Molecular Cloning and Expression of a Second Chondroitin N-Acetylgalactosaminyltransferase Involved in the Initiation and Elongation of Chondroitin/Dermatan Sulfate*

Toru UyamaDagger , Hiroshi KitagawaDagger , Junko Tanaka§, Jun-ichi Tamura§, Tomoya Ogawa, and Kazuyuki SugaharaDagger ||

From the Dagger  Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan, the § Department of Environmental Sciences, Faculty of Education and Regional Sciences, Tottori University, Tottori 680-8551, Japan, and  RIKEN (The Institute of Physical and Chemical Research), Wako-shi, Saitama 351-0198, Japan

Received for publication, September 16, 2002, and in revised form, November 7, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We identified a novel human chondroitin N-acetylgalactosaminyltransferase, designated chondroitin GalNAcT-2 after a BLAST analysis of the GenBankTM data base using the sequence of a previously described human chondroitin N-acetylgalactosaminyltransferase (chondroitin GalNAcT-1) as a probe. The new cDNA sequence contained an open reading frame encoding a protein of 542 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 60% identity to that of human chondroitin GalNAcT-1. Like chondroitin GalNAcT-1, the expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which not only transferred beta 1,4-N-acetylgalactosamine (GalNAc) from UDP-[3H]GalNAc to a polymer chondroitin representing growing chondroitin chains (beta -GalNAc transferase II activity) but also to GlcUAbeta 1-3Galbeta 1-O-C2H4NHCbz, a synthetic substrate for beta -GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein-linkage region of chondroitin sulfate. In contrast, the tetrasaccharide serine (GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser) derived from the linkage region, which is an inert acceptor substrate for chondroitin GalNAcT-1, served as an acceptor substrate. The coding region of this enzyme was divided into seven discrete exons, which is similar to the genomic organization of the chondroitin GalNAcT-1 gene, and was localized to chromosome 10q11.22. Northern blot analysis revealed that the chondroitin GalNAcT-2 gene exhibited a ubiquitous but differing expression in human tissues, and the expression pattern differed from that of chondroitin GalNAcT-1. Thus, we demonstrated redundancy in the chondroitin GalNAc transferases involved in the biosynthetic initiation and elongation of chondroitin sulfate, which is important for understanding the biosynthetic mechanisms leading to the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate and heparin/heparan sulfate chains.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chondroitin sulfate is one of the sulfated glycosaminoglycan (GAG)1 chains that are covalently attached to various core proteins as proteoglycans. Most if not all vertebrate cells produce chondroitin sulfate proteoglycans to a variable extent as major components of the connective tissue matrix, and they are also found at the surface of many cell types. GAGs play fundamental roles in growth factor signaling, cellular differentiation, morphogenesis, and pathophysiology (for reviews see Refs. 1-3). Recent studies of chondroitin/dermatan sulfate chains have shown that they play important roles in neural network formation in the developing mammalian brain (for reviews see Refs. 4 and 5) and are major inhibitory molecules affecting axon growth after spinal cord injury in the central nervous system of adult mammals (6). Although GAGs are ubiquitously expressed, they exist in discrete structural forms generated by varying patterns of sulfation and epimerization, which may account for the functional specificity of tissue-derived proteoglycans.

Both chondroitin/dermatan sulfate and heparin/heparan sulfate are produced on the so-called GAG protein linkage region (GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O attached to specific Ser residues of core proteins) common to GAGs (for reviews, see Refs. 7 and 8). Synthesis of this region is initiated by the addition of Xyl to Ser, followed by the addition of two Gal residues, and completed by the addition of GlcUA; each reaction is catalyzed by a specific glycosyltransferase (7, 8). The GAGs are built up on this linkage region tetrasaccharide by the alternate addition of N-acetylhexosamine and GlcUA residues. Chondroitin/dermatan sulfate is synthesized once GalNAc is transferred to the common linkage region, whereas heparin/heparan sulfate is formed if GlcNAc is added first. Therefore, the first hexosamine transfer is critical in determining whether chondroitin/dermatan sulfate or heparin/heparan sulfate chains are selectively assembled on the common linkage region. Although such mechanisms have long been proposed based on data from conventional structural and enzymological studies (8), the molecular mechanisms underlying the selective chain assembly of different GAG chains have not been clarified.

Recent cDNA cloning of GAG glycosyltransferases has led to unexpected developments (for reviews see Refs. 9-11), providing several important clues to help determine the molecular mechanisms of the biosynthetic sorting of chondroitin/dermatan sulfate and heparin/heparan sulfate chains, as well as the mechanisms of chain elongation and polymerization of these GAGs. The glycosyltransferases responsible for heparin/heparan sulfate biosynthesis are encoded by the EXT gene family, the hereditary multiple exostoses gene family of tumor suppressors (9, 10). The EXT gene family has five members. EXT1 and EXT2 encode heparan sulfate copolymerases, which catalyze the alternate addition of GlcNAc and GlcUA residues (12-14), whereas EXTL1, EXTL2, and EXTL3, called EXT-like genes, are highly homologous to EXT1 and EXT2 (9, 10). The human EXTL1 protein is a GlcNAc transferase II involved in elongation of the heparan sulfate chain (15); the human EXTL2 protein is a GlcNAc transferase I (16) that determines and initiates the synthesis of heparan sulfate on the common GAG-protein linkage region (17); and the human EXTL3 protein exhibits both GlcNAc transferase I and II activities (15).

Three homologous glycosyltransferases responsible for chondroitin/dermatan sulfate biosynthesis have been cloned (18-21). The first chondroitin glycosyltransferase cloned was chondroitin synthase consisting of a single large polypeptide with dual glycosyltransferase activities of GlcUA transferase II (GlcAT-II) and GalNAc transferase II (GalNAcT-II) that is responsible for synthesizing the repeating disaccharide units of chondroitin sulfate (18). Chondroitin GalNAcT-1, the second chondroitin glycosyltransferase cloned, exhibits GalNAcT-II activity for chain elongation and GalNAc transferase I (GalNAcT-I) activity that determines and initiates the synthesis of chondroitin sulfate on the common GAG-protein linkage region (19, 20, 22). Chondroitin GlcUA transferase, the third chondroitin glycosyltransferase cloned, has only GlcAT-II activity, which has been proposed to be involved in chain elongation (21). Therefore, more than three enzymes are likely responsible for chondroitin/dermatan sulfate biosynthesis, and these likely form a gene family as in the case of heparin/heparan sulfate biosynthesis. To search for additional members of the glycosyltransferase gene family involved in chondroitin/dermatan sulfate biosynthesis, the chondroitin GalNAcT-1 protein sequence was used to screen a data base. Here, we describe the cloning of a human cDNA encoding a novel chondroitin GalNAc transferase, designated chondroitin GalNAcT-2, that is similar to but distinct from chondroitin GalNAcT-1.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- UDP-[U-14C]GlcUA (285.2 mCi/mmol) and UDP-[3H]GalNAc (10 Ci/mmol) were purchased from PerkinElmer Life Sciences. Unlabeled UDP-GlcUA and UDP-GalNAc were obtained from Sigma. Chondroitin (a chemically desulfated derivative of whale cartilage chondroitin sulfate A), Acremonium sp. alpha -N-acetylgalactosaminidase (EC 3.2.1.49), and Arthrobacter aurescens chondroitinase AC-II (EC 4.2.2.5) were purchased from Seikagaku Kogyo (Tokyo, Japan). Purified alpha -thrombomodulin (23) was provided by the research institute of Dai-ichi Pure Chemicals (Tokyo, Japan) and contained the linkage tetrasaccharide GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xyl (24). The chemically synthesized linkage tetrasaccharide serines GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser, GlcUAbeta 1-3Gal(4-O-sulfate)beta 1-3Galbeta 1-4Xylbeta 1-O-Ser, GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-(Gly)Ser-(Gly-Glu), and GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser-(Gly-Trp-Pro-Asp-Gly) were chemically synthesized (25, 26). GlcUAbeta 1-3Galbeta 1-O-C2H4NHCbz was also synthesized.2 Chondro-hexasaccharide (GlcUAbeta 1-3GalNAc)3 was prepared from chondroitin as previously described (27). The SuperdexTM peptide HR10/30 column was obtained from Amersham Biosciences.

In Silico Cloning of the Novel Chondroitin GalNAcT cDNA-- A tBLASTn analysis of the GenBankTM data base using the sequence of human chondroitin GalNAcT-1 (19), revealed highly homologous clones. Analysis of one clone (GenBankTM accession number BC030268) revealed a single open reading frame with significant sequence similarity to human chondroitin GalNAcT-1. In addition, a data base search of the Human Genome Project, which recently became available, identified a genome sequence (GenBankTM accession number NT033985.2) identical to the cDNA sequence. Comparison of the cDNA and genome sequences revealed the genomic organization of the novel chondroitin GalNAcT gene.

Construction of a Soluble Form of the Novel Chondroitin GalNAcT-- A cDNA fragment of a truncated form of the novel chondroitin GalNAcT lacking the first 36 N-terminal amino acids was amplified by reverse transcription PCR with total RNA derived from G361 human melanoma cells (ATCC CRL-1424) as a template using a 5'-primer (5'-CGCGGATCCTTGTTAGGCAAATACACATTAATAAG-3') containing an in-frame BamHI site and a 3'-primer (5'-CGCGGATCCGTTTTGTGGTTCATACAGTAACGC-3') containing a BamHI site located 37 bp downstream from the stop codon. PCR was carried out with Pfu polymerase (Stratagene, La Jolla, CA) for 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 120 s in 5% (v/v) dimethyl sulfoxide. The PCR fragments were subcloned into the BamHI site of pGIR201protA (28), resulting in the fusion of the putative GalNAcT with the insulin signal sequence and the protein A sequence present in the vector. An NheI fragment containing this fusion protein sequence was inserted into the XbaI site of the expression vector pEF-BOS (29). The nucleotide sequence of the amplified cDNA was determined in a 377 DNA sequencer (PE Applied Biosystems).

Expression of a Soluble Form of the Novel Chondroitin GalNAcT and Enzyme Assays-- The expression plasmid (6.7 µg) was transfected into COS-1 cells on 100-mm plates using FuGENETM 6 (Roche Molecular Biochemicals) according to the manufacturer's instructions. Two days after transfection, 1 ml of the culture medium was collected and incubated with 10 µl of IgG-Sepharose (Amersham Biosciences) for 1 h at 4 °C. The beads recovered by centrifugation were washed with, and then resuspended in, the assay buffer and tested for GalNAcT and GlcUA transferase activities as described below. The acceptors used for GalNAcT included polymer chondroitin (167 µg), chondro-hexasaccharide (GlcUAbeta 1-3GalNAc)3 (10 nmol), alpha -thrombomodulin (1 nmol), GlcUAbeta 1-3Galbeta 1-O-C2H4NHCbz (1 or 100 nmol), GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser (1 nmol), GlcUAbeta 1-3Gal(4-O-sulfate)beta 1-3Galbeta 1- 4Xylbeta 1-O-Ser (1 nmol), GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-(Gly)Ser-(Gly-Glu) (1 nmol), and GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser-(Gly-Trp-Pro-Asp-Gly) (1 nmol), whereas that for GlcUA transferase was polymer chondroitin (167 µg). The assay mixture for GalNAcT contained 10 µl of the resuspended beads, an acceptor substrate, 8.57 µM UDP-[3H]GalNAc (3.60 × 105 dpm), 50 mM MES buffer, pH 6.5, 10 mM MnCl2, and 171 µM sodium salt of ATP in a total volume of 30 µl (30, 31). The assay mixture for GlcAT-II contained 10 µl of the resuspended beads, 167 µg of polymer chondroitin, 14.3 µM UDP-[14C]GlcUA (1.46 × 105 dpm), 50 mM sodium acetate buffer, pH 5.6, and 10 mM MnCl2 in a total volume of 30 µl (32). The reaction mixtures were incubated at 37 °C for 4 h, and then the radiolabeled products were separated from UDP-[3H]GalNAc or UDP-[14C]GlcUA by gel filtration using a syringe column packed with Sephadex G-25 (superfine), or a Superdex peptide column, or by HPLC on a Nova-Pak® C18 column (3.9 × 150 mm, Waters, Tokyo, Japan) as described previously (16, 30-33). The recovered labeled products were quantified by liquid scintillation spectrophotometry.

Identification of the Enzyme Reaction Products-- The products of the GalNAcT reaction using polymer chondroitin as the acceptor were isolated by gel filtration on a Superdex peptide column equilibrated with 0.25 M NH4HCO3, 7% 1-propanol. The radioactive fractions containing the enzyme reaction product were pooled and evaporated to dryness. The isolated GalNAcT reaction product (about 150 µg) was digested with 100 mIU of chondroitinase AC-II to assess digestibility in a total volume of 30 µl of 50 mM sodium acetate buffer, pH 6.0, at 37 °C overnight. The enzyme digest was analyzed using the Superdex peptide column described above.

The products from the GalNAcT reaction using GlcUAbeta 1-3Galbeta 1-O-C2H4NHCbz were isolated by HPLC on a Nova-Pak® C18 column (described above) in an LC-10A system (Shimadzu, Kyoto, Japan). The column was developed isocratically for 15 min with H2O at a flow rate of 1.0 ml/min at room temperature; then, 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 for 40 min with 100% methanol. The radioactive fractions containing the product were pooled and evaporated to dryness. The isolated product (about 80 pmol) was incubated with 100 mIU of chondroitinase AC-II to assess the digestibility in a total volume of 30 µl of 50 mM sodium acetate buffer, pH 6.0, at 37 °C overnight or 40 mIU of alpha -N-acetylgalactosaminidase in a total volume of 20 µl of 50 mM sodium citrate buffer, pH 4.5, at 37 °C overnight (16). The enzyme digest was analyzed using the Nova-Pak® C18 column described above.

Northern Blot Analysis-- A commercial human 12-lane multiple tissue Northern blot (Clontech) membrane was used for the analysis. The membrane was probed with a gel-purified, radiolabeled (>1 × 109 cpm/µg), 1.0-kb chondroitin GalNAcT-2-specific fragment corresponding to nucleotides 109-1123 of the chondroitin GalNAcT-2 cDNA (GenBankTM accession number AB090811).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In Silico Cloning of a Human cDNA Homologous to Chondroitin GalNAcT-1-- We recently identified and characterized human chondroitin GalNAcT-1 (19). Screening of the nonredundant data base at the National Center for Biotechnology Information (National Institutes of Health, Bethesda, MD) using the deduced amino acid sequence of human chondroitin GalNAcT-1 identified a clone (GenBankTM accession number BC030268) containing a 335-bp 5'-untranslated region, a single open reading frame of 1626 bp encoding a protein of 542 amino acids with two potential N-glycosylation sites (Fig. 1), and a 3'-untranslated region of approximately 1.8 kb with one presumptive polyadenylation signal. Northern blot analysis indicated that the mRNA was about 3.9 kb long in various human tissues (see below), suggesting that the cDNA was approximately full-length. The deduced amino acid sequence corresponded to a 62,571-Da polypeptide. The predicted translation initiation site conformed to the Kozak consensus sequence for initiation (34), and an in-frame stop codon was present upstream from the assigned ATG initiating codon. A Kyte-Doolittle hydropathy analysis (35) revealed one prominent hydrophobic segment of 19 amino acid residues in the N-terminal region, predicting that the protein has the type II transmembrane topology characteristic of many of the Golgi-localized glycosyltransferases cloned to date (Fig. 1). Data base searches indicated that the amino acid sequence displayed 60% identity to human chondroitin GalNAcT-1 (GenBankTM accession number AB071403) (Fig. 1); the highest sequence identity was found in the C-terminal domain, which has catalytic activity. Notably, each protein shared a conserved DVD motif (Fig. 1), which appears to correspond to the conserved DXD motif found in most glycosyltransferases (36). Therefore, the features of the identified protein sequence suggest that the identified gene product possesses beta 1,4-GalNAc transferase (GalNAcT-I and/or -II) activities like chondroitin GalNAcT-1. Intriguingly, a homologue of the identified human gene was found in the Drosophila genome but not in the Caenorhabditis elegans genome. The human sequence shared 38% identity with that of Drosophila (Fig. 1).


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Fig. 1.   Comparison of the predicted amino acid sequence of the novel human glycosyltransferase (chondroitin GalNAcT-2), human chondroitin GalNAcT-1 (GenBankTM accession number AB071403), and the putative chondroitin GalNAcT in Drosophila (GenBankTM accession number CG12913). The predicted amino acid sequences were aligned using the program GENETYX-MAC (version 10). Closed and shaded boxes indicate that the predicted amino acid in the alignment is identical in all three or any two sequences, respectively. Gaps introduced for maximal alignment are indicated by dashes. The putative membrane spanning domains are boxed. The conserved DXD motif is indicated by underlines. Two potential N-glycosylation sites for the human putative glycosyltransferase (chondroitin GalNAcT-2) are marked with asterisks.

Genome Organization and Chromosome Localization-- Comparison of the identified cDNA sequence with the genome sequence deposited in the Human Genome Project data base revealed the genomic structure and chromosome localization of the gene. The gene spans more than 47 kb, and the coding region of the gene is divided into seven discrete exons as shown in Fig. 2. Its genomic organization is very similar to that of the chondroitin GalNAcT-1 gene, which consists of seven discrete exons in the coding region. The intron/exon junctions follow the G(T/A)G rule (37) and are flanked by conserved sequences (data not shown). This gene is located on human chromosome 10q11.22.


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Fig. 2.   Comparison of the genomic organization of the novel human glycosyltransferase (chondroitin GalNAcT-2) and human chondroitin GalNAcT-1 genes. Exon regions are denoted by boxes. Closed boxes represent the coding sequence, and open boxes denote the 5'- and 3'-untranslated sequences. The translation initiation (ATG) and termination (TGA) codons are also shown. Black horizontal bars denote the introns.

Expression of a Soluble Form of the Novel Glycosyltransferase and Its Characterization as Chondroitin GalNAcT-2-- To facilitate the functional analysis of the putative GalNAcT, a soluble form of the protein was generated by replacing the first 36 amino acids of the putative glycosyltransferase with a cleavable insulin signal sequence and the protein A IgG-binding domain, as described under "Experimental Procedures." Then, the soluble putative glycosyltransferase was expressed in COS-1 cells as a recombinant enzyme fused with the protein A IgG-binding domain. When the expression plasmid containing the putative glycosyltransferase/protein A fusion construct was expressed in COS-1 cells, an ~95-kDa protein was secreted as shown by Western blotting using IgG (data not shown). The apparent Mr of the fused protein was reduced to about 87 kDa after N-glycosidase treatment (data not shown), suggesting that either one or both of the two potential N-linked glycosylation sites of the putative glycosyltransferase was utilized. The fused enzyme expressed in the medium was adsorbed onto IgG-Sepharose beads to eliminate endogenous glycosyltransferases, and then the enzyme-bound beads were used as an enzyme source. The bound fusion protein was assayed for glycosyltransferase activity using a variety of acceptors and either UDP-GalNAc or UDP-GlcUA as a donor substrate. GlcUAbeta 1-3Galbeta 1-O-C2H4NHCbz, which was used as an acceptor substrate for the GalNAcT-I reaction, shares the disaccharide sequence with the GAG protein linkage region tetrasaccharide. As shown in Table I, marked GalNAc transferase activity was detected with polymer chondroitin, chondro-hexasaccharide (GlcUAbeta 1-3GalNAc)3, alpha -thrombomodulin containing a linkage region tetrasaccharide, GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1 on the native core protein (24), GlcUAbeta 1-3Galbeta 1-O-C2H4NHCbz, the tetrasaccharide serine, GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser, and the two tetrasaccharide peptides, GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-(Gly)Ser-(Gly-Glu) and GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser-(Gly-Trp-Pro-Asp-Gly), but not with the sulfated tetrasaccharide serine GlcUAbeta 1-3Gal(4-O-sulfate)beta 1-3Galbeta 1-4Xylbeta 1-O-Ser, as acceptor substrates. In contrast, no GlcUA transferase activity was observed using polymer chondroitin. No detectable glycosyltransferase activity was recovered by affinity purification from a control pEF-BOS transfection sample. These findings clearly indicate that the expressed protein is a GalNAcT responsible for chondroitin biosynthesis.

                              
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Table I
Comparison of the acceptor specificity of chondroitin GalNAcT-1 and -2 secreted into culture medium by transfected COS-1 cells

To identify the GalNAcT reaction products, representative acceptor substrates, polymer chondroitin and GlcUAbeta 1-3Galbeta 1-O-C2H4NHCbz, were individually labeled via the respective transferase reaction using UDP-[3H]GalNAc as a donor substrate and the enzyme-bound beads as an enzyme source. Both labeled products were completely digested by chondroitinase AC-II, which cleaves beta 1,4-N-acetylgalactosaminidic linkages in an eliminative fashion, quantitatively yielding a 3H-labeled peak at the position of free [3H]GalNAc as demonstrated by gel filtration (Fig. 3A) or hydrophobic HPLC (Fig. 3B). In contrast, both products were inert with respect to alpha -N-acetylgalactosaminidase, which is in marked contrast to the product of the alpha 1,4-GalNAc transferase reaction catalyzed by EXTL2 (16). These findings indicate that a GalNAc residue was indeed transferred to the nonreducing terminal GlcUA of the polymer chondroitin or GlcUAbeta 1-3Galbeta 1-O-C2H4NHCbz through a beta 1-4 linkage. Combined, these results indicate that the identified protein is a novel beta 1,4-GalNAc transferase I/II involved in the initiation and elongation of chondroitin/dermatan sulfate.


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Fig. 3.   Identification of the reaction products of a novel human glycosyltransferase. A, 3H-labeled GalNAcT-II reaction products obtained using polymer chondroitin as the acceptor substrate were digested with chondroitinase AC-II as described under "Experimental Procedures." The chondroitinase AC-II digest (solid circles) or the undigested sample (solid squares) was applied to a Superdex peptide column (1.0 × 30 cm), and the radioactivity in the effluent fractions (0.4 ml each) was analyzed as described under "Experimental Procedures." The arrowhead indicates the elution position of free GalNAc. B, 3H-labeled GalNAcT-I reaction products obtained using GlcUAbeta 1-3Galbeta 1-O-C2H4NHCbz as the acceptor substrate digested with chondroitinase AC-II or alpha -N-acetylgalactosaminidase as described under "Experimental Procedures." The chondroitinase AC-II digest (solid circles), alpha -N-acetylgalactosaminidase digest (solid squares), and undigested sample (solid triangles) were analyzed by HPLC on a Nova-Pak® C18 column as described under "Experimental Procedures," and the radioactivity in the effluent fractions (2 ml each) was analyzed. The arrowhead indicates the elution position of free GalNAc.

Pattern of Chondroitin GalNAcT-2 Expression-- Northern blot mRNA analysis demonstrated a single ~3.9-kb band in all human tissues examined (Fig. 4). The gene exhibited a ubiquitous but differing expression in all the human tissues examined. Notably, the expression pattern differed from those of chondroitin GalNAcT-1 and chondroitin synthase (Fig. 4). The strongest signals were seen in the skeletal muscle, lung, and peripheral blood leukocytes. These findings are in accord with the observations that chondroitin/dermatan sulfate proteoglycans are distributed on the surfaces of most cells and in the extracellular matrices of virtually every tissue.


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Fig. 4.   Northern blot analysis of chondroitin GalNAcT-2, chondroitin GalNAcT-1, and chondroitin synthase in human tissues. Northern blots with RNA from various human tissues were hybridized with probes for chondroitin GalNAcT-2 (upper panel), chondroitin GalNAcT-1 (middle panel), or chondroitin synthase (lower panel), as described under "Experimental Procedures." Lane 1, brain; lane 2, heart; lane 3, skeletal muscle; lane 4, colon; lane 5, thymus; lane 6, spleen; lane 7, kidney; lane 8, liver; lane 9, small intestine; lane 10, placenta; lane 11, lung; lane 12, peripheral blood leukocytes.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study we identified a second human chondroitin GalNAcT, chondroitin GalNAcT-2, which is homologous to but distinct from the previously cloned chondroitin GalNAcT-1. Chondroitin GalNAcT-2 is the fourth chondroitin-synthesizing glycosyltransferase cloned to date. As summarized in Table II, the four glycosyltransferases exhibit distinct but overlapping acceptor substrate specificities, except for the two chondroitin GalNAcTs that harbor both GalNAcT-I and -II activities. Therefore, at least four distinct enzymes forming this novel gene family are responsible for initiation and elongation of chondroitin/dermatan sulfate chains. The situation is similar to that of the heparan sulfate glycosyltransferases in which five homologous EXT family members with distinct but overlapping acceptor specificity have been identified (14-16). It is still possible that other glycosyltransferases are also involved in chondroitin/dermatan sulfate biosynthesis.

                              
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Table II
Summary of the human glycosyltransferases involved in chondroitin sulfate biosynthesis

Our findings demonstrated that chondroitin GalNAcT-2, like chondroitin GalNAcT-1, possesses both GalNAcT-I and -II activities responsible for chain initiation and elongation in chondroitin/dermatan sulfate. These results are reminiscent of the heparan sulfate GlcNAc transferase encoded by human EXTL3, Drosophila DEXT3, and C. elegans rib-2, which have both GlcNAc transferase-I and -II activities responsible for chain initiation and elongation in heparin/heparan sulfate (15, 38, 39). Therefore, there are at least two types of enzymes involved in the synthesis of both chondroitin/dermatan sulfate and heparin/heparan sulfate, one for both chain initiation and elongation (chondroitin GalNAcT-1 and -2 and heparan sulfate GlcNAc transferase EXTL3, respectively) and the other for chain polymerization via the alternate transfer of GlcUA and GalNAc or GlcNAc (chondroitin synthase and heparan sulfate copolymerases EXT1 and EXT2, respectively). Intriguingly, these combined findings suggest that the assembly of the polysaccharide backbones of chondroitin sulfate and heparan sulfate shares a similar biosynthetic mechanism irrespective of the involvement of the two distinct glycosyltransferase gene families.

As shown in Table I, the two recombinant chondroitin GalNAcTs exhibited both GalNAcT-I and -II activities, and polymer chondroitin was the best substrate for both enzymes. However, chondroitin GalNAcT-2 showed broader acceptor specificity than chondroitin GalNAcT-1, as the former utilized all of the acceptor substrates tested that contained terminal beta 1,3-linked GlcUA residues except for the sulfated tetrasaccharide serine, GlcUAbeta 1-3Gal(4-O-sulfate)beta 1-3Galbeta 1-4Xylbeta 1-O-Ser (Table I). Notably, no such chondroitin GalNAcT has been reported. Chondroitin GalNAcT-2 is a unique beta 1,4-GalNAc transferase that utilizes the tetrasaccharide serine, GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser, from the linkage region as an acceptor substrate. Previously, when this compound was tested as an acceptor together with a sugar donor, UDP-GalNAc, to search for such GalNAcTs using fetal bovine sera or mouse mastocytoma cell extracts as the enzyme source, only alpha -GalNAc transferase activity was detected, resulting in the exclusive production of an alpha -GalNAc-capped pentasaccharide, GalNAcalpha 1-4GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser (27, 40, 41). Therefore, high expression of alpha -GalNAc transferase might have interfered with the identification of a beta 1,4-GalNAc transferase, such as chondroitin GalNAcT-2.

Chondroitin GalNAcT-2, like chondroitin GalNAcT-1, transfers the first beta 1,4-GalNAc residue to initiate chondroitin/dermatan sulfate chains on the common linkage region by segregating chondroitin/dermatan sulfate synthesis from heparin/heparan sulfate synthesis. One explanation for the existence of two distinct GalNAcT-I molecular species that share enzyme activity critical for the differential assembly of chondroitin and heparan chains is that they initiate chondroitin/dermatan sulfate chains on different core proteins by discriminating the amino acid sequences. The notion of the importance of amino acid sequences in heparin/heparan sulfate biosynthesis was well documented by Esko and Zhang (42) based on the specificity of GlcNAcT-I in Chinese hamster ovary cells. Artificial beta -D-xylosides with lipophilic aglycons containing two fused aromatic rings, such as estradiol and naphthalene, efficiently initiate heparin/heparan sulfate biosynthesis, whereas chondroitin/dermatan sulfate chains are initiated on less lipophilic xylosides (43, 44). Consistent with this concept were our observations that chondroitin GalNAcT-1 and -2 differed in how they transferred a GalNAc residue to an alpha -thrombomodulin proteoglycan bearing the truncated linkage region tetrasaccharide (GlcUAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1) (24), chemically synthesized disaccharides GlcUAbeta 1-3Galbeta 1-O-C2H4NHCbz, and the tetrasaccharide-peptides GlcAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser-(Gly-Trp-Pro-Asp-Gly) derived from the chondroitin sulfate/heparan sulfate attachment site of betaglycan (26, 45) and GlcAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-(Gly)Ser-(Gly-Glu) derived from the chondroitin sulfate attachment site of alpha -thrombomodulin (23, 25). The aglycone part of GlcUAbeta 1-3Galbeta 1-O-C2H4NHCbz probably mimics the required peptide sequence as observed for GlcNAc transferase I reactions catalyzed by EXTL2 and EXTL3, both of which also utilize GlcUAbeta 1-3Galbeta 1-O-C2H4NHCbz as an acceptor for heparan synthesis (15, 16). Interestingly, the chondroitin GalNAcT-2 activity was inhibited when the sulfated tetrasaccharide serine was used as an acceptor (Table I). The 4-O-sulfated Gal residue has been identified in the linkage region of chondroitin/dermatan sulfate but not in that of heparin/heparan sulfate (46, 47). Hence, it is hypothesized that it is recognized by a chondroitin GalNAcT specific for the linkage region and might be a molecular signal that promotes chondroitin chain synthesis segregating it from heparan chain (9). However, the negative results obtained in this study do not appear to support this possibility, although the function of the 4-O-sulfated Gal residue remains unclear.

In addition, the respective roles of the GalNAcT-II activity of chondroitin GalNAcT-1 and -2 in the chain polymerization of chondroitin/dermatan sulfate remain to be clarified, especially where it requires GlcAT-II as a partner. It is not known whether chondroitin synthase or chondroitin GlcUA transferase cooperates with chondroitin GalNAcT-1 and/or -2 to synthesize the repeating disaccharide region or in chain polymerization, or whether an as yet unidentified GlcAT-II with no homology to the known genes exists in addition to chondroitin synthase and chondroitin GlcUA transferase. A multimeric complex might also exist. In this context, no chondroitin chain polymerizing enzyme activity has been demonstrated in vitro for chondroitin synthase (18). If either chondroitin GalNAcT-1 or -2 is associated with chondroitin synthase, chondroitin/dermatan sulfate chains might possibly be initiated and polymerized efficiently on the tetrasaccharide core. However, such hetero-oligomeric complex formation has not been reported and is the subject of a future study.

The chondroitin GalNAcT-1 and -2 genes exhibit unique tissue-specific patterns of expression. Particularly striking is the abundant expression of the chondroitin GalNAcT-2 gene in the skeletal muscle, lung, and peripheral blood leukocytes, with modest expression in the brain, thymus, and placenta (Fig. 4). In contrast, chondroitin GalNAcT-1 is abundantly expressed in the placenta, followed by the heart and small intestine. Very little is expressed in the colon and thymus (19), similar to the pattern of chondroitin synthase expression (18). Therefore, chondroitin GalNAcT-2 may play a unique role in the biosynthesis of chondroitin sulfate in certain tissues in view of the different specificities (Table I) and distinct tissue expression of chondroitin GalNAcT-1 and -2 (Fig. 4). The production and analysis of chondroitin GalNAcT-1 or -2 knock-out mice would provide further insights into the possible distinct functions of these genes.

The genomic organization of the four cloned chondroitin glycosyltransferases involved in chondroitin backbone formation has been elucidated. The protein-coding sequences of the human chondroitin GalNAcT-1 and -2 genes are distributed among seven exons that span ~120 (19) and 30 kb (this study), respectively. Comparison of the genomic organizations of these two genes shows a quite similar genetic exon-intron organization within the coding sequences (Fig. 2). In contrast, the genomic organizations of the human chondroitin synthase and chondroitin GlcUA transferase genes are simpler; their protein-coding sequences are divided into 3 and 4 exons that span ~76 and 6 kb, respectively (18, 21). Interestingly, chromosomal assignments of the four human chondroitin glycosyltransferases (chondroitin synthase, chondroitin GalNAcT-1 and -2, and chondroitin GlcUA transferase) indicate that these genes are localized on different chromosomes, 15q26.3, 8p21.3, 10q11.22, and 7q35 (Table II), respectively, despite the significant homology in the nucleotide and amino acid sequences of the four genes. These findings suggest that the four chondroitin glycosyltransferases diverged from an ancestor gene early in evolution.

As described previously (18, 19), a homologue of human chondroitin synthase is present in C. elegans and Drosophila, which is consistent with the finding of chondroitin and chondroitin 4-sulfate in C. elegans and Drosophila, respectively (48-50). Notably, however, a homologue of human chondroitin GalNAcT-1 or -2 is absent from C. elegans but is present in "higher" species, such as Drosophila (Fig. 1). It is possible that the biosynthetic mechanism for chondroitin sulfate in C. elegans is similar to but different from that in mammals, although the conventional linkage region tetrasaccharide sequence has recently been identified not only in Drosophila but also in C. elegans (51).

The mechanism for the selective chain assembly of chondroitin/dermatan sulfate and heparin/heparan sulfate on the common linkage region tetrasaccharide has been an enigma since the discovery of the common linkage region tetrasaccharide in the mid-1960s (7, 9). cDNA probes for enzymes harboring GalNAcT-I or GlcNAc transferase I activity (15, 16, 19) are now available for investigating the biological functions of chondroitin/dermatan sulfate as well as the mechanism of selective chain assembly of chondroitin/dermatan sulfate and heparin/heparan sulfate.

    ACKNOWLEDGEMENT

We thank Y. Kato for technical assistance.

    FOOTNOTES

* This work was supported in part by the Science Research Promotion Fund of the Japan Private School Promotion Foundation and Grants-in-aid for Scientific Research C-14572086 (to H. K.) and for Scientific Research on Priority Areas 14082207 (to K. S.) from the Ministry of Education, Science, Culture, and Sports of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB090811.

|| To whom correspondence should be addressed: Dept. of Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan. Tel.: 81-78-441-7570; Fax: 81-78-441-7571; E-mail: k-sugar@kobepharma-u.ac.jp.

Published, JBC Papers in Press, November 13, 2002, DOI 10.1074/jbc.M209446200

2 J. Tamura, unpublished results.

    ABBREVIATIONS

The abbreviations used are: GAG, glycosaminoglycan; bp, base pair(s); Cbz, benzyloxycarbonyl; EXT, hereditary multiple exostoses gene; GalNAc, N-acetyl-D-galactosamine; GalNAcT, N-acetylgalactosaminyltransferase; GlcNAc, N-acetyl-D-glucosamine; GlcAT, beta 1,3-glucuronyltransferase; GlcUA, D-glucuronic acid; HPLC, high-performance liquid chromatography; MES, 2-(N-morpholino)ethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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