Souhei Mizuguchi

Chondroitin proteoglycans are involved in cell division of Caenorhabditis elegans

Glycosaminoglycans such as heparan sulphate and chondroitin sulphate are extracellular sugar chains involved in intercellular signalling. Disruptions of genes encoding enzymes that mediate glycosaminoglycan biosynthesis have severe consequences in Drosophila and mice. Mutations in the Drosophila gene sugarless, which encodes a UDP-glucose dehydrogenase, impairs developmental signalling through the Wnt family member Wingless, and signalling by the fibroblast growth factor and Hedgehog pathways. Heparan sulphate is involved in these pathways, but little is known about the involvement of chondroitin. Undersulphated and oversulphated chondroitin sulphate chains have been implicated in other biological processes, however, including adhesion of erythrocytes infected with malaria parasite to human placenta and regulation of neural development. To investigate chondroitin functions, we cloned a chondroitin synthase homologue of Caenorhabditis elegans and depleted expression of its product by RNA-mediated interference and deletion mutagenesis. Here we report that blocking chondroitin synthesis results in cytokinesis defects in early embryogenesis. Reversion of cytokinesis is often observed in chondroitin-depleted embryos, and cell division eventually stops, resulting in early embryonic death. Our findings show that chondroitin is required for embryonic cytokinesis and cell division.

Nematode chondroitin polymerizing factor showing cell-/organ-specific expression is indispensable for chondroitin synthesis and embryonic cell division.

Chondroitin polymerization was first demonstrated in vitro when human chondroitin synthase (ChSy) was coexpressed with human chondroitin polymerizing factor (ChPF), which is homologous to ChSy but has little glycosyltransferase activity. To analyze the biological function of chondroitin, the Caenorhabditis elegans ortholog of human ChSy (sqv-5) was recently cloned, and the expression of its product was depleted by RNA-mediated interference (RNAi) and deletion mutagenesis. Blocking of chondroitin synthesis resulted in defects of cytokinesis in early embryogenesis, and eventually, cell division stopped. Here, we cloned the ortholog of human ChPF in C. elegans, PAR2.4. Despite little glycosyltransferase activity of the gene product, chondroitin polymerization was demonstrated as in the case of mammals when PAR2.4 was coexpressed with cChSy in vitro. The worm phenotypes including the reversion of cytokinesis, observed after the depletion of PAR2.4 by RNAi, were very similar to the cChSy (sqv-5)-RNAi phenotypes. Thus, PAR2.4 in addition to cChSy is indispensable for the biosynthesis of chondroitin in C. elegans, and the two cooperate to synthesize chondroitin in vivo. The expression of the PAR2.4 protein was observed in seam cells, which can act as neural stem cells in early embryonic lineages. The expression was also detected in vulva and distal tip cells of the growing gonad arms from L3 through to the young adult stage. These findings are consistent with the notion that chondroitin is involved in the organogenesis of the vulva and maturation of the gonad and also indicative of an involvement in distal tip cell migration and neural development.

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

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.

Ceramide glucosyltransferase of the nematode Caenorhabditis elegans is involved in oocyte formation and in early embryonic cell division

Ceramide glucosyltransferase (Ugcg) [uridine diphosphate (UDP)-glucose:N-acylsphingosine D-glucosyltransferase or UDP-glucose ceramide glucosyltransferase (GlcT): EC 2.4.1.80] catalyzes formation of glucosylceramide (GlcCer) from ceramide and UDP-glucose. There is only one Ugcg gene in the mouse genome, which is essential in embryogenesis and brain development. The nematode Caenorhabditis elegans has three Ugcg genes (cgt-1, cgt-2 and cgt-3), and double RNAi of the cgt-1 and cgt-3 genes results in lethality at the L1 larval stage. In this study, we isolated knockout worms for the three genes and characterized the gene functions. Each gene product showed active enzymatic activity when expressed in GM95 cells deficient in glycosphingolipids (GSLs). When each gene function was disrupted, the brood size of the animal markedly decreased, and abnormal oocytes and multinucleated embryos were formed. The CGT-3 protein had the highest Ugcg activity, and knockout of its gene resulted in the severest phenotype. When cgt-3 RNAi was performed on rrf-1 worms lacking somatic RNAi machinery but with intact germline RNAi machinery, a number of abnormal oocytes and multinucleated eggs were observed, although the somatic phenotype, i.e., L1 lethal effects of cgt-1/cgt-3 RNAi, was completely suppressed. Cell surface expression of GSLs and sphingomyelin, which are important components of membrane domains, was affected in the RNAi-treated embryos. In the embryos, an abnormality in cytokinesis was also observed. From these results, we concluded that the Ugcg gene is indispensable in the germline and that an ample supply of GlcCer is needed for oocytes and fertilized eggs to maintain normal membranes and to proceed through the normal cell cycle.

Involvement of blood-group-B-active trisaccharides in Ca2+-dependent cell-cell adhesion in the Xenopus blastula

Abstract Despite their wide distribution in various organisms, no physiological roles have been proposed for the human blood-group-ABO (ABH)-active trisaccharides. Here we show that monoclonal antibodies against human blood-group-B-active trisaccharides (B-substance) completely block the Ca2+-dependent cell-cell adhesion system of frog (Xenopuslaevis) embryonic cells. Synthetic B-substance or B-active glycopeptides also disrupt the Ca2+ -dependent cell-cell adhesion. These results suggest that blood-group-B-active substances play a role in cell-cell adhesion. Blood-group-B-active substances were found as glycoproteins and as glycosphingolipids. In order to identify B-active glycoproteins active in cell-cell adhesion, we purified B-active membrane glycoproteins by two-dimensional electrophoresis and found that they are 45- to 58-kDa proteins with pI(s) ranging from 4.0 to 5.3. They are glycosylphosphatidyl inositol (GPI) anchored. Amino acid sequence analysis showed that the purified B-active GPI-anchored proteins are homologues of soluble Xenopus cortical granule lectins (CGL). The results suggest that the B-active membrane glycoproteins are GPI-anchored forms of the lectin and are directly involved in frog Ca 2+-dependent cell-cell adhesion.

Essential Roles of 3′-Phosphoadenosine 5′-Phoshosulfate Synthase in Embryonic and Larval Development of the Nematode Caenorhabditis elegans

Sulfation of biomolecules, which is widely observed from bacteria to humans, plays critical roles in many biological processes. All sulfation reactions in all organisms require activated sulfate, 3′-phosphoadenosine 5′-phosphosulfate (PAPS), as a universal donor. In animals, PAPS is synthesized from ATP and inorganic sulfate by the bifunctional enzyme, PAPS synthase. In mammals, genetic defects in PAPS synthase 2, one of two PAPS synthase isozymes, cause dwarfism disorder, but little is known about the consequences of the complete loss of PAPS synthesis. To define the developmental role of sulfation, we cloned a Caenorhabditis elegans PAPS synthase-homologous gene, pps-1, and depleted expression of its product by isolating the deletion mutant and by RNA-mediated interference. PPS-1 protein exhibits specific activity to form PAPS in vitro, and disruption of the pps-1 gene by RNAi causes pleiotropic developmental defects in muscle patterning and epithelial cell shape changes with a decrease in glycosaminoglycan sulfation. Additionally, the pps-1 null mutant exhibits larval lethality. These data suggest that sulfation is essential for normal growth and integrity of epidermis in C. elegans. Furthermore, reporter analysis showed that pps-1 is expressed in the epidermis and several gland cells but not in neurons and muscles, indicating that PAPS in the neurons and muscles is provided by other cells.

Two Golgi-resident 3′-Phosphoadenosine 5′-Phosphosulfate Transporters Play Distinct Roles in Heparan Sulfate Modifications and Embryonic and Larval Development in Caenorhabditis elegans

Synthesis of extracellular sulfated molecules requires active 3′-phosphoadenosine 5′-phosphosulfate (PAPS). For sulfation to occur, PAPS must pass through the Golgi membrane, which is facilitated by Golgi-resident PAPS transporters. Caenorhabditis elegans PAPS transporters are encoded by two genes, pst-1 and pst-2. Using the yeast heterologous expression system, we characterized PST-1 and PST-2 as PAPS transporters. We created deletion mutants to study the importance of PAPS transporter activity. The pst-1 deletion mutant exhibited defects in cuticle formation, post-embryonic seam cell development, vulval morphogenesis, cell migration, and embryogenesis. The pst-2 mutant exhibited a wild-type phenotype. The defects observed in the pst-1 mutant could be rescued by transgenic expression of pst-1 and hPAPST1 but not pst-2 or hPAPST2. Moreover, the phenotype of a pst-1;pst-2 double mutant were similar to those of the pst-1 single mutant, except that larval cuticle formation was more severely defected. Disaccharide analysis revealed that heparan sulfate from these mutants was undersulfated. Gene expression reporter analysis revealed that these PAPS transporters exhibited different tissue distributions and subcellular localizations. These data suggest that pst-1 and pst-2 play different physiological roles in heparan sulfate modification and development.

Translationally controlled tumor protein is a novel biological target for neurofibromatosis type 1-associated tumors.

Background: Loss of NF1 gene function predisposes individuals to develop NF1-associated tumors, for which there are no known biomarkers/therapeutic targets. Results: TCTP was up-regulated in NF1-associated tumors and enhanced their growth via its positive feedback to the mTOR signaling, which was inhibited by artesunate/rapamycin. Conclusion: TCTP is a novel target functionally implicated in NF1 tumorigenesis. Significance: TCTP could serve as a biomarker/therapeutic target for NF1-associated tumors. Neurofibromatosis type 1 (NF1) is an autosomal dominant disease that predisposes individuals to develop benign neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). Due to the lack of information on the molecular mechanism of NF1-associated tumor pathogenesis or biomarkers/therapeutic targets, an effective treatment for NF1 tumors has not been established. In this study, the novel NF1-associated protein, translationally controlled tumor protein (TCTP), was identified by integrated proteomics and found to be up-regulated via activated MAPK/PI3K-AKT signaling in response to growth factors in NF1-deficient Schwann cells. Immunohistochemical analysis of NF1-associated tumors revealed that the TCTP expression level correlated with tumorigenicity. In NF1-deficient MPNST cells, TCTP protein but not mRNA was down-regulated by NF1 GTPase-activating protein-related domain or MAPK/PI3K inhibitors, and this correlated with suppression of mammalian target of rapamycin (mTOR) signaling. mTOR inhibition by rapamycin also down-regulated TCTP protein expression, whereas knockdown or overexpression of TCTP suppressed or activated mTOR signaling, respectively, and affected cell viability. These results suggest that a positive feedback loop between TCTP and mTOR contributes to NF1-associated tumor formation. Last, the anti-tumor effect of artesunate, which binds to and degrades TCTP, was evaluated. Artesunate significantly suppressed the viability of MPNST cells but not normal Schwann cells, and the TCTP level inversely correlated with artesunate sensitivity. Moreover, combinational use of artesunate and rapamycin enhanced the cytotoxic effect on MPNST cells. These findings suggest that TCTP is functionally implicated in the progression of NF1-associated tumors and could serve as a biological target for their therapy.

GPI-anchor synthesis is indispensable for the germline development of the nematode Caenorhabditis elegans

Twenty-four Caenorhabditis elegans genes are involved in GPI-anchor synthesis. Based on the isolation of a deletion allele of the PIGA gene mediating the first step of GPI-anchor synthesis, GPI-anchor synthesis in somatic gonads and/or in germline is shown to be indispensable for the normal development of oocytes and eggs.

The ortholog of human solute carrier family 35 member B1 (UDP-galactose transporter-related protein 1) is involved in maintenance of ER homeostasis and essential for larval development in Caenorhabditis elegans

Although the solute carrier 35B1 (SLC35B1) is evolutionarily conserved, its functions in metazoans remain unknown. To elucidate its function, we examined developmental roles of an SLC35B1 family gene (HUT‐1: homolog of UDP‐Gal transporter) in Caenorhabditis elegans. We isolated a deletion mutant of the gene and characterized phenotypes of the mutant and hut‐1 RNAitreated worms. GFP‐HUT‐1 reporter analysis was performed to examine gene expression patterns. We also tested whether several nucleotide sugar transporters can compensate for hut‐1 deficiency. The hut‐1 deletion mutant and RNAi worms showed larval growth defect and lethality with disrupted intestinal morphology. Inactivation of hut‐1 induced chronic endoplasmic reticulum (ER) stress, and hut‐1 showed genetic interactions with the atf‐6, pek‐1, and ire‐1 genes involved in unfolded protein response signaling. ER ultrastructure and ER marker distribution in hut‐1‐deficient animals showed that HUT‐1 is required for maintenance of ER structure. Reporter analysis revealed that HUT‐1 is an ER protein ubiquitously expressed in tissues, including the intestine. Lethality and the ER stress phenotype of the mutant were rescued with the human hut‐1 ortholog UGTrel1. These results indicate important roles for hut‐1 in development and maintenance of ER homeostasis in C. elegans.—Dejima, K.,Murata, D., Mizuguchi, S., Nomura, K. H., Gengyo‐Ando, K.,Mitani, S., Kamiyama, S., Nishihara, S., Nomura, K. The ortholog of human solute carrier family 35 member B1 (UDP‐galactose transporter‐related protein 1) is involved in maintenance of ER homeostasis and essential for larval development in Caenorhabditis elegans. FASEBJ. 23, 2215–2225 (2009)