Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1)

A. Sami Saribaş, Ali Mobasseri, Pavlo Pristatsky, Xi Chen, Roger Barthelson, David Hakes, Jin Wang

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

Heparan sulfate/heparin N-deacetylase/N-sulfotransferase-1 (NDST-1) is a critical enzyme involved in heparan sulfate/heparin biosynthesis. This dual-function enzyme modifies the GlcNAc-GlcA disaccharide repeating sugar backbone to make N-sulfated heparosan. N-sulfation is an absolute requirement for the subsequent epimerization and O-sulfation steps in heparan sulfate/heparin biosynthesis. We have expressed rat liver (r) NDST-1 in Saccharomyces cerevisiae as a soluble protein. The yeast-expressed enzyme has both N-deacetylase and N-sulfotransferase activities. N-acetyl heparosan, isolated from Escherichia coli K5 polysaccharide, de-N-sulfated heparin and completely desulfated N-acetylated heparan sulfate are all good substrates for the rNDST-1. However, N-desulfated, N-acetylated heparin is a poor substrate. The rNDST-1 was partially purified on heparin Sepharose CL-6B. Purified rNDST-1 requires Mn2+ for its enzymatic activity, can utilize PAPS regenerated in vitro by the PAPS cycle (PAP plus para-nitrophenylsulfate in the presence of arylsulfotransferase IV), and with the addition of exogenous PAPS is capable of producing 60-65% N-sulfated heparosan from E. coli K5 polysaccharide or Pasteurella multocida polysaccharide.

Original languageEnglish (US)
Pages (from-to)1217-1228
Number of pages12
JournalGlycobiology
Volume14
Issue number12
DOIs
StatePublished - Dec 2004

Keywords

  • Heparan sulfate
  • K5 polysaccharide
  • NDST-1
  • PAPS cycle
  • Yeast

ASJC Scopus subject areas

  • Biochemistry

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    Saribaş, A. S., Mobasseri, A., Pristatsky, P., Chen, X., Barthelson, R., Hakes, D., & Wang, J. (2004). Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1). Glycobiology, 14(12), 1217-1228. https://doi.org/10.1093/glycob/cwh129