rib-2, a Caenorhabditis elegansHomolog of the Human Tumor Suppressor EXT Genes Encodes a Novel α1,4-N-Acetylglucosaminyltransferase Involved in the Biosynthetic Initiation and Elongation of Heparan Sulfate*

The proteins encoded by the EXT1,EXT2, and EXTL2 genes, members of the hereditary multiple exostoses gene family of tumor suppressors, are glycosyltransferases required for the heparan sulfate biosynthesis. Only two homologous genes, rib-1 and rib-2, of the mammalian EXT genes were identified in theCaenorhabditis elegans genome. Although heparan sulfate is found in C. elegans, the involvement of the rib-1 and rib-2 proteins in heparan sulfate biosynthesis remains unclear. In the present study, the substrate specificity of a soluble recombinant form of the rib-2 protein was determined and compared with those of the recombinant forms of the mammalian EXT1, EXT2, and EXTL2 proteins. The present findings revealed that the rib-2 protein was a unique α1,4-N-acetylglucosaminyltransferase involved in the biosynthetic initiation and elongation of heparan sulfate. In contrast, the findings confirmed the previous observations that both the EXT1 and EXT2 proteins were heparan sulfate copolymerases with both α1,4-N-acetylglucosaminyltransferase and β1,4-glucuronyltransferase activities, which are involved only in the elongation step of the heparan sulfate chain, and that the EXTL2 protein was an α1,4-N-acetylglucosaminyltransferase involved only in the initiation of heparan sulfate synthesis. These findings suggest that the biosynthetic mechanism of heparan sulfate inC. elegans is distinct from that reported for the mammalian system.

Heparan sulfate proteoglycans are distributed on the surfaces of most cells and in the extracellular matrices of virtually every tissue. They consist of a protein core to which heparan sulfate glycosaminoglycan (GAG) 1 chains are attached. Hepa-ran sulfate GAGs show a tremendous diversity of structures that are known to play important roles in many biological recognition events of vertebrates and invertebrates, such as cell adhesion, growth factor/cytokine action, and regulation of important signaling pathways (for reviews see Refs. 1 and 2). Their biological activities are believed to be expressed through interactions with various proteins through specific saccharide sequences. These GAGs are synthesized on the so-called GAGprotein linkage region, GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser, attached to specific Ser residues of core proteins, which is common to the GAGs including heparin/heparan sulfate and chondroitin sulfate/dermatan sulfate (for reviews see Refs. 3 and 4). The linkage region synthesis is initiated by the addition of Xyl to Ser followed by the addition of two Gal residues and is completed by the addition of GlcUA, each reaction being catalyzed by the respective specific glycosyltransferase (3,4). The GAGs are built up on this linkage region by the alternate addition of N-acetylhexosamine and GlcUA residues. Heparin/ heparan sulfate is synthesized once GlcNAc is transferred to the common linkage region, whereas chondroitin sulfate/dermatan sulfate is formed if GalNAc is first added. However, the biosynthetic sorting mechanisms for different GAG chains remain enigmatic.
Recent cDNA cloning of the glycosyltransferases involved in the GAG biosynthesis revealed that the heparin/heparan sulfate biosynthesis in mammals is associated with the EXT gene family, the hereditary multiple exostoses (HME) gene family of tumor suppressors (for a review see Ref. 5). HME is an autosomal dominant disorder characterized by cartilage-capped skeletal excrescences, which may lead to skeletal abnormalities and short stature (6). Although the exostoses represent osteochondromas that are benign bone tumors, malignant transformation into chondrosarcomas or osteosarcomas occurs in ϳ2% of HME patients (6,7). Genetic linkage of this disorder has been ascribed to three independent loci on chromosomes 8q24.1 (EXT1), 11p11-13 (EXT2), and 19p (EXT3) (8 -10). This family of EXT genes has recently been extended by the identification of three additional EXT-like genes, EXTL1, EXTL2, and EXTL3 (11)(12)(13)(14). Exostoses-derived and sporadic chondrosarcomas are attributable to the loss of heterozygosity for the markers in EXT1 and EXT2 loci (15,16), indicating that the EXT genes may encode tumor suppressors. It has been demonstrated that both mammalian EXT1 and EXT2 proteins are the heparan sulfate copolymerases that polymerize GlcUA and GlcNAc alternately (17)(18)(19), and the human EXTL2 protein is GlcNAc transferase I (20), which determines and initiates the heparan sulfate synthesis on the common GAG-protein linkage region (21).
Interestingly, the EXT gene family has also been identified in invertebrate genomes such as the nematode worm Caenorhabditis elegans (22) or the fruit fly Drosophila melanogaster (23), suggesting the presence of heparan sulfate in these organisms. In fact, heparan sulfate is found in invertebrates, including C. elegans and Drosophila. Recent studies have shown that both these genetically tractable organisms make heparan sulfate and chondroitin or chondroitin 4-sulfate (24 -26). Although analysis of mutants defective in the Drosophila EXT1 homolog, ttv, has provided some evidence that Ttv might also be a heparan sulfate polymerase (25), glycosyltransferase activities have not yet been reported for these invertebrate EXT family proteins. A data base search revealed only the two homologous genes, rib-1 and rib-2, of the mammalian EXT genes in the C. elegans genome (22). Notably, however, no C. elegans homolog of the mammalian EXTL2 gene encoding GlcNAc transferase I required for the chain initiation of heparan sulfate was found. These observations, therefore, suggested that the biosynthetic mechanism of heparan sulfate in C. elegans might be distinct from that reported for mammals. In the present study, to clarify the involvement of the rib-2 protein in heparan sulfate biosynthesis, the substrate specificity of a soluble recombinant form of the rib-2 protein was determined and compared with those of the recombinant forms of the mammalian EXT1, EXT2, and EXTL2 proteins. The findings demonstrated that the rib-2 protein was a novel and unique ␣1,4-N-acetylglucosaminyltransferase involved in the biosynthetic initiation and elongation of heparan sulfate.
Construction of Expression Vectors Encoding Soluble Forms of the Rib-2, EXT-1, EXT-2, and EXTL2 Proteins-The cDNA fragment encoding a truncated form of rib-2, lacking the first N-terminal 58 amino acids of the rib-2, was amplified by reverse transcription PCR with adult C. elegans total RNA as a template using a 5Ј primer (5Ј-CGG-GATCCGATTATGACGCGTCATGCAGTG-3Ј) containing an in-frame BamHI site and a 3Ј primer (5Ј-CGGGATCCGTAGCACCATTCGA-CAAGTGAA-3Ј) containing a BamHI site located 68 bp downstream of the stop codon. The cDNA fragment encoding a truncated form of EXT-1, lacking the first N-terminal 43 amino acids, was amplified by PCR with human EXT-1 cDNA in pcDNA 3.1 (29) as a template using a 5Ј primer (5Ј-CCGGAATTCGAATGGCTTGCACCACCCCAG-3Ј) containing an in-frame EcoRI site and a 3Ј primer (5Ј-CGCGGATCCCTG-ATGAGTGGATCTGCACTG-3Ј) containing a BamHI site located 84 bp downstream of the stop codon. The cDNA fragment encoding a truncated form of EXT-2, lacking the first N-terminal 55 amino acids, was amplified by PCR with bovine EXT-2 cDNA in pcDNA 3.1zeo (17) as a template using a 5Ј primer (5Ј-CGCGGATCCCTGGAGCGTGGAGAA-GCGCA-3Ј) containing an in-frame BamHI site and a 3Ј primer (5Ј-C-GCGGATCCTGTCCGGCATTCGTGTTGAGC-3Ј) containing a BamHI site located 40 bp downstream of the stop codon. PCR reactions were carried out with KOD DNA polymerase (TOYOBO, Tokyo, Japan) by 30 cycles of 96°C for 30 s, 58°C for 30 s, and 72°C for 60 s. The PCR fragment of rib-2 or EXT-2 was subcloned into the BamHI site of pGIR201protA (30), resulting in the fusion of rib-2 or EXT-2 to the insulin signal sequence and the protein A sequence present in the vector. The PCR fragment of EXT-1 was subcloned into the EcoRI/ BamHI site of pGIR201protA. An NheI fragment containing each of the above fusion protein sequences was inserted into the XbaI site of the expression vector pEF-BOS (31). The nucleotide sequence of the amplified cDNA was determined in a 377 DNA sequencer (PerkinElmer Life Sciences). The construction of a soluble form of EXTL2 fused with the cleavable insulin signal sequence and the protein A IgG-binding domain was carried out as described previously (20).
Identification of the Enzyme Reaction Products-The isolation of the products from the GlcNAc transferase reaction using N-acetylheparosan as an acceptor was carried out by gel filtration on a Superdex peptide column (Amersham Pharmacia Biotech) equilibrated with 0.25 M NH 4 HCO 3 /7% 1-propanol. The radioactive peak containing the enzyme reaction product was pooled and evaporated to dryness. The isolated product (about 72 pmol) was digested with 3 mIU of ␤-Nacetylhexosaminidase in a total volume of 20 l of 50 mM sodium citrate buffer, pH 4.5, or with 3 mIU of heparitinase I for testing the digestability in a total volume of 30 l of 20 mM sodium acetate buffer, pH 7.0, containing 2 mM calcium acetate at 37°C overnight. The enzyme digest was analyzed using the same Superdex peptide column as described above.
The isolation of the products from the GlcNAc transferase I reaction using GlcUA␤1-3Gal␤1-O-C 2 H 4 NHCbz was performed by HPLC on a Nova-Pak ® C18 column (3.9 ϫ 150 mm; Waters, Tokyo, Japan) in an LC-10AS system (Shimadzu Co., Kyoto, Japan). The column was developed isocratically for 15 min with H 2 O at a flow rate of 1.0 ml/min at room temperature; thereafter, a linear gradient was applied to increase the methanol concentration from 0 to 100% over a 5-min period, and the column was then developed isocratically for 40 min with 100% methanol. The radioactive peak containing the product was pooled and evaporated to dryness. The isolated product (about 74 pmol) was incubated with 1 mIU of ␤-N-acetylhexosaminidase in a total volume of 20 l of 50 mM sodium citrate buffer, pH 4.5, or with 1 mIU of heparitinase I for testing the digestability in a total volume of 30 l of 20 mM sodium acetate buffer, pH 7.0, containing 2 mM calcium acetate at 37°C overnight. The enzyme digest was analyzed using the same Nova-Pak ® C18 column as described above.

Rib-2 Expressed in COS-1 Cells Generates GlcNAc
Transferase Activities for Heparan Sulfate Synthesis-C. elegans rib-2 encodes a protein with homology to the human EXT family members, especially to the EXT2 and EXTL3 proteins (see "Discussion"). In view of the recent findings that EXT2 is a heparan sulfate-polymerase required for the heparan sulfate biosynthesis (17,19), it was important to clarify the involvement of rib-2 in the heparan sulfate biosynthesis in C. elegans. The rib-2 sequence indicated an open reading frame of 2442 bp coding for a protein of 814 amino acids, with eight potential N-glycosylation sites (22) and a type II transmembrane protein topology characteristic of many glycosyltransferases cloned to date. A soluble form of the protein encoded by rib-2 cDNA was generated by replacing the first 58 amino acids of rib-2 with the cleavable insulin signal sequence and the IgG-binding domain of protein A as described under "Experimental Procedures." When the expression plasmid containing the rib-2/protein A fusion was expressed in COS-1 cells, an approximate 130-kDa protein was secreted as shown by Western blotting using IgG (data not shown). The apparent M r of the fused protein was reduced to about 110-kDa after N-glycosidase treatment (data not shown), indicating that a few potential N-linked glycosylation sites of rib-2 were utilized.
The fused enzyme expressed in the medium was absorbed on IgG-Sepharose beads to eliminate endogenous glycosyltransferases and then the enzyme-bound beads were used as an enzyme source for further studies. The bound fusion protein was assayed for GlcUA transferase or GlcNAc transferase activities involved in the heparan sulfate biosynthesis using a variety of acceptor substrates. There is no known C. elegans homolog of the human EXTL2 gene encoding GlcNAc transferase I required for the chain initiation of heparan sulfate (see "Introduction"), which suggested that rib-2 may also have GlcNAc transferase I activity. Hence, the purified fusion protein was also assayed for GlcNAc transferase I activity, in addition to the GlcUA and GlcNAc transferase activities for the polymerization reactions. GlcUA␤1-3Gal␤1-O-C 2 H 4 NHCbz used as an acceptor substrate for the GlcNAc transferase I reaction shares the disaccharide sequence with the GAG-protein linkage region tetrasaccharide. It served as a good acceptor for the EXTL2 protein (see Table I) and was comparable with GlcUA␤1-3Gal␤1-O-naphthalenemethanol, an artificial, yet authentic oligosaccharide acceptor substrate for GlcNAc transferase I. 2 As shown in Table I, the marked GlcNAc transferase activity was detected with N-acetylheparosan oligosaccharides GlcUA␤1-(4GlcNAc␣1-4GlcUA␤1-)n and GlcUA␤1-3-Gal␤1-O-C 2 H 4 NHCbz but not with the tetrasaccharide-serine GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser as acceptor substrates, whereas no GlcUA transferase activity was observed using N-acetylheparosan oligosaccharides GlcNAc␣1-(4GlcUA-␤1-4GlcNAc␣1-)n or the pentasaccharide-serine GlcNAc␣1-4GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser. No detectable GlcNAc transferase activity was recovered by the affinity purification from the control pEF-BOS transfection sample, and it therefore seems unlikely that the results are due to an artifact or an endogenous activity.
To identify these GlcNAc transferase reaction products, the acceptor substrates, N-acetylheparosan oligosaccharides GlcUA␤1-(4GlcNAc␣1-4GlcUA␤1-)n and GlcUA␤1-3Gal␤1-O-C 2 H 4 NHCbz, were individually labeled by the respective transferase reaction using UDP-[ 3 H]GlcNAc as a donor substrate and the enzyme-bound beads as an enzyme source. Both labeled products were completely digested by heparitinase I, which cleaves an ␣1,4-N-acetylglucosaminidic linkage in an eliminative fashion, quantitatively yielding a 3 H-labeled peak at the elution position of free [ 3 H]GlcNAc, as demonstrated by gel filtration (Fig. 1A) or hydrophobic HPLC (Fig. 1B). In contrast, they were inert to the action of ␤-N-acetylhexosaminidase. These findings clearly indicated that a GlcNAc residue had been transferred exclusively to the nonreducing terminal GlcUA of N-acetylheparosan oligosaccharides or GlcUA␤1-3Gal␤1-O-C 2 H 4 NHCbz through an ␣1,4 linkage. The present findings demonstrated that rib-2 was a novel and unique ␣1,4-GlcNAc transferase involved in the biosynthetic initiation and elongation of heparan sulfate.
Comparison of Acceptor Specificity of the Rib-2, EXT1, EXT2, and EXTL2 Proteins-To distinguish the specificity of rib-2 from that of other so far characterized mammalian glycosyltransferases involved in the heparan sulfate biosynthesis, the rib-2 was compared with the soluble recombinant forms of mammalian EXT1, EXT2, and EXTL2 for their ability to utilize a variety of acceptor substrates (Table I). Comparison of rib-2 with EXT1 and EXT2 showed that they all utilized N-acetylheparosan oligosaccharides GlcUA␤1-(4GlcNAc␣1-4GlcUA␤1-)n as their preferred acceptor substrates when UDP-[ 3 H]GlcNAc was used as a donor substrate. However, both EXT1 and EXT2 exhibited a strict specificity, showing no [ 3 H]GlcNAc incorporation into GlcUA␤1-3Gal␤1-O-C 2 H 4 NHCbz. Equally striking was that both EXT1 and EXT2 showed the GlcUA transferase activity required for heparan sulfate polymerization, whereas rib-2 showed no [ 14 C]GlcUA incorporation when tested with N-acetylheparosan oligosaccharides GlcNAc␣1-(4GlcUA␤1-4GlcNAc␣1-)n or the pentasaccharide-serine GlcNAc␣1-4GlcUA␤1-3Gal␤1-3Gal␤-1-4Xyl␤1-O-Ser as acceptor substrates. Thus, both EXT1 and EXT2 have marked activities of GlcNAc transferase II and GlcUA transferase required for heparan sulfate polymerization but no GlcNAc transferase I activity for the heparan sulfate initiation. In addition, comparison of rib-2 to EXTL2 revealed  (20). Hence, each of the purified recombinant proteins was assayed for GalNAc transferase activity using UDP-[ 3 H]GalNAc as a sugar donor and the tetrasaccharideserine GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser as an acceptor. As shown in Table I, a significant GalNAc transferase activity was detected for EXTL2 but not for rib-2. These findings indicate that the rib-2 exhibits properties clearly distinct from the three mammalian glycosyltransferases characterized to date.

DISCUSSION
Although heparan sulfate is found in C. elegans, it was unknown which enzyme proteins were involved in the heparan sulfate biosynthesis (24,25). In the present study, we demonstrated that the C. elegans rib-2 protein was a unique ␣1,4-GlcNAc transferase involved in the biosynthetic initiation and elongation of heparan sulfate. As shown in Table I, rib-2, characterized in the present study, exhibited distinct but overlap-ping acceptor substrate specificities when compared with those of the mammalian glycosyltransferases responsible for the heparan sulfate biosynthesis, EXT1/EXT2 and EXTL2. Indeed, the two primary products of rib-2, GlcNAc␣1-(4GlcUA␤1-4-GlcNAc␣1-)n and GlcNAc␣1-4GlcUA␤1-3Gal␤1-, are also products of EXT1/EXT2 (17,19) and of EXTL2 (20), respectively. In addition, rib-2 appears to recognize a specific sequence in the core protein or an aglycone structure attached to the linkage region tetrasaccharide as in the case of EXTL2 (20), because the tetrasaccharide-serine, GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser derived from the linkage region, was not utilized as an acceptor substrate.
The hitherto unreported specificity revealed for rib-2 can be predicted to be required for heparan sulfate biosynthesis in C. elegans, because there is no C. elegans homolog of the mammalian EXTL2 gene that encodes GlcNAc transferase I, which is responsible for the chain initiation of heparan sulfate (21). The rib-2 protein, composed of 814 amino acids, is a similar size to the mammalian four EXT family members, EXT1, EXT2, EXTL1, and EXTL3 that have 676 -919 amino acids and is about twice the size of the other EXT member, EXTL2, which has 330 amino acids. The carboxyl terminus of the rib-2 protein shows significant homology to these members of the family and exhibits the highest homology to EXT2 and EXTL3 (44 and 58% amino acid identities, respectively). Although EXT2 encodes a bifunctional heparan sulfate-cotransferase catalyzing the GlcUA and GlcNAc transferase II reactions (17,19), the rib-2 protein exhibited only the GlcNAc transferase II activity and no GlcUA transferase activity. Instead, the rib-2 protein catalyzed the GlcNAc transferase I reaction involved in the biosynthetic initiation of heparan sulfate, whereas EXT2 did not. Based on these findings, together with the observation that the rib-2 protein is more similar to EXTL3 than EXT2, it is possible that rib-2 might be a C. elegans ortholog of mammalian EXTL3 and that EXTL3 might encode a similar ␣1,4-GlcNAc transferase involved in the biosynthetic initiation and elongation of heparan sulfate in mammals.
A data base search revealed only two homologous genes, rib-1 and rib-2, of the mammalian EXT genes in the C. elegans genome (22). In view of the finding that the rib-2 protein exhibits no GlcUA transferase activity required for the heparan sulfate chain elongation, it is possible that the rib-1 protein possesses the GlcUA transferase activity. The rib-1 protein, composed of 378 amino acids, is about half the size of the rib-2 protein and the mammalian four EXT family members, EXT1, EXT2, EXTL1, and EXTL3. Interestingly, the rib-1 protein shows significant homology to the amino termini of these family members, especially to EXT1 (45% amino acid identity). In this regard, it should be noted that EXT1, as well as EXT2, encodes bifunctional heparan sulfate-cotransferase, which catalyzes the GlcUA transferase reaction, in addition to the Glc-NAc transferase II reaction (17,19), and that EXTL2, which shows significant homology to the carboxyl termini of the other EXT family members, shows the GlcNAc transferase I activity (20). Very recently, Wei et al. (33) reported that the GlcUA transferase domain most likely resides in the amino-terminal portion of the EXT1 protein. Thus, it is suggested that the rib-1 protein might be the GlcUA transferase involved in the chain elongation of heparan sulfate. However, when a soluble recombinant form of the rib-1 protein was produced and assayed for the GlcUA transferase and also the GlcNAc transferase I and II activities under the condition used in the present study, no such glycosyltransferase activities were detected for the rib-1 protein (data not shown). In this regard, it is possible that the kinetic properties of C. elegans enzymes, rib-1 and rib-2, might be very different from those of the mammalian EXT1 and EXT2 proteins. More efforts to detect the GlcUA transferase activity for the rib-1 and rib-2 proteins are required.
Nevertheless, the findings from the present study suggest that the biosynthetic mechanism of heparan sulfate in C. elegans is distinct from that in mammals. In mammals, it was demonstrated that EXT1 and EXT2 are both GlcUA/GlcNAccotransferases that need to interact with each other to form an active heparan sulfate polymerase (18,19,34). Coexpression of the two proteins, but not mixing of separately expressed recombinant EXT1 and EXT2, yields hetero-oligomeric complexes in mammalian cells, with augmented glycosyltransferase activities (18,19). The available information suggests that neither of the two cotransferases can substitute for the other. For example, L-cell or Chinese hamster ovary cell mutants deficient in EXT1 were unable to synthesize heparan sulfate even after transfection with EXT2 (18,33). Moreover, mouse embryonic stem cells derived from EXT1 homozygous null embryos markedly reduced the formation of heparan sulfate, even though the cells contained an EXT2 ortholog (35). Thus, although it is obvious that EXT1 has GlcUA/GlcNAc transferase activities and that it functions in vivo in heparan sulfate formation, the role of EXT2 is unclear given its low activity and the lack of mutants. In addition, 1-2% contamination by EXT1 could easily account for the low level of activity seen for EXT2 in mammalian systems (Refs. 17 and 19; also see Table I) given that the enzymes interact. We have demonstrated that EXTL2 encodes GlcNAc transferase I that determines and initiates the heparan sulfate synthesis (20) but have found no EXT homolog that encodes only GlcNAc transferase I or GlcUA/GlcNAc-cotransferase in C. elegans as shown in the present study. Moreover, coexpression of the rib-1 and rib-2 proteins did not result in the promotion of the GlcNAc transferase activities or the expression of the GlcUA transferase activity required for heparan sulfate polymerization (data not shown). Although the possibility cannot be ruled out that nonhomologous genes to the EXT gene family may encode GlcUA/GlcNAc-cotransferases, these findings suggest that the biosynthetic mechanism of heparan sulfate in C. elegans is distinct from that in mammals.
Recent studies have demonstrated a critical role for heparan sulfate in growth factor signaling mediated by Wingless proteins during Drosophila development (2). In addition, a Drosophila homolog of EXT1 (encoded by ttv) was recently implicated in the Hedgehog diffusion (2). C. elegans is also a genetically tractable organism ideally suited for the analysis of glycan function during tissue assembly and morphogenesis. In fact, three proteins required for wild-type vulval invagination in C. elegans, which are encoded by sqv-3, -7, and -8 (squashed vulva), respectively, have been reported to be similar to components of a glycosylation pathway (36). sqv-3, -7, and -8 are three of the eight genes in the sqv class, identified on the basis of a common vulval invagination defect. Recently, Bulik et al. (26) showed that sqv-3, -7, and -8 mutants all affected the biosynthesis of GAGs and that sqv-3 and -8 genes encoded enzymes with Gal transferase I and GlcUA transferase I activity, respectively. Because both enzymes are required for the synthesis of the GAG-protein linkage tetrasaccharide common to all GAGs, including chondroitin sulfate and heparan sulfate (37)(38)(39), it is reasonable that sqv-3 and -8 mutants showed significantly reduced levels of both chondroitin and heparan sulfate (26). Thus, if rib-2 mutants become available, they would be useful tools for determining which GAG, heparan sulfate or chondroitin, is required for C. elegans vulval invagination.