Glycobiology Advance Access published online on November 1, 2002
Glycobiology, doi:10.1093/glycob/cwg011
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© 2002 Oxford University Press
ORIGINAL ARTICLES
1 Division of Nephrology, University Hospital St. Radboud, Nijmegen, The Netherlands A new assay was developed to measure the N-deacetylase activity of the glucosaminyl N-deacetylase/N-sulfotransferases (NDST), which are key enzymes in sulfation of heparan sulfate/heparin. The assay is based on the recognition of NDST-generated N-unsubstituted glucosamine units in Escherichia coli K5 capsular polysaccharide or in heparan sulfates by monoclonal antibody JM-403. Substrate specificity and potential product inhibition of the NDST isoforms 1 and 2 were analyzed by comparing lysates of human 293 kidney cells stable transfected with mouse NDST-1 or -2. We thus found heparan sulfates to be excellent substrates for both NDST enzymes. Both NDST-1 and -2 N-deacetylate heparan sulfate from human aorta (0.6 sulfate groups/disaccharide) with comparable high efficiency, apparent Km values of 0.35 and 0.76 µM (calculation based on [HexA]), being lower (representing a higher affinity) than those for K5 polysaccharide (13.3 and 4.7 µM respectively). Comparison of various heparan sulfate preparations and the unsulfated K5 polysaccharide as substrates indicate that both NDST-1 and -2 can differentially N-sulfate polysaccharides already modified to some extent by various other enzymes involved in HS/heparin synthesis. Both enzymes were equally inhibited by N-sulfated sequences (
Revised on September 6, 2002
Accepted on September 6, 2002
Antibody-based assay for N-deacetylase activity of heparan sulfate/heparin N-deacetylase/N-sulfotrans-ferase (NDST). Novel characteristics of NDST-1 and -2
2 Department of Medical Biochemistry and Microbiology, University of Uppsala, Sweden
3 Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden
6 sugar residues) present in N-sulfated K5, N-deacetylated N-resulfated heparan sulfate and heparin. Main findings were confirmed in the conventional N-deacetylase assay measuring the release of 3H-acetate of radiolabeled K5 or HS as substrates. We furthermore showed that NDST N-deacetylase activity in crude cell/tissue lysates can be partially blocked by endogenous HS/heparin. We speculate that in HS biosynthesis, some NDST variants initiate HS modification/sulfation reactions, while other (or the same) NDST isoforms later on fill in or extend already modified HS sequences.
Key words: biosynthesis / heparan / heparin / N-deacetylase / sulfotransferase
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