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J. Biol. Chem., Vol. 283, Issue 24, 16801-16807, June 13, 2008

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2-O-Phosphorylation of Xylose and 6-O-Sulfation of Galactose in the Protein Linkage Region of Glycosaminoglycans Influence the Glucuronyltransferase-I Activity Involved in the Linkage Region Synthesis^*

Yuko Tone, Lars C. Pedersen, Tomoko Yamamoto, Tomomi Izumikawa, Hiroshi Kitagawa, Junko Nishihara^¶, Jun-ichi Tamura^¶, Masahiko Negishi^||, and Kazuyuki Sugahara^**1

From the Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan, the ^||Laboratory of Reproductive and Developmental Toxicology and Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, the ^¶Department of Regional Environment, Faculty of Regional Sciences, Tottori University, Tottori 680-8551, Japan, and the ^**Laboratory of Proteoglycan Signaling and Therapeutics, Frontier Research Center for Post-Genomic Science and Technology, Faculty of Advanced Life Science, Hokkaido University Graduate School of Life Science, Sapporo 001-0021, Japan

Sulfated glycosaminoglycans (GAGs), including heparan sulfate and chondroitin sulfate, are synthesized on the so-called common GAG-protein linkage region (GlcUAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser) of core proteins, which is formed by the stepwise addition of monosaccharide residues by the respective specific glycosyltransferases. Glucuronyltransferase-I (GlcAT-I) is the key enzyme that completes the synthesis of this linkage region, which is a prerequisite for the conversion of core proteins to functional proteoglycans bearing GAGs. The Xyl and Gal residues in the linkage region can be modified by phosphorylation and sulfation, respectively, although the biological significance of these modifications remains to be clarified. Here we present evidence that these modifications can significantly influence the catalytic activity of GlcAT-I. Enzyme assays showed that the synthetic substrates, Gal-Gal-Xyl(2-O-phosphate)-O-Ser and Gal-Gal(6-O-sulfate)-Xyl(2-O-phosphate)-O-Ser, served as better substrates than the unmodified compound, whereas Gal(6-O-sulfate)-Gal-Xyl(2-O-phosphate)-O-Ser exhibited no acceptor activity. The crystal structure of the catalytic domain of GlcAT-I with UDP and Gal-Gal(6-O-sulfate)-Xyl(2-O-phosphate)-O-Ser bound revealed that the Xyl(2-O-phosphate)-O-Ser is disordered and the 6-O-sulfate forms interactions with Gln³¹⁸ from the second GlcAT-I monomer in the dimeric enzyme. The results indicate the possible involvement of these modifications in the processing and maturation of the growing linkage region oligosaccharide required for the assembly of GAG chains.

Received for publication, November 21, 2007 , and in revised form, April 8, 2008.

The atomic coordinates and structure factors (code 3CU0) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported, in whole or in part, by a National Institutes of Health grant from the Intramural Research Program (NIEHS). This work was supported in part by the Scientific Research Promotion Fund from the Japan Private School Promotion Foundation, Grants-in-aid for Scientific Research-B 19390025 (to H. K.) and 18390030 (to K. S.), and for Scientific Research on Priority Areas 14082207 (to K. S.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and the Core Research for Evolutional Science and Technology (CREST) program of the Japan Science and Technology (JST) Agency (to H. K. and K. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Hokkaido University Graduate School of Life Science, Sapporo 001-0021, Japan. Tel.: 81-11-706-9054; Fax: 81-11-706-9056; E-mail: k-sugar{at}sci.hokudai.ac.jp.

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