Determination of the Glycosaminoglycan-Protein Linkage Region Oligosaccharide Structures of Proteoglycans from Drosophila melanogaster and Caenorhabditis elegans *

Caenorhabditis elegans and Drosophila melanogaster are relevant models for studying the roles of glycosaminoglycans (GAG) during the development of multicellular organisms. The genome projects of these organisms have revealed the existence of multiple genes related to GAG-synthesizing enzymes. Although the putative genes encoding the enzymes that synthesize the GAG-protein linkage region have also been identified, there is no direct evidence that the GAG chains bind covalently to core proteins. This study aimed to clarify whether GAG chains in these organisms are linked to core proteins through the conventional linkage region tetrasaccharide sequence found in vertebrates and whether modifications by phosphorylation and sulfation reported for vertebrates are present also in invertebrates. The linkage region oligosaccharides were isolated from C. elegans chondroitin in addition to D. melanogasterheparan and chondroitin sulfate after digestion with the respective bacterial eliminases and were then derivatized with a fluorophore 2-aminobenzamide. Their structures were characterized by gel filtration and anion-exchange high performance liquid chromatography in conjunction with enzymatic digestion and matrix-assisted laser desorption ionization time-of-flight spectrometry, which demonstrated a uniform linkage tetrasaccharide structure of -GlcUA-Gal-Gal-Xyl- or -GlcUA-Gal-Gal-Xyl(2-O-phosphate)- for C. elegans chondroitin and D. melanogaster CS, respectively. In contrast, the unmodified and phosphorylated counterparts were demonstrated in heparan sulfate of adult flies at a molar ratio of 73:27, and in that of the immortalizedD. melanogaster S2 cell line at a molar ratio of 7:93, which suggests that the linkage region in the fruit fly first becomes phosphorylated uniformly on the Xyl residue and then dephosphorylated. It has been established here that GAG chains in bothC. elegans and D. melanogaster are synthesized on the core protein through the ubiquitous linkage region tetrasaccharide sequence, suggesting that indispensable functions of the linkage region in the GAG synthesis have been well conserved during evolution.

The nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster are ideal model organisms for studying a wide range of fundamental biological disciplines in development. The genetic studies have established that proteoglycans and their associated glycosaminoglycans (GAGs) 1 are required for normal development of these organisms (Refs. 1 and 2; for a review, see Ref. 3). The unc-52 gene encodes the nematode homolog of mammalian perlecan, the major heparan sulfate (HS) proteoglycan of extracellular matrix. The unc-52 gene plays an essential role in the myofilament assembly in the body wall muscle during the embryonic development (for a review, see Ref. 4). Glypicans are a family of HS proteoglycans that are linked to the cell surface by a glycosylphosphatidylinositol anchor, and two gene family members, division abnormally delayed (dally) and dally-like (dly), have been identified in D. melanogaster. The D. melanogaster glypicans encoded by these genes have been implicated in Wingless (Wg)-mediated patterning of the embryo and play a critical role during the development (for a review, see Ref. 5).
Compared with the genetic analysis of proteoglycans and GAGs in C. elegans and D. melanogaster, their biochemical analysis has been less advanced. GAG chains in D. melanogaster have been detected in the tissue extracts, based on the [ 35 S]sulfate incorporation and the sensitivity of the materials to chondroitinase and nitrous acid treatments (16), whereas those in C. elegans have been detected in cross-sections of all organs using an electron-dense dye in conjunction with GAG lyase digestion (17). It was also reported, based on the sensitivity to heparitinase and nitrous acid but not to chondroitinase ABC, that a single syndecan homolog is expressed as an HS proteoglycan in D. melanogaster (18). The disaccharide compositions of HS and chondroitin sulfate (CS) chains in C. elegans and D. melanogaster have recently been determined (19,20). The genome projects of C. elegans and D. melanogaster have revealed the existence of multiple genes that are putative homologs of HS-synthesizing enzymes not only for the disaccharide repeating region but also for the linkage region (for a review, see Ref. 21). Although GAGs in vertebrates are covalently linked to Ser residues of their core proteins through the common tetrasaccharide linkage region (for reviews, see Refs. 22 and 23), there is no direct evidence that these GAG chains are covalently attached to core proteins through the conventional linker in invertebrates. Notably, it was suggested that unusual N-linked heparan sulfate and chondroitin sulfate chains are produced in addition to the classical CS and HS chains by some cultured cell lines (24) and by bovine lung tissues (25).
In the present study, we isolated and characterized the protein linkage region of C. elegans chondroitin in addition to D. melanogaster HS and CS to investigate whether these GAGs in invertebrates are attached to core proteins through the conventional linkage region tetrasaccharide and whether the tetrasaccharide is modified by phosphorylation and sulfation as in vertebrates.

EXPERIMENTAL PROCEDURES
Materials-GAG lyases, sulfatases, and unsaturated CS disaccharides were obtained from Seikagaku Corp. (Tokyo, Japan). Calf intestine alkaline phosphatase (EC 3.1.3.1) of special quality for molecular biology was from Roche Molecular Biochemicals (Tokyo, Japan). Actinase E was purchased from Kaken Pharmaceutical Co. (Tokyo, Japan). Lysyl endopeptidase (Achromobacter lyticus) was from Wako Pure Chemical Industries (Osaka, Japan). 2-Aminobenzamide (2AB) was purchased from Nacalai Tesque (Kyoto, Japan). Sephadex G-50 (fine) gel and prepacked disposable PD-10 columns containing Sephadex G-25 (medium) were purchased from Amersham Biosciences (Tokyo, Japan). Sep-Pak ® Plus Accell TM QMA anion-exchange cartridges were from Waters Corp. (Milford, MA). Unsaturated disaccharides derived from heparin and HS were prepared as described previously (26). The following 2AB-derivatives of the authentic unsaturated tetra-and hexasaccharides of the GAG-protein linkage region were prepared from various CS preparations as previously described (27) . D. melanogaster cell line S2 was obtained from Dr. Hiroshi Nakato (University of Arizona, Tucson, AZ) (28). Preparation of GAG-Protein Linkage Region from C. elegans-The adult worm homogenate was prepared and lyophilized as described previously (19). The dried adult worm homogenate preparation (10 mg) was treated in 680 l of boiling water for 10 min, cooled, and digested with 0.4 mg of actinase E heat-pretreated (60°C, 30 min) in 1 ml of 0.1 M borate-sodium, pH 8.0, containing 10 mM calcium acetate. The incubation was carried out at 60°C for 48 h; an additional 0.4 mg of the enzyme was added after 24 h. Following incubation, the sample was mixed with 0.2 ml of 30% trichloroacetic acid and centrifuged. The soluble fraction was extracted with ether. The aqueous phase was neutralized using 1 M sodium carbonate and lyophilized. The dried sample was dissolved in water and subjected to gel filtration chromatography on a column of Sephadex G-50 (fine) (1 ϫ 57 cm) using 50 mM pyridine acetate, pH 5.0, as an eluent at a flow rate of 0.9 ml/min. Fractions (1.8 ml) were collected, and an aliquot (30 l) of each fraction was dried and digested with 10 mIU of chondroitinase AC-II in a total volume of 20 l of 0.25 M sodium acetate, pH 6.0, at 37°C for 15 min (29). The digests were lyophilized, derivatized with 2AB, and then analyzed by anion-exchange HPLC on an amine-bound silica PA-03 column (4.6 ϫ 250 mm, YMC Co., Kyoto, Japan) as described previously (30). The fractions containing chondroitin were pooled, and derivatized with 2AB before and after LiOH treatment. The LiOH treatment, to liberate O-linked saccharides from the core proteins, was performed as described previously (27,31). After 2AB-derivatization, the excess 2AB reagent was removed by paper chromatography and gel filtration chromatography (27). For the analysis of the HS-protein linkage region, 250 mg of the dried worm homogenate preparation was used as the starting material, and an aliquot of the final 2AB-labeled GAG preparation corresponding to 25 mg of the dried worm preparation was injected for the detection by HPLC, but in vain.
Digestions of the 2AB-derivatized Chondroitin from C. elegans with Chondroitinases ABC and AC-II-The 2AB-derivatives of the C. elegans chondroitin (0.5 mg of the dry powder of the worm homogenate as the starting material) was digested with 12.5 mIU of chondroitinase ABC in a total volume of 20 l of 50 mM Tris-HCl, pH 8.0, containing 60 mM sodium acetate at 37°C for 1 h (29). The enzymatic reaction was terminated by heating at 100°C for 1 min. The digest was mixed with 20 l of water and 10 l of 250 mM sodium acetate, pH 6.0, containing 5 mIU of chondroitinase AC-II and incubated at 37°C for 1 h. The reaction was terminated as above. Each digest was analyzed by anionexchange HPLC on a PA-03 column (30) or by gel filtration HPLC on a column of Asahipak GS320 (7.6 ϫ 500 mm) as described previously (32).

Delayed Extraction Matrix-assisted Laser Desorption Ionization Time-of-flight (DE MALDI-TOF) Mass Spectrometry (MS)-DE
MALDI-TOF mass spectra in the positive or negative ion mode of the 2AB-derivatized linkage hexasaccharide from C. elegans chondroitin were recorded on a Voyager DE-RP/Pro (PerSeptive Biosystems, Framingham, MA) in the linear mode (33). An aqueous solution of the 2AB-derivative of the linkage hexasaccharide, which was purified by gel filtration HPLC on an Asahipak GS320 column (32), was mixed with an equal volume of the aqueous solution (10 mg/ml) of a matrix, 2,5dihydroxybenzoic acid. An aliquot (1 l) of this sample-matrix mixture was placed on the sample plate well, dried under a stream of air, and analyzed.
Preparation of 2AB-derivatized GAGs from Adult Flies (D. melanogaster)-Adult flies (3.6 g) were extracted with chloroform/methanol (2:1), homogenized in acetone, and air-dried. The dried materials (790 mg) were treated in 36 ml of boiling water for 10 min, cooled, and exhaustively digested with lysyl endopeptidase using 4.3 mg (10 absorbance units) of the enzyme in 40 ml of 100 mM Tris-HCl, pH 9.0, at 37°C overnight (1 absorbance unit corresponds to the amount of the enzyme that hydrolyzes 1 mol of N-benzoyl-DL-lysine-p-nitroanilide/ min). The digest was treated with 5% trichloroacetic acid, and the acid-soluble fraction was extracted with ether. The aqueous phase was neutralized with 1 M Na 2 CO 3 and adjusted to 80% ethanol. The resultant precipitate was dissolved in water and subjected to gel filtration chromatography on a PD-10 column with 50 mM pyridine acetate, pH 5.0, as the eluent. The flow-through fraction was collected, dried, and treated with 0.5 M LiOH at 4°C overnight to liberate O-linked saccharides from the core proteins (27,34,35). The reaction was terminated by acidification with glacial acetic acid, and the sample was applied to a column (12 ml) of anion-exchange resin AG 50W-X2 (H ϩ form, Bio-Rad) pre-equilibrated with water. The unbound fraction containing the liberated O-linked saccharides was neutralized with 1 M NH 4 HCO 3 and was derivatized with 2AB (30). The 2AB-derivatized GAGs were purified by removing excess reagents by paper chromatography as described above, followed by gel filtration on a PD-10 column using 50 mM pyridine acetate, pH 5.0, as the eluent. The flow-through fractions containing 2AB-labeled GAGs were dried and subjected to anion-exchange chromatography using a Sep-Pak ® cartridge (1.0 ml) of Accell TM Plus QMA, which had been equilibrated with 300 mM sodium phosphate, pH 6.0, containing 0.015 M NaCl. After washing with the equilibration buffer, the column was eluted stepwise with the same buffer containing 0.075, 0.15, 0.5, or 1.0 M NaCl, respectively. The 0.5 M NaCl fraction, which contained essentially all the 2AB-labeled CS chains and 88% of the HS chains (see "Results"), was desalted by gel filtration on a PD-10 column using 50 mM pyridine acetate, pH 5.0, as the eluent. The flow-through fraction was dried and used for the analyses of the disaccharide composition, molecular sizes, and protein linkage regions of GAGs.
Disaccharide Composition Analysis of D. melanogaster CS and HS-The 2AB-derivatized GAGs from adult flies were purified by ion-exchange chromatography using a Sep-Pak ® cartridge of Accell TM Plus QMA, and the purified 2AB-derivatized GAG preparation was digested either with chondroitinases ABC, AC-I, or AC-II or with a mixture of heparinase and heparitinase. In the case of the S2 cell-derived GAGs, the GAG-peptide preparation was subjected to the above lyase digestions. Each digest was treated with 2AB, and the 2AB-derivatized fraction was analyzed by anion-exchange HPLC on a PA-03 column (30). In the case of the adult fly sample, chondroitinase-produced unsaturated disaccharides were detectable also by ultraviolet absorption because of the large amounts.
Gel Filtration Chromatography of CS and HS Chains from Adult Flies-The 2AB-derivatized GAG fraction prepared above from adult flies was analyzed by gel filtration chromatography on a column (10 ϫ 300 mm) of Superdex 200 eluted with 0.2 M CH 3 COONH 4 as the eluent, at a flow rate of 0.3 ml/min in an FPLC system (Amersham Biosciences). Fractions were collected at 3-min intervals, lyophilized, and digested with chondroitinase ABC or a mixture of heparinase and heparitinase (35,36). The digests were derivatized with 2AB, then analyzed by anion-exchange HPLC on an amine-bound PA-03 column (30).
HPLC Analysis of the 2AB-derivatives of the Linkage Region Oligosaccharides from Adult Flies-The 2AB-derivatized GAG fraction prepared above was digested either with chondroitinases ABC and then AC-II or with a mixture of heparinase and heparitinase as described previously (34,36). For the chondroitinase digestion, the 2AB-derivatized GAG fraction corresponding to 390 mg of the dried flies was digested successively with 75 mIU of chondroitinase ABC and 50 mIU of chondroitinase AC-II. Another aliquot of the isolated GAG fraction corresponding to 70 mg of the dried flies was used for the heparinase/ heparitinase digestion. Each digest was analyzed by anion-exchange HPLC on a PA-03 column (30).
Alkaline Phosphatase Digestion-Alkaline phosphatase treatment was carried out using 4 IU of the enzyme in a total volume of 100 l of 80 mM glycine/NaOH buffer, pH 9.9, containing 0.5 mM MgCl 2 at 37°C for 30 min (32). The enzymatic reaction was terminated by heating at 100°C for 1 min, and each enzyme digest was analyzed by anionexchange HPLC on a PA-03 column (30,32).
Preparation of 2AB-derivatized GAGs from D. melanogaster S2 Cell Line-S2 cells were grown in a spinner flask for suspension culture using the Schneider's insect medium (Invitrogen) supplemented with 10% fetal calf serum and kanamycin sulfate (60 g/ml) at 25°C. Approximately 7 ϫ 10 8 cells were homogenized with ice-cold acetone and vacuum-dried. The dried materials were boiled in water for 10 min, cooled, and exhaustively digested overnight with actinase E at 60°C as described above. The digest was treated with 5% trichloroacetic acid, and the acid-soluble fraction was extracted with ether. The aqueous phase was desalted by gel filtration chromatography on a PD-10 column. The purified GAG-peptide preparation was used for disaccharide composition analysis (see above). Another aliquot was treated with 0.5 M LiOH to liberate O-linked saccharides, and the liberated linkage oligosaccharides were derivatized with 2AB as described above.
Simultaneous LiOH Treatment of Desialylated Bovine Submaxillary Mucin and Peptide CS from Whale Cartilage-Highly purified bovine submaxillary mucin (donated by Prof. K. Kakehi, Kinki University, Osaka, Japan) was treated with 2 M acetic acid at 80°C for 3 h to remove sialic acid and neutralized with alkali, and the desialylated mucin was recovered by ethanol precipitation. This preparation (48 ng), corresponding to 60 pmol of GalNAc (37), was mixed with a purified preparation of linkage region hexasaccharide peptides (30 pmol as hexasaccharides) prepared by chondroitinase ABC digestion of peptide CS-A from whale cartilage (27), dried, and treated with 50 l of 0.5 M LiOH at 4°C for 18 h. The mixture was neutralized with acetic acid, and the sample was treated with anion-exchange resin AG 50W-X2 (H ϩ form). The unbound fraction was neutralized with 1 M NH 4 HCO 3 and was derivatized with 2AB as described above (30). One fifth of the sample was analyzed by gel filtration HPLC on a column of Asahipak GS320 (see above). Only the four expected hexasaccharide peaks (27) but no GalNAc-2AB were detected, suggesting that the GalNAc␣1-O-Ser/Thr linkage is resistant to the LiOH treatment. The desialylated mucin showed a predominant peak of GalNAc-ol on HPLC as expected when treated with 1.0 M NaBH 4 , 0.05 M NaOH in a control experiment.

RESULTS
Structural Analysis of the GAG-Protein Linkage Region of C. elegans-Previous studies have shown chondroitin and HS in C. elegans (19,20). The acetone powder of the crude homogenate of the adult worms was digested with actinase E, and the resultant GAG-peptides were purified as described in "Experimental Procedures." The GAG-peptides were treated with LiOH to liberate O-glycan chains including GAGs from the core proteins (27,31). The liberated saccharides were labeled with a fluorophore 2AB, and the excess 2AB reagent was removed by paper chromatography. The sample extracted with water was then subjected to gel filtration chromatography and monitored by fluorescence intensity (Fig. 1). The fluorescence intensity of the sample prepared without the LiOH treatment was significantly weaker, compared with that of the LiOH-treated sample, suggesting the presence of C. elegans GAG attached to core proteins via O-glycosidic bonds. The flow-through fractions of both samples were pooled (see bars in Fig. 1) and analyzed further. The samples were digested with chondroitinase ABC and/or AC-II, and the digests were analyzed by anion-exchange or gel filtration HPLC. A major peak was observed at the position of the authentic 2AB-labeled nonsulfated hexasaccharide, ⌬HexUA␣1-3GalNAc␤1-4GlcUA␤1-3Gal␤1-3-Gal␤1-4Xyl1-2AB (⌬HexUA represents 4-deoxy-␣-L-threohex-4-enepyranosyluronic acid), when a chondroitinase ABC digest of the 2AB-derivative prepared after the LiOH treatment was analyzed by anion-exchange HPLC ( Fig. 2A). No significant peaks were detected for the control sample prepared without the LiOH treatment (data not shown). Upon subsequent chondroitinase AC-II digestion, this peak was shifted to the position of the authentic 2AB-labeled nonsulfated tetrasaccharide, ⌬HexUA␣1-3Gal␤1-3Gal␤1-4Xyl1-2AB (Fig. 2B). The 2AB-labeled oligosaccharide generated by chondroitinase ABC digestion was isolated by gel filtration HPLC as shown in , respectively (HexUA, HexNAc, Hex, and Pen represent hexuronic acid, Nacetylhexosamine, hexose, and pentose, respectively). Together these results indicate that the C. elegans chondroitin is synthesized as proteoglycans, being covalently bound probably to a Ser residue of the core protein through the nonsulfated linkage region hexasaccharide, -GlcUA␤1-3GalNAc␤1-4GlcUA␤1-3-Gal␤1-3Gal␤1-4Xyl␤1-. The structure of the GAG-protein linkage region of C. elegans HS could not be detected because of the limited amount of the sample, even when a sample corresponding to as much as 25 mg of the dried worms was used for a single injection for HPLC. The failure was consistent with the previous findings that the total amounts of HS disaccharides were 0.71 nmol (19) or 310 ng (20) per 25 mg of dried worms. Another aliquot of the sample prepared in the present study showed comparable amounts of 2AB-labeled disaccharides to those from the previous studies, confirming the reproducibility of the results. An amount of the sample larger by 1 order of magnitude may be required for the chemical detection, which is not practical for currently available methods; trace amounts of HS chains labeled with 2AB would have been buried in the small bump observed in the flow-through fraction in Fig. 3.
Analyses of the Disaccharide Compositions and Molecular Sizes of the D. melanogaster CS and HS-Prior to the analysis of the GAG-protein linkage regions of D. melanogaster CS and HS, the CS and HS chains were characterized for the disaccharide compositions and molecular sizes. For this purpose and for the subsequent analysis of the linkage region, fluorescently labeled free GAG chains were prepared from the acetone powder of adult flies by lysyl endopeptidase digestion followed by LiOH treatment and derivatization with a fluorophore 2AB as described under "Experimental Procedures." The resultant 2AB-derivatized GAGs were subjected to ion-exchange chromatography using a Sep-Pak ® cartridge (1.0 ml) of Accell TM Plus QMA, which had been equilibrated with 300 mM sodium phosphate, pH 6.0, containing 0.015 M NaCl. The column was washed with the equilibration buffer and then eluted stepwise with the same buffer containing 0.075, 0.15, 0.5, or 1.0 M NaCl. The 0.5 M NaCl fraction contained essentially all the 2ABlabeled CS chains and 88% of the HS chains as examined by disaccharide analysis using chondroitinase AC-II or a mixture of heparinase and heparitinase in conjunction with anion-exchange HPLC on a PA-03 column (data not shown). The 0.5 M NaCl fraction was desalted and used for disaccharide composition analysis, which was carried out by digestion with either chondroitinase ABC or a mixture of heparinase and heparitinase, respectively, followed by anion-exchange HPLC. The results, summarized in Table I, were comparable with those obtained for the CS and HS chains from the S2 cells (see below), and are basically in agreement with the previously reported results by Toyoda et al. (20). Chondroitinase AC-I or AC-II digestion gave a comparable disaccharide composition with that obtained by chondroitinase ABC, and chondroitinase B digestion gave no appreciable disaccharides, indicating that most, if not all, of the chondroitinase ABC-degraded materials are CS but not dermatan sulfate (DS), being consistent with the previous findings (20).
The molecular sizes of the GAGs were also analyzed using the 2AB-derivatized GAG preparation by gel filtration chromatography on a column of Superdex 200. To monitor small amounts of GAGs, aliquots of individual fractions were lyophilized and digested with chondroitinase ABC or a mixture of heparinase and heparitinase, respectively. The products were then derivatized with a fluorophore 2AB and analyzed by anion-exchange HPLC as described under "Experimental Procedures." Compared with the calibration plot generated using the data obtained with size-defined commercial polysaccharides (Fig. 5, inset), the average molecular sizes of the D. melanogaster CS and HS were estimated to be 70 and 20 kDa, respectively. The C. elegans chondroitin gave a broad peak in the range of 40 -50 kDa, suggesting that the heterogeneous populations were consistent with previous observations (19). It was not possible to analyze the size of the C. elegans HS chains because of the limited amount.
Structural Analysis of the GAG-Protein Linkage Region of the D. melanogaster CS and HS Chains-From the above mentioned 2AB-derivatized GAG preparation, the 2AB-labeled tetrasaccharides derived from the CS-protein linkage region were prepared by digesting the repeating disaccharide region successively with chondroitinases ABC and then AC-II, and were analyzed by anion-exchange HPLC on a PA-03 column. Depolymerization of CS chains synthesized on the conventional protein linkage region tetrasaccharide by chondroitinase ABC results in sulfated disaccharide units and core hexasaccharides derived from the linkage region (38). Chondroitinase AC-II will degrade a linkage region hexasaccharide into a disaccharide unit and a core tetrasaccharide (39). When the 2AB-derivatized linkage region prepared by chondroitinase AC-II digestion were analyzed by anion-exchange HPLC, only a single predominant peak was observed at the elution position of the authentic 2AB-tetrasaccharide ⌬HexUA␣1-3Gal␤1-3Gal␤1-4Xyl(2-Ophosphate)1-2AB (Fig. 6B). This sample was co-eluted when co-chromatographed (data not shown) with the corresponding standard linkage tetrasaccharide (Fig. 6A), confirming the identity of the peak. Upon subsequent alkaline phosphatase digestion, the peak was shifted by 13 min to the position of the nonsulfated tetrasaccharide (data not shown), suggesting that the compound in the peak contained a phosphate group most likely on the C-2 position of the Xyl residue in the linkage region tetrasaccharide structure (32, 40 -42), and accounted for most, if not all, of the linkage regions of the CS-proteoglycans in Drosophila.
The 2AB-labeled tetrasaccharides derived from the HS-pro- tein linkage region were also analyzed by HPLC after digesting the repeating disaccharide region using a mixture of bacterial heparinase and heparitinase. Because heparitinase cleaves the innermost glucosaminidic bond of HS chains (43,44), the enzyme digestion results in various sulfated disaccharide units and linkage region core tetrasaccharides. As shown in Fig. 6C, two major peaks were observed at the elution positions of the authentic 2AB-tetrasaccharides, ⌬HexUA␣1-3Gal␤1-3-Gal␤1-4Xyl1-2AB and ⌬HexUA␣1-3Gal␤1-3Gal␤1-4Xyl(2-O-phosphate)1-2AB, in a molar ratio of 73:27. The two peaks were co-eluted with the corresponding standards upon cochromatography (data not shown). The peak, eluted at the position of ⌬HexUA-Gal-Gal-Xyl(2-O-phosphate)1-2AB, was shifted by subsequent alkaline phosphatase digestion to the elution position of the nonsulfated tetrasaccharide (data not shown), suggesting that the compound in the peak contained a phosphate group most likely on the C-2 position of the Xyl residue in the linkage region tetrasaccharide sequence. The data obtained from the analyses of the chain sizes, disaccharide compositions, and linkage region structures of the adult flies are summarized in the diagrams shown in Fig. 7.
by 2AB-derivatization of the resultant disaccharides resulted in 2AB-labeled ⌬Di-0S and ⌬Di-4S in a molar ratio of 79:21, being in good agreement with the undersulfation observed for that of adult flies. Analysis after digestion with a mixture of heparinase and heparitinase gave 2AB-labeled disaccharides with sulfation profiles comparable with those obtained for the adult flies (Table I). The disaccharide compositions obtained by digestions with chondroitinases ABC, AC-I, or AC-II were comparable, and chondroitinase B digestion gave no appreciable disaccharides, suggesting that the S2 cells produce CS but not DS. It is interesting from the molecular evolution point of view that Drosophila produces 4-O-sulfated CS but no DS which is also 4-O-sulfated on GalNAc residues, and C. elegans synthesizes only nonsulfated chondroitin. A CS/DS-specific epimerase does not appear to be expressed in these organisms. For the analysis of the CS-or HS-protein linkage region, the 2ABlabeled GAG chains were digested with chondroitinases ABC and then AC-II or a mixture of heparinase and heparitinase, respectively. The chondroitinases digest was analyzed by HPLC, and a single peak was observed at the elution position of ⌬HexUA-Gal-Gal-Xyl(2-O-phosphate)-2AB (data not shown). This peak was shifted to the elution position of the nonsulfated tetrasaccharide upon alkaline phosphatase digestion (data not shown). The 2AB-labeled oligosaccharides derived from the HS-protein linkage region were also analyzed by HPLC, and two major peaks were observed at the elution positions of the authentic 2AB-tetrasaccharides, ⌬HexUA-Gal-Gal-Xyl-2AB and ⌬HexUA-Gal-Gal-Xyl(2-O-phosphate)-2AB (data not shown), in a molar ratio of 7:93. The results from the linkage region analyses of the CS and HS chains were in agreement with the findings obtained from the analysis of adult flies, although the phosphorylated component was the predominant component in the S2 cells, which suggests that the Xyl residue first becomes uniformly phosphorylated and then dephosphorylated. DISCUSSION The genes encoding the putative enzymes that are involved in the biosynthesis of the GAG-protein linkage region have been identified in the C. elegans and D. melanogaster genomes (for a review, see Ref. 21). The product of the C. elegans sqv-3 gene, which is involved in vulval invagination and oocyte development, has 38% sequence homology to human galactosyltransferase I (14), whereas that of another gene, sqv-8, which also plays a role in vulval invagination, shows high sequence similarity to human glucuronyltransferase I (46). The catalytic activities of the expressed proteins of these cDNAs have been demonstrated in vitro (47). Xylosyltransferase, which transfers a xylose residue from UDP-Xyl to Ser residues at the GAG acceptor sites of core proteins, has also been cloned, and homologous genes are found in C. elegans and D. melanogaster genomes (48). Recently, Wilson (49) and Deepa et al. 2 detected the xylosyltransferase activity for the expressed fly homolog. Bai et al. (50) reported the cloning of the Chinese hamster ovary galactosyltransferase II cDNA and the presence of its orthologs in C. elegans and D. melanogaster genomes.
The linkage region tetrasaccharide GlcUA-Gal-Gal-Xyl structure was presumed to exist but has now been isolated for 2 S. S. Deepa, S. Yamada, and K. Sugahara, unpublished data.  Fig. 3, and its DE MALDI-TOF/MS was recorded in the negative ion mode with 2,5-dihydroxybenzoic acid as the matrix.

TABLE I
The disaccharide compositions of the D. melanogaster CS and HS chains The 2AB-derivatized GAG fraction from adult flies was prepared as described under "Experimental Procedures" after protease digestion followed by LiOH treatment and derivatization with a fluorophore 2AB. The resultant 2AB-derivatized GAGs from adult flies or the GAGpeptide preparation from the D. melanogaster S2 cells were digested either with chondroitinase ABC, AC-I, or AC-II or with a mixture of heparinase and heparitinase. Each digest was labeled with 2AB, and the 2AB-derivatized fraction was analyzed by HPLC as described under "Experimental Procedures." The results from the chondroitinase digests were comparable and those obtained with the chondroitinase ABC digests are shown. In the case of the sample for adult flies, the large amount of the sample allowed us to detect the disaccharides by ultraviolet absorbance without 2AB labeling. the first time as discrete structures from the C. elegans chondroitin and D. melanogaster HS and CS. Although it was not detected for C. elegans HS, this was most likely the result of the limited amount of HS. This is the first study that demonstrates the existence of the linkage region tetrasaccharide in invertebrates. Despite a wide distribution of GAGs in different classes of invertebrates (for a review, see Ref. 51), it has not been clarified how these GAGs are attached to the presumable core proteins. Some bacterial strains, Escherichia coli K4 and K5 and Streptococcus pyrogenes, synthesize polysaccharides with the same backbone structures as CS, heparin/HS, and hyaluronic acid, respectively (52)(53)(54). These GAG-like polymers are produced as extracellular polysaccharide capsules that serve as virulence factors. These polysaccharides are not attached to core proteins and are synthesized on the inner surface of the cytoplasmic membrane (for a review, see Ref. 55). Studies on the GAG-protein linkage region of invertebrate GAGs are limited, and the reducing end structure of the invertebrate GAG chains has not been characterized. It has now been clarified that at least the conventional linkage region tetrasaccharide GlcUA-Gal-Gal-Xyl of proteoglycans has been conserved through evolution, although other linkage region structures have not been excluded. It has been suggested that unusual N-linked HS and CS chains, which are released by peptide-Nglycosidase digestion, are produced by some cultured cell lines such as bovine pulmonary arterial endothelial (CPAE) and human erythroleukemia (K562) cell lines (24) as well as by bovine lung tissues (25). Such possibilities were not investi-gated rigorously in this study and remain to be explored. The possibility that chondroitin chains are attached to core proteins via O-linked GalNAc residues (56) will be discussed below.
In this study, phosphorylation of the Xyl residue was demonstrated in the linkage region of both CS and HS from D. melanogaster. Although only the phosphorylated structure in the former, and both phosphorylated and nonphosphorylated forms in the latter, were detected, the existence of the phosphorylated Xyl in both HS and CS chains is consistent with the previous findings (23,34). Recent simultaneous analysis of both CS and HS chains of the hybrid type proteoglycan syndecan-1 molecule has suggested the Xyl phosphorylation of both CS and HS chains (34). Structural analysis of the biosynthetic intermediate oligosaccharides formed in the cultured rat fibroblasts has suggested that dephosphorylation takes place soon after the transfer reaction of GlcUA to the trisaccharide (57)(58)(59). In vitro enzyme assays using crystallized human glucuronyltransferase I as enzyme (60) and Gal-Gal-Xyl(2-O-phosphate)-Ser as acceptor have demonstrated the higher enzyme activity with the phosphorylated acceptor substrate. 3 Together, these results suggest that the Xyl phosphorylation may play an important role for the transfer reaction of the first GlcUA residue to the linkage region trisaccharide, and may be required for the efficient maturation of the linkage region tet- rasaccharide as prerequisites for the assembly of GAG chains.
In strong contrast, the phosphorylated Xyl was not detected in the linkage region of C. elegans chondroitin in the present study. This may suggest that the glycosyltransferases involved in the synthesis of the C. elegans chondroitin have different specificities from those in higher organisms and do not require the phosphate group on the Xyl. In fact, in the C. elegans genome there is no ortholog of the recently cloned human N-acetylgalactosaminyltransferase that transfers the first Gal-NAc to the linkage region tetrasaccharide (61). Instead, the C. elegans ortholog of human chondroitin synthase (62) may possess this enzyme activity. Thus, although C. elegans chondroitin is formed on the conventional linkage region tetrasaccharide structure, the biosynthetic mechanism appears to be somewhat different from those of D. melanogaster or higher organisms including humans. In the present study no sulfation of the Gal residues was observed in the linkage region of either C. elegans chondroitin or D. melanogaster CS, although it has been reported for the linkage region of CS and DS chains from various vertebrate tissues (23,34). No sulfation of the Gal residues in the linkage region of C. elegans chondroitin may be related to the finding that chondroitin is not sulfated in this organism. No homologs of human CS/DS 4-O-sulfotransferases (63,64) are found in the C. elegans genome. In Drosophila, for the synthesis of chondroitin 4-sulfate, the 4-O-sulfation of the Gal residue in the linkage region may not be required as a prerequisite.
Although low sulfated CS has been found in various animal tissues such as human inter-␣-trypsin inhibitor (65), CS from human placenta (66), mollusc (67), and human cornea (68), reports on completely nonsulfated chondroitin chains are limited to polysaccharides from squid skin (69 -71). Although the corresponding acidic polysaccharide isolated from bovine cornea in the pioneering work by Davidson and Meyer (72) was named chondroitin, it contained reportedly a small proportion (2.1%) of sulfate groups. Despite the high chondroitin content in the C. elegans nematode (2 g/mg dry worm) (19,20), its physiological function remains to be investigated. The recent cloning of human chondroitin synthase cDNA has identified the ortholog in the C. elegans genome (36% identity) (62) and makes it possible to perform gene knockout experiments in C. elegans, which is now in progress to investigate the function of the chondroitin chain in the nematode.
Guérardel et al. (56) recently isolated the short chondroitinlike oligosaccharides from the C. elegans nematode: GalNAc-␤1-4GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl-ol, GalNAc␤1-4GlcUA-␤1-3GalNAc-ol, GlcUA␤1-3GalNAc␤1-4GlcUA␤1-3GalNAcol, and GalNAc␤1-4GlcUA␤1-3GalNAc␤1-4GlcUA␤1-3-GalNAc-ol. The first one has the same saccharide sequence with that of the CS-protein linkage region demonstrated in vertebrates, and the latter three compounds are typical chondroitin oligosaccharides from the repeating disaccharide region. However, no evidence has been presented there that these oligosaccharides were derived from polymer chondroitin. The alkaline conditions used for preparing these oligosaccharides   (Table I) and the GAG-protein linkage region (Fig. 6). 2P in the linkage region represents 2-O-phosphate. The width of each box corresponds to the proportion of each disaccharide unit shown in Table I. were standard mild conditions (1 M NaBH 4 , 100 mM NaOH), and hence decomposition of chondroitin chains by peeling reactions or random cleavage was unlikely. In addition, the large amounts of the isolated oligosaccharides appear to indicate that they were released directly from the core peptides. In the present study, such small truncated oligosaccharides, if any, would have been resistant to the LiOH treatment as confirmed by simultaneously treating desialylated bovine submaxillary mucin and a standard linkage region hexasaccharide peptide preparation followed by HPLC analysis. Only the linkage region hexasaccharides but no mucin-derived GalNAc (37) were detected after 2AB-derivatization (data not shown), suggesting that the GalNAc␣1-O-Ser/Thr linkage is resistant to the mild alkaline treatment with LiOH (see "Experimental Procedures"). The unsaturated hexasaccharide, ⌬HexUA-GalNAc-GlcUA-Gal-Gal-Xyl-2AB, was the only linkage region oligosaccharide obtained in this study after exhaustive chondroitinase ABC digestion of the C. elegans chondroitin polysaccharide (Figs. 2 and 3), and neither ⌬HexUA-GalNAc-GlcUA-GalNAc-2AB nor ⌬HexUA-GalNAc-2AB was discerned. The failure to detect even GalNAc-GlcA-Gal-Gal-Xyl-2AB (56) appears to suggest that such small truncated oligosaccharides might have escaped at the step of the gel filtration column chromatography on Sephadex G-50 after Actinase E digestion (see "Experimental Procedures"). The present findings may suggest that polymer chondroitin chains are polymerized only on the conventional common linkage region tetrasaccharide structure by putative chondroitin synthase but not on the short oligosaccharides, with a reducing terminal GalNAc residue but without the conventional linkage region, which are likely to build on the core proteins but may not be elongated to a large extent. Identification of presumable glycosyltransferases responsible for the synthesis of these oligosaccharides and isolation of glycopeptides derived from the putative chondroitin oligosaccharides with a reducing terminal GalNAc residue would give insights into the mechanism of evolution of polymer chondroitin in C. elegans.
Dally and Dly, the orthologous proteins of vertebrate glypicans, in addition to Drosophila syndecan (18), have been found as the major HS-PGs in D. melanogaster (73,74), whereas no core proteins of either CS-PGs in D. melanogaster or HS-and chondroitin-PGs in C. elegans have been identified and the Drosophila syndecan core protein does not bear CS chains (18), unlike mammalian syndecan-1 or -4 (75,76). Although C. elegans unc-52, the ortholog of the perlecan HS-PG gene, has been shown to affect muscle attachment and sarcomere organization (45), little of the biochemical nature is known for the GAG chains of this PG. Recently, Bulik et al. (47) reported that there are numerous chondroitin-modified PGs in C. elegans by Western blotting analysis using anti-⌬HexUA-GalNAc antibody after chondroitinase ABC digestion. However, because short chondroitin-like oligosaccharides, GlcUA␤1-3GalNAc-␤1-4GlcUA␤1-3GalNAc and GalNAc␤1-4GlcUA␤1-3GalNAc-␤1-4GlcUA␤1-3GalNAc, identified in C. elegans would also form the ⌬HexUA-GalNAc-structure on chondroitinase ABC digestion, the heterogeneity of the core protein of chondroitin-PG may be the result of the heterogeneous O-glycosylation by such short chondroitin-like oligosaccharides. The progress of the C. elegans genome project has revealed the genes homologous to those of the core proteins of vertebrate CS-PGs such as bamacan, aggrecan, decorin, and phosphacan. Further studies are needed for identification of the core protein(s) of the C. elegans chondroitin oligo-and polysaccharides to gain insights into the biological functions of C. elegans chondroitin oligo-and polysaccharides.