Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1).
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 Mn(2+) 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.
Saribaş
A Sami
AS
Neose Technologies, Inc., 102 Witmer Road, Horsham, PA 19044, USA. ssaribas@neose.com
Mobasseri
Ali
A
Pristatsky
Pavlo
P
Chen
Xi
X
Barthelson
Roger
R
Hakes
David
D
Wang
Jin
J
D000818:Animals; D003001:Cloning, Molecular; D008099:Liver / Q000201:enzymology; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; D010724:Phosphoadenosine Phosphosulfate; D015238:Sulfotransferases; C021350:heparitin sulfotransferase