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• W2052522729 abstract "We have previously cloned chondroitin 6-sulfotransferase (C6ST) cDNA from chick embryo chondrocytes. C6ST catalyzes sulfation of chondroitin, keratan sulfate, and sialyl N-acetyllactosamine oligosaccharides. In this study, we report the cloning and characterization of a novel sulfotransferase that catalyzes sulfation of keratan sulfate. This new sulfotransferase cDNA clone was obtained from a human fetal brain library by cross-hybridization with chick C6ST cDNA. The cDNA clone obtained contains a single open reading frame that predicts a type II transmembrane protein composed of 411 amino acid residues. When the cDNA was introduced into a eukaryotic expression vector and transfected in COS-7 cells, keratan sulfate sulfotransferase activity was overexpressed, but C6ST activity was not increased over that of the control. Structural analysis of 35S-labeled glycosaminoglycan, which was formed from keratan sulfate by the reaction with 35S-labeled 3′-phosphoadenosine 5′-phosphosulfate and the recombinant sulfotransferase, showed that keratan sulfate was sulfated at position 6 of Gal residues. On the basis of the acceptor substrate specificity, we propose keratan sulfate Gal-6-sulfotransferase (KSGal6ST) for the name of the newly cloned sulfotransferase. KSGal6ST was assigned to chromosome 11p11.1–11.2 by fluorescence in situ hybridization. Among various human adult tissues, a 2.8-kilobase message of KSGal6ST was expressed mainly in the brain. When poly(A)+ RNAs from the chick embryo cornea and brain were probed with the human KSGal6ST cDNA in Northern hybridization, a clear band with about 2.8 kilobases was detected. These observations suggest that KSGal6ST may participate in the biosynthesis of keratan sulfate in the brain and cornea. We have previously cloned chondroitin 6-sulfotransferase (C6ST) cDNA from chick embryo chondrocytes. C6ST catalyzes sulfation of chondroitin, keratan sulfate, and sialyl N-acetyllactosamine oligosaccharides. In this study, we report the cloning and characterization of a novel sulfotransferase that catalyzes sulfation of keratan sulfate. This new sulfotransferase cDNA clone was obtained from a human fetal brain library by cross-hybridization with chick C6ST cDNA. The cDNA clone obtained contains a single open reading frame that predicts a type II transmembrane protein composed of 411 amino acid residues. When the cDNA was introduced into a eukaryotic expression vector and transfected in COS-7 cells, keratan sulfate sulfotransferase activity was overexpressed, but C6ST activity was not increased over that of the control. Structural analysis of 35S-labeled glycosaminoglycan, which was formed from keratan sulfate by the reaction with 35S-labeled 3′-phosphoadenosine 5′-phosphosulfate and the recombinant sulfotransferase, showed that keratan sulfate was sulfated at position 6 of Gal residues. On the basis of the acceptor substrate specificity, we propose keratan sulfate Gal-6-sulfotransferase (KSGal6ST) for the name of the newly cloned sulfotransferase. KSGal6ST was assigned to chromosome 11p11.1–11.2 by fluorescence in situ hybridization. Among various human adult tissues, a 2.8-kilobase message of KSGal6ST was expressed mainly in the brain. When poly(A)+ RNAs from the chick embryo cornea and brain were probed with the human KSGal6ST cDNA in Northern hybridization, a clear band with about 2.8 kilobases was detected. These observations suggest that KSGal6ST may participate in the biosynthesis of keratan sulfate in the brain and cornea. Keratan sulfate proteoglycans (lumican and keratocan) are present in the cornea as the major class of proteoglycan (1Blochberger T.C. Vergnes J.-P. Hempel J. Hassell J.R. J. Biol. Chem. 1992; 267: 347-352Abstract Full Text PDF PubMed Google Scholar, 2Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) and are thought to play an important role in the corneal transparency (3Funderburgh J.L. Funderburgh M.L. Mann M.M. Conrad G.W. Biochem. Soc. Trans. 1991; 19: 871-876Crossref PubMed Scopus (47) Google Scholar). A synaptic vesicle membrane glycoprotein, SV2, has been shown to be a keratan sulfate proteoglycan (4Scranton T.W. Iwata M. Carlson S.S. J. Neurochem. 1993; 61: 29-44Crossref PubMed Scopus (80) Google Scholar). Aggrecan from the cartilage (5Wight T.N. Heinegård D.K. Hascall V.C. Hay E.D. Cell Biology of Extracellular Matrix. Plenum, New York1991: 45-78Crossref Google Scholar) and 3H1 proteoglycan from adult brain (6Rauch U. Gao P. Janetzko A. Flaccus A. Hilgenberg L. Tekotte H. Margolis R.K. Margolis R.U. J. Biol. Chem. 1991; 266: 14785-14801Abstract Full Text PDF PubMed Google Scholar) contain both chondroitin sulfate and keratan sulfate. Sulfate group of keratan sulfate appears to be important for the biological function of keratan sulfate, because degree of the sulfation of keratan sulfate increased during the corneal development (7Hart G.W. J. Biol. Chem. 1976; 251: 6513-6521Abstract Full Text PDF PubMed Google Scholar, 8Nakazawa K. Suzuki S. Wada K. Nakazawa K. J. Biochem. (Tokyo). 1995; 117: 707-718Crossref PubMed Scopus (27) Google Scholar) and undersulfated keratan sulfate is synthesized by macular corneal dystrophy (9Nakazawa K. Hassell J.R. Hascall V.C. Lohmander L.S. Newsome D.A. Krachmer J. J. Biol. Chem. 1984; 259: 13751-13757Abstract Full Text PDF PubMed Google Scholar). Keratan sulfate bears sulfate groups on both GlcNAc and Gal residues. Sulfotransferase activity responsible for the sulfation of keratan sulfate was previously reported (10Rüter E.-R. Kresse H. J. Biol. Chem. 1984; 259: 11771-11776Abstract Full Text PDF PubMed Google Scholar), but specificity of the enzyme remains obscure because no purified keratan sulfate sulfotransferase (KSST) 1The abbreviations used are: KSST, keratan sulfate sulfotransferase; C6ST, chondroitin 6-sulfotransferase; C4ST, chondroitin 4-sulfotransferase; KSGal6ST, keratan sulfate Gal-6-sulfotransferase; PAPS, 3′-phosphoadenosine 5′-phosphosulfate; (6S), 6-sulfate; HPLC, high performance liquid chromatography; GalR, d-galactitol; GlcNAcR,N-acetyl-d-glucosaminitol; CDSNS-heparin, completely desulfated N-resulfated heparin; ΔDi-OSR, 2-acetamide-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-d-galactitol; DMEM, Dulbecco's modified Eagle's medium; SSPE, sodium chloride/sodium phosphate/EDTA buffer; MOPS, 4-morpholinepropanesulfonic acid; kb, kilobase pair(s). 1The abbreviations used are: KSST, keratan sulfate sulfotransferase; C6ST, chondroitin 6-sulfotransferase; C4ST, chondroitin 4-sulfotransferase; KSGal6ST, keratan sulfate Gal-6-sulfotransferase; PAPS, 3′-phosphoadenosine 5′-phosphosulfate; (6S), 6-sulfate; HPLC, high performance liquid chromatography; GalR, d-galactitol; GlcNAcR,N-acetyl-d-glucosaminitol; CDSNS-heparin, completely desulfated N-resulfated heparin; ΔDi-OSR, 2-acetamide-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-d-galactitol; DMEM, Dulbecco's modified Eagle's medium; SSPE, sodium chloride/sodium phosphate/EDTA buffer; MOPS, 4-morpholinepropanesulfonic acid; kb, kilobase pair(s). has so far been obtained. We have previously purified and cloned chondroitin 6-sulfotransferase (C6ST) from the culture medium of chick embryo chondrocytes (11Habuchi O. Matsui Y. Kotoya Y. Aoyama Y. Yasuda Y. Noda M. J. Biol. Chem. 1993; 268: 21968-21974Abstract Full Text PDF PubMed Google Scholar, 12Fukuta 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). We found that C6ST catalyzes sulfation of chondroitin, keratan sulfate, and sialyl N-acetyllactosamine oligosaccharides (11Habuchi O. Matsui Y. Kotoya Y. Aoyama Y. Yasuda Y. Noda M. J. Biol. Chem. 1993; 268: 21968-21974Abstract Full Text PDF PubMed Google Scholar, 13Habuchi O. Hirahara Y. Uchimura K. Fukuta M. Glycobiology. 1996; 6: 51-57Crossref PubMed Scopus (45) Google Scholar,14Habuchi O. Suzuki Y. Fukuta M. Glycobiology. 1997; 7: 405-412Crossref PubMed Scopus (43) Google Scholar). This enzyme may, therefore, participate in the biosynthesis of both chondroitin sulfate and keratan sulfate in tissues such as cartilage, in which both chondroitin 6-sulfate and keratan sulfate are actively synthesized. On the other hand, in the developing cornea, keratan sulfate is actively synthesized, but synthetic activity of chondroitin 6-sulfate seems to be minimal (7Hart G.W. J. Biol. Chem. 1976; 251: 6513-6521Abstract Full Text PDF PubMed Google Scholar, 8Nakazawa K. Suzuki S. Wada K. Nakazawa K. J. Biochem. (Tokyo). 1995; 117: 707-718Crossref PubMed Scopus (27) Google Scholar). In addition, the expression of C6ST mRNA was found to be much weaker in the chick cornea compared with that in cartilage (13Habuchi O. Hirahara Y. Uchimura K. Fukuta M. Glycobiology. 1996; 6: 51-57Crossref PubMed Scopus (45) Google Scholar). These observations suggest the possible existence of a different sulfotransferase in the cornea, which catalyzes mainly the sulfation of keratan sulfate. In this study we report cloning of a novel sulfotransferase cDNA that encodes a protein with sulfotransferase activity toward keratan sulfate. This sulfotransferase transferred sulfate to position 6 of the Gal residue of keratan sulfate but showed no activity toward chondroitin. The following commercial materials were used: H235SO4 was from DuPont NEN; [3H]NaBH4 (16.3 GBq/mmol) [α-32P]dCTP (110 TBq/mmol) and Hybond N+ were from Amersham Japan, Tokyo; the fetal human brain cDNA library and human multiple tissue Northern blots were fromCLONTECH, Palo Alto, CA; unlabeled PAPS,N-acetylglucosamine 6-sulfate, and galactose 6-sulfate were from Sigma; Hiload Superdex 30 HR 16/60, DEAE-Sephadex, and fast desalting column HR 10/10 were from Pharmacia Biotech, Tokyo; chondroitinase ACII, keratanase II, chondroitin sulfate A (whale cartilage), chondroitin sulfate C (shark cartilage), dermatan sulfate, and completely desulfated N-resulfated heparin (CDSNS-heparin) were from Seikagaku Corporation, Tokyo; Partisil SAX-10 was from Whatman. Keratan sulfate from bovine cornea was a product of Seikagaku Corporation and generously donated by that company. [35S]PAPS was prepared as described previously (15Delfert D.M. Conrad H.E. Anal. Biochem. 1985; 148: 303-310Crossref PubMed Scopus (36) Google Scholar). [3H]GlcNAcR(6S) and [3H]GalR(6S) were prepared from GlcNAc(6S) and Gal(6S), respectively, by the reduction with NaB3H4 (13Habuchi O. Hirahara Y. Uchimura K. Fukuta M. Glycobiology. 1996; 6: 51-57Crossref PubMed Scopus (45) Google Scholar). Chondroitin (squid skin) was prepared as previously described (16Habuchi O. Miyata K. Biochim. Biophys. Acta. 1980; 616: 208-217Crossref PubMed Scopus (32) Google Scholar). Partially desulfated keratan sulfate (sulfate/glucosamine = 0.62) was prepared from corneal keratan sulfate according to Nagasawa et al. (17Nagasawa K. Inoue Y. Tokuyasu Y. J. Biochem. (Tokyo). 1979; 86: 1323-1329Crossref PubMed Scopus (100) Google Scholar). Solvolysis with dimethyl sulfoxide was carried out at 80 °C for 45 min. The molar ratios of Galβ1–4GlcNAc to Galβ1–4GlcNAc(6S) of the desulfated keratan sulfate, which was determined by the paper chromatographic separation of [3H]Galβ1–4AManR and [3H]Galβ1–4AManR(6S) formed after the reaction sequence of hydrazinolysis, deaminative cleavage, and reduction with NaB3H4 (27Shaklee P.N. Conrad H.E. Biochem. J. 1986; 235: 225-236Crossref PubMed Scopus (32) Google Scholar), was 0.73. A mixture of [3H]Gal(6S)β1–4GlcNAcR and [3H]Galβ1–4GlcNAcR(6S) was prepared by partial acid hydrolysis (0.1 m HCl, 100 °C, 40 min) of [3H]Gal(6S)β1–4GlcNAcR(6S) as described previously (14Habuchi O. Suzuki Y. Fukuta M. Glycobiology. 1997; 7: 405-412Crossref PubMed Scopus (43) Google Scholar). Approximately 2 × 106 plaques were screened. Hybond N+ nylon membrane (Amersham Corp.) replicas of the plaques from the λgt 11 cDNA library were fixed by the alkali fixation method recommended by the manufacturer, prehybridized in a solution containing 50% formamide, 5 × SSPE, 5 × Denhardt's solution, 0.5% SDS, and 0.04 mg/ml denatured salmon sperm DNA for 3.5 h at 42 °C. Hybridization was carried out in the same buffer containing a32P-labeled probe for 16 h at 42 °C. The radioactive probe for screening the cDNA library was prepared from chick C6ST cDNA previously reported (12Fukuta 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) by the random oligonucleotide-primed labeling method (18Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16562) Google Scholar) using [α-32P]dCTP (Amersham Corp.) and a DNA random labeling kit (Takara Shuzo). The filters were washed at 55 °C in 1 × SSPE, 0.1% SDS, and subsequently in 0.1 × SSPE, 0.1% SDS, and positive clones were detected by autoradiography. DNA from λgt 11 positive clones were isolated and cut with EcoRI, which excised the cDNA insert in a single fragment. The fragments were inserted into Bluescript plasmid, and deletion clones were prepared as described previously (19Henikoff S. Gene (Amst.). 1984; 28: 351-359Crossref PubMed Scopus (2828) Google Scholar, 20Yanisch-Perron C. Viera J. Messing J. Gene (Amst.). 1985; 33: 103-109Crossref PubMed Scopus (11410) Google Scholar) using a DNA deletion kit (Takara Shuzo). The complete nucleotide sequence was determined independently on both strands using the dideoxy chain termination method (21Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52251) Google Scholar) with [α-32P]dCTP and Sequenase (U. S. Biochemical Corp.). The DNA sequence was also determined using synthetic oligonucleotide primers. DNA sequences were compiled and analyzed using the Gene Works computer programs (IntelliGenetics). For the construction of pCXNKSGal6ST, the EcoRI fragment containing the 2415-base pair cDNA indicated in Fig. 1 A was excised from the Bluescript plasmid and ligated into the EcoRI site of pCXN2 expression vector (the pCXN2 vector was constructed by Dr. Jun-ichi Miyazaki, Department of Disease-Related Gene Regulation, Faculty of Medicine, University of Tokyo (22Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-200Crossref PubMed Scopus (4524) Google Scholar) and provided by Dr. Yasuhiro Hashimoto, Tokyo Metropolitan Institute of Medical Sciences). Recombinant plasmids were analyzed by restriction mapping using BamHI to confirm the correct orientation of pCXNKSGal6ST. The plasmid that contained the cDNA fragment in the reversed orientation was designated as pCXNKSGal6ST2 and used for control experiments. COS-7 cells (obtained from Riken Cell Bank, Tsukuba, Japan) were plated in 100-mm culture dishes at a density of 8 × 105 cells/dish. Volume of the medium was 10 ml. The medium used was DMEM containing penicillin (100 units/ml), streptomycin (50 μg/ml), and 10% fetal bovine serum (Life Technologies, Inc.), and cells were grown at 37 °C in 5% CO2, 95% air. When the cell density reached 3 × 106 cells/dish (48 h after plating), COS-7 cells were transfected with pCXNKSGal6ST or pCXNKSGal6ST2. The transfection was performed using the DEAE-dextran method (23Aruffo A. Current Protocol in Molecular Biology. John Wiley & Sons, Inc., New York1991Google Scholar). 5 ml of the prewarmed DMEM containing 10% Nu serum (Collaborative Biomedical Products) were mixed with 0.2 ml of phosphate-buffered saline containing 10 mg/ml DEAE-dextran plus a 2.5 mm chloroquine solution. 15 μg of the recombinant plasmid were mixed with the solution, and the mixture was added to the cells. The cells were incubated for 4 h in a CO2 incubator. The medium was then replaced with 5 ml of 10% dimethyl sulfoxide in phosphate-buffered saline. After the cells were left at room temperature for 2 min, the dimethyl sulfoxide solution was aspirated, and 25 ml of DMEM containing penicillin (100 units/ml), streptomycin (50 μg/ml), and 10% fetal bovine serum were added. The cells were incubated for 67 h, washed with DMEM alone, scraped, and homogenized with a Dounce homogenizer in 1.5 ml/dish of 0.25 m sucrose, 10 mm Tris-HCl, pH 7.2, and 0.5% Triton X-100. The homogenates were centrifuged at 10,000 ×g for 20 min, and the activities of C6ST, C4ST, and KSST in the supernatant fractions were measured as described below. Poly(A)+ RNAs (5 μg) prepared from chick embryo tissues were denatured in 50% formamide (v/v), 5% formaldehyde (v/v), 20 mm MOPS, pH 7.0, at 65 °C for 10 min, electrophoresed in 1.2% agarose gel containing 5% formaldehyde (v/v), and transferred to a Hybond N+ nylon membrane overnight. The RNA was fixed by baking at 80 °C for 2 h and prehybridized in a solution containing 50% formamide, 5 × SSPE, 5 × Denhardt's solution, 0.5% SDS, and 0.1 mg/ml denatured salmon sperm DNA for 3 h at 42 °C. Hybridization was carried out in the same buffer containing a32P-labeled probe for 16 h at 42 °C. The filters were washed at 65 °C in 2 × SSPE, 0.1% SDS, and subsequently in 1 × SSPE, 0.1% SDS. Human multiple tissue Northern blot filters (on which 2 μg of poly(A)+ RNAs from various adult human tissues were blotted) were processed under the same conditions described above. The membranes were exposed to x-ray film for 26 h with an intensifying screen at −80 °C. To determine the chromosomal localization of KSGal6ST, fluorescence in situhybridization was performed. Metaphase chromosomes were prepared from normal male lymphocytes using the thymidine synchronization, a bromodeoxyuridine release technique for the delineation of R- and G-bands (24Inazawa J. Saito H. Ariyama T. Abe T. Nakamura Y. Genomics. 1993; 17: 153-162Crossref PubMed Scopus (135) Google Scholar). Before hybridization, chromosomes were stained in Hoechst 33258 and irradiated with UV. A 2.4-kb cDNA shown in Fig. 1 was labeled with biotin-16-UTP by nick translation and hybridized to the denatured chromosome. The hybridization signals were detected with fluorescein isothiocyanate-avidin (Boehringer Mannheim GmbH, Mannheim, Germany), and chromosomes were counterstained with propidium iodide (1 μg/ml). The fluorescent signals were examined using epifluorescent microscope and precise positions of the signals were determined according to the G-bands delineated by Hoechst 33258 through UV filter. In the early experiments (Fig. 3), C6ST activity and KSST activity were assayed by the method described previously (11Habuchi O. Matsui Y. Kotoya Y. Aoyama Y. Yasuda Y. Noda M. J. Biol. Chem. 1993; 268: 21968-21974Abstract Full Text PDF PubMed Google Scholar). The reaction mixture used for the early experiments contained, in a final volume of 50 μl, 2.5 μmol of imidazole-HCl, pH 6.8, 1.25 μg (for chondroitin) or 3.75 μg (for keratan sulfate) of protamine chloride, 0.1 μmol of dithiothreitol, 0.025 μmol of glycosaminoglycans (as glucosamine or galactosamine), 50 pmol of [35S]PAPS (about 5.0 × 105 cpm), and enzyme. The reaction mixtures were incubated at 37 °C for 20 min, and the reaction was stopped by immersing the reaction tubes in a boiling water bath for 1 min. 35S-Labeled glycosaminoglycans were isolated by precipitation with ethanol followed by gel chromatography with a fast desalting column as described previously, and radioactivity was determined. The reaction mixtures described above developed for C6ST were, however, not optimum for KSST, and the activity of KSST was underestimated. After the optimum conditions for KSST were revealed, the following modification of the reaction mixture was adopted unless otherwise stated. 2.5 μmol of imidazole-HCl, pH 6.4, and 0.5 μmol of CaCl2 were added to the reaction mixture in place of 2.5 μmol of imidazole-HCl, pH 6.8, and protamine chloride, respectively. KSST activity and chondroitin sulfotransferase activity were determined using keratan sulfate and chondroitin, respectively, as acceptor. For determining C6ST and C4ST activity, 35S-labeled chondroitin was digested with chondroitinase ACII, and the unsaturated disaccharides formed were separated by paper chromatography. The homogenate of COS-7 cells transfected with pCXNKSGal6ST (224 mg as protein obtained from 80 10-cm dishes) was applied to a DEAE-Sephadex A-50 column (2.2 × 13 cm) equilibrated with buffer A (10 mm Tris-HCl, pH 7.2, containing 20% glycerol, 20 mm MgCl2, 2 mmCaCl2, and 10 mm 2-mercaptoethanol) containing 50 mm NaCl. After the column was washed with 500 ml of the same buffer, the absorbed materials were eluted with 0.5 mNaCl in buffer A. About 20% of KSST activity was recovered in the flow through fractions. The remaining KSST activity and all of chondroitin sulfotransferase activity were eluted in 0.5 m NaCl fractions. The flow-through fractions were pooled, dialyzed against 0.15 m NaCl in buffer A, and applied to a Heparin-Sepharose CL 6B column (1.2 × 8.0 cm) equilibrated with buffer A containing 0.15 m NaCl. The materials absorbed to the Heparin-Sepharose CL 6B column were eluted with 0.5 m NaCl in buffer A, dialyzed against buffer A containing 50 mmNaCl, and used for KSGal6ST preparation devoid of C6ST activity. As a control, the homogenate of COS-7 cells without transfection obtained from 20 10-cm dishes was also separated with DEAE-Sephadex in the same procedures as described above except that the column size, elution volume, and fraction size were reduced to one-fourth. 35S-Labeled glycosaminoglycan was prepared by incubating keratan sulfate or desulfated keratan sulfate with [35S]PAPS and the partially purified KSGal6ST (2 μg as protein) as described above for 18 h. 35S-Labeled glycosaminoglycans were separated from 35SO4and [35S]PAPS with the fast desalting column and desalted by lyophilization. The desalted samples from four reaction tubes were pooled and digested with keratanase II in the reaction mixture containing, in a final volume of 50 μl, 0.005 unit of keratanase II and 2.5 μmol of acetate buffer, pH 6.5 (25Nakazawa K. Ito M. Yamagata T. Suzuki S. Greiling H. Scott J.E. Keratan Sulfate: Chemistry and Chemical Pathology. The Biochemical Society, London1989: 99-110Google Scholar, 26Hashimoto N. Morikawa K. Kikuchi H. Yoshida K. Tokuyasu K. Abstracts of the XVth International Carbohydrate Symposium, Yokohama, Japan. The International Carbohydrate Organization, 1990: 271Google Scholar). The reaction mixtures were incubated at 37 °C for 24 h.35S-Labeled disaccharides formed after the keratanase II digestion were separated with an anion exchange HPLC. The keratanase II digests were applied to a Whatman Partisil 10-SAX column (4.5 × 25 cm) equilibrated with 5 mmKH2PO4. The column was developed with 5 mm KH2PO4 for 5 min followed by a 20-min gradient from 5 mm to 250 mm of KH2PO4. The flow rate was 1 ml/min. Under the chromatographic conditions, elution time of Gal(6S)β1–4GlcNAc(6S) and Galβ1–4GlcNAc(6S) detected by absorption at 210 nm were 14 min and 22 min, respectively. 0.5-ml fractions were collected, and 10-μl aliquots were used for determination of radioactivity. Each radioactive peak was collected, dried with a centrifuging vacuum evaporator, and redissolved in a small volume of water. Potassium phosphate was removed by Superdex 30 column chromatography, and the eluate from the Superdex 30 column was lyophilized. To determine which sulfate of Gal(6S)β1–4GlcNAcR(6S) contained 35S radioactivity, monosulfated disaccharide fraction was prepared from [35S]Gal(6S)β1–4GlcNAc(6S) with partial acid hydrolysis. [35S]Gal(6S)β1–4GlcNAc(6S) obtained by Partisil-10 SAX HPLC and Superdex 30 chromatography was reduced with NaBH4 as described elsewhere (14Habuchi O. Suzuki Y. Fukuta M. Glycobiology. 1997; 7: 405-412Crossref PubMed Scopus (43) Google Scholar), and hydrolyzed with 50 μl of 0.1 m HCl at 100 °C for 40 min. After re-N-acetylation with acetic anhydride, the hydrolysate was spotted on a strip of Whatman No. 3 and developed with a solvent described below for 48 h. The second radioactive peak, which potentially contains Galβ1–4GlcNAcR(6S), Gal(6S)β1–4GlcNAcR, Gal(6S), and SO4, was eluted and subjected to paper electrophoresis. The faster migrating peak in the paper electrophoresis was assigned as35SO4. The slower migrating peak, which potentially contains Galβ1–4GlcNAcR(6S), Gal(6S)β1–4GlcNAcR, and Gal(6S) was reduced with NaBH4 as described previously (14Habuchi O. Suzuki Y. Fukuta M. Glycobiology. 1997; 7: 405-412Crossref PubMed Scopus (43) Google Scholar) and analyzed with Partisil-10 SAX HPLC as described below. Superdex 30 16/60 column was equilibrated with 0.2 m NH4HCO3. The flow rate was 1 ml/min. 1-ml fractions were collected. Paper electrophoresis was carried out on Whatman No. 3 paper (2.5 cm x 57 cm) in pyridine/acetic acid/water (1:10:400, by volume, pH 4) at 30 V/cm for 40 min. Paper chromatography was performed on Whatman No. 3 paper (2.5 cm x 57 cm) using a solvent system, 1-butanol/acetic acid/1m NH3 (3:2:1, by volume). The dried paper strips after paper electrophoresis or paper chromatography were cut into 1.25-cm segments, which were analyzed for radioactivity by liquid scintillation counting. Separation of Gal(6S)β1–4GlcNAcRand Galβ1–4GlcNAcR(6S) was carried out by HPLC using a Whatman Partisil 10-SAX column (4.5 × 25 cm) equilibrated with 5 mm KH2PO4. The column was developed with 5 mm KH2PO4 isocratically (27Shaklee P.N. Conrad H.E. Biochem. J. 1986; 235: 225-236Crossref PubMed Scopus (32) Google Scholar). The flow rate was 1 ml/min, and the column temperature was 40 °C. 0.5-ml fractions were collected. When approximately 2 × 106 plaques of a human fetal brain cDNA library were screened using chick C6ST cDNA as a probe, two cDNA clones (1.2 and 2.4 kb) other than human C6ST cDNA clones were obtained. These clones were clearly distinguished from the C6ST clones on the autoradiogram due to their weaker signals. We will report the human C6ST cDNA elsewhere. From the nucleotide sequence, the longer cDNA clone was found to contain a whole open reading frame. The nucleotide sequence of the KSGal6ST cDNA and the predicted amino acid sequence are shown in Fig.1 A. A single open reading frame predicts a protein of 411 amino acid residues with five potential N-linked glycosylation sites. To determine the location of any transmembrane domain, a hydropathy plot was generated from the translated sequence. Analysis of the plot revealed one prominent hydrophobic segment in the amino-terminal region, 14 residues in length, that extends from amino acid residues 7–20 (Fig.1 B). Comparison of the coding sequence of human KSGal6ST with that of chick C6ST has revealed that there is 37% identity on the amino acid level (Fig. 2). There are 5 regions in which more than 6 consecutive amino acid residues are identical. Homology of N-terminal region was lower than that of the C-terminal region. No significant homology in amino acid sequence was observed between human KSGal6ST and any other sulfotransferases previously reported involving heparan sulfate N-sulfotransferase (29Hashimoto Y. Orellana A. Gil G. Hirschberg C.B. J. Biol. Chem. 1992; 267: 15744-15750Abstract Full Text PDF PubMed Google Scholar), heparan sulfate 2-sulfotransferase (30Kobayashi M. Habuchi H. Yoneda M. Habuchi O. Kimata K. J. Biol. Chem. 1997; 272: 13980-13985Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), and galactosylceramide 3′-sulfotransferase (31Honke K. Tsuda M. Hirahara Y. Ishii A. Makita A. Wada Y. J. Biol. Chem. 1997; 272: 4864-4868Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). COS-7 cells were transfected with the pCXNKSGal6ST, a recombinant plasmid containing the isolated cDNA in the mammalian expression vector pCXN2. The transfected cells were scraped at 67 h after transfection, homogenized with a buffer containing 0.5% Triton X-100, and centrifuged. Activities of C6ST, C4ST, and KSST contained in the supernatant fractions were determined. Control experiments without vector and with vector containing the cDNA in the reversed orientation (pCXNKSGal6ST2) were also done. As shown in TableI, when the cells were transfected with pCXNKSGal6ST2, KSST activity in the transfected cells was unchanged compared with nontransfected cells, whereas about 10-fold increase in KSST activity was observed in the cells transfected with pCXNKSGal6ST. In contrast, both C6ST and C4ST activities were not increased above the control, indicating that the isolated cDNA encodes a protein with KSST activity alone.Table IOverexpression of keratan sulfate sulfotransferase in COS-7 cellsPlasmidSulfotransferase activityC6STC4STKSSTpmol/min/mg proteinNone1.2 ± 0.20.2 ± 0.11.9 ± 0.1pCXNKSGal6ST1.3 ± 0.20.2 ± 0.119.9 ± 0.3pCXNKSGal6ST21.3 ± 0.30.3 ± 0.12.1 ± 0.2 Open table in a new tab C6ST activity, which was included in the cell extracts from COS-7 cells transfected with the pCXNKSGal6ST, was successfully removed by ion-exchange chromatography. The COS-7 cell extracts were applied to a DEAE-Sephadex column, and the absorbed materials were eluted with 0.5 m NaCl in buffer A. About 20% of KSST activity was recovered in the flow-through fraction, whereas chondroitin sulfotransferase activity was recovered only in 0.5m NaCl fraction (Fig.3 A). When cell extracts prepared from COS-7 cells cultured without the plasmid were applied to the DEAE-Sephadex column, no KSST activity was detected in the flow-through fraction (Fig. 3 B). These observation indicates that the KSST activity recovered in the flow-through fraction is due to the overexpressed enzyme, which is encoded by KSGal6ST cDNA. About 80% of KSST activity from the transfected COS-7 cells was eluted in the 0.5 m NaCl fraction, and this activity was much higher than the activity found in 0.5 m NaCl fraction" @default.
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• W2052522729 date "1997-12-01" @default.
• W2052522729 modified "2023-10-17" @default.
• W2052522729 title "Molecular Cloning and Characterization of Human Keratan Sulfate Gal-6-Sulfotransferase" @default.
• W2052522729 cites W1489235874 @default.
• W2052522729 cites W1497340641 @default.
• W2052522729 cites W1506057274 @default.
• W2052522729 cites W1519774322 @default.
• W2052522729 cites W1530334685 @default.
• W2052522729 cites W1535596108 @default.
• W2052522729 cites W1598171890 @default.
• W2052522729 cites W1602413321 @default.
• W2052522729 cites W1607493519 @default.
• W2052522729 cites W1751233322 @default.
• W2052522729 cites W1897011699 @default.
• W2052522729 cites W1972195598 @default.
• W2052522729 cites W1975107438 @default.
• W2052522729 cites W1975304761 @default.
• W2052522729 cites W1975484793 @default.
• W2052522729 cites W1978511926 @default.
• W2052522729 cites W1981697145 @default.
• W2052522729 cites W1991772365 @default.
• W2052522729 cites W1996128601 @default.
• W2052522729 cites W2008078894 @default.
• W2052522729 cites W2010039139 @default.
• W2052522729 cites W2014669590 @default.
• W2052522729 cites W2028622989 @default.
• W2052522729 cites W2032462421 @default.
• W2052522729 cites W2039940600 @default.
• W2052522729 cites W2048162805 @default.
• W2052522729 cites W2056602792 @default.
• W2052522729 cites W2058589009 @default.
• W2052522729 cites W2087889360 @default.
• W2052522729 cites W2096070789 @default.
• W2052522729 cites W2105237208 @default.
• W2052522729 cites W2131318175 @default.
• W2052522729 cites W2138270253 @default.
• W2052522729 cites W2168503979 @default.
• W2052522729 cites W2317332613 @default.
• W2052522729 doi "https://doi.org/10.1074/jbc.272.51.32321" @default.
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