GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate) Is the Preferred Substrate for Chondroitin N-Acetylgalactosaminyltransferase-1*

Background: The relationship between chondroitin N-acetylgalactosaminyltransferase-1 (ChGn-1) and 2-phosphoxylose phosphatase (XYLP) in controlling the number of chondroitin sulfate chains is unclear. Results: GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate) was detected in ChGn-1−/− but not in wild-type cartilage. ChGn-1-mediated addition of N-acetylgalactosamine was accompanied by rapid XYLP-dependent dephosphorylation. Conclusion: GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate) is the preferred substrate for ChGn-1. Significance: ChGn-1 and XYLP cooperatively regulate the number of CS chains. A deficiency in chondroitin N-acetylgalactosaminyltransferase-1 (ChGn-1) was previously shown to reduce the number of chondroitin sulfate (CS) chains, leading to skeletal dysplasias in mice, suggesting that ChGn-1 regulates the number of CS chains for normal cartilage development. Recently, we demonstrated that 2-phosphoxylose phosphatase (XYLP) regulates the number of CS chains by dephosphorylating the Xyl residue in the glycosaminoglycan-protein linkage region of proteoglycans. However, the relationship between ChGn-1 and XYLP in controlling the number of CS chains is not clear. In this study, we for the first time detected a phosphorylated tetrasaccharide linkage structure, GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate), in ChGn-1−/− growth plate cartilage but not in ChGn-2−/− or wild-type growth plate cartilage. In contrast, the truncated linkage tetrasaccharide GlcUAβ1–3Galβ1–3Galβ1–4Xyl was detected in wild-type, ChGn-1−/−, and ChGn-2−/− growth plate cartilage. Consistent with the findings, ChGn-1 preferentially transferred N-acetylgalactosamine to the phosphorylated tetrasaccharide linkage in vitro. Moreover, ChGn-1 and XYLP interacted with each other, and ChGn-1-mediated addition of N-acetylgalactosamine was accompanied by rapid XYLP-dependent dephosphorylation during formation of the CS linkage region. Taken together, we conclude that the phosphorylated tetrasaccharide linkage is the preferred substrate for ChGn-1 and that ChGn-1 and XYLP cooperatively regulate the number of CS chains in growth plate cartilage.

CS chains have specific functions during cartilage development, suggesting that the phosphorylation, dephosphorylation, sulfation, and number of CS chains are strictly regulated by these biosynthetic enzymes (1).
Recently, we revealed that a deficiency in ChGn-1 reduced the number of CS chains, leading to skeletal dysplasias in mice (15). In addition, we found two missense mutations in the ChGn-1 gene that were associated with a profound decrease in enzyme activity in two patients with neuropathy (16). Thus, it is suggested that ChGn-1 regulates the number of CS chains and the total amount of CS in these patients and in growth plate cartilage. More recently, we demonstrated that XYLP regulates the number of CS chains by dephosphorylating the Xyl residue in the GAG-protein linkage region of proteoglycans (PGs) (3). However, the relationship between ChGn-1 and XYLP in the biosynthesis of CS was not clear. In the present study, we report that ChGn-1 and XYLP interact with each other and that ChGn-1-mediated addition of N-acetylgalactosamine was accompanied by rapid XYLP-dependent dephosphorylation during formation of the CS linkage region.

EXPERIMENTAL PROCEDURES
Animals-Mice (C57BL/6 background) were kept under pathogen-free conditions in an environmentally controlled, clean room at the Institute of Laboratory Animals, Kobe Pharmaceutical University; animals were maintained on standard rodent food and on a 12-h light/12-h dark cycle. All animal procedures were approved by the Kobe Pharmaceutical University Committee on Animal Research and Ethics. All experiments were conducted in accordance with the institutional ethical guidelines for animal experiments and safety guidelines for gene manipulation experiments.
Isolation of Linkage Region Oligosaccharides from Mouse Growth Plate Cartilage-Growth plate cartilage CSPG was extracted from E18.5 ChGn-1 Ϫ/Ϫ , ChGn-2 Ϫ/Ϫ , and wild-type mouse embryos with 4 M guanidinium chloride and 0.05 M Tris-HCl, pH 8.0 containing proteinase inhibitors as described (15,17). The extract was centrifuged at 15,000 ϫ g for 10 min to remove insoluble material. The protein concentration of each sample was determined using a BCA protein assay kit according to the manufacturer's instructions. The CSPG fractions were precipitated with 70% ethanol containing 5% sodium acetate.
The partially purified CSPG fractions were dissolved in 1 M LiOH and incubated on a rotator at 4°C for 16 h to release the O-linked saccharides from the core proteins (18,19). After neutralization, the sample was applied to an AG 50W-X2 column (2.5-ml bed volume, H ϩ form; Bio-Rad). The flow-through fractions containing the O-linked oligosaccharide components were pooled and neutralized with 10% NH 4 HCO 3 .
Derivatization of the Isolated Oligosaccharide with 2-Aminobenzamide (2AB)-Derivatization of the oligosaccharides with 2AB was performed as described (18,20). The labeled oligosaccharides were analyzed by high performance liquid chromatography (HPLC) on an amine-bound PA-03 column as described previously (3).
Expression of Soluble Forms of ChGn-1, XYLP, FAM20B, or C4ST-2-The expression plasmids (6.0 g) for ChGn-1 (4), XYLP (3), FAM20B (2), or C4ST-2 (10) were individually 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 ChGn-1 and XYLP expression plasmids (3.0 g each) were co-transfected into COS-1 cells on 100-mm plates using FuGENE 6 as above. Two days after transfection, 1 ml of the culture medium was collected and incubated with 10 l of IgG-Sepharose (GE Healthcare) for 12 h at 4°C. The beads were recovered by centrifugation and washed with the assay buffer. The beads were then resuspended in the same buffer and tested for GalNAcT-I, phosphatase, and sulfotransferase activities as described previously (4,5,10,21). To quantify the protein absorbed onto IgG-Sepharose beads, the bound protein was eluted with 1 M acetic acid and quantified using the BCA protein assay reagent (enhanced protocol; Pierce).
In addition, phosphatase reactions were simultaneously incubated in parallel in 20-l reaction mixtures containing 5 pmol of GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl(2-O-[ 32 P]phosphate)␤1-O-TM, 0.25 mM UDP-GalNAc, 50 mM Tris buffer, pH 5.8, 10 mM MnCl 2 , and 10 l of the soluble form of XYLP-or ChGn-1/XYLPbound beads (3) as the enzyme source. Each of these mixtures was incubated at 37°C for 4 h, and the products of each reaction were then separated by gel filtration chromatography on a Superdex peptide column equilibrated with elution buffer (3). Fractions (0.4 ml each) were collected at a flow rate of 0.4 ml/min, and a liquid scintillation counter was used to measure the radioactivity.
Pulldown Assays-Pulldown assays were performed as described previously (3). The His-tagged and protein A-tagged expression vectors (3,4) were co-transfected into COS-1 cells grown on 100-mm plates. FuGENE 6 was used according to the manufacturer's instructions. Two days after transfection, 1 ml of the culture medium was collected and incubated with 10 l of Ni-NTA-agarose (Qiagen) overnight at 4°C. The beads were recovered by centrifugation, washed with TBS buffer contain-ing Tween 20 three times, and subjected to SDS-PAGE (7% gel); the separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated in PBS containing 2% skim milk and 0.1% Tween 20, then incubated with IgG antibody, and then treated with anti-mouse IgG conjugated with horseradish peroxidase (GE Healthcare). An enhanced chemiluminescence (ECL) Select Detection Reagent (GE Healthcare) was used to visualize antibody-labeled protein bands.
Preparation of Embryonic Fibroblasts-Wild-type, ChGn-1 Ϫ/Ϫ , and ChGn-2 Ϫ/Ϫ mouse embryonic fibroblasts (MEFs) were generated from homozygous intercrosses (wild type ϫ wild type, ChGn-1 Ϫ/Ϫ ϫ ChGn-1 Ϫ/Ϫ , and ChGn-2 Ϫ/Ϫ ϫ ChGn-2 Ϫ/Ϫ , respectively). Primary MEFs were harvested from embryonic day 14 embryos. Pregnant female mice were anesthetized using pentobarbital, the uteruses were isolated, and the embryos were extracted and placed into a 10-cm Petri dish. The head, limbs, and liver were then removed, and the embryos were subsequently minced and incubated at 37°C in the presence of 6 ml of 0.05% trypsin and 0.02% EDTA for 20 min in a humidified incubator. Trypsin-treated embryos were homogenized by trituration until a viscous fluid was obtained with only a few tissue clumps remaining. The homogenized embryos were again incubated in the presence of 6 ml of 0.05% trypsin and 0.02% EDTA for 20 min. After the addition of 2 ml of fetal bovine serum, the homogenized embryos were centrifuged at 100 ϫ g for 5 min. Cell pellets were suspended in fresh DMEM (Wako, Osaka, Japan) containing 10% FBS, 100 units/ml penicillin, and 100 g/ml streptomycin, and each cell suspension was then transferred to a 10-cm dish.
Chondrocyte Cultures-Immature chondrocytes were isolated from long bone cartilages of newborn (5-day-old) wildtype and ChGn-1 Ϫ/Ϫ mice as described (23) and maintained in DMEM containing 10% FBS, 100 units/ml penicillin, and 100 g/ml streptomycin. The passage 2 cultures were used for subsequent analyses including gene delivery as described below and cytokine treatment. To induce anabolic processes that are characteristic of chondrocytes, the subconfluent cultures were stimulated with 200 ng/ml recombinant human insulin-like growth factor-1 (IGF-1; R&D Systems) for 48 h. The cell harvests were then utilized either to extract total RNA or to isolate the linkage region oligosaccharides as described above. For assessment of the amounts of CS chains, GAGs from chondrocytes were prepared as described previously (7). The purified GAG fraction containing CS was digested with chondroitinase ABC at 37°C for 2 h. The digests were derivatized with the fluorophore 2AB and then analyzed via anion exchange HPLC as described above. Identification and quantification of the resulting disaccharides were achieved by comparison with authentic unsaturated CS disaccharides (Seikagaku, Tokyo, Japan).
Statistical Analysis-Data are expressed as mean Ϯ S.D. The Student's t test was used to assess statistical significance. Differences were considered to be significant with a p value less than 0.05.

Accumulation of Phosphorylated Pentasaccharide and Tetrasaccharide Linkages in ChGn-1 Ϫ/Ϫ Growth Plate Cartilage-
It was shown previously that a deficiency in ChGn-1 reduced the number of CS chains, leading to skeletal dysplasias in mice (15,24). Although these results indicated that ChGn-1 regulates the number of CS chains by transferring the first GalNAc to the tetrasaccharide in the protein linkage region of CS, ChGn-1 Ϫ/Ϫ mice still produced more than half the amount of CS present in wild-type growth plate cartilage. However, the mechanism underlying this regulation is not clear. To further examine whether linkage region maturation is influenced by ChGn-1, the linkage oligosaccharides obtained from the growth plate cartilage CSPGs of E18.5 ChGn-1 Ϫ/Ϫ mice were compared with those obtained from ChGn-2 Ϫ/Ϫ and wild-type mice. The linkage oligosaccharides in each growth plate cartilage sample were isolated after mild alkaline treatment with LiOH as described previously (3,25). The isolated oligosaccharides were derivatized with the fluorophore 2AB and analyzed using HPLC. Surprisingly, the truncated linkage tetrasaccharide GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl-2AB was detected in all growth plate cartilages examined.
was recently demonstrated to be formed by EXTL2 and considered to be a biosynthetic intermediate of an immature GAG chain (25). In addition, the truncated linkage pentasaccharide GalNAc␤1-4GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl(2-O-phosphate)-2AB was not detected in any of the growth plate cartilage tissues examined.

Regulation of Chondroitin Sulfate Chain Number
strate. As shown in Table 2, the GalNAcT-I activity of ChGn-1 for GlcUA-Gal-Gal-Xyl-(2-O-phosphate)-TM was more than 100-fold higher than for GlcUA-Gal-Gal-Xyl-TM. These results indicate that ChGn-1 preferentially transfers a GalNAc residue to the phosphorylated tetrasaccharide in vitro.
Next, we used pulldown assays to determine whether ChGn-1 and XYLP interact. For this analysis, a soluble protein A-tagged XYLP fusion protein (XYLP-ProA) and soluble His 6tagged ChGn-1 and ChGn-2 fusion proteins (ChGn-1-His and ChGn-2-His, respectively) were generated. In addition, to test the specificity of the interaction, we also performed these assays with ChGn-2. Ni-NTA-agarose was added to the culture medium to pull down the His-tagged proteins, and the proteins were separated by SDS-PAGE and blotted. No band was detected in samples from co-transfectants expressing XYLP-ProA and ChGn-2-His (Fig. 1A). However, a protein band with a molecular mass of ϳ90 kDa, corresponding to the predicted size of XYLP-ProA, was detected in samples from co-transfectants expressing XYLP-ProA and ChGn-1-His (Fig. 1A). These results indicated that XYLP and ChGn-1 interact with each other and that ChGn-1-mediated addition of GalNAc can be accompanied by rapid, XYLP-dependent dephosphorylation during the completion of linkage pentasaccharide formation in CS.
Functional Relevance of ChGn-1-mediated CS Biosynthetic Machinery in the PG Production in Chondrocytes-Among CSPGs, an aggrecan core protein is a major component of the

HexUA-GalNAc(4S)-GlcUA-Gal-Gal-Xyl-2AB
HexUA cartilage extracellular matrix and has more than 100 putative Ser-Gly CS attachment sites, although it is reported that approximately half are not occupied by CS chains (28). The distinct nature of the aggrecan core protein might indicate that the number of CS chains attached to it is tightly associated with the ChGn-1 functions. To further evaluate the biological importance of our present findings, we investigated whether the ChGn-1-mediated CS biosynthetic machinery, most likely including XYLP and C4ST-2, is actually functional in chondrocytes, which are a primary producer of aggrecan CSPG.
Chondrocytes were isolated from long bone cartilages of newborn wild-type and ChGn-1 Ϫ/Ϫ mice. Consistent with the data obtained from MEFs, XYLP was also localized in the Golgi apparatus of chondrocytes in a ChGn-1-independent fashion (Fig. 4A). In both cultures, treatment with an anabolic growth factor, IGF-1, resulted in a significant increase in the expression of cartilaginous markers Col2a1 and Acan, which encode type II collagen and aggrecan core protein, respectively (Fig. 4B).
Interestingly, IGF-1 treatment increased FAM20B expression in wild-type but not in ChGn-1 Ϫ/Ϫ chondrocyte cultures (Fig. 4C). Although the molecular basis for their different responses is currently unknown, such accelerated expression of FAM20B leads to excessive production of the phosphorylated linkage tetrasaccharide that is favorable for subsequent ChGn-1-mediated CS biosynthesis in wild-type chondrocyte cultures. In contrast, despite basal level expression of FAM20B even under the stimulatory condition by IGF-1 (Fig. 4C), a marked accumulation of the phosphorylated forms of the truncated linkage oligosaccharides was detected in ChGn-1 Ϫ/Ϫ chondrocytes. Given that the phosphorylated forms of linkage tetrasaccharide in ChGn-1 Ϫ/Ϫ chondrocytes are generated at a constant rate during CS biosynthesis, the exclusive accumulation of the phosphorylated linkage oligosaccharides could be mainly attributed to a functional uncoupling between ChGn-1 and XYLP.
We recently demonstrated that the non-reducing terminal GalNAc(4-O-sulfate) linkage structure of CS was associated with an increased number of CS chains when the enzyme source was one of several complexes comprising any two of the four ChSy family proteins (21). In addition, C4ST-2 efficiently and selectively transferred sulfate from 3Ј-phosphoadenosine 5Ј-phosphosulfate to position 4 of non-reducing terminal GalNAc linkage residues, and the number of CS chains was regulated by the expression levels of C4ST-2 and of ChGn-1 (21). Therefore, C4ST-2 is thought to play a key role in regulating levels of CS synthesized via ChGn-1. Consistent with these findings, the 4-sulfated hexasaccharide ⌬HexUA-GalNAc(4-O-sulfate)-GlcUA-Gal-Gal-Xyl-2AB was not detected in ChGn-1 Ϫ/Ϫ articular cartilage (Fig. 2). Furthermore, C4ST-2 showed no activity toward GalNAc-GlcUA-Gal-Gal-Xyl(2-Ophosphate)-TM, whereas C4ST-2 transferred sulfate to GalNAc-GlcUA-Gal-Gal-Xyl-TM. These results suggest that addition of the GalNAc residue by ChGn-1 was accompanied by rapid dephosphorylation of the Xyl residue by XYLP, and 4-O-sulfate was subsequently transferred to the GalNAc residue by C4ST-2. Therefore, the number of CS chains on specific core proteins is tightly regulated during cartilage development most likely by temporal and spatial regulation of ChGn-1, C4ST-2, and XYLP expression, and progression of cartilage diseases may result from defects in these regulatory systems.
Here, we propose that CS chains can be formed via three different pathways (Fig. 5). Biosynthesis of CS is initiated by the addition of Xyl to specific serine residues in a core protein. This event is followed by the transfer of Gal residues and transient phosphorylation of Xyl residues by FAM20B (2). Next, GlcAT-I transfers GlcUA from UDP-GlcUA to the phosphorylated trisaccharide structure Gal␤1-3Gal␤1-4Xyl(2-O-phosphate). This final step completes the formation of the linkage region. XYLP can dephosphorylate Xyl(2-O-phosphate) structures during this last step, and, as shown in Fig. 5A, interactions between GlcAT-I and XYLP facilitate these two simultaneous steps (3). Thereafter, CS polymerization onto the linkage region tetrasaccharide can be catalyzed by chondroitin (Chn) poly- merases. If ChGn-1 expression is excessively augmented, resulting in substantial amounts of XYLP being captured by ChGn-1, and free GlcAT-I is increased, biosynthetic intermediates (GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl(2-O-phosphate)) may accumulate (Fig. 5B). Under these conditions, transfer of a GalNAc residue by ChGn-1 occurs on the phosphorylated linkage tetrasaccharide, and CS polymerization by Chn polymerases can occur on the phosphorylated pentasaccharides, although the chain length of Chn formed on the phosphorylated pentasaccharides is short (Fig. 3). Alternatively, the addition of the GalNAc residue (Fig. 5C) by ChGn-1 is accompanied by rapid dephosphorylation by XYLP, and subsequent 4-O-sulfation of the GalNAc residue by C4ST-2 occurs. Then CS polymerization onto the GalNAc(4-O-sulfate) linkage pentasaccharide structure is efficiently catalyzed by Chn polymerases. Therefore, the different numbers, lengths, and structures of CS chains could be synthesized through each pathway with the CS synthesized through the pathway mediated by ChGn-1, XYLP, and C4ST-2 having a specific structure that is indispensable for cartilage development.
More recently, we showed that ChGn-1 Ϫ/Ϫ mice recover more completely from spinal cord injury than wild-type and chondroitinase ABC-treated mice (17). Our results indicate that the deficiency in ChGn-1 mediates excellent recovery from spinal cord injury by optimizing the counteracting effectors of axon regeneration. In addition, we recently reported two missense mutations in the human ChGn-1 gene, both of which were associated with a profound decrease in enzyme activity in two patients with neuropathy (16). As we demonstrated in this study, ChGn-1 cooperates with XYLP and C4ST-2 to regulate the biosynthetic fine-tuning of CS chains that play important roles in various biological/pathological events (31). In view of the modes of action for the ChGn-1-mediated biosynthetic machinery, its targets may include not only developing cartilage and glial scar formation after central nervous system injury (32,33) but also inflammation processes in which excessive CSPG production often occurs (34 -36). Further studies with a focus on the regulatory mechanisms involving activation of the individual enzymes are required for the clarification of generality and utility of the ChGn-1-mediated biosynthetic pathway(s). In conclusion, this study proposes a novel strategy for the treatment of degenerative cartilage disorders and recovery from spinal cord injury and minor trauma.