Demonstration of a Novel Gene DEXT3 of Drosophila melanogaster as the Essential N -Acetylglucosamine Transferase in the Heparan Sulfate Biosynthesis INITIATION

Hereditary multiple exostoses gene ( EXT ) family members encode glycosyltransferases required for heparan sulfate (HS) biosynthesis in humans as well as in Drosophila . In the present study, we identified a novel Drosophila EXT protein with a type II transmembrane topology and demonstrated its glycosyltransferase activities. The truncated soluble form of this new homolog designated DEXT3 transferred N -acetylglu-cosamine (GlcNAc) through an (cid:1) 1,4-linkage not only to N -acetylheparosan oligosaccharides that represent growing HS chains ( (cid:1) -GlcNAc transferase II activity) but also to GlcUA (cid:2) 1–3Gal (cid:2) 1- O -C 2 H 4 NHCbz, a synthetic substrate for (cid:1) -GlcNAc transferase I that determines and initiates HS biosynthesis. The results suggest that DEXT3 is the ortholog of human EXTL3 and Caenorhab-ditis elegans rib-2. Semiquantitative reverse tran-scriptase-PCR analysis revealed ubiquitous expression of the DEXT3 mRNA. Based on the findings of the present study and those of a recent study where a fly mutant, deficient in the botv gene identical to DEXT3 , affected HS proteoglycan-mediated developmental signalings, it is suggested that DEXT3 with the revealed glycosyltransferase activities is critically involved in HS formation in Drosophila . These results suggest the essential roles of DEXT3 , its human ortholog EXTL3 , and the elegans ortholog rib-2 in the biosynthesis of heparan sulfate and heparin, if present, in the respective organisms.

Proteoglycans are distributed ubiquitously on cell surfaces and in extracellular matrices in virtually all animal tissues and function in a variety of cellular and developmental processes such as cell adhesion, proliferation, migration, differentiation, and tissue morphogenesis (1,2). They consist of core proteins and highly negatively charged glycosaminoglycan (GAG) 1 chains. Their ability to interact with a panel of various functional proteins, such as growth factors, morphogens, cytokines, growth factor/cytokine receptors, proteases, and extracellular matrix components, is ascribable to the specific saccharide sequences in the GAG chains (3,4). GAGs can be classified into heparin/heparan sulfate (HS) and chondroitin sulfate (CS)/ dermatan sulfate (DS), which are glucosaminoglycans and galactosaminoglycans, respectively. They are synthesized on the common GAG-protein linkage region tetrasaccharide GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-bound to the specific serine residues in the core proteins (5). Following the transfer of ␣-GlcNAc as the first N-acetylhexosamine unit to this linkage region, catalyzed by GlcNAcT-I (6), HS copolymerase starts to act on and elongate the resultant nascent pentasaccharide chain by alternate addition of GlcUA and GlcNAc (reviewed in Ref. 7). In contrast, a ␤-GalNAc transfer to the linkage region triggers CS/DS biosynthesis (8,9). Formation of the repeating CS/DS disaccharide region on the linkage region is achieved by the action of chondroitin synthase through alternate additions of GlcUA and GalNAc (10).
HS polymerases have been identified as EXT1 and EXT2 proteins (11), which revealed the relation between HS synthesizing glycosyltransferases and the EXT gene family. The EXT gene family consists of five members, of whom at least EXT1 and EXT2 and probably also the others have tumor suppressor activities (12)(13)(14)(15)(16)(17). Congenital mutations of EXT1 and EXT2 are clinically associated with hereditary multiple exostoses, a benign bone tumor characterized by multiple cartilage-capped outgrowths of long bones (18 -20). Although the three other family members called EXT-like genes, designated EXTL1, EXTL2 and EXTL3, are highly homologous to EXT1 and EXT2 (14 -17) they have not been demonstrated to be linked to the genetic disorder. Purification of a unique ␣1,4-GalNAc trans-ferase encoded by EXTL2 has led us to identify this enzyme as HS-synthesizing ␣1,4-GlcNAcT-I (21), which exhibits a bifunctional ␣1,4-GalNAc/GlcNAc transferase that can determine and initiate HS synthesis. More recently, we demonstrated that EXTL3 harbors both GlcNAcT-I and GlcNAcT-II activities whereas EXTL1 displays GlcNAcT-II activity only, suggesting that EXT-like genes also play functional roles in HS biosynthesis (22). Thus, all human EXT family member proteins possess HS synthesis-related glycosyltransferase activities.
EXT proteins also exist in Drosophila melanogaster and Caenorhabditis elegans (23,24), and recent studies suggested that some of them are essential participants in forming HS in these organisms (23,25). In the fruit fly, two EXT homologs have been identified and named ttv (EXT1 ortholog) and DEXT2 (likely EXT2 ortholog) (23). The ttv mutation causes severe loss of HS (26) and thereby early embryonic death by affecting the developmental signalings that are regulated by HS proteoglycans (23). However, its glycosyltransferase activities have not been demonstrated, although accumulating circumstantial evidence strongly suggests that TTV is an HS polymerase. No glycosyltransferase activity has been revealed for DEXT2 either. A Drosophila database search for proteins related to human EXT members revealed the third homolog encoding 972 amino acids. A hydropathy plot of the amino acid sequence exhibited a type II transmembrane structure typical of many endoplasmic reticulum/Golgi resident glycosyltransferases. By expressing its recombinant soluble form in mammalian cells, we detected the glycosyltransferase activities most probably involved in HS biosynthesis, which is the first demonstration of catalytic activities of an EXT homolog in Drosophila. Intriguingly, while we were preparing the manuscript, a new segment polarity gene (27) was reported and was found to be identical to this new EXT homolog, which we named DEXT3 here. Its mutant phenotypes showed that developmental signalings regulated by HS proteoglycans were specifically impaired. Its in vitro catalytic activities revealed in this study strongly suggest that DEXT3 is critically involved in HS production in Drosophila in vivo. Preliminary results have been presented in abstract form (28).
Construction of the Expression Vector pEF-BOS/IP-An 894-bp cDNA fragment containing the insulin signal sequence and the protein A sequence was cleaved from pGIR201protA (31) by NheI digestion and then inserted into the XbaI site of the expression vector pEF-BOS (32) to prepare pEF-BOS/IP. A DNA insert of interest was ligated into the BamHI site derived from pGIR201protA for expression as described below.
Construction of a Soluble Form of the Novel Homolog DEXT3-Five corresponding Drosophila EST clones were purchased including LD21192, whose sequence was deposited as the complete mRNA sequence (AF132161) in the GenBank TM . Plasmids of the purchased EST clones were digested with several appropriate restriction endonucleases, and two clones, LD38007 and LD43838, were found to have the whole sequence of the novel EXT homolog. Unexpectedly, the LD21192 clone that we received had an entirely different sequence from AF132161. The cDNA fragment encoding a truncated form of the gene, lacking the N-terminal first 95 amino acids, was amplified with the plasmid from LD38007 as a template by PCR using a 5Ј primer (5Ј-ATTGATCATACGAAGATTTCAGCGCCATG-3Ј) containing an inframe Bcl I site and a 3Ј primer (5Ј-ATTGATCAAAGTGAAGTGGC-CGCCTTT-3Ј) containing a Bcl I site located 53 bp downstream of the stop codon. PCR was carried out with Pfu polymerase by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 6 min. The amplified fragments were digested with Bcl I and cloned into the BamHI site of pEF-BOS/IP, resulting in the fusion protein, which had the cleavable insulin signal sequence for secretion and protein A for purification of the expressed fusion protein.
Functional Expression of the Soluble Form of DEXT3 in COS-1 Cells and Enzyme Assay-The expression plasmid (6.7 g) was transfected into COS-1 cells on 100-mm plates using FuGENE TM 6 (Roche Molecular Biochemicals, Tokyo, Japan) according to the instructions supplied by the manufacturer. Two days after transfection, 1 ml of the medium was collected, and the secreted fusion protein was purified by incubation with 10 l of IgG-Sepharose (Amersham Biosciences) for 2 h at 4°C. The beads recovered by centrifugation were washed with each assay buffer described below, resuspended in the same buffer and then tested for glycosyltransferase activities. For GlcNAc transferase assays, the reaction mixture of a total volume of 20 l contained 10 l of the resuspended beads, 100 mM 2-(N-morpholino)ethanesulfonic acid-NaOH, pH 6.5, 10 mM MgCl 2 , 1 mM ATP, 250 M UDP-[ 3 H]GlcNAc (about 1.1 ϫ 10 6 dpm), and either GlcUA␤1-3Gal␤1-O-C 2 H 4 NHCbz (250 nmol), the tetrasaccharide-serine representing the GAG-protein linkage region (1 nmol) for GlcNAcT-I assay, or N-acetylheparosan oligosaccharides with the nonreducing terminal GlcUA (20 g) for GlcNAcT-II assay as acceptors. Assays for GlcAT-I (33), HS-GlcAT-II (34), ␣-GalNAc transferase (35), the ␤-GalNAc transferase (36), and the GlcUA transferase involved in CS chain elongation (CS-GlcAT-II) (37), were carried out according to the previously described methods. All the enzyme reactions were carried out at 30°C. Reaction products were quantified in a liquid scintillation counter (TRI-CARB 2900TR, Packard Co.) using a scintillation fluid containing 1.2% (w/v) 2,5-diphenyloxazole and 33% (w/v) Triton X-100.
Identification of the Enzyme Reaction Products-The products from the GlcNAcT-I reaction using GlcUA␤1-3Gal␤1-O-C 2 H 4 NHCbz were isolated by hydrophobic HPLC on a Nova-Pak C 18 column in an LC-10A system (Shimadzu Co., Kyoto, Japan) (22,25). The column was developed with H 2 O for 15 min at a flow rate of 1.0 ml/min at room temperature and thereafter a linear gradient was applied to increase the methanol concentration from 0 to 100% over a 5-min period. The column was then developed isocratically with 100% methanol for 20 min. The radioactive peak containing the product was pooled and evaporated to dryness. The dried sample (about 40 pmol) was incubated with either 9 mIU of ␤-N-acetylhexosaminidase in a total volume of 20 l of 50 mM sodium citrate buffer, pH 4.5, or with 10 mIU of heparitinase I in a total volume of 50 l of 20 mM sodium acetate buffer, pH 7.0, containing 2 mM calcium acetate at 37°C overnight. Each enzyme digest was analyzed using the same Nova-Pak C 18 column as that noted above.
GlcNAcT-II reaction products were chromatographed by gel filtration on a Superdex peptide HR 10/30 column (1.0 ϫ 30 cm) using 0.2 M NH 4 HCO 3 as an eluent. Fractions containing radioactive products were pooled and evaporated with intermittent addition of H 2 O until the ammonium salt became invisible. An aliquot (about 30 pmol) of each purified product was incubated with either 9 mIU of ␤-N-acetylhexosaminidase in a total volume of 20 l of 50 mM sodium citrate buffer, pH 4.5, or with 10 mIU of heparitinase I in a total volume of 100 l of 20 mM sodium acetate buffer, pH 7.0, containing 2 mM calcium acetate at 37°C overnight. Each digest was analyzed again using the same Superdex peptide column as described above. s, and 72°C for 60 s; for ttv, predenaturing at 94°C for 3 min and 35 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 60 s. The used primers were: 5Ј primer (5Ј-ATGGTGCCCGCCCGAAGGAAATA-3Ј) and 3Ј primer (5Ј-AAAGGGCAGCTGGGGGTAAAGGTAA-3Ј) for DEXT3, and 5Ј primer (5Ј-ATCGGAGACGCGCAACTC-3Ј) and 3Ј primer (5Ј-CAAGGGTATGTGGCTGGTG-3Ј) for ttv.

Primary Structure of the Novel EXT Homolog DEXT3-
When the human EXT family members were used as query sequences for BLASTP search in the Berkeley Drosophila Genome Project homepage (fruit fly.org), three highly homologous genes were found: ttv (CG10117, AF083889), DEXT2 (CG8433, AF145598) and the unreported EXT homolog (CG15110, AF132161) with unknown functions, which is designated DEXT3 here. The complete cDNA of the DEXT3 gene and the predicted amino acid sequence of the putative protein are presented in Fig. 1A. The cDNA consisted of a single open reading frame of 2919 bp encoding a type II transmembrane protein of 972 amino acids, which had a 56-amino acid cytoplasmic domain and one prominent hydrophobic segment that extended from amino acid residue 57 to 75 (Fig. 1B). This novel hypothetical protein showed significant amino acid identity with all human EXT family members: EXT1 (20.1%), EXT2 (25.1%), EXTL1 (17.9%), EXTL2 (22.7%), and EXTL3 (45.8%). The phylogenetic tree of the EXT family members among humans, C. elegans, and D. melanogaster was generated based on the amino acid sequences, which showed that EXTL3, Rib-2, and DEXT3 are more closely related with one another as compared with other EXT family members ( Fig. 2A). A multiple alignment of the amino acid sequences of EXTL3, Rib-2, and DEXT3 revealed that they shared markedly high amino acid sequence identity especially in the C-terminal regions (Fig. 2B), suggesting that DEXT3 may have similar glycosyltransferase activities to EXTL3 and Rib-2, both of which showed GlcNAcT-I and GlcNAcT-II activities.
DEXT3 Harbored Both GlcNAcT-I and GlcNAcT-II Activities-To clarify whether DEXT3 has glycosyltransferase activities, its recombinant soluble form was expressed. Using LD38007 cDNA as a template, the truncated form of DEXT3 was produced by replacing the N-terminal 95 amino acids of the putative cytoplasmic and transmembrane domains with a cleavable insulin signal sequence and a protein A IgG-binding domain and then expressed in COS-1 cells as a fused enzyme form. The recombinant enzyme secreted into the culture medium was purified with IgG-Sepharose beads to eliminate endogenous glycosyltransferases, and the enzyme-bound beads were used for enzyme assays with various oligosaccharides as sugar-acceptor substrates. The bound enzyme efficiently transferred GlcNAc to N-acetylheparosan oligosaccharides, which have nonreducing terminal GlcUA residues and represent growing HS chains (Table I), indicating that DEXT3 has Glc-NAcT-II activity. However, it did not transfer GlcUA to Nacetylheparosan oligosaccharides with nonreducing terminal GlcNAc residues, indicating that DEXT3 is not an HS polymerase. It did not use Gal␤1-3Gal␤1-4Xyl either, a substrate for GlcAT-I (33). It also showed significant GlcNAc transferase activity as measured with GlcUA␤1-3Gal␤1-O-C 2 H 4 NHCbz, a synthetic acceptor substrate for HS-initiating GlcNAcT-I enzyme (22,25). However, the linkage region tetrasaccharide serine GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser was not utilized as an acceptor. The significance of these seemingly discrepant results obtained with this substrate and the above synthetic acceptor will be discussed below (see "Discussion"). The soluble form of DEXT3 showed no ␣-GalNAc transferase activity (21), the unique catalytic activity of EXTL2, as detected with N-acetylchondrosine, GlcUA␤1-3GalNAc, consistent with the idea that DEXT3 is not an EXTL2 ortholog. Chon-droitin did not serve as an acceptor when UDP-[ 3 H]GalNAc or UDP-[ 14 C]GlcUA was used as a donor substrate. The culture medium from the control transfection showed no GlcNAcT-I or GlcNAcT-II activity after purification steps using IgG-Sepharose beads.
Product Identification of the Transferase Reactions-The products from the GlcNAcT-I and GlcNAcT-II reactions were characterized by digestions with an appropriate eliminase and glycosidase. The radiolabel of GlcNAcT-I products was quantitatively released by heparitinase I treatment as shown by hydrophobic HPLC on a C 18 column, where the radiolabel was eluted in the flow-through fraction as free GlcNAc (Fig. 4). ␤-N-Acetyhexosaminidase digestion did not release any radioactivity.
Heparitinase I treatment also released [ 3 H]GlcNAc from the GlcNAcT-II reaction products: the products, which originally eluted near the void volume upon gel filtration using a Superdex peptide column, shifted to the free GlcNAc position after heparitinase digestion (Fig. 3B). In contrast, 3 H-labeled products were insensitive to ␤-N-acetylhexosaminidase digestion. Since heparitinase I cleaves only an ␣1,4-glucosaminyl glucuronate linkage, these results suggest that DEXT3 transferred GlcNAc to the acceptors via an ␣1,4-linkage in both GlcNAcT-I and GlcNAcT-II reactions, suggesting that the enzyme activities are most likely involved in initiation and elongation of HS precursor chains.

RT-PCR Analysis of DEXT3 in Different Developmental Stages and in Different
Tissues-The expression profile of DEXT3 were determined by RT-PCR using a Drosophila expression panel containing first strand cDNAs from different tissues and different developmental stages. Deficiency of ttv, the Drosophila homolog of EXT1, causes severe reduction of HS and embryonic lethality during the development, which strongly suggests TTV is a Drosophila HS polymerase. Since DEXT3 showed GlcNAc-TI and GlcNAcT-II activities apparently involved in HS biosynthesis, the expression profiles of both Drosophila genes were compared.
Unexpectedly, even with the lowest cDNA concentration (1x, 1 pg of cDNA), amplification of ttv reached a saturation suggesting its abundant expression. Hence, the results obtained with only two upper concentrations (1000ϫ and 100ϫ) are displayed in Fig. 4. The ttv expression looked similar to those of housekeeping genes. The results of semiquantitative RT-PCR analysis showed that it is expressed evenly and strongly throughout the developmental stages and in both head and body from male and female adult fruit flies. In contrast, the pattern of amplified bands of DEXT3 may suggest its differential expression, although it should be substantiated by more quantitative methods. DISCUSSION Homologs of human EXT gene family members are also found in invertebrates such as D. melanogaster and C. elegans (23,24), which produce HS with disaccharide compositions similar to that of human HS (26,38,39), and recent studies have suggested the involvement of at least some of them in the HS production in these genetically tractable organisms (23,25). TTV and DEXT2 are EXT homologs identified in Drosophila and show the highest amino acid sequence identity to human EXT1 (50.4%) and EXT2 (44.8%), respectively, which suggests that ttv and DEXT2 may be corresponding orthologs of human EXT1 and EXT2 and that their functions may be similar to their counterparts. This appears to be the case, but has not yet been confirmed. According to the mutant analyses, TTV looks like a Drosophila HS polymerase with both GlcNAcT-II and GlcAT-II activities like EXT1 but in vitro catalytic activities have not been demonstrated (23). No evidence has been presented either for the involvement of DEXT2 in the HS biosynthesis in the fruit fly, partly due to lack of mutants. Recently, we proposed that human EXT-like proteins as well as EXT1 and EXT2 play important roles in the HS biosynthesis based on their in vitro catalytic activities (22). In humans, HS synthesis is initiated on the common linkage region, which is shared by chondroitin/dermatan sulfate, by the action of Glc-NAcT-I that is encoded by both EXTL2 and EXTL3 (7, 21, 22). The presence of the EXT1 and EXT2 homologs in Drosophila suggests that the HS biosynthetic mechanism is similar in both species such that GlcNAcT-I activity would also be required for initiating the HS formation in Drosophila. In the present study, by searching Drosophila databases, we identified the third Drosophila EXT homolog, DEXT3, most highly homologous to human EXTL3 (45.8% amino acids identity) and demonstrated that DEXT3 also had GlcNAcT-I and GlcNAcT-II activities as in the case of human EXTL3. These results suggested that the HS synthetic mechanism is quite similar in humans and Drosophila although only three family members have been identified in the latter.
After we identified the DEXT3 gene and characterized the glycosyltransferase activities of the expressed protein, we noticed an abstract by Lin et al. for a recent Drosophila Research Conference (27). The abstract reported a Drosophila mutant deficient in the gene named botv (brother of ttv), which shares the highest homology to human EXTL3 and was identical to DEXT3. The genetic screen was performed to isolate genes that are involved in both Wingless (Wg) and Hedgehog (Hh) signalings, and the isolated fruit fly mutant showed segment polarity phenotypes. The botv mutant phenotypes closely resemble those of the ttv mutant, suggesting that they relate to the same biological processes. Since Wg and Hh signalings are both mediated by HS chains attached to HS proteoglycans, these phenotypes likely reflect the lack of the functional saccharide sequences, implying that botv is involved in HS production. It should be emphasized that the phenotypes of the botv mutant are distinct from that of the ttv mutant. In the ttv mutant, Hh signaling is specifically impaired (23). In contrast, the botv mutation affects both Hh and Wg signalings, which should cause severer defects during Drosophila development. When The values represent the averages of two independent experiments. b ND, not detected (Ͻ 0.5 pmol/ml medium/h). c When truncated EXT1 was used for the positive control, the measured activity was 539 pmol/ml medium/h. d GlcUA␤1-4GlcNAc␣1-(4GlcUA␤1-4GlcNAc␣1)n-4GlcUA␤1-aMan R ; n, in average 7. e When truncated EXT1 was used for the positive control, the measured activity was 111 pmol/ml medium/h. f GlcNAc␣1-(4GlcUA␤1-4GlcNAc␣1)n-4GlcUA␤1-aMan R ; n, in average 7. g A mixture of GlcUA␤1-(3GalNAc␤1-4GlcUA␤1-)n and GalNAc␤1-(4GlcUA␤1-3GalNAc␤1-) n . Under the conditions, the chondroitin synthesizing enzyme system (9,36,37) as a positive control showed marked activities of CS-GlcAT-II and GalNAcT-II.
h GlcUA␤1-3GalNAc. HS is assembled on the linkage region tetrasaccharide, the first GlcNAc transfer by GlcNAcT-I allows the subsequent synthesis of the repeating disaccharide region by HS polymerase. In this regard, our results, which have demonstrated that DEXT3 has both GlcNAcT-I and GlcNAcT-II activities involved in the initiation and elongation of HS chains, are in good agreement with the phenotypes of the botv mutant. The absence of a Drosophila ortholog of human EXTL2 that encodes GlcNAcT-I may account for the phenotypes affecting both Hh and Wg signalings in the DEXT3/botv mutant, which may, in turn, suggest a possibility that human EXTL2 and EXTL3 are responsible for the HS chain initiation for distinct core proteins.
The acceptor substrate specificity revealed for DEXT3 closely resembled those of human EXTL3 and C. elegans Rib-2 in that it has both GlcNAcT-I and GlcNAcT-II activities and does not transfer GlcNAc to the linkage region tetrasaccharide-serine (Table I), which is characteristic of GlcNAcT-I. Rib-2 is a C. elegans EXT homolog, and we reported that it has both GlcNAc transferase activities related to HS biosynthesis (25). DEXT3 shows the highest homology to EXTL3 (45.8%) followed by Rib-2 (29.2%), and the homology in the C-terminal presumptive catalytic domain increases up to 78 and 50%, respectively, to the corresponding domains of EXTL3 and Rib-2 (Fig. 2B). It is therefore not surprising that DEXT3 has the same two catalytic activities. It is conceivable that these three enzyme proteins, which have identical catalytic activities and substrate specificity, are obligatorily conserved during evolution among quite different species of invertebrates and vertebrates. They must be indispensable elements in the HS biosynthetic machinery. The phenotypes of the botv mutants strongly support this idea. Hence, it is suggested that EXTL3 and Rib-2, the DEXT3 counterparts in humans and C. elegans, also have critical roles in the HS formation in the respective organisms, at least as an initiating enzyme. In addition, it is noteworthy that all three enzymes have GlcNAcT-II activity apparently related to HS chain elongation. This activity appears to be redundant between DEXT3 and HS polymerases, which have both GlcNAcT-II and HS-GlcAT-II activities that are definitely required for the synthesis of the repeating disaccharide regions in HS chains. Although it remains to be clarified whether the GlcNAcT-II activity of DEXT3 and the corresponding homologs in other species are involved in HS synthesis in vivo, the highly conserved nature of these proteins among very distant species in the evolutionary tree strongly suggests its importance in the HS synthesis. It might be a necessary component in the putative HS synthesizing enzyme complex.
It has become evident that DEXT3 plays critical roles in the heparan sulfate synthesis in Drosophila, which consequently raises a good possibility that EXTL3, its human ortholog and Rib-2, the C. elegans ortholog, also have similar roles. Indeed, we recently observed embryonic partially lethal phenotypes in C. elegans by RNA interference experiments using rib-2 dsRNA. 3 Further studies on EXTL genes should contribute to elucidation of the molecular mechanism of HS/heparin biosynthesis.