The effect of precursor structures on the action of glucosaminyl 3-O-sulfotransferase-1 and the biosynthesis of anticoagulant heparan sulfate.
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(SO(3))3(SO(3)) and GlcUA-GlcN(SO(3))3(SO(3))6(SO(3)) in both wild-type and mutant cells. In addition, IdoUA-GlcN(SO(3))3(SO(3)) and IdoUA-GlcN(SO(3))3(SO(3))6(SO(3)) accumulate in the mutant chain. These disaccharides were also observed by tagging [6-(3)H]GlcN-labeled pgsF-17 heparan sulfate in vitro with [(35)S]PAPs and purified 3-OST-1. Heparan sulfate derived from the transduced mutant also had approximately 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.
Zhang
L
L
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Lawrence
R
R
Schwartz
J J
JJ
Bai
X
X
Wei
G
G
Esko
J D
JD
Rosenberg
R D
RD
D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000494:pharmacology; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004187:Disaccharides / Q000737:chemistry; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014316:Tritium
D000925:Anticoagulants; D004187:Disaccharides; D016222:Fibroblast Growth Factor 2; D005944:Glucosamine; D006497:Heparan Sulfate; D011994:Recombinant Proteins; D015238:Sulfotransferases; D014316:Tritium; C093554:heparin-glucosamine 3-O-sulfotransferase