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Glycobiology Advance Access published online on July 14, 2004
Glycobiology, doi:10.1093/glycob/cwh129
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Received February 4, 2004
Revised July 6, 2004
Accepted July 7, 2004

ORIGINAL ARTICLES

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

A. Sami Saribas 1*, Ali Mobasseri 1, Pavlo Pristatsky 1, Xi Chen 1, Roger Barthelson 1, David Hakes 1, Jin Wang 1

1 Neose Technologies Inc., 102 Witmer Rd., Horsham PA 19044

* To whom correspondence should be addressed. E-mail: ssaribas{at}neose.com.


  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 (DNSH) and completely desulfated, N-acetylated heparan sulfate (CDSNAcHS) are all good substrates for the rNDST-1. However, N-desulfated, N-acetylated heparin (NDSNAcH) 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 (Burkart et al. (2000) J. Org. Chem. 65, 5565-5574), 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.

Keywords: Heparan sulfate; K 5 polysaccharide; NDST-1; PAPS cycle; yeast.
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[Abstract] [Full Text] [PDF]


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