EXPRESSION OF THE MOUSE MASTOCYTOMA GLUCOSAMINYL N-DEACETYLASE N-SULFOTRANSFERASE IN HUMAN KIDNEY-293-CELLS RESULTS IN INCREASED N-SULFATION OF HEPARAN-SULFATE

The biosynthesis of heparin and heparan sulfate involves a series of p olymer-modification reactions that is initiated by N-deacetylation and subsequent N-sulfation of N-acetylglucosamine residues. These reactio ns are catalyzed by a combined N-deacetylase/N-sulfotransferase. Prote ins expressing both activities have previously been purified from mous e mastocytoma, which generates heparin, and from rat liver, which prod uces heparan sulfate. In the present study, the mouse mastocytoma enzy me has been expressed in the human kidney cell line, 293, to investiga te whether it could promote modification of the endogenous heparan sul fate precursor polysaccharide into a heparin-like molecule. The N-deac etylase activity of the stably transfected cell clones was similar to 8-fold higher, on a cell-protein basis, than that of control cells, wh ile the N-sulfotransferase activity was increased similar to 2.5-fold. The amounts of glycosaminoglycans synthesized were the same in contro l and transfected cells, measured as incorporation of [H-3]-glucosamin e, whereas S-35-labeled glycosaminoglycans were similar to 50% increas ed in transfected cells, with an increased relative content of heparan sulfate. Structural analysis demonstrated that the glucosamine units of the ''heparan sulfate'' from transfected cells were almost exclusiv ely 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, nor by C-5 epimerization of D-glucuronic to L-iduronic ac id units. The implications of these findings are discussed with regard to the regulation of the biosynthetic process.