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J. Biol. Chem., Vol. 282, Issue 11, 8533-8544, March 16, 2007

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Expression of rib-1, a Caenorhabditis elegans Homolog of the Human Tumor Suppressor EXT Genes, Is Indispensable for Heparan Sulfate Synthesis and Embryonic Morphogenesis^*

Hiroshi Kitagawa²¹, Tomomi Izumikawa, Souhei Mizuguchi^¶3, Katsufumi Dejima^¶, Kazuko H. Nomura^¶2, Noriyuki Egusa, Fumiyasu Taniguchi, Jun-ichi Tamura^||, Keiko Gengyo-Ando^**, Shohei Mitani^**, Kazuya Nomura^¶, and Kazuyuki Sugahara²⁴

From the Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, the ^¶Department of Biology, Faculty of Sciences 33, Kyushu University, Fukuoka 812-8581, Core Research for Evolutional Science and Technology of Japan Science and Technology Agency, Kawaguchi Center Building, 4-1-8, Hon-cho, Kawaguchi, Saitama 332-0012, the ^||Department of Regional Environment, Faculty of Regional Sciences, Tottori University, Tottori 680-8551, and the ^**Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo 162-8666, Japan

The proteins encoded by all of the five cloned human EXT family genes (EXT1, EXT2, EXTL1, EXTL2, and EXTL3), members of the hereditary multiple exostoses gene family of tumor suppressors, are glycosyltransferases required for the biosynthesis of heparan sulfate. In the Caenorhabditis elegans genome, only two genes, rib-1 and rib-2, homologous to the mammalian EXT genes have been identified. Although rib-2 encodes an N-acetylglucosaminyltransferase involved in initiating the biosynthesis and elongation of heparan sulfate, the involvement of the protein encoded by rib-1 in the biosynthesis of heparan sulfate remains unclear. Here we report that RIB-1 is indispensable for the biosynthesis and for embryonic morphogenesis. Despite little individual glycosyltransferase activity by RIB-1, the polymerization of heparan sulfate chains was demonstrated when RIB-1 was coexpressed with RIB-2 in vitro. In addition, RIB-1 and RIB-2 were demonstrated to interact by pulldown assays. To investigate the functions of RIB-1 in vivo, we depleted the expression of rib-1 by deletion mutagenesis. The null mutant worms showed reduced synthesis of heparan sulfate and embryonic lethality. Notably, the null mutant embryos showed abnormality at the gastrulation cleft formation stage or later and arrested mainly at the 1-fold stage. Nearly 100% of the embryos died before L1 stage, although the differentiation of some of the neurons and muscle cells proceeded normally. Similar phenotypes have been observed in rib-2 null mutant embryos. Thus, RIB-1 in addition to RIB-2 is indispensable for the biosynthesis of heparan sulfate in C. elegans, and the two cooperate to synthesize heparan sulfate in vivo. These findings also show that heparan sulfate is essential for post-gastrulation morphogenic movement of embryonic cells and is indispensable for ensuring the normal spatial organization of differentiated tissues and organs.

Received for publication, December 4, 2006 , and in revised form, January 16, 2007.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB241458 [GenBank] .

^* This work was supported in part by the Science Research Promotion Fund of the Japan Private School Promotion Foundation and Grant-in-aid for Scientific Research 16590075 (to H. K.), grant-in-aid for young scientists (to S. M.), grant-in-aid for JSPS Fellows (to K. D.), and a Grant-in-aid for Scientific Research on Priority Areas 14082207 (to K. S. and to K. N.) from the Ministry of Education, Science, Culture, and Sports of Japan. 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.

² Supported by Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency.

¹ To whom correspondence may be addressed: Dept. of Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan. Tel.: 81-78-441-7570; Fax: 81-78-441-7571; E-mail: kitagawa{at}kobepharma-u.ac.jp.

³ To whom correspondence may be addressed. E-mail: smizuscb{at}mbox.nc.kyushu-u.ac.jp.

⁴ To whom correspondence may be addressed: Laboratory of Proteoglycan Signaling and Therapeutics, Graduate School of Life Science, Hokkaido University, Frontier Research Center for Post-Genomic Science and Technology, Nishi 11-choume, Kita 21-jo, Kita-ku, Sapporo, Hokkaido 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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