The biosynthesis of
heparin and
heparan sulfate involves a series of
polymer-modification reactions that is initiated by N-deacetylation and subsequent N-sulfation of
N-acetylglucosamine residues. These reactions are catalysed by a combined N-deacetylase/N-
sulfotransferase.
Proteins expressing both activities have previously been purified from mouse
mastocytoma, which generates
heparin, and from rat liver, which produces
heparan sulfate. In the present study, the mouse
mastocytoma enzyme has been expressed in the human kidney cell line, 293, to investigate whether it could promote modification of the endogenous
heparan sulfate precursor
polysaccharide into a heparan-like molecule. The N-deacetylase activity of the stably transfected cell clones as approximately 8-fold higher, on a cell-
protein basis, than that of control cells, while the N-
sulfotransferase activity was increased approximately 2.5 fold. The amounts of
glycosaminoglycans synthesized were the same in control and transfected cells, measured as incorporation of [3H]-
glucosamine, whereas 35S-labeled
glycosaminoglycans were approximately 50% increased in transfected cells, with an increased relative content of
heparin sulfate. Structural analysis demonstrated the the
glucosamine units of the "
heparan sulfate" from transfected cells were almost exclusively N-sulfated, as expected for
heparin, whereas more than half of the
glucosamine units of the control
polysaccharide remained N-acetylated. Notably, the increased N-sulfation was not accompanied by increased O-sulfation, not by C-5 epimerization of D-glucuronic to L-
iduronic acid units. The implications of these findings are discussed with regard to the regulation of the biosynthetic process.