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J. Biol. Chem., Vol. 279, Issue 14, 14087-14095, April 2, 2004
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1,3-N-Acetylgalactosaminyltransferase That Synthesizes a Unique Carbohydrate Structure, GalNAc1-3GlcNAc*
¶¶¶
From the
Glycogene Function Team, Research Center for Glycoscience (RCG), National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Frontier Research Division, Fundamental Research Department, Fujirebio, Inc., 51 Komiya-cho, Hachioji, Tokyo 192-0031, **JGS Japan Genome Solutions, Inc., 51 Komiya-cho, Hachioji, Tokyo 192-0031, Japan, the ¶Mitsui Knowledge Industry Co., Ltd., Honcho 1-Chome, Nakano-ku, Tokyo 164-8721, Japan, the ||Laboratory of Reproductive Biology, Mitsubishi Kagaku Institute of Life Sciences, 11 Minami-Ooya, Machida-shi, Tokyo 194-8511, Japan, Amersham Biosciences KK, Sanken Building, 3-25-1 Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan, and Molecular Recognition Team, Biological Information Research Center, AIST, C-6, 1-1 Higashi, Tsukuba, Ibaraki 305-8568, Japan
Received for publication, September 25, 2003 , and in revised form, January 12, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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-N-acetylgalactosaminyltransferase-II (3GalNAc-T2). The acceptor substrate specificity of 3GalNAc-T2 was examined using various oligosaccharide substrates. Gal1-3(GlcNAc1-6)GalNAc
1-O-para-nitrophenyl (core 2-pNP) was the best acceptor substrate for 3GalNAc-T2, followed by GlcNAc1-4GlcNAc1-O-benzyl, and GlcNAc1-6GalNAc1-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, m3GalNAc-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 GalNAc1-3GlcNAc1-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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1,3-glycosyltransferase (3GT)1 motifs, originally found in our previous study (1, 2), are conserved in the catalytic domain of 3GT family members. Three 3GT motifs, XIRX(S/T)W(G/L/M), (F/Y)XXXXDXD, and (E/D)DVXXGX, are commonly encoded in 3GTs that combine two sugars with a 1,3-linkage. To date, thirteen members of the 3GT family, i.e. six 1,3-galactosyltransferases (3Gal-T) (1, 3, 4), six 1,3-N-acetylglucosaminyltransferases (3Gn-T) (5, 6), and one 1,3-N-acetylgalactosaminyltransferase (3GalNAc-T) (7) have been cloned and characterized.
3Gal-T1, -T2, and -T5 transfer galactose (Gal) to the N-acetylglucosamine (GlcNAc)--R residue from UDP-Gal, resulting in the synthesis of a type 1 chain Gal1-3GlcNAc. 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 Gal to the GalNAc--R residue of GM2 resulting in the synthesis of GM1 (8). 3Gal-T5 exhibits the strongest activity for Gal transfer to GlcNAc among 3Gal-Ts, and is restrictively expressed in colon, intestine, stomach, and pancreas (1). 3Gal-T5 is responsible for the expression of the sialyl Lewis a antigen epitopes, a famous tumor marker, in cancer cells derived from such tissues (9). 3Gal-T6 catalyzes a Gal1-3Gal linkage and is responsible for the synthesis of Gal1-3Gal1-4 xylose (Xly)-O-(Ser/Thr), the core structure of proteoglycans (4).
iGn-T, cloned by the expression cloning method, can express polylactosamine on the cell surface (5). However, iGnT is not included in the 3GT family because it does not possess the 3GT motif. Four 3Gn-Ts (-T2, -T3, -T4, and -T5) have activity for the transfer of GlcNAc to the Gal--R residue from UDP-GlcNAc. Transfection experiments with each 3Gn-T showed the expression of a polylactosamine chain on the cell surface (2, 10). 3Gn-T3 was reported to effectively transfer GlcNAc to Gal with a 1,3 linkage on core 1 O-glycan (10). 3Gn-T5 transfers GlcNAc to a Gal residue of lactosylceramide with a 1,3-linkage and synthesizes lactotriaosylceramide (Lc3Cer) of glycolipid (2). 3Gn-T6 is the core 3-synthesizing enzyme that transfers GlcNAc to GalNAc1-O-Ser/Thr with a 1,3-linkage, the core 3 structure of O-glycan (6).
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, GalNAc1-3GlcNAc, on both N-glycan and O-glycan. Carbohydrate structures determined to date are being accumulated in data bases such as GlycoSuiteDB (11, 12). However, not all carbohydrate structures have been determined. Although the GalNAc1-3GlcNAc structure has not been found in mammals, there is a possibility that it exists in tissues where this new enzyme is expressed.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-benzyl (Bz) were obtained from Sigma.
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 (GenBankTM accession number BC029564
[GenBank]
), 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 5'-CCCAAGCTTGGGCCTGCAGATCAGTTGGCCTTATTTC-3' and 5'-AACGCGGATCCGCGCTGTTATCTTGCTTGACATCGACAAGGA-3' 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 CaCl2 and suspended in 200 µl 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 (13-15).
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 MnCl2, 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 µl of a basic reaction mixture containing 2.5 mM UDP-GalNAc to produce the reaction product. The reaction mixture was added to 80 µl of H2O and was filtrated using Ultrafree-MC (Millipore, Bedford, MA). A 50-µl reaction mixture was subjected to high performance liquid chromatography (HPLC) with an ODS-80Ts QA column (4.6 x 250 mm; Tosoh, Tokyo). The reaction products were eluted with 30 ml of 9% acetonitrile containing 0.1% trifluoroacetic acid and H2O 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 µl of H2O, and 45 µl of 0.1% formic acid, and added to 50 µl of methanol. The product solution was infused at a rate of 3 µl/min with a capillary voltage of 3 kV. For MALDI-TOF MS analysis, 10 pmol of the product was dried, dissolved in 1 µl of H2O, and applied.
Determination of Products of 3GalNAc-T2 using 1H 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-µl aliquot of supernatant was subjected to HPLC on an ODS-80Ts QA column (4.6 x 250 mm; Tosoh). The reaction products were eluted with 30 ml of 9% acetonitrile containing 0.1% trifluoroacetic acid and H2O 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 D2O. Then, 100 µg of the lyophilized product was dissolved again in 0.18 ml of D2O for NMR analysis. One-dimensional 1H NMR and two-dimensional COSY, TOCSY, and NOESY spectra were recorded with a DMX750 spectrometer (Bruker, Germany, 750.13 MHz for 1H 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 1H NMR chemical shifts.
Substrate Specificity of 3GalNAc-T2—A GalNAc-T assay of human 3GalNAc-T2 using the synthetic oligosaccharide was performed as follows. [14C]UDP-GalNAc (50 nCi) and the oligosaccharides as the acceptor substrates were added to 10 µl 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 µl of H2O. Radioactive products were separated from the free UDP-[14C]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 µl of H2O, 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).
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3GalNAc-T2 Transcripts in Human Tissues by Real Time PCR—For the quantification of 3GalNAc-T2 transcripts, we employed the real time PCR method, as described in detail previously (16, 17). Total RNA from various human tissues was purchased from Clontech. cDNAs were synthesized using oligo(dT)12-18 primers and the Super-Script First-Strand Synthesis system (Invitrogen). A standard curve for 3GalNAc-T2 cDNA was generated by serial dilution of a pDONRTM201 vector DNA containing the 3GalNAc-T2 gene encoding the putative catalytic domain (amino acids 35-500). The primer set and probe for 3GalNAc-T2 were as follows: forward primer, 5'-GGAGTGTTCTACGATGCCAAT-3'; reverse primer, 5'-CTGAAGCGAGCAATGAAGAG-3'; and probe, 5'-CACTGTCAAACTTTATCAGGCAGAACAAGAGG-3'. Primers, probe, and cDNA were added to the TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA), which contained all reagents for PCR. The PCR conditions included 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 10 min, and 50 cycles at 95 °C for 15 s, and 60 °C for 1 min. PCR products were measured continuously with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems).
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
[GenBank]
and BG923691
[GenBank]
) orthologous to 3GalNAc-T2 were found. The two cDNAs overlapped and encoded the full-length 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 GenBankTM under accession number AB116655
[GenBank]
. A truncated form of m3GalNAc-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 m3GalNAc-T2 (nucleotides 311-1081 in AB116655
[GenBank]
) was amplified by PCR using pFLAG-CMV3-m3GalNAc-T2 as the template. The fragment amplified by 5'-TGTGGAAGACAGGGAGG-3' and 5'-AGTCGTCATCTGTCTTGAGC-3' was inserted into the vector pCR®-Blunt IITOPO® (Invitrogen) to construct pCR-TOPO-m3GalNAc-T2. Adult mouse testis fixed in Bouin's solution was embedded in paraffin and sectioned (7 µm) for in situ hybridization analyses. After linearization of pCR-TOPO-m3GalNAc-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—h3GalNAc-T2 expressed in insect cells, sf21, was purified for immunization of mice. A polyclonal antibody against h3GalNAc-T2 was bled from the mice. The antibody cross-reacted to m3GalNAc-T2.
Recombinant 3GalNAc-T2 enzymes, which were purified from the medium of HEK293T cells transfected with the pFLAG-CMV3-mock, pFLAG-CMV3-h3GalNAc-T2 or pFLAG-CMV3-m3GalNAc-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 h3GalNAc-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-µg 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-µl 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 µl). 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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, GenBankTM accession number AL135928
[GenBank]
, 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 sequences 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 m3GalNAc-T2. The mGalNAc-T2 gene also contained at least 12 exons, and the junctions between exons and introns are at the same positions in h3GalNAc-T2 and m3GalNAc-T2 (Fig. 3).
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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 14C. 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+ and K+, respectively.
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3GalNAc-T2 Product with 1H NMR—To determine the newly formed glycosidic linkage of the 3GalNAc-T2 product, 1H NMR spectroscopy was employed. Although there were negligible weak signals from contaminants in the 1H 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 1H 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, (J1,2) 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).
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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 1,3-linkage. Results in NMR experiments thus indicated clearly that the product of 3GalNAc-T2 is GalNAc1-3GlcNAc-O-Bz.
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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 GlcNAcs examined. The activity of 3GalNAc-T2 was strongest when Gal1-3(GlcNAc1-6)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 GlcNAc1-4GlcNAc--Bz; 19% activity for GlcNAc1-6GalNAc -pNP (core 6-pNP); and little activity for GlcNAc1-3GalNAc -pNP (core 3-pNP). 3GalNAc-T2 showed faint activity toward Glc--pNP, but no activity toward other substrates examined.
Km values for UDP-GalNAc and GlcNAc--Bz were determined as 5.4 µM and 11 mM, respectively (see legend in Table I). The Km value for the donor substrate was only a little different from those values of the other glycosyltransferases. However, the Km 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.
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3GalNAc-T2 in Mouse Testis—In situ hybridization showed germ cell-specific expression of the m3GalNAc-T2 gene in adult mouse testis. The m3GalNAc-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 m3GalNAc-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.
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3GalNAc-T2 in Mouse Testis by Western Blotting—As shown in Fig. 8, a band of 
55 kDa was detected in both recombinant hGalNAc-T2 and mGalNAc-T2, but not in the mock-transfectant. This size matched the expected size of the truncated recombinant enzymes. The polyclonal antibody raised against h3GalNAc-T2 cross-reacted to m3GalNAc-T2. A nonspecific band of 52 kDa was detected. In mouse testis homogenates, a distinct band was detected at 60 kDa, which is the molecular size of full-length m3GalNAc-T2 that has a transmembrane domain. On the other hand, mouse spleen, which expressed no transcripts of m3GalNAc-T2, did not show any positive band.
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3GalNAc-T2 transferred GalNAc to various kinds of non-reducing terminal GlcNAcs. However, the GalNAc1-3GlcNAc structure has not been found in humans. To determine the acceptor preference of 3GalNAc-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, asialo/agalacto-FCF appeared as an 55 kDa band (lane 3) on Coomassie Brilliant Blue (CBB) staining. 3GalNAc-T2 effectively transferred GalNAc to asialo/agalacto-FCF, as seen from the positive band obtained with WFA (lane 7). The band mostly disappeared on treatment with glycopeptidase F (lane 8), although a faint band obtained with WFA still remained at around 45 kDa (lane 8), which was the size of the glycopeptidase F-treated FCF stained with CBB (lane 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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3GalNAc-T2 and m3GalNAc-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 (GenBankTM accession number BC029564
[GenBank]
) and m3GalNAc-T2 (AB116655
[GenBank]
) 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 GalNAc1-3GlcNAc1-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 GalNAc1-3GlcNAc structure. However, FCF is a convenient acceptor for the screening of N- and O-glycosylation (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 GalNAc1-3GlcNAc1-3Gal1-3GalNAc1-4GalNAc1-4GlcNAc1-3Man1-4Glc1-1 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 stage-specific 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 GalNAc1-3GlcNAc structure exists or not, a tool is needed for probing such a structure. We will try to establish an antibody to detect the GalNAc1-3GlcNAc 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. (25-28). 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 N-glycans. Male mice lacking the MX gene, which is predominantly 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 GalNAc1-3GlcNAc 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 m3GalNAc-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.
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FOOTNOTES |
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¶¶ To whom correspondence should be addressed: Glycogene Function Team, Research Center for Glycoscience (RCG), National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan. Tel.: 81-29-861-3200; Fax: 81-29-861-3201; E-mail: h.narimatsu{at}aist.go.jp.
1 The abbreviations used are: 3GT, 1,3-glycosyltransferase; (3)GalNAc-T, (1,3-)N-acetylgalactosaminyltransferase; GlcNAc, N-acetylglucosamine; Bz, benzyl; pNP, para-nitrophenyl; oNP, ortho-nitrophenyl; (3)Gal-T, (1,3-)galactosyltransferase; (3)Gn-T, (1,3-) N-acetylgalactosaminyltransferase; Gal, galactose; GalNAc, N-acetylgalatosamine; Xly, xylose; MS, mass spectrometry; EST, expressed sequence tag; Lc3Cer, lactotriaosylceramide; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; WFA, Wisteria floribunda agglutinin; FCF, fetal calf fetuin; HRP, horseradish peroxidase; NeuAc, neuraminic acid (sialic acid); free-mRNP, free-messenger ribonucleoprotein particles; CBB, Coomassie Brilliant Blue; ORF, open reading frame.
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