A Novel Human β1,3-N-Acetylgalactosaminyltransferase That Synthesizes a Unique Carbohydrate Structure, GalNAcβ1-3GlcNAc

We found, using a BLAST search, a novel human gene (GenBank™ accession number BC029564) that possesses β3-glycosyltransferase motifs. The full-length open reading frame consists of 500 amino acids and encodes a typical type II membrane protein. This enzyme has a domain containing β1,3-glycosyltransferase motifs, which are widely conserved in the β1,3-galactosyltransferase and β1,3-N-acetylglucosaminyltransferase families. The putative catalytic domain was expressed in human embryonic kidney 293T cells as a soluble protein. Its N-acetylgalactosaminyltransferase activity was observed when N-acetylglucosamine (GlcNAc) β1-O-benzyl was used as an acceptor substrate. The enzyme product was determined to have a β1,3-linkage by NMR spectroscopic analysis, and was therefore named β1,3-N-acetylgalactosaminyltransferase-II (β3GalNAc-T2). The acceptor substrate specificity of β3GalNAc-T2 was examined using various oligosaccharide substrates. Galβ1-3(GlcNAcβ1-6)GalNAcα1-O-para-nitrophenyl (core 2-pNP) was the best acceptor substrate for β3GalNAc-T2, followed by GlcNAcβ1-4GlcNAcβ1-O-benzyl, and GlcNAcβ1-6GalNAcα1-O-para-nitrophenyl (core 6-pNP), among the tested oligosaccharide substrates. Quantitative real time PCR analysis revealed that the β3Gal-NAc-T2 transcripts was restricted in its distribution mainly to the testis, adipose tissue, skeletal muscle, and ovary. Its putative orthologous gene, mβ3GalNAc-T2, was also found in a data base of mouse expressed sequence tags. In situ hybridization analysis with mouse testis showed that the transcripts are expressed in germ line cells. β3GalNAc-T2 efficiently transferred GalNAc to N-glycans of fetal calf fetuin, which was treated with neuraminidase and β-galactosidase. However, it showed no activity toward any glycolipid examined. Although the GalNAcβ1-3GlcNAcβ1-R structure has not been reported in humans or other mammals, we have discovered a novel human glycosyltransferase producing this structure on N- and O-glycans.

We found, using a BLAST search, a novel human gene (GenBank TM accession number BC029564) that possesses ␤3-glycosyltransferase motifs. The full-length open reading frame consists of 500 amino acids and encodes a typical type II membrane protein. This enzyme has a domain containing ␤1,3-glycosyltransferase motifs, which are widely conserved in the ␤1,3-galactosyltransferase and ␤1,3-N-acetylglucosaminyltransferase families. The putative catalytic domain was expressed in human embryonic kidney 293T cells as a soluble protein. Its N-acetylgalactosaminyltransferase activity was observed when N-acetylglucosamine (GlcNAc) ␤1-O-benzyl was used as an acceptor substrate. The enzyme product was determined to have a ␤1,3-linkage by NMR spectroscopic analysis, and was therefore named ␤1,3-Nacetylgalactosaminyltransferase-II (␤3GalNAc-T2). The acceptor substrate specificity of ␤3GalNAc-T2 was examined using various oligosaccharide substrates. Gal-␤1-3(GlcNAc␤1-6)GalNAc␣1-O-para-nitrophenyl (core 2-pNP) was the best acceptor substrate for ␤3GalNAc-T2, followed by GlcNAc␤1-4GlcNAc␤1-O-benzyl, and Glc-NAc␤1-6GalNAc␣1-O-para-nitrophenyl (core 6-pNP), among the tested oligosaccharide substrates. Quantitative real time PCR analysis revealed that the ␤3Gal-NAc-T2 transcripts was restricted in its distribution mainly to the testis, adipose tissue, skeletal muscle, and ovary. Its putative orthologous gene, m␤3GalNAc-T2, was also found in a data base of mouse expressed sequence tags. In situ hybridization analysis with mouse testis showed that the transcripts are expressed in germ line cells. ␤3GalNAc-T2 efficiently transferred GalNAc to N-glycans of fetal calf fetuin, which was treated with neuraminidase and ␤-galactosidase. However, it showed no activity toward any glycolipid examined. Although the GalNAc␤1-3GlcNAc␤1-R structure has not been reported in humans or other mammals, we have discovered a novel human glycosyltransferase producing this structure on N-and O-glycans.
␤3Gal-T1, -T2, and -T5 transfer galactose (Gal) to the Nacetylglucosamine (GlcNAc)-␤-R residue from UDP-Gal, resulting in the synthesis of a type 1 chain Gal␤1-3GlcNAc. ␤3Gal-T3 was originally considered to be a ␤3Gal-T; however, its function was found to be the transfer of N-acetylgalatosamine (GalNAc) to the Gal-␣-R residue of paragloboside with a ␤1,3-linkage and synthesis of globoside (7). ␤3Gal-T3 is not a Gal-T, so it was renamed ␤3GalNAc-T1 as a globoside synthase. ␤3Gal-T4, the GM1 synthase, efficiently transfers * This work was performed as part of the R&D Project of the Industrial Science and Technology Frontier Program (R&D for Establishment and Utilization of a Technical Infrastructure for Japanese Industry) supported by the New Energy and Industrial Technology Development Organization (NEDO). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
In this study, DNA databases were searched using the amino acid sequences of ␤3GT family members with particular attention being paid to the existence of a transmembrane domain and the conserved motifs of ␤3GTs. Thus, a new member of the ␤3GT family was found and cloned for characterization. The new enzyme was demonstrated to be active in synthesizing a unique carbohydrate structure, GalNAc␤1-3GlcNAc, on both N-glycan and O-glycan. Carbohydrate structures determined to date are being accumulated in data bases such as GlycoSuit-eDB (11,12). However, not all carbohydrate structures have been determined. Although the GalNAc␤1-3GlcNAc structure has not been found in mammals, there is a possibility that it exists in tissues where this new enzyme is expressed.
Construction and Purification of Human ␤3GalNAc-T2 Proteins Fused with FLAG Peptide-We performed a BLAST search of the data base at NCBI and identified a cDNA (GenBank TM accession number BC029564), homologous in amino acid sequence to the open reading frame (ORF) of ␤3Gal-T6. The putative catalytic domain of the enzyme (amino acids 35-500) was amplified by PCR using a single strand DNA derived from kidney total RNA (Clontech, Palo Alto, CA). The fragment amplified by 5Ј-CCCAAGCTTGGGCCTGCAGATCAGTTGGCCTTATT-TC-3Ј and 5Ј-AACGCGGATCCGCGCTGTTATCTTGCTTGACATCGA-CAAGGA-3Ј was inserted into pFLAG-CMV3 (Invitrogen, Groningen, Netherlands) to construct pFLAG-CMV3-␤3GalNAc-T2. The putative catalytic domain of ␤3GalNAc-T2 was expressed as a secreted protein fused with a FLAG peptide in HEK293T cells (a human embryonic renal cancer cell line). A 12-ml volume of culture medium was mixed with anti-FLAG M1 antibody resin (Sigma). The protein-resin mixture was washed twice with 50 mM TBS (50 nM Tris-HCl, pH 7.4, and 150 mM NaCl) containing 1 mM CaCl 2 and suspended in 200 l of each of the assay buffers.
Screening for Donor and Acceptor Substrates for ␤3GalNAc-T2-To determine a donor and acceptor substrate for ␤3GalNAc-T2, all combinations of nucleotide sugars and monosaccharides were screened by a method described previously (13)(14)(15).
N-Acetylgalactosaminyltransferase Assay-The basic reaction mixture for assaying GalNAc-T activity contained 14 mM HEPES buffer, pH 7.4, an appropriate concentration of UDP-GalNAc, 10 mM MnCl 2 , 0.4% Triton CF-54, a suitable amount of acceptor substrate, and the purified enzyme. After incubation at 37°C for 16 h, the product was analyzed by various techniques as described below.
Determination of Products of ␤3GalNAc-T2 with Mass Spectrometry (MS)-GlcNAc-Bz (10 nmol) was incubated with ␤3GalNAc-T2 in 20 l of a basic reaction mixture containing 2.5 mM UDP-GalNAc to produce the reaction product. The reaction mixture was added to 80 l of H 2 O and was filtrated using Ultrafree-MC (Millipore, Bedford, MA). A 50-l reaction mixture was subjected to high performance liquid chromatography (HPLC) with an ODS-80Ts QA column (4.6 ϫ 250 mm; Tosoh, Tokyo). The reaction products were eluted with 30 ml of 9% acetonitrile containing 0.1% trifluoroacetic acid and H 2 O at a flow rate of 1.0 ml/min at 40°C and monitored with an ultraviolet spectrophotometer (absorbance at 210 nm), the SPD-10AVP (Shimadzu, Kyoto, Japan). An additional peak was analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS (Reflex IV; Bruker Daltonics, Billerica, MA). Then, 25 pmol of the product was dissolved in 5 l of H 2 O, and 45 l of 0.1% formic acid, and added to 50 l of methanol. The product solution was infused at a rate of 3 l/min with a capillary voltage of 3 kV. For MALDI-TOF MS analysis, 10 pmol of the product was dried, dissolved in 1 l of H 2 O, and applied.
Determination of Products of ␤3GalNAc-T2 using 1 H NMR Spectra-GlcNAc-Bz (640 nmol) was incubated with ␤3GalNAc-T2 in 1 ml of a basic reaction mixture containing 2.5 mM UDP-GalNAc to produce the reaction product. A 50-l aliquot of supernatant was subjected to HPLC on an ODS-80Ts QA column (4.6 ϫ 250 mm; Tosoh). The reaction products were eluted with 30 ml of 9% acetonitrile containing 0.1% trifluoroacetic acid and H 2 O at a flow rate of 1.0 ml/min at 40°C and monitored with an ultraviolet spectrophotometer (absorbance at 210 nm), the SPD-10AVP (Shimadzu). The enzyme reaction product was purified with the HPLC technique as described above and lyophilized from D 2 O. Then, 100 g of the lyophilized product was dissolved again in 0.18 ml of D 2 O for NMR analysis. One-dimensional 1 H NMR and twodimensional COSY, TOCSY, and NOESY spectra were recorded with a DMX750 spectrometer (Bruker, Germany, 750.13 MHz for 1 H nucleus) at 25°C. The methylene proton of the Bz group in the higher field (4.557 ppm) was used as a reference for the 1 H NMR chemical shifts. Substrate Specificity of ␤3GalNAc-T2-A GalNAc-T assay of human ␤3GalNAc-T2 using the synthetic oligosaccharide was performed as follows. [ 14 C]UDP-GalNAc (50 nCi) and the oligosaccharides as the acceptor substrates were added to 10 l of the basic reaction mixture. The acceptor substrates used in this study are listed in Table I. After incubation at 37°C for 2 h, the reaction was terminated by adding 100 l of H 2 O. Radioactive products were separated from the free UDP-[ 14 C]GalNAc using a Sep-Pak Plus C18 cartridge (Waters, Milford, MA). The cartridge was activated by washing with 1 ml of 100% methanol and then washed twice with 1 ml of water. The enzyme reaction was terminated by adding 100 l of H 2 O, then the reaction mixture was applied to the equilibrated cartridge and washed twice with 1 ml of water. Elution of the radioactive product was achieved using 1 ml of 100% methanol. The eluted solution was added to 5 ml of liquid scintillation mixture (Amersham Biosciences) then the radioactivity was measured with a liquid scintillation counter (Beckman Coulter).
Construction and Purification of Mouse ␤3GalNAc-T2 Proteins Fused with FLAG Peptide-In the EST data base of mouse cDNAs, two partial cDNA sequences (BQ963315 and BG923691) orthologous to ␤3Gal-NAc-T2 were found. The two cDNAs overlapped and encoded the fulllength ORF of the mouse ␤3GalNAC-T2 (mGalNAc-T2) gene. PCR was performed to clone it using cDNAs prepared from mouse testis as a template. The full-length ORF sequence was confirmed and registered in GenBank TM under accession number AB116655. A truncated form of m␤3GalNAc-T2 was expressed in HEK293T cells with the same method described in the human ␤3GalNAc-T section.
In Situ Hybridization in Mouse Testis-The partial sequence of m␤3GalNAc-T2 (nucleotides 311-1081 in AB116655) was amplified by PCR using pFLAG-CMV3-m␤3GalNAc-T2 as the template. The fragment amplified by 5Ј-TGTGGAAGACAGGGAGG-3Ј and 5Ј-AGTCGT-CATCTGTCTTGAGC-3Ј was inserted into the vector pCR®-Blunt II-TOPO® (Invitrogen) to construct pCR-TOPO-m␤3GalNAc-T2. Adult mouse testis fixed in Bouin's solution was embedded in paraffin and sectioned (7 m) for in situ hybridization analyses. After linearization of pCR-TOPO-m␤3GalNAc-T2, sense and antisense digoxigenin-labeled RNA probes were generated using an RNA labeling kit (Roche Applied Science). Hybridization signals were detected with alkaline phosphatase-conjugated anti-digoxigenin antibody and NBT as the chromogen, as described previously (18).
Detection of ␤3GalNAc-T2 in Mouse Testis by Western blot using Polyclonal Antibody-h␤3GalNAc-T2 expressed in insect cells, sf21, was purified for immunization of mice. A polyclonal antibody against h␤3GalNAc-T2 was bled from the mice. The antibody cross-reacted to m␤3GalNAc-T2.
Recombinant ␤3GalNAc-T2 enzymes, which were purified from the medium of HEK293T cells transfected with the pFLAG-CMV3-mock, pFLAG-CMV3-h␤3GalNAc-T2 or pFLAG-CMV3-m␤3GalNAc-T2 vector, were subjected to Western blotting as controls. Tissue homogenates of mouse testis and spleen were used as experimental samples.
Each sample was subjected to 7.5% SDS-PAGE, transferred to a nitrocellulose membrane (Schleicher & Schuell), and treated with the polyclonal antibody against h␤3GalNAc-T2. The membrane was treated with anti-mouse Ig-HRP (Amersham Biosciences). The signals were detected using enhanced chemiluminescence and Hyperfilm ECL (Amersham Biosciences).
N-Acetylgalactosaminyltransferase Assay with Glycoproteins-Fetal calf fetuin (FCF) was used as the acceptor substrate for ␤3GalNAc-T2. FCF was treated with ␤-galactosidase (Streptococcus 6646K, Seikagaku Corporation, Tokyo, Japan) and neuraminidase (Nakarai Tesuque, Tokyo, Japan) in advance of the GalNAc-T assay. A 200-g amount of each glycoprotein was incubated with ␤-galactosidase (5 microunits) and neuraminidase (50 microunits) in 50 mM sodium acetate buffer (pH 6.0) at 37°C for 16 h. The reaction mixture was further incubated at 100°C for 5 min to inactivate the glycosidases. A 10-l volume of the glycosidase-treated samples was incubated with ␤3GalNAc-T2 in the basic reaction mixture containing 2.5 mM UDP-GalNAc (total volume, 20 l). One microliter of the reaction products was subjected to 12.5% SDS-PAGE, transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH) and stained with 0.1% horseradish peroxidase (HRP)-conjugated Wisteria floribunda agglutinin (WFA) (EY Laboratories, San Mateo, CA) for detection of the transferred GalNAc residue. The signals were detected using enhanced chemiluminescence and Hyperfilm ECL (Amersham Biosciences). To remove N-glycans, an aliquot of the reaction product was digested with glycopeptidase F (TaKaRa, Ohtsu, Japan) at 37°C for 16 h.

Determination of Nucleotide and Amino Acid Sequences of
␤3GalNAc-T2-We obtained a new sequence for the ␤3GT family as described under "Experimental Procedures" and named it ␤3GalNAc-T2. As shown in Fig. 1A, the ORF of ␤3GalNAc-T2 consisted of 1,500 bp encoding a predicted 500-amino acid protein with a typical type II topology, same as in the other ␤3GTs. It contained two N-glycosylation sites, a transmembrane segment of 19 residues, and a putative stem region and catalytic domain of 479 residues. The same sequence was found in a clone, GenBank TM accession number AL135928, which is located on chromosome 1. By comparison of the ␤3GalNAc-T2 cDNA sequence with the genome data base, the genomic structure of the ␤3GalNAc-T2 gene was determined (Fig. 1B). The ␤3GalNAc-T2 gene contains at least 12 exons. As shown in a phylogenetic tree ( Fig. 2A), ␤3GalNAc-T2 was most homologous to ␤3Gal-T6 in the human ␤3GT family. The three ␤3GT motifs, one of which contained a DXD motif, were conserved between ␤3GalNAc-T2 and ␤3Gal-T6 (Fig. 2B). Partial se-quences highly homologous to human ␤3GalNAc-T2 were found in the mouse EST data base. Based on the partial sequences in EST, we cloned a full-length ORF of this gene. It showed 88.4% identity in amino acid sequence to human ␤3GalNAc-T2 as shown in Fig. 3. This mouse gene product is probably an ortholog of human ␤3GalNAc-T2, and was named m␤3GalNAc-T2. The m␤GalNAc-T2 gene also contained at least 12 exons, and the junctions between exons and introns are at the same positions in h␤3GalNAc-T2 and m␤3GalNAc-T2 (Fig. 3).
Determination of Glycosyltransferase Activity of ␤3GalNAc-T2-FLAG-tagged recombinant ␤3GalNAc-T2 was purified from the supernatants of HEK293T cells as described under "Experimental Procedures." Its calculated molecular mass is 56.6 kDa, however, a major band was observed at around 60 kDa on Western blot analysis with an anti-FLAG antibody (data not shown). This result indicated that the recombinant protein is probably glycosylated in HEK293T cells. Its glycosyltransferase activities, Gal-T, Gn-T, and GalNAc-T activities, were screened using each donor labeled with 14 C. No Gal-T or Gn-T activity toward any acceptor substrate was observed (data not shown), whereas GalNAc-T activity was exhibited toward GlcNAc-␤-Bz (Table I). The recombinant protein showed faint activity toward Glc-␤-pNP, and no activity toward GlcNAc-␣-Bz (Table I).
On HPLC analysis as shown in Fig. 4, A and B, we observed a peak of an acceptor substrate (S) at 20.7 min and an additional peak (P) of the reaction product at 19.1 min when UDP-GalNAc and GlcNAc-␤-Bz were used as a donor substrate and an acceptor substrate, respectively. The peak P was isolated by reversed-phase chromatography and identified using MALDI-TOF-MS (Fig. 4C). It gave two peaks of 554.154 and 558.194 m/z as shown in Fig. 4C. The two molecular masses, 554.154 and 558.194 m/z, exactly matched those of GalNAc-linked GlcNAc-Bz with Na ϩ and K ϩ , respectively.
Determination of the Linkage of the ␤3GalNAc-T2 Product with 1 H NMR-To determine the newly formed glycosidic linkage of the ␤3GalNAc-T2 product, 1 H NMR spectroscopy was employed. Although there were negligible weak signals from contaminants in the 1 H NMR spectrum (not shown), signal integrals of five aromatic protons of Bz, two methylene protons of Bz, two anomeric protons, twelve sugar protons except anomeric protons, and six methyl protons of two N-acetyl groups corresponded well with the structure of GalNAc-GlcNAc-O-Bz. All 1 H signals could be assigned after high resolutional recordings of COSY, TOCSY, and NOESY spectra. The chemical shifts and coupling constants of the sugar component of the sample are shown in Table II. Two anomeric protons revealed signals with the same coupling constant, (J 1,2 ) 8.4 Hz, as shown in Table II. This indicates that two pyranoses in the samples are in a ␤-gluco-configuration. The anomeric resonance at 4.398 ppm was relatively broad and showed a NOE cross peak with one methylene proton of the Bz group at 4.557 ppm (not shown). On the other hand, the anomeric resonance in the higher field did not show a NOE peak with any methylene proton (not shown). This indicates that the anomeric resonance at 4.398 ppm is responsible for the anomeric proton of the substrate pyranose (␤-GlcNAc, defined as A), and that the anomeric proton at 4.381 ppm corresponds to the anomeric proton of the transferred pyranose (␤-GalNAc, defined as B).
As shown in Fig. 5, there was a weak NOE cross-peak between B1 and A3 in addition to strong inner residual NOEs between B1 and B5 and between A1 and A5. These results suggest the existence of a ␤1-3 linkage between two pyranoses. Two substrate pyranose signals, A1 and A3, were observed as relatively broadened signals, but signal broadening of A4 was not observed (see 1D spectrum in Fig. 5). The signal broadening of the substrate residue was caused by the strong restriction of the mobility of these protons and by the slow equilibrium between two conformations of the molecule by the binding to the Bz group and ␤-GalNAc group, respectively. Signal broadening of the A3 resonance supports the existence of a ␤1,3linkage. Results in NMR experiments thus indicated clearly that the product of ␤3GalNAc-T2 is GalNAc␤1-3GlcNAc-O-Bz. Substrate Specificity of ␤3GalNAc-T2-The substrate specificity of ␤3GalNAc-T2 is summarized in Table I. ␤3GalNAc-T2 showed GalNAc-transfer activity toward all kinds of non-reducing terminal GlcNAc␤s examined. The activity of ␤3GalNAc-T2 was strongest when Gal␤1-3(GlcNAc␤1-6)GalNAc-␣-pNP (core 2-pNP) was used as an acceptor substrate. Thus, the activity of ␤3GalNAc-T2 for GlcNAc-␤-Bz is presented as 100%, and all other activities are given as relative values in Table I. ␤3GalNAc-T2 exhibited 185% activity toward core 2-pNP; 29% activity for GlcNAc␤1-4GlcNAc-␤-Bz; 19% activity for GlcNAc␤1-6GalNAc ␣-pNP (core 6-pNP); and little activity for GlcNAc␤1-3GalNAc ␣-pNP (core 3-pNP). ␤3GalNAc-T2 showed faint activity toward Glc-␤-pNP, but no activity toward other substrates examined.
K m values for UDP-GalNAc and GlcNAc-␤-Bz were determined as 5.4 M and 11 mM, respectively (see legend in Table I). The K m value for the donor substrate was only a little different from those values of the other glycosyltransferases. However, the K m value for GlcNAc-␤-Bz was much higher than the values of the other enzymes. This indicated that ␤3GalNAc-T2 requires oligosaccharide, not monosaccharide, structures for acceptors.
Tissue Distribution of the ␤3GalNAc-T2 Transcripts-The expression levels of ␤3GalNAc-T2 in various human tissues were determined by quantitative real-time PCR (Fig. 6). The transcripts were most highly expressed in testis, and also highly expressed in the adipose, skeletal muscle, and ovary, in that order, although they were ubiquitously expressed in all tissues examined.
In Situ Hybridization of m␤3GalNAc-T2 in Mouse Testis-In situ hybridization showed germ cell-specific expression of the m␤3GalNAc-T2 gene in adult mouse testis. The m␤3GalNAc-T2 transcript was mainly detected in cells in middle layers of somniferous tubules at stages XII to II (Fig. 7). As shown in a and c to l in Fig. 7, the metaphase II spermatocytes and the resulting early round spermatids gave strong signals for the m␤3GalNAc-T2 transcript, in contrast, testicular somatic cells such as Sertoli and Leydig cells gave no signal. As shown in b in Fig. 7, no signal was detected in any cell when a sense probe was used. a The chemical shifts were set as the higher field signal of the benzyl methylene proton is tentatively 4.557 ppm. Detection of ␤3GalNAc-T2 in Mouse Testis by Western Blotting-As shown in Fig. 8, a band of ϳ55 kDa was detected in both recombinant h␤GalNAc-T2 and m␤GalNAc-T2, but not in the mock-transfectant. This size matched the expected size of the truncated recombinant enzymes. The polyclonal antibody raised against h␤3GalNAc-T2 cross-reacted to m␤3GalNAc-T2. A nonspecific band of ϳ52 kDa was detected. In mouse testis homogenates, a distinct band was detected at ϳ60 kDa, which is the molecular size of full-length m␤3GalNAc-T2 that has a transmembrane domain. On the other hand, mouse spleen, which expressed no transcripts of m␤3GalNAc-T2, did not show any positive band.
N-Acetylgalactosaminyltransferase Assay for Glycoproteins-As demonstrated in Table II, ␤3GalNAc-T2 transferred GalNAc to various kinds of non-reducing terminal GlcNAc␤s.
However, the GalNAc␤1-3GlcNAc structure has not been found in humans. To determine the acceptor preference of ␤3Gal-NAc-T2 for glycoproteins or glycolipids, FCF, which possesses both N-and O-glycans, and a mixture of glycolipids extracted from mouse testis and commercially available glycolipids were employed as a glycoprotein and glycolipid acceptor, respectively. ␤3GalNAc-T2 did not show any activity toward glycolipids (data not shown). As shown in Fig. 9

DISCUSSION
With the aid of bioinformatics technology, we performed the cloning of novel human and mouse glycosyltransferases, ␤3Gal-NAc-T2 and m␤3GalNAc-T2. All ␤3GTs, i.e. ␤3Gal-Ts, ␤3Gn-Ts, and ␤3GalNAc-T, share common amino acid motifs in three regions of the putative catalytic domain. Both ␤3GalNAc-T2 (GenBank TM accession number BC029564) and m␤3-GalNAc-T2 (AB116655) are typical members of the ␤3GT family having three ␤3GT motifs, including a DXD motif, and showing the topology of type II membrane proteins. Interestingly, they showed ␤3GalNAc-T activity toward non-reducing terminal GlcNAc␤ residues. Although the GalNAc␤1-3Glc-NAc␤1-R structure has not been reported in humans and other mammals, ␤3GalNAc-T2 was demonstrated to synthesize this unidentified structure on N-and O-glycans.
When we assayed for in vitro activity of ␤3GalNAc-T2 toward various oligosaccharides, we found core 2-pNP to be the best acceptor for ␤3GalNAc-T2, followed by core 6-pNP. FCF is not a physiological acceptor for ␤4GalNAc-T2 because there is no report that it has the GalNAc␤1-3GlcNAc structure. However, FCF is a convenient acceptor for the screening of N-and Oglycosylation (19 -21). ␤3GalNAc-T2 could transfer GalNAc to the GlcNAc termini on both N-glycans and O-glycans in asialo/ agalacto-FCF. It was reported that GalNAc␤1-3GlcNAc␤1-3-Gal␤1-3GalNAc␣1-4GalNAc␤1-4GlcNAc␤1-3Man␤1-4Glc-␤1-1 ceramide is present in neutral glycosphingolipids of the green-bottle fly (22). Therefore, we examined the activity of ␤3GalNAc-T2 to transfer GalNAc to glycolipids as acceptors. Neutral and acidic glycolipids extracted from mouse testis by us and commercially available glycolipids were employed as acceptors. However, no activity toward any glycolipids examined was detected (data not shown).
Interestingly, the ␤3GalNAc-T2 transcripts were most highly expressed in germ cells of mouse testis in a stagespecific manner. The transcripts were strongly expressed in primary and secondary spermatocytes and early round spermatids, but not expressed in spermatogonias, elongating or elongated spermatids, or in somatic cells, such as Leydig cells and Sertoli cells. We speculate that ␤3GalNAc-T2 may be involved in the maturation of sperm. It has been well established that spermatogenic cells show striking differences in patterns of translation from somatic cells (23). As one of these differences, the translation of some mRNA species does not necessarily occur even though the transcripts are expressed in the germ cells. Such mRNAs are mostly transcribed in undifferentiated spermatocytes, such as primary and secondary spermatocytes, and immature round spermatids (23). However, it was confirmed that the ␤GalNAc-T2 transcripts are translated in mouse testis by the Western blotting analysis in Fig. 8. The distribution of the enzyme in each tissue will be examined in detail after the establishment of a monoclonal antibody.
Although dramatic changes to carbohydrate structures during the development of testicular cells were suggested by histological analysis using a series of lectins (24), it is very difficult to determine the precise structure of such carbohydrates. In order to detect whether the GalNAc␤1-3GlcNAc structure exists or not, a tool is needed for probing such a structure. We will try to establish an antibody to detect the GalNAc␤1-3GlcNAc structure. In addition, it will be important to find physiological acceptor substrate(s), i.e. glycoprotein(s), for ␤3GalNAc-T2, which should exist in germ cells of testis.
To date, N-glycans on glycoproteins have been reported to participate in various physiological functions, such as immune reactions, cell adhesion, cell migration and fertilization, etc. (25)(26)(27)(28). Recently, Akama et al. (29) reported the importance of N-glycans for the maturation of spermatogenic cells. ␣-Mannosidase IIx, which is encoded by the MX gene, is an enzyme essential for formation of the intermediate structure of Nglycans. Male mice lacking the MX gene, which is predomi-nantly expressed in male germ cells, exhibited an almost complete loss of fertility because of impairment of the interaction between germ cells and Sertoli cells. Thus, it was demonstrated that incomplete N-glycan synthesis gives rise to a disturbance of cellular interaction during spermatogenic cell maturation. However, the complete N-glycan structure required for cellular interaction has not yet been defined. The GalNAc␤1-3GlcNAc structure at the termini of N-and O-glycans might be involved in such cellular interaction.
In summary, we found two novel members of the ␤3GT family, ␤3GalNAc-T2 and m␤3GalNAc-T2, which synthesize an unidentified carbohydrate structure. Cloning technology with the aid of bioinformatics as described in this study has the potential to discover novel glycosyltransferases, although in this report the products were not identified.