Chondroitin Sulfate Synthase-3
2003; Elsevier BV; Volume: 278; Issue: 41 Linguagem: Inglês
10.1074/jbc.m304421200
ISSN1083-351X
AutoresToshikazu Yada, Takashi Sato, Hiromi Kaseyama, Masanori Gotoh, Hiroko Iwasaki, Norihiro Kikuchi, Yeondae Kwon, Akira Togayachi, Takashi Kudo, Hideto Watanabe, Hisashi Narimatsu, Koji Kimata,
Tópico(s)Polysaccharides Composition and Applications
ResumoRecently, it has become evident that chondroitin sulfate (CS) glycosyltransferases, which transfer glucuronic acid and/or N-acetylgalactosamine residues from each UDP-sugar to the nonreducing terminus of the CS chain, form a gene family. We report here a novel human gene (GenBank™ accession number AB086062) that possesses a sequence homologous with the human chondroitin sulfate synthase-1 (CSS1) gene, formerly known as chondroitin synthase. The full-length open reading frame consists of 882 amino acids and encodes a typical type II membrane protein. This enzyme contains a β3-glycosyltransferase motif and a β4-glycosyltransferase motif similar to that found in CSS1. Both the enzymes were expressed in COS-7 cells as soluble proteins, and their enzymatic natures were characterized. Both glucuronyltransferase and N-acetylgalactosaminyltransferase activities were observed when chondroitin, CS polymer, and their corresponding oligosaccharides were used as the acceptor substrates, but no polymerization reaction was observed as in the case of CSS1. The new enzyme was thus designated chondroitin sulfate synthase-3 (CSS3). However, the specific activity of CSS3 was much lower than that of CSS1. The reaction products were shown to have a GlcUAβ1–3GalNAc linkage and a GalNAcβ1–4GlcUA linkage in the nonreducing terminus of chondroitin resulting from glucuronyltransferase activity and N-acetylgalactosaminyltransferase activity, respectively. Quantitative real time PCR analysis revealed that the transcript level of CSS3 was much lower than that of CSS1, although it was ubiquitously expressed in various human tissues. These results indicate that CSS3 is a glycosyltransferase having both glucuronyltransferase and N-acetylgalactosaminyltransferase activities. It may make a contribution to CS biosynthesis that differs from that of CSS1. Recently, it has become evident that chondroitin sulfate (CS) glycosyltransferases, which transfer glucuronic acid and/or N-acetylgalactosamine residues from each UDP-sugar to the nonreducing terminus of the CS chain, form a gene family. We report here a novel human gene (GenBank™ accession number AB086062) that possesses a sequence homologous with the human chondroitin sulfate synthase-1 (CSS1) gene, formerly known as chondroitin synthase. The full-length open reading frame consists of 882 amino acids and encodes a typical type II membrane protein. This enzyme contains a β3-glycosyltransferase motif and a β4-glycosyltransferase motif similar to that found in CSS1. Both the enzymes were expressed in COS-7 cells as soluble proteins, and their enzymatic natures were characterized. Both glucuronyltransferase and N-acetylgalactosaminyltransferase activities were observed when chondroitin, CS polymer, and their corresponding oligosaccharides were used as the acceptor substrates, but no polymerization reaction was observed as in the case of CSS1. The new enzyme was thus designated chondroitin sulfate synthase-3 (CSS3). However, the specific activity of CSS3 was much lower than that of CSS1. The reaction products were shown to have a GlcUAβ1–3GalNAc linkage and a GalNAcβ1–4GlcUA linkage in the nonreducing terminus of chondroitin resulting from glucuronyltransferase activity and N-acetylgalactosaminyltransferase activity, respectively. Quantitative real time PCR analysis revealed that the transcript level of CSS3 was much lower than that of CSS1, although it was ubiquitously expressed in various human tissues. These results indicate that CSS3 is a glycosyltransferase having both glucuronyltransferase and N-acetylgalactosaminyltransferase activities. It may make a contribution to CS biosynthesis that differs from that of CSS1. Chondroitin sulfate (CS) 1The abbreviations used are: CS, chondroitin sulfate; CSS, chondroitin sulfate synthase; HS, heparan sulfate; GlcUA, glucuronic acid; (β3)GlcUA-T, (β1,3-)glucuronyltransferase; (β3 or β4)Gal-T, (β1,3 or β1,4-)galactosyltransferase; (β4)GalNAc-T, (β1,4-)N-acetylgalactosaminyltransferase; (α4)Gn-T, (α1,4-)N-acetyglucosaminyltransferase; EST, expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MES, 2-(N-morpholino)ethanesulfonic acid.1The abbreviations used are: CS, chondroitin sulfate; CSS, chondroitin sulfate synthase; HS, heparan sulfate; GlcUA, glucuronic acid; (β3)GlcUA-T, (β1,3-)glucuronyltransferase; (β3 or β4)Gal-T, (β1,3 or β1,4-)galactosyltransferase; (β4)GalNAc-T, (β1,4-)N-acetylgalactosaminyltransferase; (α4)Gn-T, (α1,4-)N-acetyglucosaminyltransferase; EST, expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MES, 2-(N-morpholino)ethanesulfonic acid. proteoglycans are located in the extracellular matrix and on cell surfaces in various kinds of human tissues. Some CS proteoglycans provide high osmotic pressure and water retention, and others may modulate cell adhesion to the extracellular matrix, proliferation, and morphogenesis (1Schwartz N.B. Domowicz M. Glycobiology. 2002; 12: R57-R68Crossref PubMed Scopus (102) Google Scholar, 2Bandtlow C.E. Zimmermann D.R. Physiol. Rev. 2000; 80: 1267-1290Crossref PubMed Scopus (529) Google Scholar). The biosynthetic assembly of chondroitin sulfate proteoglycans is characterized by the following sequential processes: (i) synthesis of the core protein; (ii) xylosylation of specific Ser moieties of the core protein; (iii) addition of two Gal residues to the Xyl; (iv) completion of common tetrasaccharide linkage region by the addition of a glucuronic acid (GlcUA) residue; (v) addition of an GalNAc residue to initiate the chondroitin/dermatan sulfate biosynthesis; (vi) repeated addition of GlcUA residues alternating with GalNAc residues to grow the large heteropolymer glycosaminoglycan chains; and (vii) modification of these growing glycosaminoglycan chains by variable O-sulfation and by variable epimerization of GlcUA to IdoUA.The assembly of the linkage region on the core protein followed by glycosaminoglycan polymerization and modification occurs in the intracellular membrane system, which is composed of the endoplasmic reticulum and Golgi apparatus (3Kimata K. Okayama M. Ooira A. Suzuki S. Mol. Cell. Biochem. 1973; 1: 211-228Crossref PubMed Scopus (48) Google Scholar, 4Silbert J.E. Sugumaran G. IUBMB Life. 2002; 54: 177-186Crossref PubMed Scopus (250) Google Scholar). With the exception of the polysaccharide chain-initiating Xyl transferase, which is found in the endoplasmic reticulum (5Vertel B.M. Walters L.M. Flay N. Kearns A.E. Schwartz N.B. J. Biol. Chem. 1993; 268: 11105-11112Abstract Full Text PDF PubMed Google Scholar), all of the enzymes are firmly attached to the Golgi membranes and may work in an orchestrated manner. Some of these biosynthetic enzymes are found in serum or in the culture medium of the cells (4Silbert J.E. Sugumaran G. IUBMB Life. 2002; 54: 177-186Crossref PubMed Scopus (250) Google Scholar, 6Gotting C. Kuhn J. Zahn R. Brinkmann T. Kleesiek K. J. Mol. Biol. 2000; 304: 517-528Crossref PubMed Scopus (197) Google Scholar). The enzymes responsible for the synthesis of the linkage regions in proteoglycans, Xyl transferase (6Gotting C. Kuhn J. Zahn R. Brinkmann T. Kleesiek K. J. Mol. Biol. 2000; 304: 517-528Crossref PubMed Scopus (197) Google Scholar), Gal transferase I (7Almeida R. Levery S.B. Mandel U. Kresse H. Schwientek T. Bennett E.P. Clausen H. J. Biol. Chem. 1999; 274: 26165-26171Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 8Okajima T. Yoshida K. Kondo T. Furukawa K. J. Biol. Chem. 1999; 274: 22915-22918Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), Gal transferase II (9Bai X. Zhou D. Brown J.R. Crawford B.E. Hennet T. Esko J.D. J. Biol. Chem. 2001; 276: 48189-48195Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), as well as GlcUA transferase I (10Kitagawa H. Tone Y. Tamura J. Neumann K.W. Ogawa T. Oka S. Kawasaki T. Sugahara K. J. Biol. Chem. 1998; 273: 6615-6618Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 11Wei G. Bai X. Sarkar A.K. Esko J.D. J. Biol. Chem. 1999; 274: 7857-7864Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), which act sequentially to transfer Xyl, Gal, Gal, and GlcUA from their respective sugar nucleotide precursors to the acceptor core protein, have been cloned. We have been interested in characterizing the modification reactions, especially sulfations, because specific regional structures created by these modifications allow chondroitin sulfate to interact with other molecules, including cytokines. The modifications also allow regulation of the assembly and activities of other proteins in extracellular and pericellular matrices (12Maimone M.M. Tollefsen D.M. J. Biol. Chem. 1990; 265: 18263-18271Abstract Full Text PDF PubMed Google Scholar– 17Fried M. Duffy P.E. Science. 1996; 272: 1502-1504Crossref PubMed Scopus (930) Google Scholar). With the exception of chondroitin C5-epimerase, most of modifying enzymes in chondroitin sulfate biosynthesis have been cloned, such as chondroitin O-sulfotransferases, including chondroitin 4-O-sulfotransferase (18Yamauchi S. Mita S. Matsubara T. Fukuta M. Habuchi H. Kimata K. Habuchi O. J. Biol. Chem. 2000; 275: 8975-8981Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), chondroitin 6-O-sulfotransferase (19Uchimura 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 (143) Google Scholar), uronyl 2-O-sulfotransferase (20Kobayashi M. Sugumaran G. Liu J. Shworak N.W. Silbert J.E. Rosenberg R.D. J. Biol. Chem. 1999; 274: 10474-10480Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (21Ohtake S. Ito Y. Fukuta M. Habuchi O. J. Biol. Chem. 2001; 276: 43894-43900Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The sulfation of chondroitin sulfate ordinarily proceeds along with polymerization at the Golgi apparatus. Thus, in order to address control mechanisms of the sulfation reaction, the enzymes involved in the chain synthesis should also be studied, especially the chondroitin sulfate elongation enzymes.Recent progress with the human genome project and the expansion of other data bases, such as expressed sequence tags (ESTs) and full-length cDNAs, have enabled a wider search for novel genes that are homologous to known genes. Kitagawa et al. (22Kitagawa H. Uyama T. Sugahara K. J. Biol. Chem. 2001; 276: 38721-38726Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) identified a human chondroitin synthase from the HUGE (human unidentified gene-encoded large proteins) protein data base by screening with the keywords: “one trans-membrane domain” and “galactosyltransferase family.” This enzyme had the dual glycosyltransferase activities of glucuronyltransferase II (GlcUAT-II) and N-acetylgalactosaminyltransferase II (GalNAcT-II) that are responsible for synthesizing the repeated disaccharide units of chondroitin sulfate (22Kitagawa H. Uyama T. Sugahara K. J. Biol. Chem. 2001; 276: 38721-38726Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). By a similar search of the data base for homologues, five enzymes were found, including chondroitin synthase, which have been cloned and characterized. Thus the enzyme that contributes to the synthesis of repeated disaccharide units on chondroitin sulfate was designated chondroitin sulfate synthase-1 (CSS1), and the others were named CSGalNAcT-1, CSGalN-AcT-2, CSGlcUAT, and CSS2. Chondroitin sulfate GalNAcT-1 (CSGalNAcT-1) and chondroitin sulfate GalNAcT-2 (CSGalN-AcT-2), the second and fourth chondroitin sulfate glycosyltransferases cloned, respectively, exhibit both GalNAcT-II activity for chain elongation and GalNAcT-I activity that determines and initiates the synthesis of the common linkage region of chondroitin sulfate (23Uyama T. Kitagawa H. Tanaka J. Tamura J.I. Ogawa T. Sugahara K. J. Biol. Chem. 2002; 278: 3072-3078Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 24Sato T. Gotoh M. Kiyohara K. Akashima T. Iwasaki H. Kameyama A. Mochizuki H. Yada T. Inaba N. Togayachi A. Kudo T. Asada M. Watanabe H. Imamura T. Kimata K. Narimatsu H. J. Biol. Chem. 2003; 278: 3063-3071Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 25Gotoh M. Sato T. Akashima T. Iwasaki H. Kameyama A. Mochizuki H. Yada T. Inaba N. Zhang Y. Kikuchi N. Kwon Y.D. Togayachi A. Kudo T. Nishihara S. Watanabe H. Kimata K. Narimatsu H. J. Biol. Chem. 2002; 277: 38189-38196Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 26Uyama T. Kitagawa H. Tamura J.-i. Sugahara K. J. Biol. Chem. 2002; 277: 8841-8846Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Chondroitin sulfate GlcUA transferase (CSGlcUAT), the third chondroitin sulfate glycosyltransferase cloned, has only GlcUAT-II activity, which is involved in chain elongation (27Gotoh M. Yada T. Sato T. Akashima T. Iwasaki H. Mochizuki H. Inaba N. Togayachi A. Kudo T. Watanabe H. Kimata K. Narimatsu H. J. Biol. Chem. 2002; 277: 38179-38188Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Chondroitin sulfate synthase-2, the fifth chondroitin sulfate glycosyltransferase cloned, has both GlcUAT-II and GalNAcT-II activities, which are responsible for synthesis of the repeated disaccharide units of chondroitin sulfate (41Yada T. Gotoh M. Sato T. Shionyu M. Go M. Kaseyama H. Iwasaki H. Kikuchi N. Kwon Y.-D. Togayachi A. Kudo T. Watanabe H. Narimatsu H. Kimata K. J. Biol. Chem. 2003; 278: 30235-30245Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Therefore, more than five enzymes are likely responsible for chondroitin/dermatan sulfate biosynthesis, and they form a gene family analogous to the EXT family for heparin/heparan sulfate biosynthesis (28Zak B.M. Crawford B.E. Esko J.D. Biochim. Biophys. Acta. 2002; 1573: 346-355Crossref PubMed Scopus (145) Google Scholar).In the present study, a search of several data bases using the amino acid sequences of CS glycosyltransferases revealed a novel gene whose product was characterized as the sixth enzyme having a high homology to CSS1. Interestingly, as implied by its homology to CSS1, this enzyme, designated CSS3, shows both GlcUAT-II and GalNAcT-II activities toward the nonreducing terminal residue of chondroitin/chondroitin sulfate, which has a specific linkage structure but shows no polymerization reaction activity. However, its expression level was much lower than that of CSS1, and its specific activity was about one-tenth the activity of CSS1.EXPERIMENTAL PROCEDURESMaterials—UDP-[14C]GlcUA (313 mCi/mmol) and UDP-[3H]Gal (20 Ci/mmol) were purchased from ICN Biomedicals (Irvine, CA) and ARC (St. Louis, MO), respectively. UDP-[3H]GalNAc (7.0 Ci/mmol) and UDP-[14C]GlcNAc (200 mCi/mmol) were from PerkinElmer Life Sciences. Chondroitin (a chemically desulfated derivative of whale cartilage chondroitin sulfate A), chondroitin sulfate A (whale cartilage), dermatan sulfate (pig skin), chondroitin sulfate C (shark cartilage), chondroitin sulfate D (shark cartilage), chondroitin sulfate E (squid cartilage), hyaluronan (rooster comb), heparan sulfate (pig aorta), α-N-acetylgalactosaminidase (EC 3.2.1.49 from Acremonium sp.), and chondroitinase ACII (EC 4.2.2.5 from Arthrobacter aurescens) were from Seikagaku Corp. (Tokyo, Japan). Testicular hyaluronidase (EC 3.2.1.35, H6254, type V from sheep testes), β-glucuronidase (EC 3.2.1.31, G0501, type B-10, from bovine liver), heparin (bovine intestine), Galβ1–3Gal-NAcα-O-benzyl, d-GlcUAβ-O-4-nitrophenly, anti-FLAG BioM2 antibody, anti-FLAG M2-agarose gel, and pFLAG-CMV1 were from Sigma. The pcDNA3.1 plasmid was from Invitrogen. The Superdex™ Peptide HR10/30 column, HiLoad 16/60 Superdex 30-pg column, Fast Desalting column HR10/10, and PD10 desalting column were purchased from Amersham Biosciences. N-Acetyl heparosan, GlcNAcα-O-benzyl, GlcNAcβ-O-benzyl, Galα-O-benzyl, Galβ-O-benzyl, GalNAcα-O-benzyl, GalNAcβ-O-benzyl, Galβ1–3Galβ1–4Xylβ1-O-methoxyphenyl, GlcUA-β1–3Galβ1–3Galβ1–4Xylβ1-O-methoxyphenyl, and Galβ1–4GlcNAc-β1–3Galβ1–4GlcNAc were kindly provided by Seikagaku Corp.Cloning of CSS3 and Construction of CSS3 and CSS1 Expression Vectors—A BLAST search of the EST databases was performed using the amino acid sequences of the cloned human chondroitin sulfate glycosyltransferases, CSS1 (GenBank™ accession number AB023207), CSS2 (GenBank™ accession number AB086063), CSGlcUAT (GenBank™ accession number AB037823), CSGalNAcT-1(GenBank™ accession number AB081516), and CSGalNAcT-2 (GenBank™ accession number AB079252) as a query, and a novel EST clone was found (GenBank™ accession number AC004219). As the sequence was not complete, a GENSCAN search of human genomic databases was performed. The predicted sequence was confirmed by PCR with the following two sets of primers: set 1, 5′-ATGGCTGTGCGCTCTCGCCGCCCGT-3′ and 5′-CGTCCCCGCTGCCGTTGTGGCTACT-3′; set 2, 5′-AGTAGCCACAACGGCAGCGGGGACG-3′ and 5′-TCAGGAGAGAGTTCGATTGTACCT-3′ (GenBank™ accession number AB086062). The putative catalytic domain of CSS3 (amino acids 130–883) was expressed as a secreted protein fused with a FLAG peptide in COS-7 cells. An ∼2.3-kb DNA fragment was amplified by PCR using the Marathon-Ready™ cDNA derived from human brain (Clontech) as a template, with two primers, 5′-CCCAAGCTTGCCGAGGGGGAGCCCGA-3′ and 5′-GCTCTAGACTGTCAGGAGAGAGTTCGATT-3′. The amplified fragment was inserted between the HindIII and XbaI sites of pFLAG-CMV-1. The putative catalytic domain of CSS1 (amino acids 47–802) was expressed as a secreted protein fused with a FLAG peptide in COS-7 cells. An ∼2.3-kb DNA fragment was amplified by PCR using a cDNA clone, Kazusa DNA Research Institute number, KIAA0990, with 5′-AAGGAAAAAAGCGGCCGCGGGCTGCCGGTCCGGGCAG-3′ and 5′-GCTCTAGACATTAGGCTGTCCTCACTGA-3′. The amplified fragment was inserted between the NotI and XbaI sites of pFLAG-CMV-1.Purification of FLAG-tagged Recombinant Enzymes from Culture Supernatant—COS-7 cells (ATCC CRL-1651) were co-transfected with the expression plasmid and pcDNA3.1 by using TransFast™ (Promega, Madison, WI) according to the manufacturer's instructions. Stable transfectants were selected using 600 μg/ml G418 in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine serum (HyClone Laboratories, Logan, UT), 100 μg/ml streptomycin sulfate, and 100 units/ml penicillin G and cloned by limiting dilution. Cloned cell lines were tested for synthesis and secretion of the recombinant protein by immunoprecipitation and Western blotting of supernatants using an anti-FLAG BioM2 antibody (Sigma). The secreted enzyme was purified by affinity chromatography using an anti-FLAG M2-agarose gel (Sigma). The conditioned medium and gel were mixed overnight at 4 °C and centrifuged for 5 min, and the supernatant was aspirated. The gel was washed five times with 10 ml of 20% (v/v) glycerol in 50 mm Tris-HCl, pH 7.4, and resuspended in the same buffer containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A) to produce a 50% slurry. The immobilized enzyme was stable at 4 °C for at least 4 weeks. The amount of recombinant protein recovered was estimated by immunoblotting. It was separated by SDS-PAGE (10% gel), transferred onto polyvinylidene difluoride membranes (Immobilon, Millipore), and probed with an anti-FLAG peptide antibody BioM2 (Sigma). FLAG-tagged bacterial alkaline phosphatase (FLAG-BAP, molecular mass 49 kDa) was used as a standard to estimate the relative amount, as described previously (27Gotoh M. Yada T. Sato T. Akashima T. Iwasaki H. Mochizuki H. Inaba N. Togayachi A. Kudo T. Watanabe H. Kimata K. Narimatsu H. J. Biol. Chem. 2002; 277: 38179-38188Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Blotting and probing were performed according to the manufacturer's instructions, followed by horseradish peroxidase-conjugated streptavidin. Immune complexes were detected as positive bands using the ECL detection system (Amersham Biosciences) with 10-s exposures. The CSS3, CSS1, and BAP protein bands were quantified by densitometric scanning of the digitized image using NIH image (version 1.61) software. The standard curve for each substrate was generated by increasing the amount of FLAG-tagged BAP protein on the same blotting membrane as the CSS samples. The band intensity and the concentration of the recombinant CSS proteins (90 kDa) in the medium exhibited a linear correlation. The amounts of recombinant CSS proteins could therefore be estimated accurately from the standard curve, which was generated using known amounts of FLAG-tagged BAP protein (49 kDa). The amount of recombinant enzyme protein is expressed in arbitrary units, with each unit of intensity equivalent to 10 ng of FLAG-BAP protein (27Gotoh M. Yada T. Sato T. Akashima T. Iwasaki H. Mochizuki H. Inaba N. Togayachi A. Kudo T. Watanabe H. Kimata K. Narimatsu H. J. Biol. Chem. 2002; 277: 38179-38188Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar).Preparation of Acceptor Substrate—Glycosaminoglycan polymers were purchased from Seikagaku Corp. For the GlcUAT-II assay, chondroitin sulfate A–E, chondroitin, hyaluronan, heparan sulfate, and N-acetyl heparosan were digested with β-glucuronidase prior to the assay (27Gotoh M. Yada T. Sato T. Akashima T. Iwasaki H. Mochizuki H. Inaba N. Togayachi A. Kudo T. Watanabe H. Kimata K. Narimatsu H. J. Biol. Chem. 2002; 277: 38179-38188Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Even- and odd-numbered oligosaccharides of chondroitin sulfate, chondroitin, and hyaluronan were prepared as described previously (27Gotoh M. Yada T. Sato T. Akashima T. Iwasaki H. Mochizuki H. Inaba N. Togayachi A. Kudo T. Watanabe H. Kimata K. Narimatsu H. J. Biol. Chem. 2002; 277: 38179-38188Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar).Glycosyltransferase Assays—The glycosyl transferase activities were investigated using radioactive forms of UDP-GlcUA, UDP-GalNAc, UDP-GlcNAc, UDP-Gal, and various acceptor saccharide substrates, including chondroitin polymer, various chondroitin sulfate isoforms, hyaluronan, heparan sulfate, heparin (100 μg each), oligosaccharides of chondroitin, chondroitin sulfate isoforms, and hyaluronan (1 nmol each). The standard reaction mixture for GalNAcT-II contained 10 μl of the resuspended beads and acceptor substrate, 0.32 nmol of UDP-[3H]GalNAc (6.66 × 105 dpm), 50 mm MES, pH 6.2, and 10 mm MnCl2 in a total volume of 30 μl. The reaction mixture for GlcUAT-II contained 10 μl of the resuspended gel and the acceptor substrate, 0.307 nmol of UDP-[14C]GlcUA (2.22 × 105 dpm), 50 mm MES, pH 5.8 or 6.2, and 20 mm MnCl2 in a total volume of 30 μl. The reaction mixtures were incubated at 37 °C for 2 h with mixing. The reaction mixture for CSS3 and CSS1 polymerization reactions contained 10 μl of the resuspended gel and 1 nmol of CS-C10, 10 nmol of UDP-[3H]GalNAc (2.77 × 105 dpm), 10 nmol of UDP-GlcUA, 50 mm MES, pH 6.2, and 10 mm MnCl2 in a total volume of 30 μl. The reaction mixture for polymerization reactions carried out by Escherichia coli K4 strain chondroitin polymerase contained 1 nmol of CS-C10, 50 mm Tris-HCl, pH 7.2, 20 mm MnCl2, 0.1 m (NH4)2SO4, 1 m ethylene glycol, 10 nmol of UDP-[3H]Gal-NAc (2.77 × 105 dpm), 10 nmol of UDP-GlcUA, and 1 μg of the enzyme preparation in a total volume of 30 μl. The reaction mixtures were incubated at 37 °C (for CSS3 and CSS1) or at 30 °C (for E. coli K4 strain chondroitin polymerase) overnight. The reaction was then stopped by boiling for 5 min, and radiolabeled products were separated from free UDP-[3H]GalNAc or UDP-[14C]GlcUA by gel filtration by using the Superdex™ Peptide HR10/30 column (10 × 300 mm) with 0.2 m NaCl as an eluant or by using the HiLoad 16/60 Superdex 30-pg column (16 × 600 mm) with 0.2 m NH4HCO3 as an eluant (27Gotoh M. Yada T. Sato T. Akashima T. Iwasaki H. Mochizuki H. Inaba N. Togayachi A. Kudo T. Watanabe H. Kimata K. Narimatsu H. J. Biol. Chem. 2002; 277: 38179-38188Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The labeled products recovered were quantified by liquid scintillation counting. For the acceptor oligosaccharide substrates with an aromatic residue (methoxyphenyl-, benzyl-, or 4-nitrophenyl-) at the reducing terminus, the reaction products were diluted with 1 ml of 0.5 m NaCl and applied to a Sep-Pak C18 cartridge (100 mg; Waters, Milford, MA) (27Gotoh M. Yada T. Sato T. Akashima T. Iwasaki H. Mochizuki H. Inaba N. Togayachi A. Kudo T. Watanabe H. Kimata K. Narimatsu H. J. Biol. Chem. 2002; 277: 38179-38188Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The cartridge was washed with 3 ml of 0.5 m NaCl and then 3 ml of water, the product was eluted with 50% methanol, and the radioactivity of all fractions was measured by liquid scintillation counting.Identification of the Enzyme Reaction Products—Each product of the GlcUAT-II reaction using chondroitin or CS11 and of the GalNAcT-II reaction using chondroitin was isolated by gel filtration column chromatography using the Superdex™ Peptide HR10/30 column. The radioactive peak containing the product was pooled and desalted with the Fast Desalting column HR10/10 using distilled water as an eluant, and was lyophilized. In order to identify the linkage structure, the dried sample (about 20 pmol of radiolabeled material) from GlcUAT-II reaction was incubated with one of the following: 1) 100 milliunits of chondroitinase ACII in a total volume of 100 μl of 100 mm Tris-HCl, pH 7.4, containing 30 mm sodium acetate at 37 °C overnight; or 2) 1 unit of β-glucuronidase in a total volume of 100 μl of 100 mm sodium acetate buffer, pH 5.0, at 37 °C overnight. To confirm the linkage structure, assays were carried out to determine whether the product could serve as an acceptor for E. coli K4 strain chondroitin polymerase, which synthesizes chondroitin, and if the resultant products could be digested completely with chondroitinase ACII (29Ninomiya T. Sugiura N. Tawada A. Sugimoto K. Watanabe H. Kimata K. J. Biol. Chem. 2002; 277: 21567-21575Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) Briefly, 20 pmol of the radiolabeled material was lyophilized and served as a substrate for E. coli K4 strain chondroitin polymerase. The reaction was performed at 30 °C overnight in a 50-μl solution containing 50 mm Tris-HCl, pH 7.2, 20 mm MnCl2, 0.1 m (NH4)2SO4, 1 m ethylene glycol, 20 pmol of radiolabeled [14C]CS12, 30 nmol each of UDP-GlcUA and UDP-GalNAc, and 0.8 μg of the enzyme preparation. The solution was then boiled for 5 min to stop the reaction. The radioactive peak containing the product was pooled and desalted with the Fast Desalting column HR10/10 using distilled water as an eluant and was then lyophilized. The dried sample (about 20 pmol of radiolabeled material) containing the E. coli K4 strain chondroitin polymerase reaction products was incubated with 100 milliunits of chondroitinase ACII in a total volume of 100 μl of 100 mm Tris-HCl, pH 7.4, containing 30 mm sodium acetate at 37 °C overnight. The enzyme digests were analyzed using the same Superdex™ Peptide HR10/30 column described above. In order to identify the linkage, the dried sample (about 20 pmol of radiolabeled material) from GalNAcT-II reactions was incubated with 100 milliunits of chondroitinase ACII in a total volume of 100 μl of 100 mm Tris-HCl, pH 7.4, containing 30 mm sodium acetate at 37 °C overnight, or with 100 milliunits of α-N-acetylgalactosaminidase in a total volume of 100 μl of 50 mm sodium citrate buffer, pH 4.5, at 37 °C overnight. The enzyme digests were analyzed using the same Superdex™ Peptide HR10/30 column described above.Quantitative Analysis of the CSS3 Transcript in Human Tissues by Real Time PCR—For quantification of CSS3 transcripts, the real time PCR method was employed, as described in detail previously (30Iwai 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: 12802-12809Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Marathon Ready cDNA derived from various human tissues was purchased from Clontech. Standard curves for the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, were generated by serial dilution of pCR2.1 (Invitrogen) DNA containing the GAPDH gene. The primer set and probe for CSS3 are as follows: the forward primer, 5′-CCCAGAAAAAGTCCTTCATGATG-3′, and the reverse primer, 5′-AACTCTTCTAATTTGTCACCTTTGATGTAG-3′, and the probe, 5′-ATGAGTGGTTCATGCGC-3′, which contains a minor groove binder. The primer sets and probes for CSS1 are as follows: the forward primer for CSS1, 5′-AGTGTGTCTGGTCTTATGAGATGCA-3′, and the reverse primer for CSS1, 5′-AGCTGTGGAGCCTGTACTGGTAG-3′, and the probe for CSS1, 5′-ATGAGAATTACGAGCAGAAC-3′, which contains a minor groove binder. PCR products were measured continuously with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). The relative amounts of the transcripts were normalized to the amount of GAPDH transcript in the same cDNA sample.RESULTSMolecular Cloning of CSS3 and Determination of Its Nucleotide and Amino Acid Sequences—A BLAST search of the EST data bases was performed using the amino acid sequences of the cloned human chondroitin sulfate glycosyltransferases as a query, and a novel EST was found (GenBank™ accession number AC004219). As the sequence was incomplete, a GENSCAN search of human genomic data bases was performed. The predicted sequence was confirmed by PCR with two sets of primers as follows: set 1, 5′-ATGGCTGTGCGCTCTCGCCGCCCGT-3′ and 5′-CGTCCCCGCTGCCGTTGTGGCTACT-3′; set 2, 5′-AGTAGCCACAACGGCAGCGGGGA
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