The Effect of Precursor Structures on the Action of Glucosaminyl 3-O-Sulfotransferase-1 and the Biosynthesis of Anticoagulant Heparan Sulfate

Abstract

To understand how 2-O-sulfation of uronic acid residues influences the biosynthesis of anticoagulant heparan sulfate, the cDNA encoding glucosaminyl 3-O-sulfotransferase-1 (3-OST-1) was introduced into wild-type Chinese hamster ovary cells and mutant pgsF-17 cells, which are defective in 2-O-sulfation. 3-OST-1-transduced cells gained the ability to bind to antithrombin. Structural analysis of the heparan sulfate chains showed that 3-OST-1 generates sequences containing GlcUA-GlcN(SO3)3(SO3) and GlcUA-GlcN(SO 3)3(SO3)6(SO3) in both wild-type and mutant cells. In addition, IdoUAGlcN(SO3)3(SO3) and IdoUA- GlcN(SO3)3(SO3)6(SO3) accumulate in the mutant chain. These disaccharides were also observed by tagging [6- 3H]GlcN-labeled pgsF-17 heparan sulfate in vitro with [ 35S]PAPs and purified 3-OST-1. Heparan sulfate derived from the transduced mutant also had ∼2-fold higher affinity for antithrombin than heparan sulfate derived from the transduced wild-type cells, and it inactivated factor Xa more efficiently. This study demonstrates for the first time that (i) 3-O-sulfation by 3-OST-1 can occur independently of the 2-O-sulfation of uronic acids, (ii) 2-O-sulfation usually occurs before 3-O-sulfation, (iii) 2-O-sulfation blocks the action of 3-OST-1 at glucosamine residues located to the reducing side of IdoUA units, and (iv) that alternative antithrombin-binding structures can be made in the absence of 2-O-sulfation.