title: Enzymatic synthesis of chondroitin sulfate E by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid cartilage.
abstract: Chondroitin sulfate E (CS-E), a chondroitin sulfate isomer containing GlcAbeta1-3GalNAc(4,6-SO(4)) repeating unit, was found in various mammalian cells in addition to squid cartilage and is predicted to have several physiological functions in various mammalian systems such as mast cell maturation, regulation of procoagulant activity of monocytes, and binding to midkine or chemokines. To clarify the physiological functions of GalNAc(4,6-SO(4)) repeating unit, preparation of CS-E with a defined content of GalNAc(4,6-SO(4)) residues is important. We report here the in vitro synthesis of CS-E from chondrotin sulfate A (CS-A) by the purified squid N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) which catalyzed transfer of sulfate from 3(')-phosphoadenosine-5(')-phosphosulfate to position 6 of GalNAc(4SO(4)) residues of CS-A and dermatan sulfate (DS). When CS-A was used as an acceptor, about half of GalNAc(4SO(4)) residues, on average, were converted to GalNAc(4,6-SO(4)) residues. Anion exchange chromatography of the CS-E synthesized in vitro showed marked heterogeneity in negative charge; the proportion of GalNAc(4,6-SO(4)) in the most negative fraction exceeded 70% of the total sulfated repeating units. GalNAc4S-6ST also catalyzed the synthesis of oversulfated DS with GalNAc(4,6-SO(4)) residues from DS. Squid GalNAc4S-6ST thus should provide a useful tool for preparing CS-E and oversulfated DS with a defined proportion of GalNAc(4,6-SO(4)) residues.
authors.affiliation: Department of Life Science, Aichi University of Education, Igaya-cho, Kariya, Aichi 448-8542, Japan. ohabuchi@auecc.aichi-edu.ac.jp
authors.forename: Osami
authors.initials: O
authors.lastname: Habuchi
authors.forename: Rina
authors.initials: R
authors.lastname: Moroi
authors.forename: Shiori
authors.initials: S
authors.lastname: Ohtake
mesh_terms: D000818:Animals; D002356:Cartilage / Q000201:enzymology*; D002809:Chondroitin Sulfates / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D002810:Chondroitinases and Chondroitin Lyases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods; D049832:Decapodiformes; D003871:Dermatan Sulfate / Q000737:chemistry / Q000378:metabolism; D004187:Disaccharides / Q000032:analysis / Q000737:chemistry; D051381:Rats; D013429:Sulfatases / Q000378:metabolism; D013431:Sulfates / Q000737:chemistry; D015238:Sulfotransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014907:Whales
chemical_list: D004187:Disaccharides; D013431:Sulfates; D013462:Sulfur Radioisotopes; C038142:chondroitin sulfate glycosaminoglycan; D003871:Dermatan Sulfate; D002809:Chondroitin Sulfates; C416670:N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase; D015238:Sulfotransferases; D013429:Sulfatases; C046341:chondro-6-sulfatase; D002810:Chondroitinases and Chondroitin Lyases