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A Rapid, Nonradioactive Assay for Measuring Heparan Sulfate C-5 Epimerase Activity Using Hydrogen/Deuterium Exchange-Mass Spectrometry

  • Ponnusamy BabuEmail author
  • Xylophone V. Victor
  • Karthik Raman
  • Balagurunathan Kuberan
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1229)

Abstract

Heparin and heparan sulfate (HS) glycosaminoglycans have important roles in anticoagulation, human development, and human diseases. HS C5-epimerase, which catalyzes the epimerization of GlcA to IdoA, is a crucial enzyme involved in the biosynthesis of heparin-related biomolecules. Here, we describe a detailed method for measuring the total activity of HS C5-epimerase that involves the following steps: H/D exchange upon epimerization of the substrate with HS C5-epimerase, low-pH nitrous acid treatment of the substrate, the separation of low-pH nitrous acid-cleaved disaccharides using HPLC, and mass spectrometry analysis. This nonradioactive method is rapid and sensitive and, importantly, allows us to study the reversible nature of HS C5-epimerase.

Key words

Glycosaminoglycans Heparan sulfate C5-epimerase H/D exchange Mass spectrometry 

Notes

Acknowledgements

This work was supported in part by NIH grants (P01HL107152 and R01GM075168) to B.K. and by the NIH fellowship F31CA168198 to K.R. P.B. thanks Centre for Cellular and Molecular Platforms for support.

References

  1. 1.
    Chen R, Mias GI, Li-Pook-Than J, Jiang L, Lam HY, Miriami E, Karczewski KJ, Hariharan M, Dewey FE, Cheng Y, Clark MJ, Im H, Habegger L, Balasubramanian S, O’Huallachain M, Dudley JT, Hillenmeyer S, Haraksingh R, Sharon D, Euskirchen G, Lacroute P, Bettinger K, Boyle AP, Kasowski M, Grubert F, Seki S, Garcia M, Whirl-Carrillo M, Gallardo M, Blasco MA, Greenberg PL, Snyder P, Klein TE, Altman RB, Butte AJ, Ashley EA, Gerstein M, Nadeau KC, Tang H, Snyder M (2012) Personal omics profiling reveals dynamic molecular and medical phenotypes. Cell 148:1293–1307PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Walsh GM, Rogalski JC, Klockenbusch C, Kast J (2010) Mass spectrometry-based proteomics in biomedical research: emerging technologies and future strategies. Expert Rev Mol Med 12:e30PubMedCrossRefGoogle Scholar
  3. 3.
    Liesener A, Karst U (2005) Monitoring enzymatic conversions by mass spectrometry: a critical review. Anal Bioanal Chem 382:1451–1464PubMedCrossRefGoogle Scholar
  4. 4.
    Wu J, Takayama S, Wong CH, Siuzdak G (1997) Quantitative electrospray mass spectrometry for the rapid assay of enzyme inhibitors. Chem Biol 4:653–657PubMedCrossRefGoogle Scholar
  5. 5.
    Babu P, Victor XV, Nelsen E, Nguyen TK, Raman K, Kuberan B (2011) Hydrogen/deuterium exchange-LC-MS approach to characterize the action of heparan sulfate C5-epimerase. Anal Bioanal Chem 401:237–244PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Esko JD, Selleck SB (2002) Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu Rev Biochem 71:435–471PubMedCrossRefGoogle Scholar
  7. 7.
    Li JP, Gong F, Hagner-McWhirter A, Forsberg E, Abrink M, Kisilevsky R, Zhang X, Lindahl U (2003) Targeted disruption of a murine glucuronyl C5-epimerase gene results in heparan sulfate lacking L-iduronic acid and in neonatal lethality. J Biol Chem 278:28363–28366PubMedCrossRefGoogle Scholar
  8. 8.
    Guerrini M, Beccati D, Shriver Z, Naggi A, Viswanathan K, Bisio A, Capila I, Lansing JC, Guglieri S, Fraser B, Al-Hakim A, Gunay NS, Zhang Z, Robinson L, Buhse L, Nasr M, Woodcock J, Langer R, Venkataraman G, Linhardt RJ, Casu B, Torri G, Sasisekharan R (2008) Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events. Nat Biotechnol 26:669–675PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Naggi A, Torri G, Casu B, Oreste P, Zoppetti G, Li JP, Lindahl U (2001) Toward a biotechnological heparin through combined chemical and enzymatic modification of the Escherichia coli K5 polysaccharide. Semin Thromb Hemost 27:437–443PubMedCrossRefGoogle Scholar
  10. 10.
    Xu Y, Masuko S, Takieddin M, Xu H, Liu R, Jing J, Mousa SA, Linhardt RJ, Liu J (2011) Chemoenzymatic synthesis of homogeneous ultralow molecular weight heparins. Science 334:498–501PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Hagner-McWhirter A, Hannesson HH, Campbell P, Westley J, Roden L, Lindahl U, Li JP (2000) Biosynthesis of heparin/heparan sulfate: kinetic studies of the glucuronyl C5-epimerase with N-sulfated derivatives of the Escherichia coli K5 capsular polysaccharide as substrates. Glycobiology 10:159–171PubMedCrossRefGoogle Scholar
  12. 12.
    Raedts J, Lundgren M, Kengen SW, Li JP, van der Oost J (2013) A novel bacterial enzyme with D-glucuronyl C5-epimerase activity. J Biol Chem 288:24332–24339PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    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 287:20996–21002PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Li J, Hagner-McWhirter A, Kjellen L, Palgi J, Jalkanen M, Lindahl U (1997) Biosynthesis of heparin/heparan sulfate. cDNA cloning and expression of D-glucuronyl C5-epimerase from bovine lung. J Biol Chem 272:28158–28163PubMedCrossRefGoogle Scholar
  15. 15.
    Shively JE, Conrad HE (1970) Stoichiometry of the nitrous acid deaminative cleavage of model amino sugar glycosides and glycosaminoglycuronans. Biochemistry 9:33–43PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Ponnusamy Babu
    • 1
    Email author
  • Xylophone V. Victor
    • 2
    • 3
  • Karthik Raman
    • 2
    • 3
  • Balagurunathan Kuberan
    • 2
    • 3
  1. 1.Glycomics and GlycoproteomicsCentre for Cellular and Molecular Platforms, NCBS-TIFRBangaloreIndia
  2. 2.Department of Medicinal ChemistryUniversity of UtahSalt Lake CityUSA
  3. 3.Department of BioengineeringUniversity of UtahSalt Lake CityUSA

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