N-sulfotestosteronan, a novel substrate for heparan sulfate 6-O-sulfotransferases and its analysis by oxidative degradation.

Testosteronan, an unusual glycosaminoglycan (GAG) first isolated from the microbe Comamonas testosteroni, was enzymatically synthesized in vitro by transferring uridine diphosphate sugars on β-p-nitrophenyl glucuronide acceptor. After chemically converting testosteronan to N-sulfotestosteronan it was tested as a substrate for sulfotransferases involved in the biosynthesis of the GAG, heparan sulfate. Studies using (35) S-labeled 3'-phosphoadenosine-5'-phosphosulfate (PAPS) showed that only 6-O-sulfotransferases acted on N-sulfotestosteronan. An oxidative depolymerization reaction was explored to generate oligosaccharides from (34) S-labeled 6-O-sulfo-N-sulfotestosteroran using (34) S-labeled PAPS because testosteronan was resistant to all of the tested GAG-degrading enzymes. Liquid chromotography-mass spectrometric analysis of the oxidatively depolymerized polysaccharides confirmed the incorporation of (34) S into ∼14% of the glucosamine residues. Nuclear magnetic resonance spectroscopy also showed that the sulfo groups were transferred to ∼20% of the 6-hydroxyl groups in the glucosamine residue of N-sulfotestosteronan. The bioactivity of 6-O-sulfo-N-sulfotestosteronan was examined by performing protein-binding studies with fibroblast growth factors and antithrombin (AT) III using a surface plasmon resonance competition assay. The introduction of 6-O-sulfo groups enhanced N-sulfotestosteronan binding to the fibroblast growth factors, but not to AT III.