Abstract
A previous study identified a Chinese hamster ovary cell mutant, pgsE-606, which is defective in the N-sulfotransferase that catalyzes one of the initial polymer-modification reactions in the biosynthesis of heparan sulfate (Bame, K. J., and Esko, J. D. (1989) J. Biol. Chem. 264, 8059-8065). The structure of heparan sulfate generated by these cells reflects a 3-5-fold reduction in enzyme activity. The mutant produces heparan sulfate with half the content of N-sulfated glucosamine residues of that produced by wild-type cells and a more sparse distribution of N-sulfated residues. The present study demonstrates corresponding reductions in the proportion of 6-O-sulfated glucosamine residues (41% reduction) and the content of L-iduronic acid (51% reduction). The amount of 2-O-sulfated L-iduronic acid declines more dramatically (from 25% of total L-iduronic acid in the wild type to 8.4% in the mutant). Enzymatic assay of mixed O-sulfotransferases showed that the mutant has more activity than the wild type. Previous studies on the biosynthesis of heparin/heparan sulfate in cell-free systems point to a pivotal role of N-sulfation in determining the extent of the subsequent polymer-modification reactions. The present study shows that this concept also applies to heparan sulfate biosynthesis in the intact cell.
Highlights
A previous study identified a Chinese hamster ovary The GlcN units maybe either N-acetylated or N-sulfated, cell mutant, pgsE-606, which is defective in the Nsulfotransferase that catalyzes oneof the initial polymer-modification reactions in the biosynthesis of heparan sulfate
Previous studies on the biosynthesis of heparinbeparansulfateincell-free systems point to a pivotal role of N-sulfation in determining the extent of the subsequent polymer-modification reactions
The present study shows that this concept applies to heparan sulfate biosynthesis in the intact cell
Summary
COORDINATION OF POLYMER-MODIFICATION REACTIONSIN A CHINESE HAMSTER OVARY CELL MUTANTDEFECTIVEINN-SULFOTRANSFERASE*. A previous study identified a Chinese hamster ovary The GlcN units maybe either N-acetylated or N-sulfated, cell mutant, pgsE-606, which is defective in the Nsulfotransferase that catalyzes oneof the initial polymer-modification reactions in the biosynthesis of heparan sulfate In general terms the N-acetylated regions are rich in GlcA units and poor in 0-sulfate groups whereas the N-sulfated regions show high proportions of IdoA and 0-sulfated residues The former structural elements are preferentially found iduronic acid (51%reduction). Regulated N-deacetylation/ P-10 (Bio-Rad) was described previously (Bame and Esko, 1989).For. N-sulfation during the initial phase of polymer modification accounts for the structural difference between heparin and heparan sulfate. Samples were eluted with a gradient of KH'PO, described a Chinese hamster ovary cell mutant, pgsE-606, which produces undersulfated heparan sulfate because of a deficiency in N-sulfotransferase(Bameand Esko, 1989). The glycosaminoglycan was first N-deacetylated by treatment with hydrazine/hydrazine sulfate for 2 h at 100 "C as reported by Hook et
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