The Tumor Suppressor EXT-like Gene EXTL2 Encodes an alpha 1, 4-N-Acetylhexosaminyltransferase That Transfers N-Acetylgalactosamine and N-Acetylglucosamine to the Common Glycosaminoglycan-Protein Linkage Region
THE KEY ENZYME FOR THE CHAIN INITIATION OF HEPARAN SULFATE*

Hiroshi Kitagawa, Hiromi Shimakawa, and Kazuyuki SugaharaDagger

From the Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We previously demonstrated a unique alpha -N-acetylgalactosaminyltransferase that transferred N-acetylgalactosamine (GalNAc) to the tetrasaccharide-serine, GlcAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser (GlcA represents glucuronic acid), derived from the common glycosaminoglycan-protein linkage region, through an alpha 1,4-linkage. In this study, we purified the enzyme from the serum-free culture medium of a human sarcoma cell line. Peptide sequence analysis of the purified enzyme revealed 100% identity to the multiple exostoses-like gene EXTL2/EXTR2, a member of the hereditary multiple exostoses (EXT) gene family of tumor suppressors. The expression of a soluble recombinant form of the protein produced an active enzyme, which transferred alpha -GalNAc from UDP-[3H]GalNAc to various acceptor substrates including GlcAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser. Interestingly, the enzyme also catalyzed the transfer of N-acetylglucosamine (GlcNAc) from UDP-[3H]GlcNAc to GlcAbeta 1-3Galbeta 1-O-naphthalenemethanol, which was the acceptor substrate for the previously described GlcNAc transferase I involved in the biosynthetic initiation of heparan sulfate. The GlcNAc transferase reaction product was sensitive to the action of heparitinase I, establishing the identity of the enzyme to be alpha 1,4-GlcNAc transferase. These results altogether indicate that EXTL2/EXTR2 encodes the alpha 1,4-N-acetylhexosaminyltransferase that transfers GalNAc/GlcNAc to the tetrasaccharide representing the common glycosaminoglycan-protein linkage region and that is most likely the critical enzyme that determines and initiates the heparin/heparan sulfate synthesis, separating it from the chondroitin sulfate/dermatan sulfate synthesis.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Sulfated glycosaminoglycans (GAGs),1 including heparin/heparan sulfate and chondroitin sulfate/dermatan sulfate, are distributed on the surfaces of most cells and in the extracellular matrices of virtually every tissue. They are implicated in the regulation and maintenance of cell proliferation, cytodifferentiation, and tissue morphogenesis, exhibiting their biological activities by interacting with various proteins through specific saccharide sequences. They are synthesized as proteoglycans, on specific Ser residues in the so-called GAG-protein linkage region, GlcAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser, which is common to the GAGs (for reviews, see Refs. 1 and 2). The linkage region synthesis is initiated by the addition of Xyl to Ser followed by the addition of two Gal residues and is completed by the addition of GlcA, each reaction being catalyzed by a specific glycosyltransferase (1, 2). The GAGs are built up on this linkage region by the alternating addition of N-acetylhexosamine and GlcA residues. Heparin/heparan sulfate is synthesized once GlcNAc is transferred to the common linkage region, whereas chondroitin sulfate/dermatan sulfate is formed if GalNAc is first added. However, biosynthetic sorting mechanisms of different GAG chains remain enigmatic. Although at least eight different kinds of glycosyltransferase reactions are required to synthesize these GAGs, only the GlcA transferase that completes the tetrasaccharide linkage region and the heparan sulfate-polymerase that polymerizes GlcA and GlcNAc have been cloned (3, 4).

Recent cDNA cloning of the latter enzyme of bovine origin revealed its 94% sequence identity to human EXT2, a member of the hereditary multiple exostoses (EXT) gene family of tumor suppressors (4). EXT is an autosomal dominant disorder characterized by cartilage-capped skeletal excrescences, which may lead to skeletal abnormalities and short stature (5). Although the exostoses represent osteochondromas that are benign bone tumors, malignant transformation into chondrosarcomas or osteosarcoma occurs in approximately 2% of EXT patients (5, 6). Genetic linkage of this disorder has been ascribed to three independent loci on chromosomes 8q24.1 (EXT1), 11p11-13 (EXT2), and 19p (EXT3) (7-9). This family of EXT genes has recently been extended by the identification of three additional EXT-like genes, EXTL1, EXTL2/EXTR2, and EXTL3/EXTR1 (10-13). Sporadic and exostoses-derived chondrosarcomas are attributable to the loss of heterozygosity for the markers in EXT1 and EXT2 loci (14, 15), indicating that the genes responsible for EXTs and the EXT-like genes may encode tumor suppressors.

While searching for the key enzyme involved in biosynthetic sorting of chondroitin sulfate/dermatan sulfate from heparin/heparan sulfate, we found a novel alpha -N-acetylgalactosaminyltransferase (alpha -GalNAcT) in fetal bovine sera and in the mouse mastocytoma cells, which transferred an alpha -GalNAc residue to GlcAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser derived from the GAG-protein linkage region (16, 17). In addition, Miura and Freeze (18) detected a substantial amount of alpha -GalNAcT activity in Golgi fractions prepared from several cells. The enzyme activity was also found in the culture medium of a human sarcoma cell line and was an alpha 1,4-GalNAc transferase (19). Mysteriously, however, the structure of the reaction product has not been found in any natural GAG chain. In this study, we purified the enzyme and demonstrated 100% peptide sequence identity to the protein encoded by the multiple exostoses-like gene EXTL2/EXTR2 (10, 11), a unique member of the EXT gene family. A recombinant enzyme had a dual catalytic activity of alpha 1,4-GalNAc transferase and alpha 1,4-GlcNAc transferase that determines and initiates the heparan sulfate synthesis on the common GAG-protein linkage region (20).

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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- UDP-[3H]GalNAc (10 Ci/mmol) and UDP-[3H]GlcNAc (30 Ci/mmol) were purchased from NEN Life Science Products. Unlabeled UDP-GalNAc and UDP-GlcNAc were obtained from Sigma. Flavobacterium heparinum heparitinase I, jack bean beta -N-acetylhexosaminidase, and Acremonium sp. alpha -N-acetylgalactosaminidase were purchased from Seikagaku Corp. (Tokyo, Japan). The chemically synthesized linkage tetrasaccharide-serine GlcAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser (21) was provided by T. Ogawa (RIKEN, The Insitute of Physical and Chemical Research, Saitama, Japan). N-Acetylchondrosine GlcAbeta 1-3GalNAc was a gift from K. Yoshida (Seikagaku Corp.). GlcAbeta 1-3Galbeta 1-O-naphthalenemethanol (20) was provided by J. D. Esko (University of California, San Diego, CA).

Enzyme Purification and Peptide Sequencing-- The alpha -GalNAcT was purified from the serum-free culture medium of a human sarcoma (malignant fibrous histiocytoma) cell line using an extension of the procedure described previously (19). The details of the isolation procedure will be reported elsewhere.2 The purified alpha -GalNAcT was resolved by SDS-polyacrylamide gel electrophoresis transferred to a polyvinylidene difluoride membrane (Bio-Rad, Tokyo, Japan), and the resolved protein bands were stained with Coomassie Brilliant Blue (Sigma). The membrane strip containing the major protein band of 38 kDa was excised and was subjected to an NH2-terminal amino acid sequence analysis (Takara, Kyoto, Japan).

Construction of a Soluble Form of the Enzyme-- A truncated form of alpha -GalNAcT, lacking the first NH2-terminal 57 amino acids of EXTL2/EXTR2 (10, 11), was amplified with human fetal liver cDNA (CLONTECH) as a template by PCR using a 5' primer (5'-CGGGATCCCAGGGCAAGTCCACCAT-3') containing an in-frame BamHI site and a 3' primer (5'-CGGGATCCAAGCTACTCAAATGCCAAGCA-3') containing an in-frame BamHI site located 54 base pairs downstream of the stop codon. PCR reactions were carried out with Pfu polymerase (Stratagene, La Jolla, CA) by 30 cycles of 96 °C for 30 s, 55 °C for 30 s, and 72 °C for 75 s. The PCR fragment was subcloned into the BamHI site of pGIR201protA (22), resulting in the fusion of alpha -GalNAcT to the insulin signal sequence and the protein A sequence present in the vector. A NheI fragment containing the fusion protein was inserted into the XbaI site of the expression vector pSVL (Amersham Pharmacia Biotech, Tokyo, Japan).

Expression of the Soluble Enzyme and the Assay-- The expression plasmid (6 µg) was transfected into COS-1 cells on 100-mm plates using FuGENETM 6 (Roche Molecular Biochemicals, Tokyo, Japan) according to the instructions provided by the manufacturer. Two days after transfection, 1 ml of the culture medium was collected and incubated with 10 µl of IgG-Sepharose (Amersham Pharmacia Biotech) for 1 h at 4 °C. The beads recovered by centrifugation were washed with and then resuspended in the assay buffer and tested for alpha -GalNAcT and GlcNAc transferase-I (GlcNAcT-I) activities using the tetrasaccharide-serine (1 nmol) representing the GAG-protein linkage region, N-acetylchondrosine (5 nmol), and GlcAbeta 1-3Galbeta 1-O-naphthalenemethanol (250 nmol) as acceptor substrates as described (16, 19, 20).

Identification of the Enzyme Reaction Products-- The isolation of the products from the alpha -GalNAcT reaction using N-acetylchondrosine as an acceptor was carried out by gel filtration on a Superdex 30 column (Amersham Pharmacia Biotech) equilibrated with 0.25 M NH4HCO3, 7% 1-propanol. The radioactive peak containing the product was pooled and evaporated to dryness. The isolated product (about 10 pmol) was digested with 15 mIU of beta -N-acetylhexosaminidase or 39 mIU of alpha -N-acetylgalactosaminidase in a total volume of 20 µl of 50 mM sodium citrate buffer, pH 4.5, respectively, at 37 °C overnight. The enzyme digest was analyzed using the same Superdex 30 column as that noted above.

The isolation of the products from the GlcNAcT-I reaction using GlcAbeta 1-3Galbeta 1-O-naphthalenemethanol was performed by HPLC on a Nova-Pak® C18 column (3.9 × 150 mm; Waters, Tokyo, Japan) in an LC-10AS system (Shimadzu Co., Kyoto, Japan). The column was developed isocratically for 15 min with H2O at a flow rate of 1.0 ml/min at room temperature; thereafter, a linear gradient was applied to increase the methanol concentration from 0 to 100% over a 5-min period, and the column was then developed isocratically for 40 min with 100% methanol. The radioactive peak containing the product was pooled and evaporated to dryness. The isolated product (about 74 pmol) was incubated with 14 mIU of beta -N-acetylhexosaminidase in a total volume of 20 µl of 50 mM sodium citrate buffer, pH 4.5, or with 3 mIU of heparitinase I for testing the digestability in a total volume of 30 µl of 20 mM sodium acetate buffer, pH 7.0, containing 2 mM Ca(OAc)2 at 37 °C overnight. The enzyme digest was analyzed using the same Nova-Pak® C18 column as that noted above.

Expression Levels of the Enzyme in Human Tissues-- Human Multiple Tissue cDNA Panels (CLONTECH) were used for the analysis. The manufacturer normalizes each cDNA sample against six housekeeping genes. To verify this, we determined the levels of amplification of the glyceraldehyde-3-phosphate dehydrogenase, whose transcript is always present in the tissues at a constant level. The amplification reaction was carried out in a total volume of 50 µl using the 5' primer, 5'-ACCACTGTCCATGCCATCAC-3', and the 3' primer, 5'-TCCACAACACGGTTGCTGTA-3', by 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s. A 10-µl aliquot of the amplified products was visualized by electrophoresis on a 1.0% agarose gel containing ethidium bromide. Using the normalized cDNA input, we then performed the amplification of a transcript, using a serial number of cycles (25-30-35) to find the conditions for a semiquantitative amplification. The best results were obtained by carrying out 30 cycles of 96 °C for 30 s, 55 °C for 30 s, and 72 °C for 75 s using the 5' and 3' primers described above, which were designed to span the two introns in the EXTL2 gene (10) to discriminate a PCR product amplified from cDNA from, if any, one amplified from contaminating genomic DNA. PCR products were then visualized by electrophoresis on a 1.0% agarose gel containing ethidium bromide. To confirm that the amplified DNAs were derived from the EXTL2 mRNA, the amplified fragments were gel-purified, subcloned into the pGEM®-T Easy vector (Promega, Madison, WI), and sequenced. The nucleotide sequences of the amplified DNAs were identical to that of the human EXTL2 cDNA (10) (data not shown).

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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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The Enzyme Is Encoded by a Tumor Suppressor-like Gene-- alpha -GalNAcT was purified from the serum-free culture medium of a human sarcoma (malignant fibrous histiocytoma) cell line to near homogeneity mainly by affinity chromatographies on heparin-Sepharose and UDP-hexanolamine-Sepharose. Upon SDS-polyacrylamide gel electrophoresis under reducing conditions, the purified alpha -GalNAcT preparation gave a major protein band of 38-kDa (data not shown), which was excised and subjected to NH2-terminal amino acid sequence analysis. The obtained sequence of thirty amino acid residues (Fig. 1) was 100% identical to that of residues 54-83 of the protein encoded by EXTL2/EXTR2 except for the unidentified amino acid corresponding to the N-glycosylation site (10, 11), as demonstrated by data base searches. The EXTL2/EXTR2 sequence indicated a single open reading frame of 990-base pair coding for a protein of 330 amino acids, including one potential N-glycosylation site (10, 11), which has a type II transmembrane protein topology characteristic of many other glycosyltransferases cloned to date. Since the NH2-terminal amino acid sequence of the purified alpha -GalNAcT was found 54 amino acids away from the putative start site for translation of EXTL2/EXTR2, the purified alpha -GalNAcT seems to be a truncated form that has lost its transmembrane domain and subsequently been released from the enzyme-producing cell, as has been observed for several other glycosyltransferases (23).


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Fig. 1.   Comparison of the NH2-terminal amino acid sequence of the purified alpha -GalNAcT with the corresponding EXTL2/EXTR2 sequence. X represents an unidentified amino acid residue that is most likely glycosylated, and one potential N-glycosylation site is marked by an asterisk.

The Expressed Enzyme Is an Active alpha -GalNAc Transferase-- A soluble form of the protein encoded by EXTL2/EXTR2 cDNA was generated by replacing the first 57 amino acids of EXTL2/EXTR2 with the cleavable insulin signal sequence and the IgG-binding domain of protein A as described under "Experimental Procedures." When the expression plasmid containing the EXTL2/protein A fusion was expressed in COS-1 cells, an approximately 66-kDa protein was secreted (data not shown). The apparent molecular mass of the fused protein was reduced to 60-kDa after N-glycosidase treatment (data not shown), indicating that the one potential N-linked glycosylation site of EXTL2/EXTR2 is being utilized. The fused enzyme expressed in the medium was absorbed on IgG-Sepharose beads to eliminate endogenous glycosyltransferases and then the enzyme-bound beads were used as an enzyme source for further studies. The bound fusion protein was assayed for alpha -GalNAcT activity using a variety of acceptor substrates. As shown in Table I, marked glycosyltransferase activity was detected with N-acetylchondrosine (GlcAbeta 1-3GalNAc), GlcAbeta 1-3Galbeta 1-O-naphthalenemethanol, and the tetrasaccharide-serine representing the GAG-protein linkage region as the acceptor substrates. In addition, no detectable alpha -GalNAcT activity was recovered by the affinity purification from the control pSVL transfection sample. To identify the alpha -GalNAcT reaction products, N-acetylchondrosine was labeled by an enzyme reaction using UDP-[3H]GalNAc as a donor substrate and the enzyme-bound beads as an enzyme source. The labeled products were completely digested by alpha -N-acetylgalactosaminidase, but not by beta -N-acetylhexosaminidase, quantitatively yielding a 3H-labeled peak at the elution position of free [3H]GalNAc, as demonstrated by gel filtration (Fig. 2A). These results indicated that a GalNAc residue had been transferred to N-acetylchondrosine through an alpha -linkage and that the expressed protein was alpha 1,4-GalNAc transferase.

                              
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Table I
Acceptor specificity of the alpha 1,4-N-acetylhexosaminyltransferase secreted into the culture medium by transfected COS-1 cells


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Fig. 2.   Characterization of the alpha -GalNAcT and GlcNAc transferase reaction products using various glycosidases. A, 3H-labeled alpha -GalNAcT reaction products recovered from a Superdex 30 column were subjected to digestion with alpha -N-acetylgalactosaminidase or beta -N-acetylhexosaminidase as described under "Experimental Procedures." The alpha -N-acetylgalactosaminidase digest (), beta -N-acetylhexosaminidase digest (open circle ), or the undigested sample (black-square) was applied to a column of Superdex 30 (1.6 × 60 cm), and the respective effluent fractions (1 ml each) were analyzed for radioactivity. V0 and Vt were around fractions 40 and 120, respectively (not shown). An arrow indicates the elution position of free GalNAc. B, isolated 3H-labeled GlcNAc transferase reaction products were subjected to digestion with heparitinase I or beta -N-acetylhexosaminidase as described under "Experimental Procedures." The heparitinase I digest (open circle ), beta -N-acetylhexosaminidase digest (), or the undigested sample (black-square) was analyzed by HPLC on a Nova-Pak® C18 column as described, and the respective effluent fractions (2 ml each) were analyzed for radioactivity. An arrow indicates the elution position of free GlcNAc.

alpha 1,4-GlcNAc Transferase for Heparan Sulfate Synthesis-- In view of the recent findings that EXT1 and EXT2, the carboxyl halves of both of which shared significant homology with EXTL2/EXTR2, were heparan sulfate-polymerases required for the heparan sulfate biosynthesis (4), it was of particular interest to investigate the involvement of EXTL2/EXTR2 in the heparan sulfate biosynthesis. Although our enzyme showed an alpha -GalNAcT activity toward the tetrasaccharide-serine derived from the common GAG-protein linkage region, the structure of the reaction product has not been found in naturally occurring GAG chains, which prompted us to hypothesize that the enzyme may have an alpha -GlcNAc tansferase activity toward the linkage region structure taking into account the tetrasaccharide structure of the acceptor substrate for the alpha -GalNAcT activity. Hence, the purified fusion protein was assayed for GlcNAc transferase activity using UDP-[3H]GlcNAc as a sugar donor and two oligosaccharide acceptor substrates whose structures represent the GAG-protein linkage region. As shown in Table I, a significant GlcNAc transferase activity was detected with GlcAbeta 1-3Galbeta 1-O-naphthalenemethanol as an acceptor, whereas no activity was detected using the linkage tetrasaccharide-serine. The observed substrate specificity was consistent with that reported for GlcNAcT-I (17, 20), which is involved in the heparan sulfate biosynthesis. No detectable GlcNAc transferase activity was recovered by affinity purification from a control pSVL transfection sample, excluding the possibility of an artifact or of an endogenous origin of the activity. To identify the GlcNAc transferase reaction products, GlcAbeta 1-3Galbeta 1-O-naphthalenemethanol was labeled with [3H]GlcNAc using the enzyme bound to beads. The labeled products were completely digested by heparitinase I that cleaves an alpha 1,4-glucosaminide linkage in an eliminative fashion, quantitatively yielding a 3H-labeled peak at the position of free [3H]GlcNAc, as demonstrated by HPLC (Fig. 2B), whereas they were inert to the action of beta -N-acetylhexosaminidase. These results clearly indicated that a GlcNAc residue had been transferred exclusively to the nonreducing terminal GlcA of GlcAbeta 1-3Galbeta 1-O-naphthalenemethanol through an alpha 1,4 linkage. Taken together, the present findings demonstrated that EXTL2/EXTR2 is an alpha 1,4-N-acetylhexosaminyltransferase that transfers GalNAc/GlcNAc to the artificial yet authentic oligosaccharide acceptor substrate for GlcNAcT-I (20). The failure of the tetrasaccharide-serine derived from the GAG-protein linkage region to serve as an acceptor (Table I) is discussed below.

Ubiquitous Expression of the Gene in Human Tissues-- To screen tissue expression of the alpha 1,4-N-acetylhexosaminyltransferase, we used PCR-based methods with normalized cDNA pools. A single amplified DNA of the expected size (875 base pairs) was obtained from each cDNA preparation of the 18 adult and 8 fetal human tissues examined, although the amounts of the amplified cDNAs varied (Fig. 3), indicating that the gene was ubiquitously expressed.


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Fig. 3.   Differential expression of the alpha 1,4-N-acetylhexosaminyltransferase gene in various human tissues. The procedures used are described under "Experimental Procedures."


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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In the present study, we demonstrated that EXTL2/EXTR2 encoded enzyme with a dual catalytic activity of alpha -GalNAcT and GlcNAcT-I, i.e. an alpha 1,4-N-acetylhexosaminyltransferase that transferred GalNAc/GlcNAc to the core oligosaccharide representing the GAG-protein linkage region. Thus, the enzyme turned out to be identical to the previously described GlcNAcT-I that determines and initiates the biosynthesis of heparan sulfate (20) and most likely heparin as well. The gene, like other EXT gene family members, was found ubiquitously expressed in virtually every human tissue examined (Fig. 3), which is in accordance with the observations that heparan sulfate proteoglycans are distributed on the surfaces of most cells and the extracellular matrices in virtually every tissue. In view of the present findings of the involvement of EXTL2/EXTR2 in the heparan sulfate biosynthesis together with those of Lind et al. (4), who recently reported that EXT1 and EXT2 both encoded a heparan sulfate-polymerase required for the heparan sulfate biosynthesis, the expression of heparan sulfate seems to play an important role in the tumor suppressor function although the precise mechanism remains unclear.

The initial characterization of crude GlcNAcT-I preparations showed that the enzyme exhibited strict specificity toward GlcAbeta 1-3Galbeta 1-O-naphthalenemethanol (20), GlcAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-naphthalenemethanol, or GlcAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-benzyl (24). Neither N-acetylheparosan (-4GlcAbeta 1-4GlcNAcalpha 1-)n nor the tetrasaccharide-serine, GlcAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser derived from the linkage region, was utilized as an acceptor substrate (17, 20). Hence, it was suggested that the transfer of the first GlcNAc residue to the linkage tetrasaccharide primer is mediated by GlcNAcT-I, distinct from the enzyme that has been termed heparan sulfate-polymerase involved in the formation of the repeating disaccharide units of heparan sulfate (20) and that GlcNAcT-I directly recognizes a specific sequence in the core protein or an aglycone structure attached to the linkage tetrasaccharide (24). These hypotheses have now been proven by the molecular identification and characterization of both enzymes in a recent study (4) and in the present study. The molecular similarity of the two enzymes is consistent with the fact that both enzymes have alpha 1,4-GlcNAc transferase activities and recognize the terminal beta -GlcA moiety of their acceptor substrates.

The enzyme protein of the alpha 1,4-N-acetylhexosaminyltransferase composed of 330 amino acids is about half the size of the other EXT family members that have 676~919 amino acids. The variation in size is due to differences on the amino-terminal side of the protein. The protein shows significant homology with the carboxyl termini of the other members of the family. Based on the facts that both EXT1 and EXT2 encode a bifunctional heparan sulfate-polymerase catalyzing the GlcA and GlcNAc transferase reactions and that EXTL2/EXTR2 encodes GlcNAcT-I, it is reasonable to assume that the carboxyl-terminal side of EXT1 and EXT2 is the domain catalyzing the GlcNAc transferase reaction. A phylogenetic tree of the EXT gene family based on the conserved carboxyl-terminal amino acids shows a close relationship between EXT2 and EXTL3/EXTR1 and between EXT1 and EXTL1 (13), suggesting that the remaining two members, EXTL1 and EXTL3/EXTR1 might be heparan sulfate-polymerases. Notably, the EXTL2/EXTR2 gene is the most divergent, suggesting that this unique gene family member was the first to split from a common ancestor during evolution and evolved separately from the others (13).

The possible role of the alpha -GalNAcT activity of the enzyme in the GAG biosynthesis remains unclear, since no alpha -GalNAc-capped structure has been reported in naturally occurring GAG chains. In our recent study, the alpha -GalNAc-capped pentasaccharide serine GalNAcalpha 1-4GlcAbeta 1-3Galbeta 1-3Galbeta 1-4Xylbeta 1-O-Ser, a reaction product of the alpha -GalNAcT, was not utilized as an acceptor for the glucuronyltransferase involved in the chondroitin sulfate biosynthesis (25), suggesting that the addition of an alpha -GalNAc residue may serve as a stop signal that precludes further chain elongation. The cDNA now provides an essential tool for investigating the biological functions of the alpha -GalNAcT reaction products, if any, in naturally occurring glycoconjugates as well as heparan sulfate with regard to the tumor suppressor activity.

The EXTL2 has been assigned to chromosome 1p11-p12, and this region has been found to be involved in chromosomal rearrangements in a variety of tumors as described (10). In view of the tumor suppressor capacity of the EXT genes, EXTL2 might also be a serious candidate gene involved in one of the tumors associated with this region. In this regard, many different types of tumors are associated with undersulfation and distinct changes in the sulfation pattern of the heparan sulfate structure (Ref. 26 and the references therein), and recent studies have demonstrated a critical role for heparan sulfate in growth factor signaling mediated by Wingless proteins during Drosophila development (27). In addition, Drosophila homologues of EXT1 and heparan sulfate 2-O-sulfotransferase (encoded by ttv and pipe, respectively) were recently implicated in the Hedgehog diffusion and the formation of embryonic dorsal-ventral polarity, respectively (28, 29). Thus, considering the probability that deletion of the gene would cause the complete elimination of heparan sulfate and heparin unless functional redundancy with other genes exists, it is likely that germ line mutations inactivating the enzymatic activity result in embryonic lethality and that somatic mutations cause much more serious defects than those caused by EXT1 and EXT2, leading to the progression of various tumors or to lethal disorders. In fact, congenital deficiency in heparan sulfate even only in enterocytes results in severe clinical problems and eventually death (30).

    ACKNOWLEDGEMENTS

We thank Drs. T. Ogawa, J. Esko, and K. Yoshida for the gifts of the enzyme substrates. We also thank Dr. Satomi Nadanaka for developing the HPLC conditions used for the GlcNAcT-I assay.

    FOOTNOTES

* This work was supported in part by a Research Grant from the Research Foundation for Pharmaceutical Sciences (to H. K.), a Grant-in-aid for Scientific Research (B) 09470509 (to K. S.) and a Grant-in-aid for Scientific Research on Priority Areas 10178102 (to K. S.) from the Ministry of Education, Science, Sports, and Culture 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.

Dagger 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{at}kobepharma-u.ac.jp.

2 H. Shimakawa, Y. Kano, H. Kitagawa, H. Okabe, and K. Sugahara, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: GAG, glycosaminoglycan; EXT, hereditary multiple exostoses; GalNAc, N-acetylgalactosamine; alpha -GalNAcT, alpha 1,4-N-acetylgalactosaminyltransferase (16); GlcNAc, N-acetylglucosamine; GlcNAcT-I, alpha 1,4-N-acetylglucosaminyltransferase-I (20); GlcA, D-glucuronic acid; HPLC, high-performance liquid chromatography; PCR, polymerase chain reaction.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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