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

12 Citations (Scopus)

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

Fingerprint

Sulfotransferases
Heparitin Sulfate
Yeast
Polysaccharides
Heparin
Yeasts
tyrosine-ester sulfotransferase
Biosynthesis
Enzymes
Pasteurella multocida
Disaccharides
Saccharomyces cerevisiae
Substrates
Sugars
Liver
Escherichia coli
Rats
heparosan
Proteins
capsular polysaccharide K5

Keywords

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

ASJC Scopus subject areas

  • Biochemistry

Cite this

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

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 journalArticle

Saribaş, AS, 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, vol. 14, no. 12, pp. 1217-1228. https://doi.org/10.1093/glycob/cwh129
Saribaş, A. Sami ; Mobasseri, Ali ; Pristatsky, Pavlo ; Chen, Xi ; Barthelson, Roger ; Hakes, David ; Wang, Jin. / Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1). In: Glycobiology. 2004 ; Vol. 14, No. 12. pp. 1217-1228.
@article{163a041a22b24838a3862b353bde0b03,
title = "Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1)",
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.",
keywords = "Heparan sulfate, K5 polysaccharide, NDST-1, PAPS cycle, Yeast",
author = "Saribaş, {A. Sami} and Ali Mobasseri and Pavlo Pristatsky and Xi Chen and Roger Barthelson and David Hakes and Jin Wang",
year = "2004",
month = "12",
doi = "10.1093/glycob/cwh129",
language = "English (US)",
volume = "14",
pages = "1217--1228",
journal = "Glycobiology",
issn = "0959-6658",
publisher = "Oxford University Press",
number = "12",

}

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 -