Functional analysis of conserved cysteines in heparan sulfate N-deacetylase-N-sulfotransferases

N-Deacetylase-N-sulfotransferases (NDANST) catalyze the two initial modifications of the polysaccharide precursor in the biosynthesis of heparin and heparan sulfate. These modifications are the gating steps in establishing growth factor protein-binding domains of these glycosaminoglycans. We have undertaken a structure-activity analysis of the 841-amino acid Golgi-luminal portion of the rat liver NDANST to localize the two enzymatic functions. Each activity can be assayed in vitro independently of the other when provided with the appropriate substrate, and N-ethylmaleimide treatment selectively inactivates the deacetylase activity. In this study, dithiothreitol treatment of the rat liver NDANST was shown to inactivate the sulfotransferase function, while stimulating deacetylase activity 2-3-fold over the native protein. Site-directed mutagenesis of the eight cysteine (Cys) residues in the rat liver NDANST that are conserved in the mouse mastocytoma protein produced three important findings regarding the localization of each enzymatic function: 1) derivatization of Cys486 with N-ethylmaleimide resulted in total inactivation of the deacetylase activity based on steric hindrance of the active site (this residue was shown not to be involved in enzymatic catalysis), 2) substitution of either Cys159 or Cys486 with alanine resulted in enhanced activity of the deacetylase to the level obtained by dithiothreitol treatment, and 3) alanine substitution of Cys818 or Cys828 completely inactivated the sulfotransferase activity, while substitution of Cys586 or Cys601 resulted in a 90% loss in activity. These findings suggest that the two enzymatic domains within the NDANST localize to different portions of the protein, with two disulfide pairs toward the COOH-terminal half of the protein necessary for the sulfotransferase activity, and Cys residues within the NH2-terminal half influencing or located near the active site of the deacetylase functionality.