Abstract
To understand the mechanisms that control anticoagulant heparan sulfate (HSact) biosynthesis, we previously showed that HSact production in the F9 system is determined by the abundance of 3-O-sulfotransferase-1 as well as the size of the HSact precursor pool. In this study, HSact 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 HSact precursor chain. At least five out of six 3-O-sulfate-tagged oligosaccharides in HSact precursors bind AT, whereas none of 3-O-sulfate-tagged oligosaccharides from HSinact precursors bind AT. When treated with low pH nitrous or heparitinase, 3-O-sulfate-tagged HSact and HSinact precursors exhibit clearly different structural features. 3-O-Sulfate-tagged HSact hexasaccharides were AT affinity purified and sequenced by chemical and enzymatic degradations. The 3-O-sulfate-tagged HSact hexasaccharides exhibited the following structures, DeltaUA-[6-3H]GlcNAc6S-GlcUA-[6-3H]GlcNS3(35)S+/-6S-++ +IdceA2S-[6-3H]Glc NS6S. 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 HSact 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 HSact and HSinact 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.
Highlights
Different heparin/heparan sulfate (HS)1 sequences bind to a large number of growth factors and cytokines (1– 6), enzymes (7), protease inhibitors (8 –12), virus proteins (13), and selectins (14)
To understand the mechanisms that control anticoagulant heparan sulfate (HSact) biosynthesis, we previously showed that HSact production in the F9 system is determined by the abundance of 3-O-sulfotransferase-1 as well as the size of the HSact precursor pool
HS precursor structures are acted upon by a unique sulfotransferase, which is positioned at the end of the biosynthetic pathways and whose levels control the concentration of the specific HS component that is the product of the biosynthetic pathway
Summary
Cell Culture—F9 cells were grown on gelatin (Sigma) coated (0.1%) tissue culture dishes in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated calf serum (Irvine Scientific), penicillin G (100 units/ml), and streptomycin sulfate (100 g/ml) under an atmosphere of 5% CO2, 95% air and 100% relative humidity. After washing the pellets with 0.5 ml of 75% ethanol, 3-O-sulfate-tagged HSact and HSinact precursors were separated by AT affinity assay as described below. After adding 100 g of chondroitin sulfate as cold carrier to 3-O-sulfate-tagged HSact precursors, the pooled 3-O-sulfatetagged HSact and HSinact precursors were cleaned by phenol/chloroform extraction followed by DEAE-Sepharose chromatography and ethanol precipitation. After washing the pellets with 0.5 ml of 75% ethanol and dried briefly by Speed-Vac, 3-O-sulfate-tagged HSact and HSinact precursors were resuspended in H2O and used for chemical and enzymatic structural studies. The digestion of 3-O-sulfate-tagged HSact and HSinact precursors was carried out in 100 l of 40 mM ammonium acetate (pH 7.0) containing 1 mM CaCl2 with 2 milliunits of enzyme or 2 milliunits of each heparitinase I, heparitinase II, and heparinase. The 6-O-sulfatase digestion was completed in 2 days at 37 °C as monitored by polyamine HPLC as described above. 100 l of 6-O-sulfatase-treated materials was diluted with 300 l of H2O. 2 l of 16 M acetic acid and 5 l of ␣-N-acetylglucosaminidase (3.5 g) were added (pH 4.2). ␣-N-Acetylglucosaminidase digestion was completed overnight at 37 °C as monitored by polyamine HPLC
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