title: N-sulfotestosteronan, a novel substrate for heparan sulfate 6-O-sulfotransferases and its analysis by oxidative degradation.
abstract: 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.
authors.affiliation: College of Food Science and Technology, Ocean University of China, Qingdao, People's Republic of China, 266003; Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY, 12180; Department of Biology, Rensselaer Polytechnic Institute, Troy, NY, 12180; Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180; Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180.
authors.forename: Guoyun
authors.initials: G
authors.lastname: Li
authors.forename: Sayaka
authors.initials: S
authors.lastname: Masuko
authors.forename: Dixy E
authors.initials: DE
authors.lastname: Green
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Xu
authors.forename: Lingyun
authors.initials: L
authors.lastname: Li
authors.forename: Fuming
authors.initials: F
authors.lastname: Zhang
authors.forename: Changhu
authors.initials: C
authors.lastname: Xue
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.forename: Paul L
authors.initials: PL
authors.lastname: DeAngelis
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
mesh_terms: D006025:Glycosaminoglycans / Q000737:chemistry; D006497:Heparan Sulfate / Q000737:chemistry; D013058:Mass Spectrometry; D009844:Oligosaccharides; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D009844:Oligosaccharides
keywords: bacterial polysaccharide; heparan sulfate; protein-binding; sulfonation; sulfotransferases