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• W2107155846 abstract "N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) catalyzes the transfer of sulfate from adenosine 3′-phosphate,5′-phosphosulfate to the C-6 position of the non-reducing GlcNAc. Three human GlcNAc6STs, namely GlcNAc6ST-1, GlcNAc6ST-2 (HEC-GlcNAc6ST), and GlcNAc6ST-3 (I-GlcNAc6ST), were produced as fusion proteins to protein A, and their substrate specificities as well as their enzymological properties were determined. Both GlcNAc6ST-1 and GlcNAc6ST-2 efficiently utilized the following oligosaccharide structures as acceptors: GlcNAcβ1–6[Galβ1–3]GalNAc-pNP (core 2), GlcNAcβ1–6ManOMe, and GlcNAcβ1–2Man. The ratios of activities to these substrates were not significantly different between the two enzymes. However, GlcNAc6ST-2 but not GlcNAc6ST-1 acted on core 3 of GlcNAcβ1–3GalNAc-pNP. GlcNAc6ST-3 used only the core 2 structure among the above mentioned oligosaccharide structures. The ability of GlcNAc6ST-1 to sulfate core 2 structure as efficiently as GlcNAc6ST-2 is consistent with the view that GlcNAc6ST-1 is also involved in the synthesis of l-selectin ligand. Indeed, cells doubly transfected with GlcNAc6ST-1 and fucosyltransferase VII cDNAs supported the rolling of L-selectin-expressing cells. The activity of GlcNAc6ST-2 on core 3 and its expression in mucinous adenocarcinoma suggested that this enzyme corresponds to the sulfotransferase, which is specifically expressed in mucinous adenocarcinoma (Seko, A., Sumiya, J., Yonezawa, S., Nagata, K., and Yamashita, K. (2000) Glycobiology 10, 919–929). N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) catalyzes the transfer of sulfate from adenosine 3′-phosphate,5′-phosphosulfate to the C-6 position of the non-reducing GlcNAc. Three human GlcNAc6STs, namely GlcNAc6ST-1, GlcNAc6ST-2 (HEC-GlcNAc6ST), and GlcNAc6ST-3 (I-GlcNAc6ST), were produced as fusion proteins to protein A, and their substrate specificities as well as their enzymological properties were determined. Both GlcNAc6ST-1 and GlcNAc6ST-2 efficiently utilized the following oligosaccharide structures as acceptors: GlcNAcβ1–6[Galβ1–3]GalNAc-pNP (core 2), GlcNAcβ1–6ManOMe, and GlcNAcβ1–2Man. The ratios of activities to these substrates were not significantly different between the two enzymes. However, GlcNAc6ST-2 but not GlcNAc6ST-1 acted on core 3 of GlcNAcβ1–3GalNAc-pNP. GlcNAc6ST-3 used only the core 2 structure among the above mentioned oligosaccharide structures. The ability of GlcNAc6ST-1 to sulfate core 2 structure as efficiently as GlcNAc6ST-2 is consistent with the view that GlcNAc6ST-1 is also involved in the synthesis of l-selectin ligand. Indeed, cells doubly transfected with GlcNAc6ST-1 and fucosyltransferase VII cDNAs supported the rolling of L-selectin-expressing cells. The activity of GlcNAc6ST-2 on core 3 and its expression in mucinous adenocarcinoma suggested that this enzyme corresponds to the sulfotransferase, which is specifically expressed in mucinous adenocarcinoma (Seko, A., Sumiya, J., Yonezawa, S., Nagata, K., and Yamashita, K. (2000) Glycobiology 10, 919–929). N-acetylglucosamine N-acetylglucosamine-6-O-sulfotransferase glyceraldehyde-3-phosphate dehydrogenase adenosine 3′-phosphate,5′-phosphosulfate methyl 6-O-(2-acetamido-2-deoxy-β-O-glucopyranosyl)-α-d-mannopyranoside GlcNAcβ1–6[Galβ1–3]GalNAc-pNP GlcNAcβ1–3GalNAc-pNP thin layer chromatography reverse transcription fucosyltransferase VII galactose/N-acetylgalactosamine/N-acetylglucosamine-6-O-sulfotransferase high endothelial cell Sulfated sugar residues of glycoconjugates have been shown to elicit diverse biological functions (1Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1680) Google Scholar, 2Hooper L.V. Manzella S.M. Baenzinger J.U. FASEB J. 1996; 10: 1137-1146Crossref PubMed Scopus (83) Google Scholar, 3Muramatsu T. J. Biochem. 2000; 127: 171-176Crossref PubMed Scopus (62) Google Scholar, 4Perrimon N. Bernfield M. Nature. 2000; 404: 725-728Crossref PubMed Scopus (662) Google Scholar). The addition of a sulfate group on the carbohydrate chains is catalyzed by various sulfotransferases with rigorous acceptor specificities (5Habuchi O. Biochim. Biophys. Acta. 2000; 1474: 115-127Crossref PubMed Scopus (161) Google Scholar). Recent studies (5Habuchi O. Biochim. Biophys. Acta. 2000; 1474: 115-127Crossref PubMed Scopus (161) Google Scholar, 6Hemmerich S. Rosen S.D. Glycobiology. 2000; 10: 849-856Crossref PubMed Scopus (106) Google Scholar) have achieved purification and molecular cloning of a series of glycoconjugate sulfotransferases, which are classified into several groups with regard to substrate specificities and primary structures. 6-O-Sulfation of N-acetylglucosamine (GlcNAc)1 residues, which occurs during the synthesis of keratan sulfate (1Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1680) Google Scholar) and is also observed in N-linked (7Spiro R.G. Bhoyroo V.D. J. Biol. Chem. 1988; 263: 14351-14358Abstract Full Text PDF PubMed Google Scholar) and O-linked (8Lo-Guidice J.M. Wieruszeski J.M. Lemoine J. Verbert A. Roussel P. Lamblin G. J. Biol. Chem. 1994; 269: 18794-18813Abstract Full Text PDF PubMed Google Scholar, 9Rosen S.D. Am. J. Pathol. 1999; 155: 1013-1020Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) glycans, is catalyzed by GlcNAc-6-O-sulfotransferase (GlcNAc6ST). The enzymatic activities of GlcNAc6ST in the rat liver (10Spiro R.G. Yasumoto Y. Bhoyroo V. Biochem. J. 1996; 319: 209-216Crossref PubMed Scopus (45) Google Scholar), human respiratory mucosa (11Degroote S. Lo-Guidice J.M. Strecker G. Ducourouble M.P. Roussel P. Lamblin G. J. Biol. Chem. 1997; 272: 29493-29501Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and porcine lymph nodes (12Bowman K.G. Hemmerich S. Bhakta S. Singer M.S. Bistrup A. Rosen S.D. Bertozzi C.R. Chem. Biol. 1998; 5: 447-460Abstract Full Text PDF PubMed Scopus (44) Google Scholar) were characterized, and it was suggested that GlcNAc6ST recognizes non-reducing GlcNAc but not internal GlcNAc residues inN-acetyllactosamine-based oligosaccharides. We have previously cloned mouse (13Uchimura K. Muramatsu H. Kadomatsu K. Fan Q.W. Kurosawa N. Mitsuoka C. Kannagi R. Habuchi O. Muramatsu T. J. Biol. Chem. 1998; 273: 22577-22583Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and human (14Uchimura K. Muramatsu H. Kaname T. Ogawa H. Yamakawa T. Fan Q.W. Mitsuoka C. Kannagi R. Habuchi O. Yokoyama I. Yamamura K. Ozaki T. Nakagawara A. Kadomatsu K. Muramatsu T. J. Biochem. 1998; 124: 670-678Crossref PubMed Scopus (93) Google Scholar) GlcNAc6STs based on their sequence similarity to chondroitin 6-sulfotransferase (15Fukuta M. Uchimura K. Nakashima K. Kato M. Kimata K. Shinomura T. Habuchi O. J. Biol. Chem. 1995; 270: 18575-18580Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 16Uchimura K. Kadomatsu K. Fan Q.W. Muramatsu H. Kurosawa N. Kaname T. Yamamura K. Fukuta M. Habuchi O. Muramatsu T. Glycobiology. 1998; 8: 489-496Crossref PubMed Scopus (43) Google Scholar). In our previous study (13Uchimura K. Muramatsu H. Kadomatsu K. Fan Q.W. Kurosawa N. Mitsuoka C. Kannagi R. Habuchi O. Muramatsu T. J. Biol. Chem. 1998; 273: 22577-22583Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), N-acetyllactosamine oligomer, GlcNAcβ1–3Galβ1–4GlcNAc, was used as an acceptor for the assay of the expressed enzyme. Subsequently, another member of the GlcNAc6ST family was isolated and designated as HEC-GlcNAc6ST/LSST (17Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.R. Huang C.C. Kannagi R. Rosen S.D. Hemmerich S. J. Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (247) Google Scholar, 18Hiraoka N. Petryniak B. Nakayama J. Tsuboi S. Suzuki M. Yeh J.C. Izawa D. Tanaka T. Miyasaka M. Lowe J.B. Fukuda M. Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). HEC-GlcNAc6ST/LSST is preferentially expressed in high endothelial venules. Both GlcNAc6STs are involved in the synthesis of sialyl-6-sulfo-Lewis-X, a key structure in l-selectin ligands (17Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.R. Huang C.C. Kannagi R. Rosen S.D. Hemmerich S. J. Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (247) Google Scholar, 18Hiraoka N. Petryniak B. Nakayama J. Tsuboi S. Suzuki M. Yeh J.C. Izawa D. Tanaka T. Miyasaka M. Lowe J.B. Fukuda M. Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 19Kimura N. Mitsuoka C. Kanamori A. Hiraiwa N. Uchimura K. Muramatsu T. Tamatani T. Kansas G.S. Kannagi R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4530-4535Crossref PubMed Scopus (116) Google Scholar). The third member of the GlcNAc6ST family, I-GlcNAc6ST, is preferentially expressed in the intestinal tissues (20Lee J.K. Bhakta S. Rosen S.D. Hemmerich S. Biochem. Biophys. Res. Commun. 1999; 263: 543-549Crossref PubMed Scopus (80) Google Scholar), whereas the fourth member, C6ST-2/GST-5/GlcNAc6ST-4, is expressed in many organs (21Kitagawa H. Fujita M. Ito N. Sugahara K. J. Biol. Chem. 2000; 275: 21075-21080Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 22Bhakta S. Bartes A. Bowman K.G. Kao W.-M. Polsky I. Lee J.K. Cook B.N. Bruehl R.E. Rosen S.D. Bertozzi C.R. Hemmerich S. J. Biol. Chem. 2000; 275: 40226-40234Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 23Uchimura K. Fasakhany F. Kadomatsu K. Matsukawa T. Yamakawa T. Kurosawa N. Muramatsu T. Biochem. Biophys. Res. Commun. 2000; 274: 291-296Crossref PubMed Scopus (38) Google Scholar) as GlcNAc6ST (13Uchimura K. Muramatsu H. Kadomatsu K. Fan Q.W. Kurosawa N. Mitsuoka C. Kannagi R. Habuchi O. Muramatsu T. J. Biol. Chem. 1998; 273: 22577-22583Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 14Uchimura K. Muramatsu H. Kaname T. Ogawa H. Yamakawa T. Fan Q.W. Mitsuoka C. Kannagi R. Habuchi O. Yokoyama I. Yamamura K. Ozaki T. Nakagawara A. Kadomatsu K. Muramatsu T. J. Biochem. 1998; 124: 670-678Crossref PubMed Scopus (93) Google Scholar). Recently, a fifth member, C-GlcNAc6ST, expressed in the human cornea was found (24Akama T.O. Nishida K. Nakayama J. Watanabe H. Ozaki K. Nakamura T. Dota A. Kawasaki S. Inoue Y. Maeda N. Yamamoto S. Fujiwara T. Thonar E.J.-M.A. Shimomura Y. Kinoshita S. Tanigami A. Fukuda M.N. Nat. Genet. 2000; 26: 237-241Crossref PubMed Scopus (224) Google Scholar). The mutation in the enzyme gene has been identified as a cause of macular cornial dystrophy. Despite the highly significant roles of GlcNAc6STs, their specificities and enzymological properties have not been elucidated. In the investigation reported here, we produced protein A fusion proteins of human GlcNAc6STs and comparatively analyzed their substrate specificities as well as enzymological properties. The human GlcNAc6STs studied were GlcNAc6ST (14Uchimura K. Muramatsu H. Kaname T. Ogawa H. Yamakawa T. Fan Q.W. Mitsuoka C. Kannagi R. Habuchi O. Yokoyama I. Yamamura K. Ozaki T. Nakagawara A. Kadomatsu K. Muramatsu T. J. Biochem. 1998; 124: 670-678Crossref PubMed Scopus (93) Google Scholar, 25Li X. Tedder T.F. Genomics. 1999; 55: 345-347Crossref PubMed Scopus (47) Google Scholar), HEC-GlcNAc6ST (17Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.R. Huang C.C. Kannagi R. Rosen S.D. Hemmerich S. J. Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (247) Google Scholar), and I-GlcNAc6ST (20Lee J.K. Bhakta S. Rosen S.D. Hemmerich S. Biochem. Biophys. Res. Commun. 1999; 263: 543-549Crossref PubMed Scopus (80) Google Scholar) (for the sake of simplicity, here we call these enzymes according to the order of cloning, GlcNAc6ST-1, GlcNAc6ST-2, and GlcNAc6ST-3, respectively). The following materials were obtained commercially from the sources indicated. [35S]PAPS (1.9 Ci/mmol) was from PerkinElmer Life Sciences; GlcNAcβ1–6ManOMe, 6-O-sulfated GlcNAc, and 3-O-sulfated GlcNAc were from Sigma; GlcNAcβ1–2Man was from Dextra Laboratories (Reading, UK); GlcNAcβ1–6[Galβ1–3]GalNAc-pNP and GlcNAcβ1–3GalNAc-pNP were from Toronto Research Chemicals (North York, Ontario, Canada); and Streptococcus β-galactosidase and jack bean β-N-acetylhexosaminidase were from Seikagaku Corporation (Tokyo, Japan). GlcNAcβ1–3Galβ1–4Glc and GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc were prepared from Galβ1–4GlcNAcβ1–3Galβ1–4Glc and Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc, respectively, by β-galactosidase digestion as described previously (13Uchimura K. Muramatsu H. Kadomatsu K. Fan Q.W. Kurosawa N. Mitsuoka C. Kannagi R. Habuchi O. Muramatsu T. J. Biol. Chem. 1998; 273: 22577-22583Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The human HEC-GlcNAc6ST cDNA (17Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.R. Huang C.C. Kannagi R. Rosen S.D. Hemmerich S. J. Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (247) Google Scholar) containing the open reading frame and a FLAG-tag sequence at its 5′ region was introduced into the HindIII site of the pcDNA3.1 vector (Invitrogen). The constructed vector, pcDNA3.1(+)/FLAG-GST-3, and recombinant plasmids, pcDNA3-GlcNAc6ST (13Uchimura K. Muramatsu H. Kadomatsu K. Fan Q.W. Kurosawa N. Mitsuoka C. Kannagi R. Habuchi O. Muramatsu T. J. Biol. Chem. 1998; 273: 22577-22583Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and pcDNA3.1-human GST-4 (20Lee J.K. Bhakta S. Rosen S.D. Hemmerich S. Biochem. Biophys. Res. Commun. 1999; 263: 543-549Crossref PubMed Scopus (80) Google Scholar), were used as templates for PCR to amplify truncated forms of GlcNAc6STs. To amplify the cDNAs encoding the truncated forms of GlcNAc6ST-1 lacking the first 35 amino acids, GlcNAc6ST-2 lacking the first 29 amino acids, or GlcNAc6ST-3 lacking the first 32 amino acids, 5′ and 3′ primer sets were used, respectively, as follows: a 5′ primer containing an in-frame EcoRI site (5′-TGGAATTCCTGCAGCAGTGCAACCCCGAT-3′) and a 3′ primer containing anEcoRI site (5′-GAGAATTCTTAGAGACGGGGCTTCCGA-3′); a 5′ primer containing an in-frame EcoRV site (5′-ATGTACGATATCCAACATCAGCTCCCTGTCT-3′) and a 3′ primer containing anEcoRV site (5′-CGAAGCGATATCCTTAGTGGATTTGCTCAGGGAC-3′); and a 5′ primer containing an in-frame EcoRI site (5′-CTGAATTCTCATCCCCAGCCGGCGGCGAG-3′) and a 3′ primer containing anEcoRI site (5′-CTGAATTCTCAGTCAGGCGATGCCCAGCT-3′). The amplification was carried out at 94 °C for 3 min with 35 cycles of 94 °C for 0.5 min, 56 °C for 0.5 min, and 72 °C for 1 min. The amplified fragments of GlcNAc6ST-1 and GlcNAc6ST-3 were subcloned into the EcoRI site of the fusion vector pcDSA (26Kojima N. Yoshida Y. Kurosawa N. Lee Y.-C. Tsuji S. FEBS Lett. 1995; 360: 1-4Crossref PubMed Scopus (104) Google Scholar) to yield pcDSA-GlcNAc6ST1 and pcDSA-GlcNAc6ST3, respectively. The amplified fragment of GlcNAc6ST-2 was digested with EcoRV and then subcloned into the blunted EcoRI site of the pcDSA. The obtained plasmid was named pcDSA-GlcNAc6ST2. In all cases, the plasmids encoded fusion proteins of GlcNAc6STs to the IgM signal sequence and protein A in the N-terminal region. COS-7 cells on 10-cm dishes were transfected with 4 μg of relevant plasmids using LipofectAMINE PLUS (Invitrogen) according to manufacturer instructions. After 24 h of culture in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, the medium was replaced with Dulbecco's modified Eagle's medium containing 2% IgG-free fetal calf serum. The cells were cultured for an additional 48 h. Subsequently, the culture medium was collected, and the protein A fusion GlcNAc6STs expressed in the medium were adsorbed to IgG-Sepharose (10 μl of resin/10 ml of culture medium) at 4 °C for 3 h. The resin was collected by centrifugation and washed three times with Dulbecco's phosphate-buffered saline. Finally, the resin was suspended in 30 μl of 50 mm Tris-HCl, pH 7.5, and used as the enzyme. The standard reaction mixture contained 1 μmol of Tris-HCl, pH 7.5, 0.2 μmol of MnCl2, 0.04 μmol of AMP, 2 μmol of NaF, 20 nmol of oligosaccharides, 150 pmol of [35S]PAPS (1.5 × 106 cpm), 0.05% of Triton X-100, and 1 μl of the fusion protein suspension in a final volume of 20 μl. After incubation at 30 °C for 1 h, aliquots of 2 μl of the reaction mixture were applied to TLC plates, which were then developed with ethanol-pyridine-n-butyl alcohol-water-acetate (100:10:10:30:3 (v/v)), and the radioactivity was visualized with a BAS2000 bioimaging analyzer (Fuji Film, Tokyo, Japan). For analysis of substrate specificity, half-aliquots of the reaction mixture were subjected to Superdex 30 gel chromatography to confirm the radioactivity of the35S-labeled products as described previously (13Uchimura K. Muramatsu H. Kadomatsu K. Fan Q.W. Kurosawa N. Mitsuoka C. Kannagi R. Habuchi O. Muramatsu T. J. Biol. Chem. 1998; 273: 22577-22583Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Digestion with jack bean N-acetylhexosaminidase and TLC of the products were performed as described previously (23Uchimura K. Fasakhany F. Kadomatsu K. Matsukawa T. Yamakawa T. Kurosawa N. Muramatsu T. Biochem. Biophys. Res. Commun. 2000; 274: 291-296Crossref PubMed Scopus (38) Google Scholar). Sulfotransferase reactions of GlcNAc6ST-1, GlcNAc6ST-2, and GlcNAc6ST-3 toward various oligosaccharides analyzed in this study proceeded linearly up to 4 h under the standard assay conditions. Surgical specimens were obtained from two colon cancer patients during surgery and processed as described previously (27Kumamoto K. Goto Y. Sekikawa K. Takenoshita S. Ishida N. Kawakita M. Kannagi R. Cancer Res. 2001; 61: 4620-4627PubMed Google Scholar). These cases were histologically diagnosed as mucinous adenocarcinoma. Malignant and non-malignant portions of the specimen were used for RNA extraction. Sample was frozen rapidly and stored at −80 °C until extraction of total RNA. The specimen was powdered in liquid nitrogen, and total cellular RNA was extracted with guanidine isothiocyanate and purified by cesium chloride gradient centrifugation. Cultured human colon cancer cell lines LS180 and LS174T, which have characteristics of mucinous adenocarcinoma (28Tom B.H. Rutzky L.P. Oyasu R. Tomita J.T. Goldenberg D.M. Kahan B.D. J. Natl. Cancer Inst. 1977; 58: 1507-1512Crossref PubMed Scopus (36) Google Scholar, 29Bresalier R.S. Niv Y. Byrd J.C. Duh Q.Y. Toribara N.W. Rockwell R.W. Dahiya R. Kim Y.S. J. Clin. Invest. 1991; 87: 1037-1045Crossref PubMed Scopus (196) Google Scholar) were maintained in RPMI 1640 medium (Invitrogen) containing 10% fetal calf serum at 37 °C in a humidified atmosphere of 5% CO2 in air. Total RNA was isolated from 1 × 106 cells according to the acid guanidinium thiocyanate-chloroform extraction method using an Isogen kit (Nippon Gene, Tokyo, Japan), and the amount of RNA was quantitated by spectrophotometry at 260 nm. The first strand cDNA was synthesized by reverse transcription of the extracted total RNA (5 μg) using the first strand cDNA synthesis kit (Invitrogen) according to manufacturer protocol. One microliter of the retrotranscription reaction was subjected to PCR amplification using sulfotransferases primers. For detection of GlcNAc6ST-1 mRNA, the upstream primer 5′-CTTAAGGTCATCCACTTGGTGCG-3′ and the downstream primer 5′-GGGTCTTGCTGAGGTCTTTGACC-3′ were used. For GlcNAc6ST-2 detection, the upstream primer 5′-GCAGCATGAGCAGAAACTCAAG-3′ and the downstream primer 5′-TCCAGGTAGACAGAAGATCCAG-3′ were used. For GlcNAc6ST-3, the upstream primer 5′-CAAGACAGTGACAGTGCTCC-3′ and the downstream primer 5′-TACGTCCTGCTTGCTGATGG-3′ were used. The cycle numbers most suitable for semiquantitative RT-PCR were determined in preliminary experiments, i.e. 30 cycles for GlcNAc6ST-1, 35 cycles for GlcNAc6ST-2, and 30 cycles for GlcNAc6ST-3. As an internal control for the RT-PCR analysis, the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) transcripts were amplified from the same cDNA samples. The upstream and downstream for G3PDH genes were 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ and 5′-CATGTGGGCCATGAGGTCCACCAC-3′, respectively. Aliquots of each reaction product were fractionated by electrophoresis through a 2% agarose gel containing ethidium bromide. The intensities of the bands were quantified by the Densitograph apparatus and software (AE-6920WLSA, ATTO, Tokyo, Japan). The production of 300.19 cells stably expressing l-selectin or E-selectin has been described previously (30Kansas G.S. Ley K. Munro J.M. Tedder T.F. J. Exp. Med. 1993; 177: 833-838Crossref PubMed Scopus (185) Google Scholar). The interaction of 300.19/l-selectin cells was assayed in a parallel plate flow chamber (Glycotech, Rockville, MD). Monolayers of LS12 cells (19Kimura N. Mitsuoka C. Kanamori A. Hiraiwa N. Uchimura K. Muramatsu T. Tamatani T. Kansas G.S. Kannagi R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4530-4535Crossref PubMed Scopus (116) Google Scholar) were grown in 35-mm tissue culture plates and served as the rolling substrate. LS12 cells are cloned ECV304 cells stably transfected with both GlcNAc6ST-1 and fucosyltransferase VII (Fuc-T VII) cDNAs and have previously been shown to significantly adhere to l-selectin-expressing cells in non-static monolayer adhesion assays (19Kimura N. Mitsuoka C. Kanamori A. Hiraiwa N. Uchimura K. Muramatsu T. Tamatani T. Kansas G.S. Kannagi R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4530-4535Crossref PubMed Scopus (116) Google Scholar). Cells were introduced into the flow chamber at a concentration of 106cells/ml in a buffer of RPMI 1640 medium supplemented with 0.1% serum. The shear stress in the flow chamber was maintained constant at 1.0 dyne/cm2 using a syringe pump (Harvard Apparatus, Holliston, MA), and images were obtained using a Nikon Eclipse TE300-inverted microscope (Nikon, Tokyo, Japan). Data analysis was performed using Celltrak software developed by Compix (Cranberry Township, PA) as described previously (31Knibbs R.N. Craig R.A. Natsuka S. Chang A. Cameron M. Lowe J.B. Stoolman L.M. J. Cell Biol. 1996; 133: 911-920Crossref PubMed Scopus (158) Google Scholar, 32Snapp K.R. Heitzig C.E. Ellies L.G. Marth J.D. Kansas G.S. Blood. 2001; 97: 3806-3811Crossref PubMed Scopus (77) Google Scholar). A rolling event is defined as a rolling cell that can be tracked between sequential images separated by a defined time delay. The time defined here is 0.5 sec. Velocities of individual rolling cells were also simultaneously obtained during analysis, and the mean rolling velocity of the rolling cell population was determined. The total number of rolling events and velocities were collected for 50–100 sequential images. Each experiment was repeated at least four times. COS-7 cells stably transfected with Fuc-T VII and/or GlcNAc6ST-1 were prepared using pRC/CMV-FT VII and/or pIRES1hyg-6ST-1 by employing methods similar to those described for ECV304 transfectant cells (19Kimura N. Mitsuoka C. Kanamori A. Hiraiwa N. Uchimura K. Muramatsu T. Tamatani T. Kansas G.S. Kannagi R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4530-4535Crossref PubMed Scopus (116) Google Scholar). Rolling assays for COS-7 transfectant cells were performed using the flow chamber as described by Lawrence et al. (33Lawrence M.B. Mclntire L.V. Eskin S.G. Blood. 1987; 70: 1284-1290Crossref PubMed Google Scholar), a syringe pump (Model 944, Harvard Apparatus), and an Olympus IX70−22FL/PH inverted microscope equipped with air-curtain incubator (Model IX-IBM, Olympus, Tokyo, Japan) at a constant shear stress of 1.0 dyne/cm2. The digital images were recorded with a digital video camera system (Model CS-220, Olympus). Expression of L-selectin ligands on the transfected cells was ascertained by flow cytometric analyses using the G152 antibody (murine IgM) specific to sialyl-6-sulfo-Lewis-X according to the methods described previously (19Kimura N. Mitsuoka C. Kanamori A. Hiraiwa N. Uchimura K. Muramatsu T. Tamatani T. Kansas G.S. Kannagi R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4530-4535Crossref PubMed Scopus (116) Google Scholar). We compared enzymological properties of GlcNAc6ST-1, -2, and -3 to determine whether there are significant differences among the three enzymes. The activities of GlcNAc6ST-1 and GlcNAc6ST-2 were measured by using GlcNAcβ1–6ManOMe as a substrate, whereas core 2 was used as a substrate of GlcNAc6ST-3. GlcNAc6ST-1, -2, and -3 showed an optimum pH of approximately 7.5–8.0. GlcNAc6ST-3 activity was higher in sodium HEPES than Tris-HCl buffer, whereas GlcNAc6ST-1 and GlcNAc6ST-2 showed better activity in Tris-HCl buffer. With respect to the effects of divalent cations at a concentration of 10 mm, GlcNAc6ST-1 activity was stimulated 1.8-fold by the addition of Mn2+, whereas Mn2+ showed no effects on GlcNAc6ST-2 and inhibited GlcNAc6ST-3 to one-third of the activity without it. Ca2+ and Cu2+ strongly inhibited all three enzymes. Co2+ showed strong inhibitory effects on GlcNAc6ST-2 and GlcNAc6ST-3 but not on GlcNAc6ST-1. The Km value of GlcNAc6ST-1 for GlcNAcβ1–6ManOMe as an acceptor was lower than that of GlcNAc6ST-2 (TableI), indicating that GlcNAc6ST-1 has a higher affinity to mannosyl-linked GlcNAc residues than GlcNAc6ST-2. Km values for the core 2 oligosaccharide were the same between GlcNAc6ST-1 and GlcNAc6ST-2, whereas the value for GlcNAc6ST-3 was the lowest. GlcNAc6ST-1 had the lowest Km value for PAPS as the sulfate donor among the three enzymes (Table I). Both protein A-fused soluble GlcNAc6ST-1 and full-length GlcNAc6ST-1 as microsomal fractions of the cells transfected with pcDNA3-GlcNAc6ST showed the same enzymological properties, namely kinetical parameters and effects of pH and divalent cations (data not shown). Because microsomal fraction gave only weak activities of GlcNAc6ST-2 and GlcNAc6ST-3, production of the fusion protein became necessary to determine the activities of these enzymes precisely. Thus, fusion proteins were used to compare the properties of the three GlcNAc6STs.Table IComparison of KM values for sulfate acceptor and donor among GlcNAc6ST-1, GlcNAc6ST-2, and GlcNAc6ST-3SubstrateKMvalues1-aData are representative of one of two series of independent experiments, which exhibited essentially identical results.GlcNAc6ST-1GlcNAc6ST-2GlcNAc6ST-3mMmMmMGlcNAcβ1–6ManOMe0.21.14n.d.1-bn.d., not determined.GlcNAcβ1–6[Galβ1–3]GalNAc-pNP (core 2)0.410.410.2PAPS0.00121-cThe acceptor substrate was GlcNAcβ1–6ManOMe.0.00591-cThe acceptor substrate was GlcNAcβ1–6ManOMe.0.0141-dThe acceptor substrate was GlcNAcβ1–6[Galβ1–3]GalNAc-pNP (core 2).1-a Data are representative of one of two series of independent experiments, which exhibited essentially identical results.1-b n.d., not determined.1-c The acceptor substrate was GlcNAcβ1–6ManOMe.1-d The acceptor substrate was GlcNAcβ1–6[Galβ1–3]GalNAc-pNP (core 2). Open table in a new tab The series of oligosaccharides shown in TableII was tested as acceptors to evaluate the substrate specificities of GlcNAc6ST-1, -2, and -3. The TLC of 35S-labeled products revealed that both GlcNAc6ST-1 and GlcNAc6ST-2 utilized efficiently GlcNAcβ1–6Man, GlcNAcβ1–2Man, and core 2 oligosaccharide (GlcNAcβ1–6[Galβ1–3]GalNAc-pNP) as acceptors (Fig.1, Table II). As microsomal fractions of the cells transfected with the expression vector, GlcNAc6ST-1 showed the same specificity as that of the protein A-fused enzyme (data not shown). As compared with GlcNAc6ST-1, GlcNAc6ST-2 showed higher activity against GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc. The most notable difference was that GlcNAc6ST-2 used core 3 oligosaccharide (GlcNAcβ1–3GalNAc-pNP) as an acceptor, whereas GlcNAc6ST-1 did not (Fig. 1 and Table II). In contrast, GlcNAc6ST-3 acted only on core 2 and did not act on GlcNAcβ1–6Man, GlcNAcβ1–2Man, core 3 oligosaccharide, or GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Gal β1–4GlcNAc. Quantitative analysis revealed that both GlcNAc6ST-1 and GlcNAc6ST-2 utilized core 2 oligosaccharide, GlcNAcβ1–6Man, and GlcNAcβ1–2Man structures with similar velocities (Table II).Table IIComparison of the substrate specificities of GlcNAc6ST-1, GlcNAc6ST-2, and GlcNAc6ST-3 secreted into the culture medium by transfected COS-7 cellsAcceptorEnzyme activity2-aThe values represent the averages of two independent experiments. pmol/h/ml of medium (%)2-bThe percentage of the activity compared to that of GlcNAcβ1–6ManOMe is also shown.GlcNAc6ST-1GlcNAc6ST-2GlcNAc6ST-3GlcNAcβ1–6ManOMe20.6 (100)6.8 (100)N.D.2-cN.D. indicates <0.1 pmol/h/ml of medium.GlcNAcβ1–2Man26.3 (128)10.3 (151)N.D.2-cN.D. indicates <0.1 pmol/h/ml of medium.GlcNAcβ1–6[Galβ1–3]GalNAc-pNP (core 2)39.3 (191)9.9 (145)5.72-dThe actual observed radioactivities were approximately 30,000 cpm, whereas the assay without the enzyme or with IgG-Sepharose exposed to the culture supernatant of mock-transfected cells gave values <500 cpm.GlcNAcβ1–3GalNAc-pNP (core 3)N.D.2-cN.D. indicates <0.1 pmol/h/ml of medium.12.5 (184)N.D.2-cN.D. indicates <0.1 pmol/h/ml of medium.GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc4.3d (21)5.0d (73)N.D.2-cN.D. indicates <0.1 pmol/h/ml of medium.2-a The values represent the averages of two independent experiments.2-b The percentage of the activity compared to that of GlcNAcβ1–6ManOMe is also shown.2-c N.D. indicates <0.1 pmol/h/ml of medium.2-d The actual observed radioactivities were approximately 30,000 cpm, whereas the assay without the enzyme or with IgG-Sepharose exposed to the culture supernatant of mock-transfected cells gave values <500 cpm. Open table in a new tab Among three GlcNAc6STs examined in this study, only GlcNAc6ST-2 acted on core 3. Because Yamashita and co-workers (34Seko A. Sumiya J. Yonezawa S. Nagata K. Yamashita K. Glycobiology. 2000; 10: 919-929Crossref PubMed Scopus (19) Google Scholar) recently reported that a GlcNAc6ST acting on core 3 is specifically expressed in mucinous adenocarcinoma of the colon, we examined whether GlcNAc6ST-2 was expressed in the tumor. Ind" @default.
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• W2107155846 title "Specificities ofN-Acetylglucosamine-6-O-sulfotransferases in Relation to L-selectin Ligand Synthesis and Tumor-associated Enzyme Expression" @default.
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• W2107155846 doi "https://doi.org/10.1074/jbc.m106587200" @default.
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