Molecular Cloning and Characterization of a Human Uronyl 2-Sulfotransferase That Sulfates Iduronyl and Glucuronyl Residues in Dermatan/Chondroitin Sulfate

Abstract

A partial-length human cDNA with a predicted amino acid sequence homologous to a previously described heparan sulfate iduronyl 2-sulfotransferase (Kobayashi, M., Habuchi, H., Yoneda, M., Habuchi, O., and Kimata, K. (1997) J. Biol. Chem. 272, 13980–13985) was obtained by searching the expressed sequence-tagged data bank. Northern blot analysis was performed using this homologous cDNA as a probe, which demonstrated ubiquitous expression of messages of 5.1 and 2.0 kilobases in a number of human tissues and in several human cancer cell lines. Since the human lymphoma Raji cell line had the highest level of expression, it was used to isolate a full-length cDNA clone. The full-length cDNA was found to contain an open reading frame that predicted a type II transmembrane protein composed of 406 amino acid residues. The cDNA in a baculovirus expression vector was expressed in Sf9 insect cells, and cell extracts were then incubated together with 3′-phosphoadenosine 5′-phospho[35S]sulfate and potential glycosaminoglycan acceptors. This demonstrated substantial sulfotransferase activity with dermatan sulfate, a small degree of activity with chondroitin sulfate, but no sulfotransferase activity with desulfated N-resulfated heparin. Analysis of [35S]sulfate-labeled disaccharide products of chondroitin ABC, chondroitin AC, and chondroitin B lyase treatment demonstrated that the enzyme only transferred sulfate to the 2-position of uronyl residues, which were preponderantly iduronyl residues in dermatan sulfate, but some lesser transfer to glucuronyl residues of chondroitin sulfate. A partial-length human cDNA with a predicted amino acid sequence homologous to a previously described heparan sulfate iduronyl 2-sulfotransferase (Kobayashi, M., Habuchi, H., Yoneda, M., Habuchi, O., and Kimata, K. (1997) J. Biol. Chem. 272, 13980–13985) was obtained by searching the expressed sequence-tagged data bank. Northern blot analysis was performed using this homologous cDNA as a probe, which demonstrated ubiquitous expression of messages of 5.1 and 2.0 kilobases in a number of human tissues and in several human cancer cell lines. Since the human lymphoma Raji cell line had the highest level of expression, it was used to isolate a full-length cDNA clone. The full-length cDNA was found to contain an open reading frame that predicted a type II transmembrane protein composed of 406 amino acid residues. The cDNA in a baculovirus expression vector was expressed in Sf9 insect cells, and cell extracts were then incubated together with 3′-phosphoadenosine 5′-phospho[35S]sulfate and potential glycosaminoglycan acceptors. This demonstrated substantial sulfotransferase activity with dermatan sulfate, a small degree of activity with chondroitin sulfate, but no sulfotransferase activity with desulfated N-resulfated heparin. Analysis of [35S]sulfate-labeled disaccharide products of chondroitin ABC, chondroitin AC, and chondroitin B lyase treatment demonstrated that the enzyme only transferred sulfate to the 2-position of uronyl residues, which were preponderantly iduronyl residues in dermatan sulfate, but some lesser transfer to glucuronyl residues of chondroitin sulfate. d-glucuronic acid 3′-phosphoadenosine 5′-phosphosulfate Chinese hamster ovary expressed sequence-tagged polymerase chain reaction sodium citrate-sodium chloride high performance liquid chromatography completely desulfated andN-resulfated-heparin 4-deoxy-α-threo-hex-4-enepyranosyluronic acid 2-acetamido-2-deoxy-3-O-(β-d-Glc-4-enepyranosyluronic acid-2-O-sulfo)-d-galactose 4S, 2-acetamido-2-deoxy-3-O-(β-d-Glc-4-enepyranosyluronic acid-2-O-sulfo)-4-O-sulfo-d-galactose 6S, 2-acetamido-2-deoxy-3-O-(β-d-Glc-4-enepyranosyluronic acid-2-O-sulfo)-6-O-sulfo-d-galactose 4,6S, 2-acetamido-2-deoxy-3-O-(β-d-Glc-4-enepyranosyluronic acid-2-O-sulfo)-4-O-sulfo-6-O-sulfo-d-galactose base pair(s) kilobase pair(s) Dermatan sulfate is a glycosaminoglycan polysaccharide consisting of N-acetylgalactosamine (GalNAc) residues alternating with varying proportions of glucuronyl (GlcA)1 and iduronyl (IdceA) residues that are formed from the GlcA by epimerization during polymerization and GalNAc 4-sulfation (1Malmström A. Fransson L.-Å. Höök M. Lindahl U. J. Biol. Chem. 1975; 250: 3419-3425Abstract Full Text PDF PubMed Google Scholar, 2Malmström A. J. Biol. Chem. 1981; 259: 161-165Google Scholar, 3Silbert J.E. Glycoconj. J. 1996; 13: 907-912Crossref PubMed Scopus (16) Google Scholar, 4Silbert J.E. Silbert C.K. Humphries D.E. Sugumaran G. Scott J.E. Dermatan Sulfate Proteoglycans: Chemistry, Biology, Chemical Pathology. Portland Press Ltd., London1993: 147-158Google Scholar). Thus dermatan sulfate can be considered as a variant of chondroitin 4-sulfate, containing some IdceA as well as GlcA, with the IdceA only found next to 4-sulfated GalNAc residues (5Silbert J.E. Palmer M.E. Humphries D.E. Silbert C.K. J. Biol. Chem. 1986; 261: 13397-13400Abstract Full Text PDF PubMed Google Scholar). In addition the IdceA of dermatan sulfate is frequently 2-sulfated (6Fransson L.-Å. Cheng F. Yoshida K. Heinegård D. Malmström A. Schmidtchen A. Scott J.E. Dermatan Sulfate Proteoglycans: Chemistry, Biology, Chemical Pathology. Portland Press Ltd., London1993: 11-25Google Scholar). Some 2-sulfation of GlcA on chondroitin sulfate has also been found but only next to GalNAc 6-sulfate rather than GalNAc 4-sulfate. Proteoglycans containing dermatan sulfate are ubiquitously present in most tissues, where the dermatan sulfate portion may be involved in various biological activities presumably relating in great part to its fine structure. Activities include interaction with heparin cofactor II (7Maimone M.M. Tollefsen D.M. J. Biol. Chem. 1990; 265: 18263-18271Abstract Full Text PDF PubMed Google Scholar, 8Mascellani G. Liverani L. Bianchini P. Parma B. Torri G. Bisio A. Guerrini M. Casu B. Biochem. J. 1993; 296: 639-648Crossref PubMed Scopus (61) Google Scholar) requiring repeating 2-sulfated iduronyl-containing disaccharide units (7Maimone M.M. Tollefsen D.M. J. Biol. Chem. 1990; 265: 18263-18271Abstract Full Text PDF PubMed Google Scholar), hepatocyte growth factor/scatter factor (9Lyon M. Deakin J.A. Rahmoune H. Fernig D.G. Nakamura T. Gallagher J.T. J. Biol. Chem. 1998; 273: 271-278Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), and promotion of fibroblast growth factor-2 during wound repair (10Penc S.F. Pomahac B. Winkler T. Dorschner R.A. Eriksson E. Herndon M. Gallo R.L. J. Biol. Chem. 1998; 273: 28116-28121Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Although there is little information concerning detailed biological activities based on the structural diversity of the dermatan sulfate, the 2-sulfation of IdceA would appear to be of special interest. The only galactosaminoglycan sulfotransferase that has been cloned to date is a chondroitin 6-sulfotransferase (11Fukuta 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). However, several glucosaminoglycan sulfotransferases have been cloned (12Hashimoto Y. Orellana A. Gil G. Hirschberg C.B. J. Biol. Chem. 1992; 267: 15744-15750Abstract Full Text PDF PubMed Google Scholar, 13Orellana A. Hirschberg C.B. Wei Z. Swiedler S.J. Ishihara M. J. Biol. Chem. 1994; 269: 2270-2276Abstract Full Text PDF PubMed Google Scholar, 14Eriksson I.D. Sandbäck D. Ek B. Lindahl U. Kjellén L. J. Biol. Chem. 1994; 269: 10438-10443Abstract Full Text PDF PubMed Google Scholar, 15Kobayashi 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, 16Shworak N.W. Liu J. Fritze L.M.S. Schwartz J.J. Zhang L. Logeart D. Rosenberg R.D. J. Biol. Chem. 1997; 272: 28008-28019Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 17Habuchi H. Kobayashi M. Kimata K. J. Biol. Chem. 1998; 273: 9208-9213Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 18Shworak N.W. Liu J. Petros L.M. Zhang L. Kobayashi M. Copeland N.G. Jenkins N.A. Rosenberg R.D. J. Biol. Chem. 1999; 274: 5170-5184Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 19Fukuta M. Inazawa J. Torii T. Tsuzuki K. Shimada E. Habuchi O. J. Biol. Chem. 1997; 272: 32321-32328Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), including an IdceA 2-sulfotransferase for heparan sulfate (15Kobayashi 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, 20Kobayashi M. Habuchi H. Habuchi O. Saito M. Kimata K. J. Biol. Chem. 1996; 271: 7645-7653Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). It seemed likely that this enzyme would have similarities to IdceA 2-sulfotransferase for dermatan sulfate. Therefore, in an attempt to find such an IdceA 2-sulfotransferase, we employed the heparan sulfate IdceA 2-sulfotransferase sequence to obtain a related expressed sequence-tagged (EST) clone. This provided for the molecular cloning of a human cDNA which we found to encode a uronyl 2-sulfotransferase. We have found this enzyme to have no 2-sulfotransferase activity with heparan sulfate but to be involved in the sulfation of the IdceA residues of dermatan sulfate with some lesser activity in 2-sulfation of GlcA residues in chondroitin sulfate. The National Center for Biotechnology Information (NCBI) data bank of I.M.A.G.E. Consortium (Lawrence Livermore National Laboratory) EST cDNA clones (21Lennon G. Auffray C. Polymeropoulo M. Soares M.B. Genomics. 1996; 33: 151-152Crossref PubMed Scopus (1089) Google Scholar) was probed with a deduced CHO cell heparan sulfate 2- sulfotransferase sequence (15Kobayashi 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). Clone ID HE9MJ06 was obtained from the TIGR/ATCC Special Collection (ATCC). This was a partial-length EST clone from a 9-week-old human embryo, in which only 248 bp of sequence (positions 473–720, Fig. 1) was present in the data base (22Adams M.D. Kerlavage A.R. Fleischmann R.D. Fuldner R.A. Bult C.J. Lee N.H. Kirkness E.F. Weinstock K.G. Gocayne J.D. White O. Sutton G. Blake J.A. Brandon R.C. Man-Wai C. Clayton R.A. et al.Nature. 1995; 377: 3-174PubMed Google Scholar). The cDNA from this clone was inserted into the EcoRI and XhoI sites of the Bluescript SK− plasmid and carried in an Escherichia coli host strain. The pBluescript plasmid DNA was purified from the host bacteria using QIAfilter plasmid kits (Qiagen) and used to prepare a PCR probe consisting of a 614-bp fragment at positions 473–1086 as shown in Fig.1 using 20-bp oligonucleotides of both ends as primers. The PCR was carried out in a 50-μl volume containing 0.5 μm each primer, 40 ng of the template, 0.2 mm each dNTP, and 1.25 units of Taq2000TM DNA polymerase (Stratagene) under the conditions of 30 cycles of denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, and extension at 72 °C for 1 min. The products were subjected to agarose gel electrophoresis, and an amplified DNA band of ∼600 bp was excised, recovered from the gel using QIAEX II (Qiagen), and radiolabeled for the probe using [α-32P]dATP and a Prime-It II random primer labeling kit (Stratagene). To obtain full-length clones, we first scanned tissue-specific and cell type-specific expression of the cDNA by Northern blot hybridization using the32P-labeled PCR probe. Tissue type-specific expression of presumptive 2-sulfotransferase cDNA was analyzed with human RNA Master BlotTM and human Multiple Tissue Northern (MTNTM) Blot (CLONTECH) membranes and cell type-specific expression with a human cancer cell line Multiple Tissue Northern (MTNTM) Blot (CLONTECH) membrane. Each Northern analysis was carried out according to the manufacturer's protocol with some modifications. The Master Blot membrane was prehybridized in an ExpressHyb solution containing 100 μg/ml denatured sheared salmon testes DNA (Sigma) at 65 °C for 30 min, hybridized in the same solution containing the denatured32P-labeled probe, 6 μg/ml human Cot-1 DNA (Boehringer Mannheim), and 0.2× SSC at 65 °C for 16 h, and washed four times with 2× SSC, 1% SDS at 65 °C for 20 min, twice with 0.1× SSC, 0.5% SDS at 55 °C for 20 min. The human and cancer cell line MTN Blot membranes were prehybridized in an ExpressHyb at 68 °C for 30 min, hybridized in the same solution containing the32P-labeled probe at 68 °C for 16 h, and washed twice with 2× SSC, 0.05% SDS at 22 °C for 20 min, twice with 0.1× SSC, 0.1% SDS at 50 °C for 20 min. The membranes were then exposed to x-ray film with an intensifying screen at −80 °C. The human lymphoma 5′-STRETCH PLUS cDNA library (CLONTECH) was constructed from mRNA from Burkitt's lymphoma Raji cell line atEcoRI-cloning sites of the λgt11 vector by the priming method with oligo(dT) and random primers. The host strain Y1090r− cells were infected with phage from the library, plated at 4 × 105 plaque-forming units/dish, and approximately 1.2 × 106 plaques were screened. Colony/Plaque screenTM (NEN Life Science Products) membrane replicas of the plaques were fixed by the rapid autoclave method recommended by the manufacturer, prehybridized in an ExpressHybTMhybridization solution (CLONTECH) for 30 min at 68 °C, and hybridized in the same solution containing the denatured32P-labeled probe at 68 °C according to the manufacturer's protocol with a 16-h modification. The filters were washed twice with 2× sodium citrate-sodium chloride (SCC), 0.05% sodium dodecyl sulfate (SDS) for 20 min at 22 °C, and then twice with 0.1× SCC, 0.1% SDS for 20 min at 50 °C. The positive clones were detected by autoradiography. Plaque solutions from the positive clones were initially characterized with LD-insert screening amplimer sets (CLONTECH) according to the manufacturer's PCR protocol. The resultant PCR products were subjected to agarose gel electrophoresis to determine the sizes of the inserts, recovered from the gel, and sequenced. λgt11 DNA of the clones was isolated from its plate lysate using a Qiagen lambda kit (Qiagen), subcloned into pcDNA3 vector (Invitrogen) at the EcoRI sites, and sequenced again to confirm sequence data. For DNA sequencing, the 5′ and 3′ insert regions were enzymatically sequenced from flanking primer sites of the respective PCR fragments or vectors. The remaining sequences of both strands were obtained with internally priming oligonucleotides. Primers were spaced no more than 400 bp apart with a 200-bp offset between sense and antisense strands. Automated fluorescence sequencing was performed with Perkin-Elmer Applied Biosystems models 373A and 477 DNA Sequencers. The DNA sequence files obtained were aligned and compiled with Sequencher (Gene Codes Corp.) and GENETYX-MAC (Software Development Corp.) computer programs. Sequence comparison searches were performed on the data bases of GenBankTM, EMBL, PDB, SwissProt, SPupdate, PIR, and dbEST. ThePvuII-EcoRI fragment containing the coding region from positions 193 to 1,382 shown in Fig. 1 was excised from the λgt11 cloning vector, blunted with T4 DNA polymerase, and ligated into the StuI site of the pFASTBACTM HTa plasmid (Life Technologies, Inc.). The recombinant bacmid-sulfotransferase (presumptive) molecules were then produced by Tn7-mediated site-specific transposition when MAX EFFICIENCY DH10BACTMcompetent cells (Life Technologies, Inc.) were transformed with the recombinant pFASTBAC HTa donor plasmid according to the manufacturer's instructions. The recombinant molecules were isolated and analyzed by agarose gel electrophoresis and PCR with vector and gene-specific primers to confirm the presence of bacmid high molecular weight DNA and the correct orientation of the inserted cDNA. Recombinant bacmid-heparan sulfate 2-sulfotransferase DNA that contained the entire coding region of its cDNA (SacII-AflII fragment) (15Kobayashi 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) was also constructed and used for control experiments. cDNA was expressed using a BAC-TO-BACTM HT baculovirus expression system (Life Technologies, Inc.) according to the manufacturer's instructions with slight modifications. Sf9 insect cells (Invitrogen) seeded onto 35-mm culture dishes containing 2 ml of SF-900 II SFM (Life Technologies, Inc.) were transfected with bacmid-sulfotransferase (presumptive) or bacmid-heparan sulfate 2-sulfotransferase using CELLFECTINTM reagent (Life Technologies, Inc.). The medium was replaced with Grace's insect medium (Invitrogen), 10% fetal bovine serum (JRH Biosciences), and the culture was continued for another 3 days at 27 °C. The spent medium was centrifuged for 5 min at 500 × g to obtain the virus-containing supernatant as viral stock. 150-mm Petri dishes of Sf9 cells were then infected by each recombinant viral stock and incubated at 27 °C for 3 days. After collecting the spent medium, the 150-mm Petri dishes of infected Sf9 cells were washed with phosphate-buffered saline, scraped, and homogenized in a 3-ml solution of 10 mmTris-HCl, pH 7.4, 0.5% (w/v) Triton X-100, 0.15 m NaCl, 10 mm MgCl2, 2 mm CaCl2, 20% (v/v) glycerol, and a mixture of protease inhibitors (5 μm Nα-p-tosyl-l-lysine chloromethyl ketone, 3 μm N-tosyl-l-phenylalanine chloromethyl ketone, 30 μm phenylmethylsulfonyl fluoride, and 3 μmpepstatin A) as described previously (15Kobayashi 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, 20Kobayashi M. Habuchi H. Habuchi O. Saito M. Kimata K. J. Biol. Chem. 1996; 271: 7645-7653Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). After 1 h of gentle stirring at 4 °C, the homogenate was centrifuged at 4 °C for 30 min at 10,000 × g. Sulfotransferase activities in the supernatant fractions (cell extracts) were measured as described below. Protein contents of the cell extracts were estimated by a micro-BCA protein assay reagent kit (Pierce) using bovine serum albumin as a standard. Completely desulfated and N-resulfated heparin (CDSNS-heparin) and shark cartilage chondroitin sulfate C (4-sulfate:6-sulfate, 10:90) were obtained from Seikagaku; porcine skin dermatan sulfate was obtained from Sigma. Chemical desulfation (23Nagasawa K. Inoue Y. Tokuyasu Y. J. Biochem. (Tokyo). 1979; 86: 1323-1329Crossref PubMed Scopus (101) Google Scholar) was used to obtain dermatan and chondroitin, which resulted in apparent complete desulfation of the chondroitin but left small amounts of 4-sulfate on the dermatan. A standard reaction mixture (25 μl) contained 1.25 μmol of imidazole HCl, pH 6.8, 1.88 μg of protamine chloride, 12.5 μg of glycosaminoglycan, 0.5 nmol (2.5 μCi/nmol) of 3′-phosphoadenosine 5′-phospho[35S]sulfate (NEN Life Science Products, Inc.), and 5 μl of the cell extract. After incubation at 37 °C for 30 min, the reaction mixtures were directly spotted on Whatman No. 1 paper and chromatographed in ethanol, 1 m ammonium acetate (5:2 (v/v)) overnight. The origins, which contained the sulfated products, were assayed for radioactivity as described previously (24Sugumaran G. Humphries D.E. Silbert J.E. Methods Enzymol. 1989; 169: 428-434Google Scholar). In order to obtain sufficient labeled products for detailed analyses, higher specific activity 5′-phospho[35S]sulfate (∼150 μCi/nmol) prepared as described previously (25Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) was used. After phenol:chloroform:isoamyl alcohol (25:24:1) treatment and subsequent ethanol precipitation, the 35S-labeled glycosaminoglycans were digested with protease-free chondroitin ABC lyase (20 milliunits/μg substrate), chondroitin AC I lyase (10 milliunits/μg substrate), or chondroitin B lyase (100 milliunits/μg substrate) (Seikagaku Corp.) for 16 h at 37 °C (30 °C for B lyase) and boiled at 100 °C for 1 min to terminate the reaction. Products (∼9,000 cpm) were applied on Bio-Gel P-2 (Bio-Rad) columns (0.75 × 200 cm) that were equilibrated and eluted with 0.1m ammonium bicarbonate at a flow rate of 4 ml/h and assayed for radioactivity. of the boiled ABC lyase mixtures were then incubated for an 16 h at 37 °C with (10 milliunits/μg by K. Corp.) J. Biochem. PubMed Scopus Google Scholar), (30 milliunits/μg substrate), or (30 milliunits/μg (Seikagaku cpm) and treatment were then analyzed by high performance liquid chromatography on a of II × cm) together with disaccharide (Seikagaku Corp.) and eluted with a from 16 to a at a flow rate of K. Y. T. P. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). of 0.5 ml were and with 6 ml of and the radioactivity was In order to screen for IdceA the of EST cDNA clones was probed with the deduced amino acid sequence of CHO cell heparan sulfate IdceA 2-sulfotransferase cDNA (15Kobayashi 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). described under a human partial-length cDNA Clone ID was a related The cDNA from this clone was (positions in as shown in Fig. The 614-bp (positions PCR probe for library screening and Northern hybridization was as described under 1.2 × 106 plaques of a λgt11 human lymphoma Raji cell cDNA library were using this PCR fragment as a probe, in positive insert of clones were and sequenced as described under but only to have the complete coding sequence of the presumptive The sequence of this clone was found to contain and a in frame at 56 from the first A open reading frame at the first predicted a protein of 406 amino acid residues with a molecular of with analysis J. J. Biol. PubMed Scopus Google Scholar) of the predicted amino acid sequence of the presumptive sulfotransferase that it had a type II membrane protein in the residues in at positions 1 and of the sequence of this human presumptive sulfotransferase with CHO cell heparan sulfate IdceA 2-sulfotransferase number (15Kobayashi 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 at the amino acid In acid residues from to for a sequence in the region of which the 5′-phosphosulfate its and to the of using on Y. M. Biochem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Y. M. 1998; PubMed Scopus Google Scholar, Y. M. Biol. 1997; PubMed Scopus Google Scholar) was no at the In addition there was and with a protein from number K. C. M. J. J. M. T. J. A. M. Z. et al.Nature. 1994; PubMed Scopus Google Scholar) protein number B. PubMed Google Scholar) It little sequence and no sequence with glycosaminoglycan sulfotransferase previously (11Fukuta 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, Y. Orellana A. Gil G. Hirschberg C.B. J. Biol. Chem. 1992; 267: 15744-15750Abstract Full Text PDF PubMed Google Scholar, 13Orellana A. Hirschberg C.B. Wei Z. Swiedler S.J. Ishihara M. J. Biol. Chem. 1994; 269: 2270-2276Abstract Full Text PDF PubMed Google Scholar, 14Eriksson I.D. Sandbäck D. Ek B. Lindahl U. Kjellén L. J. Biol. Chem. 1994; 269: 10438-10443Abstract Full Text PDF PubMed Google Scholar, 16Shworak N.W. Liu J. Fritze L.M.S. Schwartz J.J. Zhang L. Logeart D. Rosenberg R.D. J. Biol. Chem. 1997; 272: 28008-28019Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 17Habuchi H. Kobayashi M. Kimata K. J. Biol. Chem. 1998; 273: 9208-9213Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 18Shworak N.W. Liu J. Petros L.M. Zhang L. Kobayashi M. Copeland N.G. Jenkins N.A. Rosenberg R.D. J. Biol. Chem. 1999; 274: 5170-5184Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 19Fukuta M. Inazawa J. Torii T. Tsuzuki K. Shimada E. Habuchi O. J. Biol. Chem. 1997; 272: 32321-32328Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) than the heparan sulfate IdceA 2-sulfotransferase (15Kobayashi 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), that it was also most likely an IdceA 2-sulfotransferase. from cells infected with recombinant viral containing the 2-sulfotransferase of from 5′-phospho[35S]sulfate into dermatan sulfate, with lesser into dermatan and chondroitin sulfate, and no into was also no into by this cell that by control cell which contained heparan sulfate sulfotransferase infected with recombinant viral containing the bacmid-heparan sulfate IdceA 2-sulfotransferase of sulfate only into with into dermatan sulfate, or chondroitin sulfate. that the isolated cDNA a protein that presumably had dermatan IdceA 2-sulfotransferase activity with lesser amounts of chondroitin GlcA 2-sulfotransferase of uronyl sulfate cell desulfated dermatan sulfate with small of chondroitin sulfate C with 10% desulfated chondroitin sulfate with no desulfated N-resulfated uronyl sulfate desulfated dermatan sulfate with small of chondroitin sulfate C with 10% desulfated chondroitin sulfate with no Completely desulfated N-resulfated heparin. in a RNA Master Blot analysis demonstrated ubiquitous expression of the a of human tissues MTN Blot membrane analysis A) demonstrated a band of 5.1 and a band of 2.0 for human Analysis with the human cancer cell line MTN Blot 4 the same with and Burkitt's lymphoma Raji cell line the For this the cDNA library of this lymphoma cell line was as the cDNA to isolate the present sulfate glycosaminoglycans were digested with chondroitin ABC lyase and with chondroitin ABC lyase by disaccharide or The were then analyzed by on a II as described under