Habuchi Osami, Moroi Rina, Ohtake Shiori
Department of Life Science, Aichi University of Education, Igaya-cho, Kariya, Aichi 448-8542, Japan.
Anal Biochem. 2002 Nov 15;310(2):129-36. doi: 10.1016/s0003-2697(02)00277-4.
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.
硫酸软骨素E（CS-E）是一种含有GlcAbeta1-3GalNAc(4,6-SO(4))重复单元的硫酸软骨素异构体，除了在鱿鱼软骨中发现外，还存在于各种哺乳动物细胞中，预计在各种哺乳动物系统中具有多种生理功能，如肥大细胞成熟、调节单核细胞的促凝血活性以及与中期因子或趋化因子结合。为了阐明GalNAc(4,6-SO(4))重复单元的生理功能，制备具有确定含量的GalNAc(4,6-SO(4))残基的CS-E很重要。我们在此报告通过纯化的鱿鱼N-乙酰半乳糖胺4-硫酸酯6-O-硫酸转移酶（GalNAc4S-6ST）从硫酸软骨素A（CS-A）体外合成CS-E，该酶催化硫酸从3(')-磷酸腺苷-5(')-磷酸硫酸转移至CS-A和硫酸皮肤素（DS）的GalNAc(4SO(4))残基的6位。当CS-A用作受体时，平均约一半的GalNAc(4SO(4))残基被转化为GalNAc(4,6-SO(4))残基。体外合成的CS-E的阴离子交换色谱显示负电荷存在明显异质性；最负级分中GalNAc(4,6-SO(4))的比例超过总硫酸化重复单元的70%。GalNAc4S-6ST还催化从DS合成带有GalNAc(4,6-SO(4))残基的过度硫酸化DS。因此，鱿鱼GalNAc4S-6ST应为制备具有确定比例的GalNAc(4,6-SO(4))残基的CS-E和过度硫酸化DS提供有用工具。
