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.

Chondroitin sulfate proteoglycans (CS-PGs) 1 are universally distributed glycoproteins consisting of CS chains substituted on core proteins and are located in the extracellular matrices and on cell surfaces in various kinds of human tissues. Some CS-PGs modulate cell adhesion, cell proliferation, and morphogenesis (for reviews, see Refs. 1 and 2). We have revealed that chondroitin is required for normal cell division and cytokinesis at an early developmental stage in Caenorhabditis elegans (3). In addition, recent studies have demonstrated that CS chains are major inhibitory molecules affecting axon growth after spinal cord injury in the central nervous system of adult mammals (4), but that they can also stimulate neuronal differentiation in a structure-dependent fashion (2). Thus, it is imperative to elucidate the mechanism of the biosynthesis of chondroitin and CS for a better understanding of various developmental processes in lower organisms through mammals.
The biosynthesis of CS is initiated by the addition of Xyl to specific serine residues in the core protein followed by the sequential addition of two Gal residues and a GlcUA residue, forming the tetrasaccharide linkage structure GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser. Then, chondroitin polymerization with alternating GalNAc and GlcUA takes place by the action of a complex consisting of chondroitin synthase (ChSy) (5) and chondroitin polymerizing factor (ChPF), a unique protein factor required for the polymerization (6). Also, the functionally redundant, multiple glycosyltransferases involved in chondroitin biosynthesis have been cloned (7)(8)(9)(10)(11). This redundancy makes it difficult to investigate the mechanism of chondroitin biosynthesis by gene knockout or characterization of individual glycosyltransferases.
To clarify the mechanism of chondroitin biosynthesis in vivo, * This work was supported in part part by grants from the Science Research Promotion Fund of the Japan Private School Promotion Foundation and the Kato Memorial Bioscience Foundation (to H. K.) and Grants-in-aid for Scientific Research C16590075 (to H. K.) and B16390026 (to K. S.) and 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM  we have been using lower organisms such as C. elegans because they are predicted to have few glycosyltransferases and a simple mechanism for production of the chondroitin chain. C. elegans is one of the most tractable model animals because all genomic DNA sequences and all cell lineages are known. In addition, it is easy to knock down specific genes using the reverse genetic method, RNA-mediated interference (RNAi) (12). Furthermore, C. elegans produces a large amount of nonsulfated chondroitin but not CS (13,14). Recently, we cloned a ChSy ortholog (cChSy or sqv-5) in C. elegans and depleted expression of its product using RNAi methods (3). cChSy-RNAitreated worms showed defects of cytokinesis in early embryogenesis, and cell division eventually stopped as a result of depletion of chondroitin (3,15).
Speculating that the fundamental mechanism underlying the biosynthesis of chondroitin in C. elegans might be similar to that in humans, we hypothesized that an ortholog of ChPF might exist in C. elegans. Screening of a data base using the amino acid sequence of human ChPF identified a candidate protein, PAR2.4. In addition, it was predicted that only two genes, cChSy and PAR2. 4 3 and GalNAc␤1-4(GlcUA␤1-3GalNAc) 3 were prepared from chondroitin as described previously (16,17). A Superdex TM peptide HR10/30 column and Sephadex LH-20 were obtained from Amersham Biosciences.
Chemical stirring at 0°C. One hour later, the reaction mixture was evaporated to dryness. To the stirred solution of the residue in a mixture of 10.5 ml of MeOH and 3.5 ml of H 2 O was added 2.5 ml of 0.107 M sodium methoxide dropwise at 0°C. The reaction mixture was neutralized with 50% AcOH after 17 h, and the volatiles were removed under diminished pressure. The residue was passed through a column of Sephadex LH-20 for gel permeation using 1% AcOH as eluent. The fractions containing the disaccharide were evaporated, and the residue was purified by passing through a column of Dowex® AG50W (H ϩ ) using MeOH-H 2 O (8:1, v/v) as eluent. The fractions containing the disaccharide were freeze-dried to give the final amorphous compound in 88% yield (75.6 mg). [␣] D was measured at 25°C to give Ϫ16.3°(c 0.48, in H 2 O). The 1 H-NMR spectrum was measured on a JEOL ECP 500-MHz spectrometer (JEOL, Tokyo) at a probe temperature of 25°C, and the chemical shifts are given in ppm relative to tetramethylsilane but were measured relative to internal tert-BuOH (1. Molecular Cloning of PAR2.4 -A tBLASTn analysis of the Gen-Bank TM data base, using the sequence of human ChSy (5), identified a highly homologous clone, PAR2.4 (GenBank TM accession number U00025). Using WormBase, the cDNA sequence was obtained (accession number WBGene 00019802).
Construction of a Soluble Form of PAR 2.4 and cChSy-A cDNA fragment of a truncated form of PAR2.4, lacking the first 52 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 5Ј-primer (5Ј-CGG-GATCCGTTCTTGAACCATCGGCACTGG-3Ј) containing a BamHI site and a 3Ј-primer (5Ј-CGGGATCCGAGTAGAAGGGTGTTCATTTTTCG-3Ј) containing a BamHI site and a stop codon. In the case of cChSy, the cDNA fragment encoding a truncated form of cChSy, lacking the first amino-terminal 43 amino acids of cChSy (AB088397), was amplified by reverse transcription-PCR with adult C. elegans total RNA as a template using a 5Ј-primer (5Ј-CGGGATCCGGCGGTTTCGATTATCTC-GAT-3Ј) containing an in-frame BamHI site and a 3Ј-primer (5Ј-CGG-GATCCGGTGGAAAACCGGCAAGGC-3Ј) containing a BamHI site located 21 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 (19), resulting in the fusion of the insulin signal sequence and the protein A sequence present in the vector as described previously (3).
Expression of a Soluble Form of PAR 2.4 and Enzyme Assays-The expression plasmid (6.0 g) was transfected into COS-1 cells on 100-mm plates using FuGENE TM 6 (Roche Applied Science) according to the manufacturer's instructions. For co-transfection experiments, the PAR2.4 and cChSy expression plasmids (3.0 g of each) (3) were co-transfected into COS-1 cells on 100-mm plates using FuGENE TM 6 as above. After a 2-day culture at 30°C, 1 ml of the culture medium was collected and incubated with 10 l of IgG-Sepharose (Amersham Biosciences) for 1 h at 4°C. The beads were recovered by centrifugation, washed, resuspended in the assay buffer described below, and then tested for GalNAcT-II transferase activity using 5 nmol of the chondroitin-derived hexasaccharide as an accepter and UDP-GalNAc as a sugar donor and tested for GlcAT-II transferase activity using 5 nmol of the chondroitin-derived heptasaccharide and UDP-GlcUA, as described previously (3,16,20). A polymerization reaction using 100 nmol of GlcUA␤1-3Gal␤1-O-C 2 H 4 NHCbz as an acceptor was conducted in incubation mixtures containing the following in a total volume of 20 l, i.e. 0.25 mM UDP-GalNAc, 0.25 mM UDP-[ 14 C]GlcUA (1.35 ϫ 10 6 dpm), 100 mM MES buffer, pH 6.2, 10 mM MnCl 2 , and 10 l of suspended beads. The mixtures were incubated at 26°C overnight, and the 14 C-labeled products were separated by gel filtration chromatography on a Superdex peptide column equilibrated and eluted with 0.2 M NH 4 HCO 3 . 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.
Characterization of the Reaction Products-Products of polymerization reactions on GlcUA␤1-3Gal␤1-O-C 2 H 4 NHCbz were isolated by gel filtration on a Superdex peptide column with 0.2 M NH 4 HCO 3 as the eluent. The radioactive peak containing the co-transfection reaction product was pooled and evaporated to dryness. The [ 14 C]GlcUA-labeled oligosaccharide chains were exhaustively digested with 100 mIU of chondroitinase AC-II in a total volume of 30 l of 50 mM sodium acetate buffer, pH 6.0, at 37°C overnight. The enzyme digest was analyzed using the same Superdex peptide column as described above.
RNAi by Feeding-The protocol for RNAi-by-feeding was based on described methods (21,22). We obtained a cDNA clone, yk356a9, corresponding to the PAR2.4 full-length cDNA from the Expression Sequence Tag project (23). Cloned fragments were re-amplified with a T3 (5Ј-AATTAACCCTCACTAAAGGGAAC-3Ј) and a T7 (5Ј-GTAATAC-GACTCACTATAGGGCGA-3Ј) primer. PCR was carried out with LA Taq polymerase (TaKaRa, Kusatsu, Japan) for 30 cycles of 94°C for 30 s, 58°C for 30 s, and 68°C for 180 s. The PCR fragment was subcloned into the L4440 (pPD129.36) vector, and the subcloned plasmid was transformed into Escherichia coli HT115 (DE3). A single colony of HT115 (DE3) containing the plasmid was grown in the LB culture medium for 8 h and seeded onto NGM agar plates (80 l/plate), which were incubated at 37°C overnight. Following the addition of 10 mM isopropyl ␤-D-thiogalactopyranoside (80 l/plate), the cells were cultivated for 4 h to induce the expression of double-stranded RNA (dsRNA). Mixed-stage hermaphrodites were sown on these plates, and dsRNA was introduced into the nematode by feeding.
Western Blotting-The worms were collected by centrifugation after being washed with the M9 buffer (23). The collected C. elegans pellet (50 mg) was sonicated and immediately heated at 95°C for 5 min to inactivate proteases, and then the supernatant fluid was subjected by chondroitinase ABC digestion, and the digests were resolved on 10% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and incubated for 3 h with an anti-⌬Di-0S antibody (1-B-5; Seikagaku Corp., Tokyo, Japan). The anti-chondroitin antibody was diluted 1:5,000 with 25 mM Tris-buffered saline. The bound antibody was detected with F(abЈ) 2 against mouse IgG conjugated to alkaline phosphatase (EY Laboratories) as described (3).
Analysis of Glycosaminoglycans-Glycosaminoglycans were prepared from 24 mg of dried homogenates of RNAi-treated nematodes. The unsaturated disaccharides were produced by enzymatic digestion 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 (HPLC) as described previously (3,13). It should be noted that the amount of heparan sulfate (HS) in C. elegans was so small (13) that 100 g of shark cartilage chondroitin 6-O-sulfate (Seikagaku Corp.), which contained a negligible proportion of non-sulfated disaccharides, was added as a carrier after the borohydride treatment but before the purification steps.
Transgenic Constructs-The expression vector pFX_DsRedXT or pFX_ EGFPT is composed of Bluescript (Stratagene, Palo Alto, CA) with an additional multiple cloning site followed by DsRed or enhanced GFP (EGFP) cDNA (Clontech) and about 1 kb of 3Ј-untranslated region from the unc-86 gene for poly(A) signal. 2 Each component was introduced into Bluescript by the serial addition of DNA fragments with restriction sites as follows. The multiple cloning site from pGEX-4T3 (Amersham Biosciences) was added between the KpnI and NotI sites and further modified by inserting an oligonucleotide sequence 5Ј-CCATATGTTAACTGGG-GAGTCGACGCCCCAGCTGACTAGTGG-3Ј between the BamHI site and the NotI site to make two XcmI sites. The DsRed and EGFP cDNAs were amplified by PCR using pDsRed-Express-1 and pEGFP-N1 cDNAs as templates, respectively, and subcloned into the NotI and BglII sites. The 3Ј-untranslated region from unc-86 was prepared by digestion of the C. elegans unc-86 genomic DNA subcloned into Bluescript with BglII and SacI sites and was added between the BglII and SacI sites. The vector containing two XcmI sites in the multiple cloning site was digested with XcmI to make a thymidine overhang in 3Ј-terminus at both ends for the TA cloning strategy (24), as described below.
The PAR 2.4 reporter gene plasmid was constructed using the vector pFX_DsRedXT. The translational fusion construct contained a 4.4-kb genomic fragment including the 1.0-kb potential promoter region. The fragment was amplified with C. elegans genomic DNA as a template using a 5Ј-primer (5Ј-ATTTTGGTTTATCGATTGAGCA-3Ј) and a 3Јprimer (5Ј-TTTTTCGTGGAATAACAATTTTG-3Ј) located just before the stop codon. 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 300 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. The sqv-5 (cChSy) reporter gene plasmid was constructed using the vector pFX_EGFPT as above. The translational fusion construct contained a 6.0-kb genomic fragment including the 5.0-kb potential promoter region. The fragment was amplified with C. elegans genomic DNA as a template using a 5Ј-primer (5Ј-GCG-TAGTCAATCGGCGTCTCAC-3Ј) and a 3Ј-primer (5Ј-TCGTCGTCT-GCTCGCAAGAACC-3Ј) located in exon 3. PCR was carried out with Platinum TaqDNA polymerase High Fidelity for 28 cycles of 94°C for 30 s, 58°C for 30 s, and 68°C for 420 s followed by adenine addition with Taq polymerase at 72°C for 15 min. The PCR fragment was inserted into the pFX_EGFPT vector using the TA cloning strategy to fuse the coding sequence region of EGFP. PAR2.4::DsRed and cChSy::EGFP constructs were co-injected into the distal gonad of a wild type N2 strain. Transgenic lines containing reporter constructs were isolated. Live transgenic worms were paralyzed with a 50 mM sodium azide solution and placed on an 8-well printed microscope slide glass (Matsunami Glass) and examined by four-dimensional microscopy (a DMRXA full automatic microscope with differential interference contrast (DIC) and fluorescent optics, Leica) as described (3). The images were processed using MetaMorph software (version 4.6, Universal Imaging).

RESULTS
Molecular Cloning of PAR2.4 -Screening of the non-redundant data base at the NCBI, National Institutes of Health (Bethesda, MD), using the deduced amino acid sequence of human ChSy, identified a few homologs in C. elegans. One of them, designated PAR2.4 (WormBase accession PAR2.4), contained a 5Ј-untranslated region of 133 bp, a single open reading frame of 2,412 bp coding for a protein of 804 amino acids with three potential N-glycosylation sites (Fig. 1), and a 3Ј-untranslated region of 308 bp. The predicted translation initiation site conformed to the Kozak consensus sequence for initiation (26). A Kyte-Doolittle hydropathy analysis (27) revealed one prominent hydrophobic segment of 16 amino acid residues in the NH 2 -terminal region, predicting that the protein has a type II transmembrane topology (Fig. 1). Notably, the identified protein did not have the conserved DXD motif found in most glycosyltransferases (28). Data base searches suggested that the amino acid sequence displayed 28, 27, and 16% identity to human chondroitin GlcAT (8), ChPF (6), and ChSy (5), respectively (Fig. 1). Thus, the features of the identified protein sequence suggest that the gene product is involved in the biosynthesis of chondroitin in C. elegans but would not possess glycosyltransferase activity.
Coexpression and Characterization of PAR 2.4 -To facilitate the functional analysis of PAR2.4, a soluble form of the protein was generated by replacing the first 52 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 chondro-hexasaccharide (GlcUA␤1-3GalNAc) 3 or chondro-heptasaccharide GalNAc␤1-4(GlcUA␤1-3GalNAc) 3 as a sugar accepter and either UDP-GalNAc or UDP-GlcUA as a sugar donor substrate, neither GalNAcT-II nor GlcAT-II activity was detected (Table I). However, coexpression of the soluble PAR2.4 with the soluble cChSy augmented the GalNAcT-II activity of cChSy over 20-fold and clearly showed GlcAT-II activity, which had not been detected when only cChSy was expressed. Notably, these effects of coexpression were not due to differences in the expression levels of these proteins, as assessed by Western blot analysis (data not shown). These results are analogous to the findings recently made for human ChPF and ChSy. Thus, we asked whether the coexpression of PAR2.4 and cChSy could result in the expression of polymerization activity. Incubations of the coexpressed proteins with GlcUA␤1-3Gal␤1-O-C 2 H 4 NHCbz as an acceptor in the presence of UDP-GalNAc and UDP-[ 14 C]GlcUA yielded radiolabeled polymer chondroitin chains as shown in Fig. 2. The reaction products were analyzed by gel chromatography using a column of Superdex peptide. The synthesized saccharide chains were around 20-mer long. The radioactive peaks containing co-transfectant reaction products were digested with chondroitinase AC-II and subjected to gel chromatography as described under "Experimental Procedures." The radiolabeled products were completely digested by chondroitinase AC-II, quantitatively yielding a 14 C-labeled peak at the position of   (GlcUA␤1-3GalNAc) 3 was used as an acceptor substrate. b GalNAc␤1-4(GlcUA␤1-3GalNAc) 3 was used as an acceptor substrate.

Depletion of Chondroitin by PAR 2.4-RNAi
Treatment-Embryogenesis in C. elegans takes only 14 h from fertilization to hatching at 20°C. Gastrulation begins at the 28-cell stage, and cell division and organogenesis continue until about 6 h after fertilization. During the next 6 h, the embryo changes from a spheroid to an elongated shape, and during the final 2 h, the pharynx begins to pump, and the eggshell is softened enzymatically so that the early L1 (the first larval stage) worm can hatch. After hatching, the animal passes through four larval stages (L1-L4), and at the end of each larval developmental stage, nematodes undergo a molt in which a new cuticle is formed and the old cuticle is shed. The larva takes 2-3 days to grow into an adult.
To understand the roles of the PAR2.4 gene in the nematode C. elegans, we examined phenotypes caused by PAR2.4-RNAi experiments. After 12 h of treatment (feeding) with PAR2.4 dsRNA, most of the F1 progeny of the treated worms showed wild-type phenotypes. About 30% of the worms treated with the dsRNA for 20 h and 80 -90% of those treated for 48 h died as embryos. F2 worms treated with the dsRNA for more than 60 h died at a rate of nearly 100%. Early embryonic development of the RAR2.4-RNAi-treated worms (F2 or F3 worms) was monitored with a four-dimensional microscopy (a multifocal timelapse image recording system). The process of early embryonic cell death was identical to that observed for the ChSy-RNAitreated worms. An apparent inversion of cytokinesis due to incomplete cytokinesis was observed as reported for ChSy RNAi (see supplementary movies), and embryos failed to complete cytokinesis, became multinucleated, and died (Fig. 3A). Although nucleic division proceeded without cytokinesis, normal chromosome partition also seemed to be affected by GFPtagged chromosomes in the RNAi-treated worms (data not shown). The results strongly suggest that chondroitin synthesis is severely affected in PAR2.4-RNAi-treated worms. Western blot analysis of proteins of RNAi-treated worms showed a complete lack of chondroitn PG in the worms treated with the dsRNA for more than 60 h (Fig. 3B) as reported for cChSy (sqv-5)-RNAi in the nematode C. elegans (3).
Analysis of the Disaccharide Composition of C. elegans Treated with PAR2.4 RNAi-In a previous study (3), the cChSy-RNAi F1 worm showed a 73% decrease in chondroitin and a 112% increase in HS. Thus, the glycosaminoglycan content of PAR2.4-RNAi-treated F1 worms was determined by chondroitinase or heparin lyase digestion followed by HPLC as described under "Experimental Procedures." PAR2.4-RNAitreated F1 worms showed a 54% decrease in chondroitin and a 68% increase in HS (Table II). In addition, the disaccharides of the HS from the RNAi-treated F1 worms were less sulfated than those of the wild-type worms (Table III). These results were similar to those obtained for the cChSy-RNAi F1 worms (3).
Coexpression of EGFP-cChSy and DsRed-PAR2.4 Fusion Protein in Wild-type Worms-To characterize the potential expression of the nematode PAR2.4 gene, we constructed a transcriptional reporter that fuses the gene (DsRed) encoding DsRed fluorescent protein to the putative promoter/enhancer region located upstream of the PAR2.4 sequence as described under "Experimental Procedures." Expression of PAR2.4::DsRed proteins was first observed in the late embryonic stages or in the L1 stage and continued to adulthood. Expression of the proteins in the vulva and in distal tip cells (DTC) was observed in the late L3 or young adult stage, when this organ and these cells are formed. In adult worms, expression of the reporter was seen in vulval cells (Fig. 4, c and d) and distal tip cells (Fig. 4, e and f), and an especially strong expression was observed in the lateral zone. To identify the cells expressing PAR2.4 in this region, we injected the PAR2.4::DsRed reporter transgene into gonads of the JR667 strain that expresses GFP in the nuclei of seam cells, which are in the cell lineage of neuronal stem cells and are glia-like neuron-supporting cells (29). Strong PAR2.4::DsRed signals were found in the cytoplasm of seam cells expressing strong GFP signals (Fig. 4, a and b), which led us to conclude that PAR2.4 proteins are strongly expressed in these cells. Next, we asked whether the PAR2. 4 and ChSy genes could be expressed in the same cells or not. To identify cells expressing the ChSy gene in the nematode, we constructed a ChSy::EGFP transgene with a full-length coding sequence and the putative promoter/enhancer region located 5 kb upstream of the cChSy sequence. When the cChSy reporter construct and the PAR2.4::DsRed reporter construct were injected simultaneously into gonads of N 2 worms, both ChSy::EGFP and PAR2.4 were expressed in the head and body region of adult worms. Both proteins were expressed in the  cytoplasm of the same cells at least in some cells as shown in Fig. 4, g-j, although coexpression of these two genes was not obvious in other cells and a more detailed study of expression patterns is necessary to investigate whether only the PAR2.4 or cChSy gene is expressed alone in some cells.

DISCUSSION
In previous studies, we revealed that the enzyme complex consisting of human ChSy (5) and ChPF (6) could polymerize chondroitin chains in vitro. In this study, we showed that PAR2.4 is the C. elegans ortholog of human ChPF, and only two genes, PAR2.4 and sqv-5 (cChSy), are required for chondroitin biosynthesis. In addition, we demonstrated for the first time that cChSy and PAR2.4 are indispensable for the biosynthesis of chondroitin chains in vivo, whereas PAR2.4 showed little glycosyltransferase activity like ChPF (6). Although the features of the cChSy protein sequence are similar to those of the human ChSy sequence, the catalytic activities of cChSy resemble those of the human chondroitin GalNAcT-1 (7,8) and -2 (9, 10), which exhibit GalNAcT-I activity for chain initiation and GalNAcT-II activity for chain elongation of CS, respectively, but not GlcAT-II activity. Coexpression of cChSy with PAR2.4 yielded a ϳ20-fold increase in the GalNAcT-II activity when compared with the expression of cChSy alone, and the GlcAT-II activity, which was not detected when only cChSy was expressed, could be detected here (see Table I). In addition, the complex showed chondroitin polymerization activity. These results suggested that the mechanism of chondroitin biosynthesis in C. elegans is quite similar to that in humans and that both ChSy and ChPF are indispensable for the biosynthesis of chondroitin chains in the nematode as well as in humans. Thus, PAR2.4 has been identified as a C. elegans ortholog of human ChPF and named here polymerizing factor for chondroitin-1 (pfc-1).
The mechanism for the biosynthesis of chondroitin is reminiscent of that for HS. The repeating disaccharide region of HS is synthesized by glycosyltransferases encoded by EXT1 (30) and EXT2 (31), which form an enzyme complex (HS polymerase) (32,33). These genes are involved in the hereditary multiple exostoses, which is an autosomal dominant disorder characterized by the formation of a cartilage-capped tumor, caused by mutations in either EXT1 or EXT2 (34). Thus, it has been suggested that EXT1 and EXT2 cannot have redundant roles in the biosynthesis of HS, which is similar to the observation that PAR2.4 (pfc-1)-or cChSy (sqv-5)-RNAi in C. elegans resulted in a reduction of chondroitin and the RNAi-treated worms showed similar phenotypes. Therefore, both PAR2.4 and cChSy have non-redundant functions as EXT1 and EXT2 do. In addition, these observations suggest that the mechanism for the biosynthesis of chondroitin is quite similar to that for HS.
Analytical data on the expression of PAR2.4 and cChSy in C. elegans suggest that both proteins are localized in seam cells, which can act as stem cells to produce neurons in early embryonic lineages, whereas differentiated and fused seam cells are responsible for production of the cuticular structures. PAR2.4 is expressed in such seam cells from the L1 stage through to adulthood (data not shown). In addition, the expression of PAR2.4 was also detected in DTC and vulva from L3 through to the young adult stage. DTC are located at the tip of the growing gonad arms and are required for the formation of the two U-shaped arms of gonads. Various mutant strains defective in the migration of DTC have been isolated and studied extensively. Since the inhibition of chondroitin synthesis by sqv-5 (cChSy) RNAi or deletion mutagenesis of sqv-5 (cChSy) resulted in an abnormal migration of gonad arms (mig phenotypes) with low penetrance (3) with cell-specific promoters is necessary, and it is also important to identify chondroitin PG core proteins in these cells. Studies along these lines are in progress in our laboratory.