title: Expression of rib-1, a Caenorhabditis elegans homolog of the human tumor suppressor EXT genes, is indispensable for heparan sulfate synthesis and embryonic morphogenesis.
abstract: 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.
authors.affiliation: Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan. kitagawa@kobepharma-u.ac.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kitagawa
authors.forename: Tomomi
authors.initials: T
authors.lastname: Izumikawa
authors.forename: Souhei
authors.initials: S
authors.lastname: Mizuguchi
authors.forename: Katsufumi
authors.initials: K
authors.lastname: Dejima
authors.forename: Kazuko H
authors.initials: KH
authors.lastname: Nomura
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Egusa
authors.forename: Fumiyasu
authors.initials: F
authors.lastname: Taniguchi
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Tamura
authors.forename: Keiko
authors.initials: K
authors.lastname: Gengyo-Ando
authors.forename: Shohei
authors.initials: S
authors.lastname: Mitani
authors.forename: Kazuya
authors.initials: K
authors.lastname: Nomura
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Sugahara
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins / Q000096:biosynthesis / Q000502:physiology*; D002454:Cell Differentiation; D002522:Chlorocebus aethiops; D005260:Female; D005775:Gastrula / Q000378:metabolism; D005786:Gene Expression Regulation; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000502:physiology
chemical_list: D029742:Caenorhabditis elegans Proteins; D006497:Heparan Sulfate; D025521:Tumor Suppressor Proteins; C518898:Rib-1 protein, C elegans