Skip to main content
Book cover

Glycosaminoglycans pp 3–11Cite as

One-Pot Enzymatic Synthesis of Heparin from N-Sulfoheparosan

Part of the Methods in Molecular Biology book series (MIMB,volume 2303)

Abstract

Heparin, a glycosaminoglycan-based anticoagulant drug, is prepared as an extract of animal tissues. Heparosan, an Escherichia coli (E. coli) K5 capsular polysaccharide with the structure →4)-β-D-glucuronic acid (1 → 4)-β-D-N-acetylglucosamine (1→, corresponds to the precursor backbone in the Golgi-based biosynthesis of heparin. Anticoagulant heparin is prepared in a one-pot synthesis using a chemically prepared derivative of heparosan called N-sulfoheparosan (NSH), recombinant Golgi enzymes expressed in E. coli, and the 3-phosphoadenosine-5-phosphosulfate (PAPS) cofactor.

Key words

  • Heparin
  • Heparan sulfate
  • Heparosan
  • Enzymes
  • 3-Phosphoadenosine-5-phosphosulfate
  • Biosynthesis

This is a preview of subscription content, access via your institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-0716-1398-6_1
  • Chapter length: 9 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   169.00
Price excludes VAT (USA)
  • ISBN: 978-1-0716-1398-6
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   219.99
Price excludes VAT (USA)
Hardcover Book
USD   299.99
Price excludes VAT (USA)
Learn about institutional subscriptions
Fig. 1

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Bhaskar U, Sterner E, Hickey AM, Onishi A, Zhang F, Dordick JS, Linhardt RJ (2012) Engineering of routes to heparin and related polysaccharides. Appl Microbiol Biot 93:1–16

    CrossRef  Google Scholar 

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

    CrossRef  CAS  Google Scholar 

  3. Bigelow WG (1990) Mysterious heparin: the key to open heart surgery. McGraw-Hill Ryerson, Toronto, ON

    Google Scholar 

  4. Keire D, Mulloy B, Chase C, Al-Hakim A, Cairatti D, Gray E, Hogwood J, Morris TS, Mourão P, Da Luz Carvalho Soares M, Szajek A (2015) Diversifying the global heparin supply chain: reintroduction of bovine heparin in the United States. Pharm Technol 39(11):28–35

    Google Scholar 

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

    CrossRef  CAS  Google Scholar 

  6. Szajek AY, Chess E, Johansen K, Gratzl G, Gray E, Keire D, Linhardt RJ, Liu J, Morris T, Mulloy B, Nasr M, Shriver Z, Torralba P, Viskov C, Williams R, Woodcock J, Workman W, Al-Hakim A (2016) The US regulatory and pharmacopeia response to the global heparin contamination crisis. Nat Biot 34:625–630

    CrossRef  CAS  Google Scholar 

  7. Lindahl U (2014) A personal voyage through the proteoglycan field. Matrix Biol 35:3–7

    CrossRef  CAS  Google Scholar 

  8. Li JP, Gong F, El Darwish K, Jalkanen M, Lindahl U (2001) Characterization of the D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate. J Biol Chem 276:20069–20077

    CrossRef  CAS  Google Scholar 

  9. Zhang L, Beeler DL, Lawrence R, Lech M, Liu J, Davis JC, Shriver Z, Sasisekharan R, Rosenberg RD (2001) 6-O-sulfotransferase-1 represents a critical enzyme in the anticoagulant heparan sulfate biosynthetic pathway. J Biol Chem 276:42311–42321

    CrossRef  CAS  Google Scholar 

  10. Kuberan B, Lech M, Borjigin J, Rosenberg RD (2004) Light-induced 3-O-sulfotransferase expression alters pineal heparan sulfate fine structure. A surprising link to circadian rhythm. J Biol Chem 279:5053–5054

    CrossRef  CAS  Google Scholar 

  11. Chen J, Avci FY, Muñoz EM, McDowell LM, Chen M, Pedersen LC, Zhang L, Linhardt RJ, Liu J (2005) Enzymatic redesigning of biologically active heparan sulfate. J Biol Chem 280:42817–42825

    CrossRef  CAS  Google Scholar 

  12. Zhang X, Pagadala V, Jester HM, Lim AM, Pham TQ, Goulas AMP, Liu J, Linhardt RJ (2017) Chemoenzymatic synthesis of heparan sulfate and heparin oligosaccharides and NMR analysis: paving the way to a diverse library for glycobiologists. Chem Sci 8:7883–8466

    CrossRef  Google Scholar 

  13. Vaidyanathan D, Williams A, Dordick JS, Koffas MAG, Linhardt RJ (2016) Engineered heparins as new anticoagulant drugs. Bioeng Transl Med 2:17–30

    CrossRef  Google Scholar 

  14. Xu Y, Chandarajoti K, Zhang X, Pagadala V, Dou W, Hoppensteadt DM, Sparkenbaugh E, Cooley B, Daily S, Key NS, Severynse-Stevens D, Fareed J, Linhardt RJ, Pawlinski R, Liu J (2017) Synthetic oligosaccharides can replace animal-sourced low-molecular weight heparins. Sci Transl Med 9:eaan5954

    CrossRef  Google Scholar 

  15. Bhaskar U, Li G, Fu L, Onishi A, Suflita M, Dordick JS, Linhardt RJ (2015) Combinatorial one-pot chemoenzymatic synthesis of heparin. Carbohydr Polym 122:399–407

    CrossRef  CAS  Google Scholar 

  16. Wang Z, Ly M, Zhang F, Zhong W, Suen A, Dordick JS, Linhardt RJ (2010) E. coli K5 fermentation and the preparation of heparosan, a bioengineered heparin precursor. Biotechnol Bioeng 107:968–977

    Google Scholar 

  17. Wang Z, Li J, Cheong S, Bhaskar U, Akihiro O, Zhang F, Dordick JS, Linhardt RJ (2011) Response surface optimization of the heparosan N-deacetylation in producing bioengineered heparin. J Biotechnol 156:188–196

    CrossRef  CAS  Google Scholar 

  18. Zhang J, Suflita M, Li G, Zhong W, Li L, Dordick JS, Linhardt RJ, Zhang F (2015) High cell density cultivation of recombinant Escherichia coli strains expressing 2-O-sulfotransferase and C5-epimerase for the production of bioengineered heparin. Appl Biochem Biotechnol 175(6):2986–2995

    CrossRef  CAS  Google Scholar 

  19. Bhaskar U, Paul P, Lin L, Rosa MD, Dordick JS, Linhardt RJ (2013) High cell density cultivation of a recombinant E. coli strain expressing a key enzyme in bioengineered heparin production. Appl Microbiol Biotechnol 97(9):3893–3900

    CrossRef  Google Scholar 

  20. Zhang J, Suflita M, Fiaschetti CM, Li G, Li L, Zhang F, Dordick JS, Linhardt RJ (2015) High cell density cultivation of a recombinant Escherichia coli strain expressing a 6-O-sulfotransferase for the production of bioengineered heparin. J Appl Microbiol 118(1):92–98

    CrossRef  CAS  Google Scholar 

  21. Jin W, Li S, Chen J, Liu B, Li J, Li X, Zhang F, Linhardt RJ, Zhong W (2018) Increased soluble heterologous expression of a rat brain 3-O-sulfotransferase1 – a key enzyme for heparin biosynthesis. Protein Expr Purif 151:23–29

    CrossRef  CAS  Google Scholar 

  22. Lohse DL, Linhardt RJ (1992) Purification and characterization of heparin lyases from flavobacterium heparinum. J Biol Chem 267:24347–24355

    CrossRef  CAS  Google Scholar 

  23. Yang B, Chang Y, Weyers AM, Sterner E, Linhardt RJ (2012) Disaccharide analysis of glycosaminoglycan mixtures by ultra-performance liquid chromatography-mass spectrometry. J Chromatogr A 1225:91–98

    CrossRef  CAS  Google Scholar 

  24. Li G, Steppich J, Wang Z, Sun Y, Xue C, Linhardt RJ, Li L (2014) Bottom-up LMWH analysis using LC-FTMS for extensive characterization. Anal Chem 86:6626–6632

    CrossRef  CAS  Google Scholar 

  25. Sterner E, Li L, Paul P, Beaudet JM, Liu J, Linhardt RJ, Dordick JS (2014) Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity. Anal Bioanal Chem 406:525–536

    CrossRef  CAS  Google Scholar 

  26. Linhardt RJ (1994) Analysis of glycosaminoglycans with polysaccharide lyases. In: Varki A (ed) Current protocols in molecular biology, analysis of glycoconjugates, vol 2. Wiley Interscience, Hoboken, NJ, pp 17.13.17–17.13.32

    Google Scholar 

Download references

Acknowledgments

The authors are grateful for funding from the National Institutes of Health in the form of grants # DK111958 and CA231074.

Author information

Authors and Affiliations

  1. Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA

    Li Fu & Robert J. Linhardt

Authors
  1. Li Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert J. Linhardt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Robert J. Linhardt .

Editor information

Editors and Affiliations

  1. Departments of Biology, Bioengineering, and Medicinal Chemistry, University of Utah, Salt Lake City, UT, USA

    Kuberan Balagurunathan

  2. Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA

    Hiroshi Nakato

  3. Department of Medicinal Chemistry and Institute for Structural Biology, Drug Discovery and Development, Virginia Commonwealth University, Richmond, VA, USA

    Umesh Desai

  4. Department of Neurobiology & Department of Nutrition & Integrative Physiology, University of Utah, Salt Lake City, UT, USA

    Yukio Saijoh

Rights and permissions

Reprints and Permissions

Copyright information

© 2022 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Fu, L., Linhardt, R.J. (2022). One-Pot Enzymatic Synthesis of Heparin from N-Sulfoheparosan. In: Balagurunathan, K., Nakato, H., Desai, U., Saijoh, Y. (eds) Glycosaminoglycans. Methods in Molecular Biology, vol 2303. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1398-6_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1398-6_1

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1397-9

  • Online ISBN: 978-1-0716-1398-6

  • eBook Packages: Springer Protocols