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 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.
Cheung
W F
WF
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
Eriksson
I
I
Kusche-Gullberg
M
M
Lindhal
U
U
Kjellén
L
L
D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D012515:Mast-Cell Sarcoma / Q000201:enzymology* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D000581:Amidohydrolases; D006497:Heparan Sulfate; D011994:Recombinant Proteins; D015238:Sulfotransferases; C021350:heparitin sulfotransferase