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• W2152447911 abstract "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™ 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. With the aid of bioinformatics technology, it is now possible to find candidate genes for glycosyltransferases that are distributed in relatively few tissues, are expressed at very low levels, or synthesize unknown structures. The β1,3-glycosyltransferase (β3GT) 1The abbreviations used are: β3GT, β1,3-glycosyltransferase; (β3)GalNAc-T, (β1,3-)N-acetylgalactosaminyltransferase; GlcNAc, N-acetylglucosamine; Bz, benzyl; pNP, para-nitrophenyl; oNP, ortho-nitrophenyl; (β3)Gal-T, (β1,3-)galactosyltransferase; (β3)Gn-T, (β1,3-) 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. 1The abbreviations used are: β3GT, β1,3-glycosyltransferase; (β3)GalNAc-T, (β1,3-)N-acetylgalactosaminyltransferase; GlcNAc, N-acetylglucosamine; Bz, benzyl; pNP, para-nitrophenyl; oNP, ortho-nitrophenyl; (β3)Gal-T, (β1,3-)galactosyltransferase; (β3)Gn-T, (β1,3-) 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. motifs, originally found in our previous study (1Isshiki S. Togayachi A. Kudo T. Nishihara S. Watanabe M. Kubota T. Kitajima M. Shiraishi N. Sasaki K. Andoh T. Narimatsu H. J. Biol. Chem. 1999; 274: 12499-12507Google Scholar, 2Togayachi A. Akashima T. Ookubo R. Kudo T. Nishihara S. Iwasaki H. Natsume A. Mio H. Inokuchi J. Irimura T. Sasaki K. Narimatsu H. J. Biol. Chem. 2001; 276: 22032-22040Google Scholar), are conserved in the catalytic domain of β3GT family members. Three β3GT motifs, XIRX(S/T)W(G/L/M), (F/Y)XXXXDXD, and (E/D)DVXXGX, are commonly encoded in β3GTs that combine two sugars with a β1,3-linkage. To date, thirteen members of the β3GT family, i.e. six β1,3-galactosyltransferases (β3Gal-T) (1Isshiki S. Togayachi A. Kudo T. Nishihara S. Watanabe M. Kubota T. Kitajima M. Shiraishi N. Sasaki K. Andoh T. Narimatsu H. J. Biol. Chem. 1999; 274: 12499-12507Google Scholar, 3Zhou D. Berger E.G. Hennet T. Eur. J. Biochem. 1999; 263: 571-576Google Scholar, 4Bai X. Zhou D. Brown J.R. Crawford B.E. Hennet T. Esko J.D. J. Biol. Chem. 2001; 276: 48189-48195Google Scholar), six β1,3-N-acetylglucosaminyltransferases (β3Gn-T) (5Sasaki K. Kurata-Miura K. Ujita M. Angata K. Nakagawa S. Sekine S. Nishi T. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14294-14299Google Scholar, 6Iwai T. Inaba N. Naundorf A. Zhang Y. Gotoh M. Iwasaki H. Kudo T. Togayachi A. Ishizuka Y. Nakanishi H. Narimatsu H. J. Biol. Chem. 2002; 277: 12802-12809Google Scholar), and one β1,3-N-acetylgalactosaminyltransferase (β3GalNAc-T) (7Okajima T. Nakamura Y. Uchikawa M. Haslam D.B. Numata S.I. Furukawa K. Urano T. J. Biol. Chem. 2000; 275: 40498-40503Google Scholar) have been cloned and characterized. β3Gal-T1, -T2, and -T5 transfer galactose (Gal) to the N-acetylglucosamine (GlcNAc)-β-R residue from UDP-Gal, resulting in the synthesis of a type 1 chain Galβ1-3GlcNAc. β3Gal-T3 was originally considered to be a β3Gal-T; however, its function was found to be the transfer of N-acetylgalatosamine (GalNAc) to the Gal-α-R residue of paragloboside with a β1,3-linkage and synthesis of globoside (7Okajima T. Nakamura Y. Uchikawa M. Haslam D.B. Numata S.I. Furukawa K. Urano T. J. Biol. Chem. 2000; 275: 40498-40503Google Scholar). β3Gal-T3 is not a Gal-T, so it was renamed β3GalNAc-T1 as a globoside synthase. β3Gal-T4, the GM1 synthase, efficiently transfers Gal to the GalNAc-β-R residue of GM2 resulting in the synthesis of GM1 (8Amado M. Almeida R. Carneiro F. Levery S.B. Holmes E.H. Nomoto M. Hollingsworth M.A. Hassan H. Schwientek T. Nielsen P.A. Bennett E.P. Clausen H. J. Biol. Chem. 1998; 273: 12770-12778Google Scholar). β3Gal-T5 exhibits the strongest activity for Gal transfer to GlcNAc among β3Gal-Ts, and is restrictively expressed in colon, intestine, stomach, and pancreas (1Isshiki S. Togayachi A. Kudo T. Nishihara S. Watanabe M. Kubota T. Kitajima M. Shiraishi N. Sasaki K. Andoh T. Narimatsu H. J. Biol. Chem. 1999; 274: 12499-12507Google Scholar). β3Gal-T5 is responsible for the expression of the sialyl Lewis a antigen epitopes, a famous tumor marker, in cancer cells derived from such tissues (9Isshiki S. Kudo T. Nishihara S. Ikehara Y. Togayachi A. Furuya A. Shitara K. Kubota T. Watanabe M. Kitajima M. Narimatsu H. J. Biol. Chem. 2003; Google Scholar). β3Gal-T6 catalyzes a Galβ1-3Gal linkage and is responsible for the synthesis of Galβ1-3Galβ1-4 xylose (Xly)α-O-(Ser/Thr), the core structure of proteoglycans (4Bai X. Zhou D. Brown J.R. Crawford B.E. Hennet T. Esko J.D. J. Biol. Chem. 2001; 276: 48189-48195Google Scholar). iGn-T, cloned by the expression cloning method, can express polylactosamine on the cell surface (5Sasaki K. Kurata-Miura K. Ujita M. Angata K. Nakagawa S. Sekine S. Nishi T. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14294-14299Google Scholar). However, iGnT is not included in the β3GT family because it does not possess the β3GT motif. Four β3Gn-Ts (-T2, -T3, -T4, and -T5) have activity for the transfer of GlcNAc to the Gal-β-R residue from UDP-GlcNAc. Transfection experiments with each β3Gn-T showed the expression of a polylactosamine chain on the cell surface (2Togayachi A. Akashima T. Ookubo R. Kudo T. Nishihara S. Iwasaki H. Natsume A. Mio H. Inokuchi J. Irimura T. Sasaki K. Narimatsu H. J. Biol. Chem. 2001; 276: 22032-22040Google Scholar, 10Shiraishi N. Natsume A. Togayachi A. Endo T. Akashima T. Yamada Y. Imai N. Nakagawa S. Koizumi S. Sekine S. Narimatsu H. Sasaki K. J. Biol. Chem. 2001; 276: 3498-3507Google Scholar). β3Gn-T3 was reported to effectively transfer GlcNAc to Gal with a β1,3 linkage on core 1 O-glycan (10Shiraishi N. Natsume A. Togayachi A. Endo T. Akashima T. Yamada Y. Imai N. Nakagawa S. Koizumi S. Sekine S. Narimatsu H. Sasaki K. J. Biol. Chem. 2001; 276: 3498-3507Google Scholar). β3Gn-T5 transfers GlcNAc to a Gal residue of lactosylceramide with a β1,3-linkage and synthesizes lactotriaosylceramide (Lc3Cer) of glycolipid (2Togayachi A. Akashima T. Ookubo R. Kudo T. Nishihara S. Iwasaki H. Natsume A. Mio H. Inokuchi J. Irimura T. Sasaki K. Narimatsu H. J. Biol. Chem. 2001; 276: 22032-22040Google Scholar). β3Gn-T6 is the core 3-synthesizing enzyme that transfers GlcNAc to GalNAcα1-O-Ser/Thr with a β1,3-linkage, the core 3 structure of O-glycan (6Iwai T. Inaba N. Naundorf A. Zhang Y. Gotoh M. Iwasaki H. Kudo T. Togayachi A. Ishizuka Y. Nakanishi H. Narimatsu H. J. Biol. Chem. 2002; 277: 12802-12809Google Scholar). In this study, DNA databases were searched using the amino acid sequences of β3GT family members with particular attention being paid to the existence of a transmembrane domain and the conserved motifs of β3GTs. Thus, a new member of the β3GT family was found and cloned for characterization. The new enzyme was demonstrated to be active in synthesizing a unique carbohydrate structure, GalNAcβ1-3GlcNAc, on both N-glycan and O-glycan. Carbohydrate structures determined to date are being accumulated in data bases such as GlycoSuiteDB (11Cooper C.A. Harrison M.J. Wilkins M.R. Packer N.H. Nucleic Acids Res. 2001; 29: 332-335Google Scholar, 12Cooper C.A. Joshi H.J. Harrison M.J. Wilkins M.R. Packer N.H. Nucleic Acids Res. 2003; 31: 511-513Google Scholar). However, not all carbohydrate structures have been determined. Although the GalNAcβ1-3GlcNAc structure has not been found in mammals, there is a possibility that it exists in tissues where this new enzyme is expressed. Materials—UDP-[14C]GalNAc was purchased from Amersham Biosciences (Amersham Place, UK) and UDP-[3H]GalNAc from ICN Biomedicals (Irvine, CA). UDP-GalNAc and GlcNAc-β-benzyl (Bz) were obtained from Sigma. Construction and Purification of Human β3GalNAc-T2 Proteins Fused with FLAG Peptide—We performed a BLAST search of the data base at NCBI and identified a cDNA (GenBank™ accession number BC029564), homologous in amino acid sequence to the open reading frame (ORF) of β3Gal-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-β3GalNAc-T2. The putative catalytic domain of β3GalNAc-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 β3GalNAc-T2—To determine a donor and acceptor substrate for β3GalNAc-T2, all combinations of nucleotide sugars and monosaccharides were screened by a method described previously (13Hosomi O. Takeya A. Kogure T. Jpn. J. Med. Sci. Biol. 1989; 42: 77-82Google Scholar, 14Holmes E.H. Arch Biochem. Biophys. 1989; 270: 630-646Google Scholar, 15Taga S. Tetaud C. Mangeney M. Tursz T. Wiels J. Biochim. Biophys. Acta. 1995; 1254: 56-65Google Scholar). 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 β3GalNAc-T2 with Mass Spectrometry (MS)—GlcNAc-Bz (10 nmol) was incubated with β3GalNAc-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 × 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 β3GalNAc-T2 using 1H NMR Spectra—GlcNAc-Bz (640 nmol) was incubated with β3GalNAc-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 × 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 β3GalNAc-T2—A GalNAc-T assay of human β3GalNAc-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).Table ISubstrate specificity of β3GalNAc-T2Acceptor substrates%GlcNAc-α-BzNDaND, not detected.GlcNAc-β-Bz100Gal-α-pNPNDGal-β-oNPNDGal-NAc-α-BzNDGalNAc-β-pNPNDGlc-α-pNPNDGlc-β-pNP2GlcA-β-pNPNDFuc-α-pNPNDXyl-α-pNPNDXyl-β-pNPNDMan-α-pNPNDLactoside-BzbLactoside, Galβ1-4Glc.NDLac-CercLac-Cer, Lactosylceramide (Galβ1-4Glcβ1-1 ceramide).NDGal-CerdGal-Cer, galactocenebroside type 1 (Galβ1-1 ceramide).NDParaglobosideeParagloboside, Galα1-4Galβ1-4Glcβ1-1 ceramide.NDGlobosidefGloboside, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1 ceramide.NDGalβ1-4GalNAc-α-pNPNDGalβ1-3GlcNAc-β-pNPNDGlcNAcβ1-4GlcNAc-β-Bz29Galβ1-3GalNAcα(core1)-pNPNDGalβ1-3(GlcNAcβ1-6)GalNAcα(core2)-pNP185GlcNAcβ1-3GalNAcα(core3)-pNP8GlcNAcβ1-6GalNAcα(core6)-pNP19a ND, not detected.b Lactoside, Galβ1-4Glc.c Lac-Cer, Lactosylceramide (Galβ1-4Glcβ1-1 ceramide).d Gal-Cer, galactocenebroside type 1 (Galβ1-1 ceramide).e Paragloboside, Galα1-4Galβ1-4Glcβ1-1 ceramide.f Globoside, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1 ceramide. Open table in a new tab Quantitative Analysis of the β3GalNAc-T2 Transcripts in Human Tissues by Real Time PCR—For the quantification of β3GalNAc-T2 transcripts, we employed the real time PCR method, as described in detail previously (16Gibson U.E. Heid C.A. Williams P.M. Genome Res. 1996; 6: 995-1001Google Scholar, 17Heid C.A. Stevens J. Livak K.J. Williams P.M. Genome Res. 1996; 6: 986-994Google Scholar). 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 β3GalNAc-T2 cDNA was generated by serial dilution of a pDONR™201 vector DNA containing the β3GalNAc-T2 gene encoding the putative catalytic domain (amino acids 35-500). The primer set and probe for β3GalNAc-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 β3GalNAc-T2 Proteins Fused with FLAG Peptide—In the EST data base of mouse cDNAs, two partial cDNA sequences (BQ963315 and BG923691) orthologous to β3GalNAc-T2 were found. The two cDNAs overlapped and encoded the full-length ORF of the mouse β3GalNAC-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™ under accession number AB116655. A truncated form of mβ3GalNAc-T2 was expressed in HEK293T cells with the same method described in the human β3GalNAc-T section. In Situ Hybridization in Mouse Testis—The partial sequence of mβ3GalNAc-T2 (nucleotides 311-1081 in AB116655) was amplified by PCR using pFLAG-CMV3-mβ3GalNAc-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-mβ3GalNAc-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-mβ3GalNAc-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 (18Fujiwara Y. Komiya T. Kawabata H. Sato M. Fujimoto H. Furusawa M. Noce T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12258-12262Google Scholar). Detection of β3GalNAc-T2 in Mouse Testis by Western blot using Polyclonal Antibody—hβ3GalNAc-T2 expressed in insect cells, sf21, was purified for immunization of mice. A polyclonal antibody against hβ3GalNAc-T2 was bled from the mice. The antibody cross-reacted to mβ3GalNAc-T2. Recombinant β3GalNAc-T2 enzymes, which were purified from the medium of HEK293T cells transfected with the pFLAG-CMV3-mock, pFLAG-CMV3-hβ3GalNAc-T2 or pFLAG-CMV3-mβ3GalNAc-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 hβ3GalNAc-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 β3GalNAc-T2. FCF was treated with β-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 β-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 β3GalNAc-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. Determination of Nucleotide and Amino Acid Sequences of β3GalNAc-T2—We obtained a new sequence for the β3GT family as described under “Experimental Procedures” and named it β3GalNAc-T2. As shown in Fig. 1A, the ORF of β3GalNAc-T2 consisted of 1,500 bp encoding a predicted 500-amino acid protein with a typical type II topology, same as in the other β3GTs. 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™ accession number AL135928, which is located on chromosome 1. By comparison of the β3GalNAc-T2 cDNA sequence with the genome data base, the genomic structure of the β3GalNAc-T2 gene was determined (Fig. 1B). The β3GalNAc-T2 gene contains at least 12 exons. As shown in a phylogenetic tree (Fig. 2A), β3GalNAc-T2 was most homologous to β3Gal-T6 in the human β3GT family. The three β3GT motifs, one of which contained a DXD motif, were conserved between β3GalNAc-T2 and β3Gal-T6 (Fig. 2B). Partial sequences highly homologous to human β3GalNAc-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 β3GalNAc-T2 as shown in Fig. 3. This mouse gene product is probably an ortholog of human β3GalNAc-T2, and was named mβ3GalNAc-T2. The mβGalNAc-T2 gene also contained at least 12 exons, and the junctions between exons and introns are at the same positions in hβ3GalNAc-T2 and mβ3GalNAc-T2 (Fig. 3).Fig. 2A phylogenetic tree of human β3GTs and an alignment of β3Gal-T motifs of β3GalNAc-T2 with those of β3Gal-T6. A, phylogenetic tree of human β3GTs and mβ3GalNAc-T2. The numbers at the right represent the references reported. B, alignment of three β3Gal-T motifs of β3GalNAc-T2 with those of β3Gal-T6. Identical amino acids are shown with asterisks.View Large Image Figure ViewerDownload (PPT)Fig. 3Alignment of amino acid sequences of human β3GalNAc-T2 and mouse β3GalNAc-T2. Identical amino acids are indicated with asterisks. Junctions between exons are indicated with triangles.View Large Image Figure ViewerDownload (PPT) Determination of Glycosyltransferase Activity of β3GalNAc-T2—FLAG-tagged recombinant β3GalNAc-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-β-Bz (Table I). The recombinant protein showed faint activity toward Glc-β-pNP, and no activity toward GlcNAc-α-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-β-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. Determination of the Linkage of the β3GalNAc-T2 Product with 1H NMR—To determine the newly formed glycosidic linkage of the β3GalNAc-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 β-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 (β-GlcNAc, defined as A), and that the anomeric proton at 4.381 ppm corresponds to the anomeric proton of the transferred pyranose (β-GalNAc, defined as B).Table IIChemical shift (ppm) and coupling constant (Hz) of sugar CH protons in the β3GalNAc-T2 productβ3GalNAc-T2 productGlcNAc (A)GalNAc (B)1H Chemical shifts (ppm)δ14.398aThe chemical shifts were set as the higher field signal of the benzyl methylene proton is tentatively 4.557 ppm.4.381aThe chemical shifts were set as the higher field signal of the benzyl methylene proton is tentatively 4.557 ppm.δ23.6873.711aThe chemical shifts were set as the higher field signal of the benzyl methylene proton is tentatively 4.557 ppm.δ33.599aThe chemical shifts were set as the higher field signal of the benzyl methylene proton is tentatively 4.557 ppm.3.655δ43.435aThe chemical shifts were set as the higher field signal of the benzyl methylene proton is t" @default.
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• W2152447911 title "A Novel Human β1,3-N-Acetylgalactosaminyltransferase That Synthesizes a Unique Carbohydrate Structure, GalNAcβ1-3GlcNAc" @default.
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