Expression of the Mouse Mastocytoma Glucosaminyl N-Deacetylase/N-Sulfotransferase in Human Kidney 293 Cells Results in Increased N-Sulfation of Heparan Sulfate

Abstract

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.

NDST2 (N-Deacetylase/N-Sulfotransferase-2) Enzyme Regulates Heparan Sulfate Chain Length

“…More Total HS and Longer HS Chains in Cells Overexpressing NDST2-As shown previously (22,23), overexpression of NDST2 in HEK 293 cells results in increased HS N-sulfation ( Fig. 2A).…”

“…Cells-HEK 293 cells with a stable overexpression of murine His 6 -tagged NDST2 and His 6 -tagged NDST1, respectively, and HEK 293 cells expressing NDST2 without His 6 tag have previously been described (22,34). To generate NDST1/NDST2-overexpressing cells, NDST2 (His 6 tag)-overexpressing cells were transfected with an Ndst1/pBud CE4.1 plasmid construct using Lipofectamine TM 2000 (Invitrogen) according to the manufacturer's protocol.…”

The Putative Heparin-specific N-AcetylglucosaminylN-Deacetylase/N-Sulfotransferase Also Occurs in Non-heparin-producing Cells

“…We believe that the purified enzyme from mastocytoma (17), in a manner analogous to the purified one from rat liver (27), has lost during the many purification steps its ability to N-deacetylate N-acetylglucosamine of heparin or heparan sulfate in vitro; in the presence of polycations, this activity is regained in assays in vitro. When a soluble chimera of the heparin/heparan sulfate N-acetylglucosaminyl N-deacetylase/N-sulfotransferase of either MST cells or rat liver with protein A is expressed and secreted, thus allowing a rapid, one-step purification of the enzyme by its binding to IgG beads both proteins have both catalytic activities in assays in vitro without need of polycations (14,15).…”

“…Up to now, proteins from rat liver (13,14), cell lines derived from a mastocytoma tumor (MST) 1 (15), and the tumor itself (16,17) have been cloned and shown to catalyze the N-deacetylation/N-sulfation of N-acetylglucosamine of heparin/heparan sulfate. The protein from MST cells and the mastocytoma tumor are virtually identical and have 70% of amino acid sequence identity with the one from rat liver with significant sequence differences toward their amino terminus (13)(14)(15)(16).…”

Lowered Expression of Heparan Sulfate/Heparin Biosynthesis Enzyme N-Deacetylase/N-Sulfotransferase 1 Results in Increased Sulfation of Mast Cell Heparin

“…As discussed above, NDST1 appears to have a vital role for HS synthesis in most cells whereas NDST2 is crucial for heparin sulfation in MCs. We also know that the degree of N-sulfation depends both on isoform and the level of enzyme expressed; overexpression of NDST1 and NDST2 in 293 cells both result in increased HS N-sulfation, but the level of N-sulfation reached is higher in NDST2-overexpressing cells (40,43). Investigating the roles of NDST1 and NDST2 in liver HS biosynthesis, we previously could conclude that in the absence of NDST1 (and NDST3 and 4, not expressed in the liver), NDST2 was responsible for HS N-sulfation (21).…”