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J. Biol. Chem., Vol. 282, Issue 11, 8533-8544, March 16, 2007
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21¶3¶¶2****¶24
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
Received for publication, December 4, 2006 , and in revised form, January 16, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1-3Gal1-3Gal1-4Xyl1-), which is assembled by the stepwise transfer of monosaccharides from respective UDP-sugars to a Ser residue of the core proteins and/or the naked nonreducing terminus through the actions of the respective specific glycosyltransferases. Next, chain elongation, which results in the formation of the repeating disaccharide region of HS, occurs with GlcNAc and GlcUA transferred alternately by the actions of HS copolymerases. Finally, HS chains are modified by GlcNAc N-deacetylase, GlcUA epimerase, and sulfotransferases (for reviews see Refs. 3 and 4). Recently, studies involving cDNA cloning of the genes encoding these enzymes and genetic analyses using knock-out mice, Zebrafish, fruit flies, and nematodes have led to unanticipated findings of interesting biological phenotypes (for a review see Ref. 5).
In humans, polymerization of the repeating disaccharide region in HS occurs through the actions of an enzyme complex consisting of EXT1 (6) and EXT2 (7) of the EXT (exostosin) gene family (8-11). These genes were first identified as causative of a genetic bone disorder, hereditary multiple exostoses (HME), and subsequently demonstrated to function as tumor suppressor genes. HME is an autosomal dominant disorder characterized by the formation of a cartilage-capped tumor, caused by mutations in either EXT1 or EXT2 (12). The family of EXT genes has been extended to include three EXT-Like genes, EXTL1, EXTL2, and EXTL3, all of which have GlcNAc transferase activities, likely involved in HS synthesis (4, 13, 14).
HS is present in genetically tractable model animals as well (15-17), and EXT genes are well conserved in mammals, Zebrafish (Danio rerio) (ext1, ext2/dackel, and extl3/boxer), Drosophila melanogaster (ttv, sotv, and botv) and Caenorhabditis elegans (rib-1 and rib-2) (18-28). In Zebrafish dackel and boxer mutants, which are defective in ext2 and extl3, respectively, some dorsal retinal ganglion cell axons inappropriately project into the optic tract (19). Even though Dackel and Boxer have not been demonstrated to have glycosyltransferase activities, the amount of HS was drastically reduced in the dackel and boxer mutants, suggesting that both genes are required for its production (19). In addition, Drosophila homologs of EXT genes, tout-velu (ttv), sister of tout-velu (sotv), and brother of tout-velu (botv), which correspond to vertebrate EXT1, EXT2, and EXTL3, respectively, are involved in Hh, Wg, and Dpp signaling (20-23). Biochemical and immunohistochemical studies in Drosophila have revealed that HS levels are dramatically reduced in the absence of ttv, sotv, or botv (21-23). We recently demonstrated in vitro that the polymerization is performed by a complex of TTV and SOTV (TTV-SOTV) as an enzyme source (25). However, TTV-SOTV exhibited no GlcNAc transferase-I (GlcNAcT-I) activity required for the initiation of HS, indicating that BOTV, which corresponds to human EXTL3 and possesses GlcNAcT-I activity (24), is indispensable for the biosynthesis of HS chains in Drosophila. Thus, all three EXT members in Drosophila, TTV, SOTV, and BOTV, are required for the biosynthesis of a full-length HS.
In C. elegans, rib-1 and rib-2 are most homologous to EXT1 and EXTL3, respectively, among the human EXT genes (26). rib-2 mutants exhibit developmental delay and egg-laying defects, which are most likely caused by a reduction in HS (28). Although rib-2 encodes a GlcNAc transferase involved in initiating the biosynthesis of HS and in the elongation of HS chains (27), the involvement of the protein encoded by rib-1 in HS biosynthesis remains unclear. In this study, we investigated the polymerization of HS using recombinant soluble forms of RIB-1 and RIB-2 to elucidate the mechanism behind the biosynthesis of HS in C. elegans, and we demonstrated that the polymerization was performed by a complex of RIB-1 and RIB-2 as an enzyme source. In addition, to investigate functions of RIB-1 in vivo, we depleted the expression of rib-1 by deletion mutagenesis. Here we demonstrate that in addition to rib-2, rib-1 is indispensable for the biosynthesis of HS and embryonic morphogenesis in C. elegans.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Single Worm PCR—Single animals were picked up with a platinum wire and placed individually in a 10-µl drop of lysis buffer (500 µg/ml proteinase K in 25 mM Tris-HCl, pH 8.5, 50 mM KCl, 0.5% Tween 20, 1 mM EDTA) in a 200-µl PCR tube. The drops were spun down to the bottom of the tube by brief centrifugation, frozen (-80 °C, 10 min), and heated (50 °C, 30 min followed by 95 °C, 20 min). After cooling to room temperature, 1-2 µl of the drops was used as a template for single worm PCR for the tm516 and tm710 deletion alleles with the primers described above.
Materials—UDP-[U-14C]GlcUA (285.2 mCi/mmol) and UDP-[3H]GlcNAc (60 Ci/mmol) were purchased from PerkinElmer Life Sciences. Unlabeled UDP-GlcUA and UDP-GlcNAc were obtained from Sigma. Arthrobacter aurescens chondroitinase ABC and Flavobacterium heparinum heparitinases I and II were purchased from Seikagaku Corp. (Tokyo, Japan). N-Acetylheparosan oligosaccharides derived from the capsular polysaccharide of Escherichia coli K5 (30) were gifts from Marion Kusche-Gullberg and Ulf Lindahl, Uppsala University, Uppsala, Sweden. SuperdexTM 75 HR10/30 and SuperdexTM Peptide HR10/30 columns were supplied by GE Healthcare.
Construction of Soluble Forms of RIB-1 and RIB-2—A cDNA fragment of a truncated form of RIB-1, lacking the first 17 amino-terminal amino acids, including the putative cytoplasmic and transmembrane domains, was amplified by reverse transcription-PCR with adult C. elegans total RNA as a template using a 5'-primer (5'-GAAGATCTGTTCGGTTACAAAATCCAACTATTG-3') containing a BglII site and a 3'-primer (5'-GAAGATCTAGATCGGGATTGAACCTTATGG-3') containing a BglII site located 22 bp downstream of the stop codon. In the case of RIB-2, the cDNA fragment encoding a truncated form of RIB-2, lacking the first amino-terminal 38 amino acids of RIB-2 (AB077851 [GenBank] ), was amplified by reverse transcription-PCR with adult C. elegans total RNA as a template using a 5'-primer (5'-CGGGATCCTCCTTCTCCGAGCCTTCCTG-3') containing a BamHI site and a 3'-primer (5'-CGGGATCCGTAGCACCATTCGA-3') containing a BamHI site located 68 bp downstream of the stop codon. PCR was carried out with KOD polymerase (Toyobo, Osaka, Japan) for 32 cycles of 94 °C for 30 s, 60 °C for 30 s, and 68 °C for 180 s in 5% (v/v) dimethyl sulfoxide. The PCR fragment was subcloned into the BamHI site of pGIR201protA (31), resulting in the fusion of the insulin signal sequence and the protein A sequence present in the vector as described previously (25). The nucleotide sequence of the amplified cDNA was determined in a 377 DNA sequencer (Applied Biosystems).
Expression of Soluble Forms of RIB-1 and RIB-2 and Enzyme Assays—The expression plasmid (6.0 µg) was transfected into COS-1 cells on 100-mm plates using FuGENETM 6 (Roche Applied Science) according to the manufacturer's instructions. For cotransfection experiments, the RIB-1 and RIB-2 expression plasmids (3.0 µg each) (27) were cotransfected into COS-1 cells on 100-mm plates using FuGENETM 6 as above. After a 2-day culture at 30 °C, 1 ml of the culture medium was collected and incubated with 10 µl of IgG-Sepharose (GE Healthcare) for 1 h at 4 °C. The beads were recovered by centrifugation, washed with and then resuspended in each of the assay buffers described below, and tested for GlcNAcT-II transferase activity using 10 µg of N-acetylheparosan oligosaccharides, GlcUA1-[4GlcNAc
1-4GlcUA-]n, and for GlcAT-II activity using 10 µg of GlcNAc1-[4GlcUA1-4GlcNAc1-]n as an acceptor, respectively, as described previously (25). The assay mixture for GlcNAcT contained 10 µl of the resuspended beads, an acceptor substrate, 0.25 mM UDP-[3H]GlcNAc (8.21 x 105 dpm), 100 mM MES buffer, pH 5.8, and 10 mM MnCl2 in a total volume of 20 µl. The assay mixture for GlcAT-II activity contained 10 µl of the resuspended beads, an acceptor substrate, 0.25 mM UDP-[14C]GlcUA (5.66 x 105 dpm), 100 mM MES buffer, pH 5.8, and 10 mM MnCl2 in a total volume of 20 µl. A polymerization reaction using 10 µg of N-acetylheparosan oligosaccharides, GlcNAc1-[4GlcUA1-4GlcNAc1-]n, as an acceptor was conducted in incubation mixtures containing, in a total volume of 20 µl, 0.25 mM UDP-GlcNAc, 0.25 mM UDP-[14C]GlcUA (1.35 x 106 dpm), 100 mM MES buffer, pH 5.8, 10 mM MnCl2, and 10 µl of suspended beads. The mixtures were incubated at 26 °C overnight, and then radiolabeled products were separated from UDP-[3H]GlcNAc or UDP-[14C]GlcUA by gel filtration chromatography on a column of SuperdexTM 75 equilibrated and eluted with 0.2 M NH4HCO3. Fractions (0.4 ml each) were collected at a flow rate of 0.4 ml/min, and the measurement of radioactivity was carried out by liquid scintillation counting.
Western Blot Analysis—After 3 days of culture at 28 °C, the culture medium was collected and incubated with 10 µl of IgG-Sepharose (GE Healthcare) for 1 h at 4 °C. The beads recovered by centrifugation were washed with phosphate-buffered saline and then resolved on 7.5% SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and incubated for 1 h with mouse IgG antibody. The mouse IgG antibody was diluted 1:1,000 with 25 mM Tris-buffered saline. The bound antibody was detected with anti-mouse IgG conjugated to horseradish peroxidase. Proteins bound to the antibody were visualized with an enhanced chemiluminescence (ECL) advance kit (GE Healthcare).
Characterization of the Reaction Products—Products of polymerization reactions on N-acetylheparosan oligosaccharides, GlcUA1-[4GlcNAc1-4GlcUA-]n, or GlcUA1-3Gal1-O-C2H4NHCbz were isolated by gel filtration on a column of SuperdexTM 75 or SuperdexTM Peptide, respectively, with 0.2 M NH4HCO3 as the eluent. The radioactive peak containing the cotransfection reaction product was pooled and evaporated dry. The [14C]GlcUA-labeled oligosaccharide chains were exhaustively digested with 3 mIU of heparitinase I in a total volume of 20 µl of 50 mM sodium acetate buffer, pH 7.0, containing 2 mM calcium acetate at 37 °C overnight. The enzyme digest was analyzed using the same SuperdexTM 75 or SuperdexTM Peptide column as described above.
Analysis of Glycosaminoglycans—Freshly cultured nematodes were sonicated with a GE-70 ultrasonic processor (Branson Ultrasonics) and freeze-dried. The dried samples (295.2 mg of wild type or 386.3 mg of mutant nematode) were extracted with acetone and then treated with 6 ml of 1.0 M NaBH4, 0.05 M NaOH at 4 °C for 20 h. The reaction mixture was neutralized with acetic acid to stop the reaction. The samples were adjusted to 5% trichloroacetic acid and centrifuged. The soluble fraction was extracted with ether. As shown previously (15, 32, 33), the amount of HS in C. elegans was so small that 100 µg of shark cartilage chondroitin 6-O-sulfate (Seikagaku Corp.), which contains a negligible proportion of nonsulfated disaccharides, was added as a carrier. The aqueous phase was adjusted to 80% ethanol. The resultant precipitate was dissolved in 50 mM pyridine acetate and subjected to gel filtration on a PD-10 column using 50 mM pyridine acetate as an eluent. The flow-through fraction was collected and evaporated dry. The dried samples were dissolved in water and applied to a column (7 ml) of cation-exchange resin AG 50W-X2 (H+ form; Bio-Rad) pre-equilibrated with water. The unbound fraction containing the liberated O-linked saccharides was neutralized with 1 M NH4HCO3. The purified glycosaminoglycan fraction was digested with chondroitinase ABC or a mixture of heparitinases I and II, and then the digests were derivatized with 2-aminobenzamide and analyzed by high performance liquid chromatography as described previously (34).
Pulldown Assays—The cDNA fragment of a truncated form of RIB-2, lacking the first 38 amino-terminal amino acids of RIB-2, was amplified using a 5'-primer (5'-CGGGATCCCTCCTTCTCCGAGCCTTCC-3') containing an in-frame BamHI site and a 3'-primer (5'-CGGGATCCGTAGCACCATTCGA-3') containing a BamHI site located 68 bp downstream of the stop codon. The DNA fragment was inserted into the expression vector pcDNA3Ins-His, resulting in the fusion of the protein with the insulin signal sequence and His6 sequence present in the vector. The rib-2 construct and the protein A-tagged RIB-1 expression vector were introduced into COS-1 cells on 100-mm plates using FuGENETM 6 (Roche Applied Science) according to the manufacturer's instructions. Two days after the cotransfection, 1 ml of the culture medium was collected and incubated with 10 µl of Ni-NTA-agarose (Qiagen) overnight at 4 °C. The beads recovered by centrifugation were washed with Tris-buffered saline buffer containing Tween 20 three times and subjected to SDS-PAGE (7% gel), and proteins were transferred to a polyvinylidene difluoride membrane. The membrane, after blocking in phosphate-buffered saline containing 2% skim milk and 0.1% Tween 20, was incubated with IgG antibody and then treated with anti-mouse IgG conjugated with horseradish peroxidase (GE Healthcare). Proteins bound to the antibody were visualized with an ECL advance kit (GE Healthcare).
Transgenic Construct—The tph-1 (ZK1290.2) reporter gene plasmid was constructed using the vector pFX_DsRedXT (33, 34). The translational fusion construct contained a 3.7-kb genomic fragment, including the 3.1-kb potential promoter region. The fragment was amplified with C. elegans genomic DNA as a template using a 5'-primer (5'-GAGTGCAAGACTATTAGGGAGT-3') and a 3'-primer (5'-AGTACGAGTTGGTGTGAAGAG-3') located at the start of the 3rd exon. PCR was carried out with Platinum TaqDNA polymerase high fidelity (Invitrogen) for 28 cycles of 94 °C for 30 s, 58 °C for 30 s, and 68 °C for 240 s followed by adenine addition with Taq polymerase at 72 °C for 15 min. The PCR fragment was inserted into pFX_ DsRedXT using the TA cloning strategy to fuse the coding sequence region of DsRed.
Phenotypic Analysis—We determined the embryonic phenotypes and extent of lethality of the rib-1/rib-2 mutant worms by monitoring embryonic development with four-dimensional microscopy (a DMRXA full automatic microscope with differential interference contrast and fluorescence optics; Leica) (32) followed by single worm PCR to confirm the genotype. A total of 92 rib-1-/- worms was monitored from the start of gastrulation until they had reached a terminal phenotype and scored. Phenotypes were also monitored and scored by using a high resolution dissecting microscope (Olympus SZX12) as described previously (36).
Transgenic Rescue Experiment—To rescue the rib-1 deletion mutant tm516, a transgenic rescue construct was created using the C. elegans expression vector (pFX_LVT-R03G5.1) constructed from PFX_venusT cloning vector (35), in which eft-4 (R03G5.1; elongation factor-1 ) promoter (3,616 bp from X:7820004 to X:7823619) was inserted into the XcmI site in the multicloning site, which is located upstream of the venus open reading frame. The rib-1 cDNA was reverse-transcribed with SuperScript II reverse transcriptase (Invitrogen) from the total RNA prepared from mixed stage worms (36) and amplified with PCR using a 5'-primer (5'-GTGGCGGCCGCATGCAAAATGTAATGAAAT-3') containing the start codon and a 3'-primer (5'-CATGCGGCCGCCTTGATGATCTTCCAGCTC-3') located before the stop codon. PCR was carried out with Platinum TaqDNA polymerase high fidelity (Invitrogen) for 30 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 90 s. The full-length cDNA was inserted into a NotI site and placed under the control of the eft-4 promoter, and the sequence was verified by using Prism 3130 Genetic analyzer (Applied Biosystems, Inc.). The rescue of the rib-1 mutant strain (tm516) with the full-length cDNA was performed by microinjecting the rescue construct into distal gonads as described (36). The concentration of injected DNA was 39 µg/ml, and no coinjection marker was used in the experiment.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To facilitate the functional analysis of RIB-1, a soluble, protein A-tagged form of the protein was generated by replacing the first 17 amino acids with a cleavable insulin signal sequence and a protein A IgG-binding domain as described under "Experimental Procedures." Then the soluble protein was expressed in COS-1 cells at 30 °C as a recombinant protein fused with the protein A IgG-binding domain. The fusion protein secreted in the medium was adsorbed onto IgG-Sepharose beads for purification to eliminate endogenous glycosyltransferases, and then the protein-bound beads were used as an enzyme source. Although the bound fusion protein was assayed for glycosyltransferase activity at 26 °C using N-acetylheparosan oligosaccharides, [GlcUA-GlcNAc]n, or GlcNAc-[GlcUA-GlcNAc]n, as a sugar acceptor and either UDP-GlcNAc or UDP-GlcUA as a sugar donor substrate, neither GlcNAcT-II nor GlcAT-II activity was detected (Table 1). However, coexpression of the soluble RIB-1 with the soluble RIB-2 clearly showed GlcAT-II activity, which had not been detected when only RIB-2 was expressed. Notably, these effects of coexpression were not because of differences in the levels of these proteins, as assessed by Western blot analysis (Fig. 2). These results are analogous to the findings previously made for human EXT1 and EXT2 (9). Hence, we examined whether the coexpression of RIB-1 and RIB-2 could result in the expression of polymerizing activity. Incubation of the coexpressed proteins with N-acetylheparosan oligosaccharides, [GlcUA-GlcNAc]n, with the nonreducing terminal GlcUA as an acceptor in the presence of UDP-GlcNAc and UDP-[14C]GlcUA yielded radiolabeled polymer N-acetylheparosan chains. The reaction products were analyzed by gel chromatography using a column of SuperdexTM 75 as shown in Fig. 3A. The synthesized saccharide chains were around 20-mer in length. The radioactive peaks containing cotransfectant reaction products were digested with heparitinase I and subjected to gel chromatography as described under "Experimental Procedures." The radiolabeled products were completely digested, quantitatively yielding a 14C-labeled peak at the position of [14C]GlcUA1-3GlcNAc, indicating that the polymerization occurred on N-acetylheparosan when RIB-1 and RIB-2 were coexpressed (Fig. 3A).
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1-3Gal1-O-C2H4NHCbz, was used. The linkage region analog, GlcUA1-3Gal1-O-C2H4NHCbz, was incubated with the coexpressed RIB-1 and RIB-2, and the reaction products were analyzed by gel chromatography using a column of SuperdexTM Peptide as described under "Experimental Procedures." As shown in Fig. 3B, HS polymerization was demonstrated, and the synthesized saccharide chains were completely digested by heparitinase I. These results indicated that two C. elegans EXT proteins, RIB-1 and RIB-2, together could achieve polymerization on a linkage region.
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80 kDa, corresponding to RIB-1-ProA, was detected for the cotransfectant of RIB-1-ProA and RIB-2-His (Fig. 4, lane 2). As expected, the coexpression of SQV-5 (cChSy)-ProA and CPF-1-His gave a single band with a molecular mass of 130 kDa, corresponding to SQV-5-ProA (Fig. 4, lane 4). These results indicated that RIB-1 and RIB-2 physiologically interact with each other.
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Almost all (99.3% (n = 1622)) of the rib-1-/- fertilized eggs laid by rib-1-/- adult worms showed embryonic lethality and died before the L1 stage. The remaining 0.7% worms hatched and showed abnormal head or tail structures and died just after hatching at L1 (Figs. 5D and 6C). In some cases, axon migration patterns of adult worms hatched from heterozygous worms were severely distorted (Fig. 5F), especially in hermaphrodite-specific neurons (HSNs) (26.5%, n = 34). Two axon neuronal processes from HSNs showed midline crossover defects, whereas in normal N2 worms, they run parallel with each other (Fig. 5G). This phenotype is similar to the HSN abnormality reported on the knock-out of the HS glucuronyl C5-epimerase gene (hse-5) in C. elegans (39). For the Egl phenotype, further detailed study is necessary to know whether the phenotype is related to defects in egg-laying neurons, muscle cell functions, or abnormal vulva structure as found in the protruding vulva.
To determine at what point abnormal development is detected, we monitored 92 rib-1-/- embryos by four-dimensional microscopy. Ingression of E-daughter cells seemed to be normal and blastocele was formed. The first abnormality was detected at the "closure of ventral cleft" stage, and worms with an abnormal ventral cleft (80%, n = 10) were observed along with worms with a normal ventral cleft (20%). In severe cases (class I, Fig. 7A), oozing of cells and prominent bulges were observed in various regions of embryos. These phenotypes strongly suggest that normal epidermal enclosure was severely affected (40). All class I embryos were arrested at 1-1.25-fold stages and died (68.4% n = 92). In some cases (class II, Fig. 7B), embryos continued to elongate to the 1.5-fold stage at which point they failed to elongate further and died (21.1%). Oozing of cells from ventral midline region was clearly observed in these embryos. In other cases (class III), embryos developed into 1.5-2.5-fold stages and died (10.5%).Oozing of a few cells was observed. In class I to class III rib-1-/- F1 embryos, active apoptosis was observed after intestinal differentiation (Fig. 6, A and B). Judging from active contractile movement (twitching) in these rib-1-/- embryos (92.4%, n = 92), at least some of the contractile muscle tissues seemed to differentiate normally. In some cases, the differentiation of a pharyngeal bulb-like structure in the head region was observed, and muscles frequently contracted (see Ref. 41 and data not shown). To examine the relative timing of neuronal differentiation and rib-1-/- embryonic death, we crossed rib-1+/- worms with the pan-neuronal GFP reporter strain NW1229 (made and deposited to CGC by J. Culotti), and we examined the differentiation of neuronal cells in rib-1-/- integrated Ex[F25B3.3::gfp; dpy-20(+)] embryos. The rib-1-/- embryos confirmed to be null mutants by single worm PCR with rib-1 primer pairs showed active neuronal GFP fluorescence (Figs. 5B and 6D). Similar results were obtained in transgenic worms expressing DsRed under the control of the tph-1 promoter. In the transgenic N2 worms, differentiated NSM serotonergic neurons were usually stained brightly, and in some worms the differentiated hermaphrodite-specific HSNs were also stained brightly (see Ref. 42 and data not shown). In the transgenic rib-1-/- Ex[tph-1::DsRed] worms, bright neuronal fluorescence of possible NSM pair neurons (NSML/NSMR) and neuronal processes were observed in developmentally arrested embryos, suggesting the normal differentiation of these neuronal cells in rib-1-/- worms (Fig. 6, E and F). In these null mutant embryos, morphogenic movement stopped in the early stages of elongation and development appeared to be arrested (Fig. 7, A-C). Although some of the neurons and muscle cells seemed to differentiate in the null mutant embryos as described above, further detailed study is necessary to know whether any other cells (including neuronal and muscle cells) show various degrees of defects in the embryos or not.
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-integrin) and pat-3 (-integrin), which are essential for muscle-body wall integration (43), suggesting possible interactions with HS proteoglycans.
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Reduction of HS Synthesis in rib-1 Knock-out Worms—We next examined the amount of HS and chondroitin in mass cultures of tm516 worms. Because it was impractical to isolate large quantities of rib-1-/- worms because of their lethal condition, plate culture samples of tm516 worms possibly containing rib-1+/+, rib-1+/-, and rib-1-/- worms were used for biochemical analysis. The expected ratio of worms containing these genotypes is 1:2:1. Because rib-1-/- worms are expected to die in the later stages of embryogenesis, the rib-1 gene dose in the mass culture is estimated to be 66% that in wild type N2 worms. As shown in Table 2, direct measurements revealed that the amount of HS in the mass culture was reduced to 63% that in the wild type mass culture, whereas the amount of chondroitin was increased to 143%. Although the change in chondroitin was unexpected, comparable increases have been observed in rib-2 mutants (28), and an analogous increase in HS was observed in sqv-5 (cChSy)- or pfc-1-RNAi-treated worms deficient in chondroitin biosynthesis (32, 33), suggesting levels of HS and chondroitin are interdependent in vivo. In addition, the disaccharide composition of the HS in the mass culture was comparable with that in the wild type mass culture (Table 3). These findings were similar to those obtained for the rib-2 deletion mutant (28) and strongly suggest that the rib-1 mutant is specifically defective in the synthesis of HS and not chondroitin in the nematode C. elegans.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Although five EXT family genes (EXT1, EXT2, EXTL1, EXTL2, and EXTL3) have been identified in the human genome, only two homologs, rib-1 and rib-2, have been found in the C. elegans genome. Previously, we revealed that rib-2 encodes GlcNAcT involved in initiating the biosynthesis and in the elongation of HS but not GlcAT-II required for lengthening the HS chains (27). Because HS is found in C. elegans (15, 16), it was predicted that rib-1 encodes the GlcAT-II. However, when a soluble recombinant form of RIB-1 was produced and assayed for GlcAT-II and also GlcNAcT activities, no such glycosyl-transferase activities were detected. In this study, despite little glycosyltransferase activity by RIB-1, we showed that the polymerization of HS in C. elegans was achieved by an enzyme complex composed of RIB-1 and RIB-2, which is analogous to the findings made previously for the human EXT1 and EXT2 protein complex (9). In this regard, it should be noted, however, that RIB-1 is unique among members of the EXT family identified to date in that the protein, composed of 382 amino acids, is about half the size of RIB-2 and shows significant homology only to the amino termini of EXT family members, especially to EXT1 (45% amino acid identity, see Fig. 1A). Moreover, RIB-2 is most homologous to human EXTL3 (not to EXT2) (see Fig. 1B). In fact, as mentioned above, RIB-2 harbors both GlcNAcT-I and GlcNAcT-II activities involved in the chain initiation and elongation of HS, and its acceptor specificity resembles that of human EXTL3 (12, 27), suggesting that rib-2 is a C. elegans EXTL3 ortholog (Fig. 1B). These results together indicate that the mechanism of HS biosynthesis in C. elegans is similar but distinct from that in humans and that both RIB-1 and RIB-2 are indispensable for the biosynthesis of HS chains in the nematode.
The human EXT1 and EXT2 genes are involved in HME, an autosomal dominant disorder characterized by the formation of a cartilage-capped tumor, caused by mutations in either EXT1 or EXT2 (12). The family of EXT genes has been extended with the identification of three additional EXT-like genes, EXTL1, EXTL2, and EXTL3 (4). Although the EXT-like genes are predicted to be involved in the synthesis of HS on the basis of the enzymatic activities of their products in vitro (13, 14), these genes are not linked with HME and have not been associated with any disease pathology, and so their functions in vivo have not been determined. In fact, the polymerization of HS on the linkage analog is achieved by an enzyme complex of EXT1 and EXT2, without the aid of EXTL proteins (8-10). Because the polymerization of HS was achieved with low efficiency by a complex of RIB-1, the EXT1 counterpart in C. elegans, and RIB-2, the EXTL3 counterpart in C. elegans, and the amount of HS in C. elegans was much smaller than that of chondroitin (15, 16) (see Table 2), it is likely that EXT1 and EXTL3 can also cooperate to polymerize HS chains albeit with low efficiency in mammals.
EXT1- and EXT2-deficient mice generated by gene targeting failed to undergo gastrulation and died by embryonic day 8.5, pointing to the early essential role for HS in mammalian embryonic development. In fact, embryonic stem cells from EXT1- and EXT2-deficient mice showed a loss of HS (44, 45). Interestingly, it has been reported that EXT1 mutant mice generated by the gene trap method (EXT1Gt/Gt) survive to embryonic day 14.5 (46). In addition, Yamada et al. (47) showed that the embryonic fibroblasts established from mice with the gene trap mutation in EXT1 produce short HS chains as compared with those from wild type mice. The gene trap cassette is inserted between exons 1 and 2 in the EXT1 gene. Exon 1 encodes 43% of all the amino acids (i.e. 320 of 746), indicating that the mutant EXT1 still contains almost half of the amino-terminal side of the protein. Thus, they suggested that the presence of HS in the gene trap mouse could be a result of residual transferase activity in the amino-terminal part of the protein. As mentioned above, the RIB-1 protein, composed of 382 amino acids, is about half the size of EXT1 and shows significant homology only to the amino termini of the EXT family members, especially to EXT1. Therefore, it is possible that the mutant EXT1 protein formed in the gene trap mouse can interact with EXTL3 and that these two proteins might cooperate to produce short HS chains.
The mechanism for the biosynthesis of HS is reminiscent of that for chondroitin in C. elegans. The repeating disaccharide region of chondroitin is synthesized by glycosyltransferases encoded by sqv-5 (cChSy) (32, 48) and pfc-1 (PAR2.4) (33), which form an enzyme complex (chondroitin polymerase) (see Fig. 3, lane 4). RNAi depletion of SQV-5 or PFC-1 in C. elegans resulted in a reduction of chondroitin, and the RNAi-treated worms showed similar phenotypes, such as cytokinetic defects in early embryogenesis (32, 33). Thus, it has been suggested that sqv-5 and pfc-1 cannot have redundant roles in the biosynthesis of chondroitin, which is similar to the observation that rib-1 or rib-2 null mutant worms showed reduced synthesis of HS and similar phenotypes, such as post-gastrulation and egg-laying defects. Therefore, both RIB-1 and RIB-2 have nonredundant functions as do SQV-5 (cChSy) and PFC-1 (PAR2.4). In addition, these observations suggest that the mechanism for the biosynthesis of HS is similar to that for chondroitin in C. elegans and clearly indicate that the functions of HS are different from those of chondroitin in C. elegans.
Several recent studies in C. elegans have greatly expanded our understanding of how specific modifications to HS can affect different signaling pathways in different tissues (2). In this study and in the related study of Morio et al. (28), mutations in rib-1- and rib-2-encoded proteins involved in the polymerization of HS affected post-gastrulation morphogenic movement and the subsequent elongation in F1 embryos as well as egg-laying and other phenotypes in P0 worms. We detected defects in ventral enclosure and epithelial morphogenesis in rib-1-/- F1 worms, and the results suggest the possible involvement of HS in epidermal morphogenesis as well. In this respect, Franks et al. (41) reported genetic interactions between pyr-1 and rib-1/rib-2 system. They also showed that the rib-1 and rib-2 system play vital roles in body elongation and pharyngeal isthmus morphogenesis. These results strongly indicate the indispensable roles of HS in post-gastrulation and the subsequent morphogenesis.
In contrast, HS-modifying enzymes are not required for viability in C. elegans.Bülow and Hobert (39) isolated mutants of the HS 6-O-sulfotransferase, the glucuronyl C5-epimerase, and the HS 2-O-sulfotransferase, and they found each to be viable and fertile, indicating a more specialized function of the modification to HS. Further analyses showed that each of the mutant worms exhibits distinct yet overlapping axonal and cellular guidance defects in specific classes of neurons. Hence, it has been proposed that individual modifications to HS in a given area or on specific neurons give rise to a "sugar code" that regulates axonal guidance by modulating specific receptor-ligand interactions (39). Additional regional specificity in HS modification patterns can be brought about by modifying core proteins, such as syndecan (49-51). However, no studies have yet addressed the fundamental questions of whether signaling molecules interact differently with specifically modified HS sequences and how these sequences affect particular signaling pathways. The development of robust and highly sensitive sequencing methods for HS chains will provide definitive answers to these questions in the future.
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* 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.
5 The abbreviations used are: HS, heparan sulfate; GFP, green fluorescent protein; GlcAT, glucuronyltransferase; HSN, hermaphrodite-specific neuron; HME, hereditary multiple exostoses; MES, 2-(N-morpholino)ethanesulfonic acid; ECL, enhanced chemiluminescence; Ni-NTA, nickel-nitrilotriacetic acid; NSM, neurosecretory motor; RNAi, RNA interference; DIC, differential interference contrast.
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