title: LC-MS and LC-MS/MS studies of incorporation of 34SO3 into glycosaminoglycan chains by sulfotransferases.
abstract: The specificities of glycosaminoglycan (GAG) modification enzymes, particularly sulfotransferases, and the locations and concentrations of these enzymes in the Golgi apparatus give rise to the mature GAG polysaccharides that bind protein ligands. We studied the substrate specificities of sulfotransferases with a stable isotopically labeled donor substrate, 3'-phosphoadenosine-5'-phosphosulfate. The sulfate incorporated by in vitro sulfation using recombinant sulfotransferases was easily distinguished from those previously present on the GAG chains using mass spectrometry. The enrichment of the [M + 2] isotopic peak caused by (34)S incorporation, and the [M + 2]/[M + 1] ratio, provided reliable and sensitive measures of the degree of in vitro sulfation. It was found that both CHST3 and CHST15 have higher activities at the non-reducing end (NRE) units of chondroitin sulfate, particularly those terminating with a GalNAc monosaccharide. In contrast, both NDST1 and HS6ST1 showed lower activities at the NRE of heparan sulfate (HS) chains than at the interior of the chain. Contrary to the traditional view of HS biosynthesis processes, NDST1 also showed activity on O-sulfated GlcNAc residues.
authors.affiliation: Department of Biochemistry and Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Shi
authors.forename: Chun
authors.initials: C
authors.lastname: Shao
authors.forename: Yang
authors.initials: Y
authors.lastname: Mao
authors.forename: Yu
authors.initials: Y
authors.lastname: Huang
authors.forename: Zhengliang L
authors.initials: ZL
authors.lastname: Wu
authors.forename: Joseph
authors.initials: J
authors.lastname: Zaia
mesh_terms: D002809:Chondroitin Sulfates / Q000737:chemistry*; D002853:Chromatography, Liquid; D006025:Glycosaminoglycans / Q000737:chemistry*; D006497:Heparan Sulfate / Q000737:chemistry*; D013058:Mass Spectrometry; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry*; D013460:Sulfur Isotopes; D013461:Sulfur Oxides; D000097811:Carbohydrate Sulfotransferases
chemical_list: D002809:Chondroitin Sulfates; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D015238:Sulfotransferases; D013460:Sulfur Isotopes; D013461:Sulfur Oxides; D000097811:Carbohydrate Sulfotransferases; C020341:glucosaminoglycans; C011118:sulfur trioxide
keywords: chondroitin sulfate; glycosaminoglycan; heparan sulfate; mass spectrometry; sulfotransferase