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 language | English (US) |
|---|---|
| Pages (from-to) | 1217-1228 |
| Number of pages | 12 |
| Journal | Glycobiology |
| Volume | 14 |
| Issue number | 12 |
| DOIs | |
| State | Published - Dec 2004 |
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Keywords
- Heparan sulfate
- K5 polysaccharide
- NDST-1
- PAPS cycle
- Yeast
ASJC Scopus subject areas
- Biochemistry
Cite this
Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1). / Saribaş, A. Sami; Mobasseri, Ali; Pristatsky, Pavlo; Chen, Xi; Barthelson, Roger; Hakes, David; Wang, Jin.
In: Glycobiology, Vol. 14, No. 12, 12.2004, p. 1217-1228.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1)
AU - Saribaş, A. Sami
AU - Mobasseri, Ali
AU - Pristatsky, Pavlo
AU - Chen, Xi
AU - Barthelson, Roger
AU - Hakes, David
AU - Wang, Jin
PY - 2004/12
Y1 - 2004/12
N2 - 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.
AB - 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.
KW - Heparan sulfate
KW - K5 polysaccharide
KW - NDST-1
KW - PAPS cycle
KW - Yeast
UR - http://www.scopus.com/inward/record.url?scp=9744240193&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=9744240193&partnerID=8YFLogxK
U2 - 10.1093/glycob/cwh129
DO - 10.1093/glycob/cwh129
M3 - Article
VL - 14
SP - 1217
EP - 1228
JO - Glycobiology
JF - Glycobiology
SN - 0959-6658
IS - 12
ER -