3-OST-3) involved in the biosynthesis of an entry receptor for herpes simplex virus 1. J Biol Chem 279:25789–25797

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Eric Sterner, Priscilla Paul, Robert J. Linhardt & Jonathan S. Dordick

  2. Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Lingyun Li, Julie M. Beaudet & Robert J. Linhardt

  3. Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Robert J. Linhardt & Jonathan S. Dordick

  4. Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Robert J. Linhardt & Jonathan S. Dordick

  5. Department of Material Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Jonathan S. Dordick

  6. Department of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, 27599, USA

    Jian Liu

Authors
  1. Eric Sterner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lingyun Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Priscilla Paul
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Julie M. Beaudet
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jian Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Robert J. Linhardt
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jonathan S. Dordick
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Robert J. Linhardt or Jonathan S. Dordick.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 855 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sterner, E., Li, L., Paul, P. et al. Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity. Anal Bioanal Chem 406, 525–536 (2014). https://doi.org/10.1007/s00216-013-7470-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-013-7470-4

Keywords

Search

Navigation