Anticoagulant heparan sulfate precursor structures in F9 embryonal carcinoma cells

Abstract

To understand the mechanisms that control anticoagulant heparan sulfate (HS(act)) biosynthesis, we previously showed that HS(act) production in the F9 system is determined by the abundance of 3-O-sulfotransferase-1 as well as the size of the HS(act) precursor pool. In this study, HS(act) precursor structures have been studied by characterizing [6-3H]GlcN metabolically labeled F9 HS tagged with 3-O-sulfates in vitro by 3'-phosphoadenosine 5'- phospho-35S and purified 3-O-sulfotransferase-1. This later in vitro labeling allows the regions of HS destined to become the antithrombin (AT)- binding sites to be tagged for subsequent structural studies. It was shown that six 3-O-sulfation sites exist per HS(act) precursor chain. At least five out of six 3-O-sulfate-tagged oligosaccharides in HS(act) precursors bind AT, whereas none of 3-O-sulfate-tagged oligosaccharides from HS(inact) precursors bind AT. When treated with low pH nitrous or heparitinase, 3-O-sulfate- tagged HS(act) and HS(inact) precursors exhibit clearly different structural features. 3-O-Sulfate-tagged HS(act) hexasaccharides were AT affinity purified and sequenced by chemical and enzymatic degradations. The 3-O- sulfate-tagged HS(act) hexasaccharides exhibited the following structures, ΔUA-[6-3H]GlcNAc6S-GlcUA-[6-3H]GlcNS335S+6S-IdceA2S-[6-3H]GlcNS6S. The underlined 6- and 3-O-sulfates constitute the most critical groups for AT binding in view of the fact that the precursor hexasaccharides possess all the elements for AT binding except for the 3-O-sulfate moiety. The presence of five potential AT-binding precursor hexasaccharides in all HS(act) precursor chains demonstrates for the first time the processive assembly of specific sequence in HS. The difference in structures around potential 3-O- sulfate acceptor sites in HS(act) and HS(inact) precursors suggests that these precursors might be generated by different concerted assembly mechanisms in the same cell. This study permits us to understand better the nature of the HS biosynthetic pathway that leads to the generation of specific saccharide sequences.