Chondroitin Sulfate Synthase-2
2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês
10.1074/jbc.m303657200
ISSN1083-351X
AutoresToshikazu Yada, Masanori Gotoh, Takashi Sato, Masafumi Shionyu, Mitiko Gō, Hiromi Kaseyama, Hiroko Iwasaki, Norihiro Kikuchi, Yeondae Kwon, Akira Togayachi, Takashi Kudo, Hideto Watanabe, Hisashi Narimatsu, Koji Kimata,
Tópico(s)Polysaccharides Composition and Applications
ResumoChondroitin sulfate is found in a variety of tissues as proteoglycans and consists of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues with sulfate residues at various places. We found a novel human gene (GenBank™ accession number AB086063) that possesses a sequence homologous with the human chondroitin sulfate glucuronyltransferase gene which we recently cloned and characterized. The full-length open reading frame encodes a typical type II membrane protein comprising 775 amino acids. The protein had a domain containing β3-glycosyltransferase motif but lacked a typical β4-glycosyltransferase motif, which is the same as chondroitin sulfate glucuronyltransferase, whereas chondroitin synthase had both domains. The putative catalytic domain was expressed in COS-7 cells as a soluble enzyme. Surprisingly, both glucuronyltransferase and N-acetylgalactosaminyltransferase activities were observed when chondroitin, chondroitin sulfate, and their oligosaccharides were used as the acceptor substrates. The reaction products were identified to have the linkage of GlcUAβ1–3GalNAc and GalNAcβ1–4GlcUA at the non-reducing terminus of chondroitin for glucuronyltransferase activity and N-acetylgalactosaminyltransferase activity, respectively. Quantitative real time PCR analysis revealed that the transcripts were ubiquitously expressed in various human tissues but highly expressed in the pancreas, ovary, placenta, small intestine, and stomach. These results indicate that this enzyme could synthesize chondroitin sulfate chains as a chondroitin sulfate synthase that has both glucuronyltransferase and N-acetylgalactosaminyltransferase activities. Sequence analysis based on three-dimensional structure revealed the presence of not typical but significant β4-glycosyltransferase architecture. Chondroitin sulfate is found in a variety of tissues as proteoglycans and consists of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues with sulfate residues at various places. We found a novel human gene (GenBank™ accession number AB086063) that possesses a sequence homologous with the human chondroitin sulfate glucuronyltransferase gene which we recently cloned and characterized. The full-length open reading frame encodes a typical type II membrane protein comprising 775 amino acids. The protein had a domain containing β3-glycosyltransferase motif but lacked a typical β4-glycosyltransferase motif, which is the same as chondroitin sulfate glucuronyltransferase, whereas chondroitin synthase had both domains. The putative catalytic domain was expressed in COS-7 cells as a soluble enzyme. Surprisingly, both glucuronyltransferase and N-acetylgalactosaminyltransferase activities were observed when chondroitin, chondroitin sulfate, and their oligosaccharides were used as the acceptor substrates. The reaction products were identified to have the linkage of GlcUAβ1–3GalNAc and GalNAcβ1–4GlcUA at the non-reducing terminus of chondroitin for glucuronyltransferase activity and N-acetylgalactosaminyltransferase activity, respectively. Quantitative real time PCR analysis revealed that the transcripts were ubiquitously expressed in various human tissues but highly expressed in the pancreas, ovary, placenta, small intestine, and stomach. These results indicate that this enzyme could synthesize chondroitin sulfate chains as a chondroitin sulfate synthase that has both glucuronyltransferase and N-acetylgalactosaminyltransferase activities. Sequence analysis based on three-dimensional structure revealed the presence of not typical but significant β4-glycosyltransferase architecture. Chondroitin sulfate (CS) 1The abbreviations used are: CS, chondroitin sulfate; (β3)GlcAT, (β1,3)-glucuronyltransferase; (β3 or β4)-Gal-T, (β1,3 or β1,4)-galactosyltransferase; (β4)GalNAc-T, (β1,4)-N-acetylgalactosaminyltransferase; EST, expressed sequence tag; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; MES, 2-(N-morpholino)ethanesulfonic acid. proteoglycans are located in the extracellular matrix and on cell surfaces of various kinds of human tissues. Some of the chondroitin sulfate proteoglycans provide high osmotic pressure and water retention, and others modulate not only cell adhesion to extracellular matrix, cell migration, cell proliferation, and morphogenesis but also some cytokine signals (1Schwartz N.B. Domowicz M. Glycobiology. 2002; 12: 57R-68RCrossref PubMed Scopus (104) Google Scholar, 2Bandtlow C.E. Zimmermann D.R. Physiol. Rev. 2000; 80: 1267-1290Crossref PubMed Scopus (540) Google Scholar). A few general features of the biosynthetic assembly of chondroitin sulfate proteoglycans are as follows: (i) the sequential 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 a common tetrasaccharide linkage region by addition of a GlcUA residue; (v) addition of GalNAc residue to initiate the chondroitin/dermatan sulfate biosynthesis; (vi) repeated addition of GlcUA residues alternating with GalNAc residues to grow to the large heteropolymer glycosaminoglycan chains; and (vii) modification of these growing glycosaminoglycan chains by O-sulfation at various places, and by epimerization of some of GlcUA residues to IdoUA residues. The assembly of the linkage region on the core protein followed by glycosaminoglycan polymerization and modification occurs in the intracellular membrane system 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 (257) Google Scholar). With the exception of the polysaccharide chain-initiating Xyl transferase, which is found partially in the endoplasmic reticulum (5Vertel B.M. Walters L.M. Flay N. Kearns A.E. Schwartz N.B. J. Biol. Chem. 1993; 268: 11105-11111Abstract Full Text PDF PubMed Google Scholar), all the enzymes are firmly attached to the Golgi membranes and may work in an orchestrated manner, but some are found in serum or the culture medium of cells (4Silbert J.E. Sugumaran G. IUBMB Life. 2002; 54: 177-186Crossref PubMed Scopus (257) Google Scholar, 6Inoue H. Otsu K. Yoneda M. Kimata K. Suzuki S. Nakanishi Y. J. Biol. Chem. 1986; 261: 4460-4469Abstract Full Text PDF PubMed Google Scholar). The enzymes responsible for the synthesis of the linkage region of proteoglycans, Xyl transferase (7Gotting C. Kuhn J. Zahn R. Brinkmann T. Kleesiek K. J. Mol. Biol. 2000; 304: 517-528Crossref PubMed Scopus (203) Google Scholar), Gal transferase I (8Almeida 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 (203) Google Scholar, 9Okajima T. Yoshida K. Kondo T. Furukawa K. J. Biol. Chem. 1999; 274: 22915-22918Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), Gal transferase II (10Bai 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 (142) Google Scholar), and GlcUA transferase I (11Kitagawa 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 (158) Google Scholar, 12Wei G. Bai X. Sarkar A.K. Esko J.D. J. Biol. Chem. 1999; 274: 7857-7864Abstract Full Text Full Text PDF PubMed Scopus (55) 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 the modification reactions, especially sulfations, because specific regional structures raised by the modifications determine the capacity of chondroitin sulfate to interact with other molecules including cytokines and regulate their assembly and activities in extracellular and pericellular matrices (13Maimone M.M. Tollefsen D.M. J. Biol. Chem. 1990; 265: 18263-18271Abstract Full Text PDF PubMed Google Scholar, 14Lyon 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, 15Razin E. Stevens R.L. Akiyama F. Schmid K. Austen K.F. J. Biol. Chem. 1982; 257: 7229-7236Abstract Full Text PDF PubMed Google Scholar, 16Maeda N. Ichihara-Tanaka K. Kimura T. Kadomatsu K. Muramatsu T. Noda M. J. Biol. Chem. 1999; 274: 12474-12479Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 17Naujokas M.F. Morin M. Anderson M.S. Peterson M. Miller J. Cell. 1993; 74: 257-268Abstract Full Text PDF PubMed Scopus (202) Google Scholar, 18Fried M. Duffy P.E. Science. 1996; 272: 1502-1504Crossref PubMed Scopus (940) Google Scholar). Except for chondroitin C5-epimerase, most of modifying enzymes for chondroitin sulfate biosynthesis, such as chondroitin O-sulfotransferases including chondroitin 4-O-sulfotransferase (19Yamauchi 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 (99) Google Scholar), chondroitin 6-O-sulfotransferase (20Uchimura 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), uronyl 2-O-sulfotransferase (21Kobayashi 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 (130) Google Scholar), and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (22Ohtake S. Ito Y. Fukuta M. Habuchi O. J. Biol. Chem. 2001; 276: 43894-43900Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), have been cloned. The sulfation of chondroitin sulfate ordinarily proceeds together with polymerization at the Golgi apparatus. Thus, in order to address control mechanisms of the sulfation, we should also study the enzymes for the chain synthesis, especially 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 has enabled the search for novel genes that are homologous to known genes. Kitagawa et al. (23Kitagawa H. Uyama T. Sugahara K. J. Biol. Chem. 2001; 276: 38721-38726Abstract Full Text Full Text PDF PubMed Scopus (182) 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 transmembrane domain" and "galactosyltransferase family." This enzyme had the dual glycosyltransferase activities of glucuronyltransferase II (GlcAT-II) and N-acetylgalactosaminyltransferase II (GalNAcT-II) responsible for synthesizing the repeated disaccharide units of chondroitin sulfate (23Kitagawa H. Uyama T. Sugahara K. J. Biol. Chem. 2001; 276: 38721-38726Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). By a similar homology search of the data bases, four enzymes including chondroitin synthase have further been cloned and characterized. Chondroitin sulfate GalNAcT-1 (CSGalNAcT-1) and chondroitin sulfate GalNAcT-2 (CSGalNAcT-2), the second and fourth chondroitin glycosyltransferases cloned, respectively, exhibit both GalNAcT-II activity for chain elongation and GalNAcT-I activity that determine and initiate the synthesis of chondroitin sulfate in the common linkage region (24Uyama 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 (97) Google Scholar, 25Sato 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. 2002; 278: 3063-3071Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 26Gotoh 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 (66) Google Scholar, 27Uyama T. Kitagawa H. Tamura Ji J. Sugahara K. J. Biol. Chem. 2002; 277: 8841-8846Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Chondroitin sulfate GlcUA transferase (CSGlcAT), the third chondroitin glycosyltransferase cloned, has only GlcAT-II activity, which has been proposed to be involved in chain elongation (28Gotoh 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 (64) Google Scholar). Therefore, more than four enzymes are likely responsible for chondroitin/dermatan sulfate biosynthesis, and they form a gene family, like the EXT family for heparin/heparan sulfate biosynthesis (29Zak B.M. Crawford B.E. Esko J.D. Biochim. Biophys. Acta. 2002; 1573: 346-355Crossref PubMed Scopus (147) Google Scholar). In the present study, a search of the data bases using the amino acid sequence of CSGlcAT revealed a novel gene whose product was characterized as the fifth enzyme to possess high homology with CSGlcAT. Interestingly, despite its homology with CSGlcAT, this enzyme, designated CSS2, shows both GlcAT-II and GalNAcT-II activity toward the non-reducing terminal residue of chondroitin/chondroitin sulfate with the specific linkage structure. Materials—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), Galβ1–3GalNAcα-O-benzyl, d-GlcUAβ-O-4-nitrophenyl, anti-FLAG BioM2 antibody, anti-FLAG M2-agarose gel, and pFLAG-CMV1 were from Sigma. A pcDNA3.1 was from Invitrogen. A 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, GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-methoxyphenyl, and Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc were kindly provided by Seikagaku Corp. Construction of CSS2 Expression Vector—We performed a BLAST search of the EST data bases using the amino acid sequence of the cloned human CSGlcAT as a query, and we found a novel EST clone (GenBank™ accession number NM_018590) (28Gotoh 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 (64) Google Scholar). As the sequence was not complete, a GeneScan search was performed on the human genomic data bases. The predicted sequence was confirmed by PCR with two primers, 5′-ACTCCTCTGGCTGCTCTGGGGGTTCG-3′ and 5′-TCTGGTTTTGGGGGAGAAGTGG-3′ (GenBank™ accession number AB086063). The putative catalytic domain of the enzyme (amino acids 97–775) was expressed as a secreted protein fused with a FLAG peptide in COS-7 cells. An ∼2.0-kb DNA fragment was amplified by PCR using the Marathon-Ready™ cDNA derived from human brain (Clontech), as a template, and two primers, 5′-GGAATTCCGGCCAGGCCGCCAAAAAGGC-3′ and 5′-CGGGATCCTCAGGTGCTGTTGCCCTGCTCC-3′. The amplified fragment was inserted between EcoRI and BamHI sites of pFLAG-CMV-1 (Sigma). Purification of FLAG-tagged Recombinant Enzyme from Culture Supernatants—COS-7 cells (ATCC CRL-1651) were co-transfected with the expression plasmid and pcDNA3.1 using TransFast™ (Promega, Madison, WI) according to the manufacturer's instructions. The stable transfectants were selected with 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 using an anti-FLAG BioM2 antibody (Sigma). The secreted enzyme was purified by affinity chromatography using anti-FLAG M2-agarose gel (Sigma). The conditioned medium and gel were mixed overnight at 4 °C and centrifuged for 5 min, and the supernatants were 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 (28Gotoh 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 (64) Google Scholar). The immobilized enzyme was stable at 4 °C for at least 4 weeks. The amount of recombinant protein recovered was estimated by immunoblotting. FLAG-tagged bacterial alkaline phosphatase (Met-FLAG-BAP, molecular mass of 49 kDa) was used as a standard to estimate the relative amount, as described previously (28Gotoh 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 (64) Google Scholar). The amount of recombinant enzyme protein is expressed in arbitrary units, with each unit of intensity equivalent to 10 ng of FLAG-tagged BAP protein (28Gotoh 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 (64) Google Scholar). Preparation of Acceptor Substrate—Glycosaminoglycan polymers were purchased from Seikagaku Corp. For GlcAT-II assay, chondroitin sulfate A–E, chondroitin, hyaluronan, heparan sulfate, and N-acetyl-heparosan were digested with β-glucuronidase prior to the assay (28Gotoh 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 (64) Google Scholar). Briefly, 1 mg of each polymer was digested with 100 units of β-glucuronidase in a total volume of 1 ml of 100 mm sodium acetate buffer, pH 5.0, at 37 °C overnight. The digests were then boiled for 20 min; the denatured enzyme was removed by trichloroacetic acid precipitation from the resultant supernatants that were neutralized with sodium hydroxide, and the glycosaminoglycans were recovered by ethanol precipitation. The glycosaminoglycans were redissolved with distilled water of a concentration of 10 mg/ml. Oligosaccharides of chondroitin sulfate, chondroitin, and hyaluronan were prepared as described previously (28Gotoh 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 (64) Google Scholar). Briefly, for the preparation of the oligosaccharides with even numbers (4-, 6-, 8-, 10-, 12-, and 14-saccharides), each polymer (10 mg) was partially digested with 1,000 turbidity reducing units of testicular hyaluronidase in 1 ml of 0.1 m sodium acetate buffer, pH 5.2, containing 0.15 m NaCl at 37 °C for the appropriate incubation times. For the preparation of the oligosaccharides with odd numbers (5-, 7-, 9-, 11-, and 13-saccharides), the hyaluronidase digests were boiled for 20 min and further digested with 1,000 units of β-glucuronidase (EC 3.2.1.31, from bovine liver, Sigma) at 37 °C for 24 h. After inactivation of the enzyme by boiling for 10 min and centrifuging at 10,000 × g for 20 min at 4 °C, the supernatants of the digests were fractionated on the HiLoad 16/60 Superdex 30-pg column (16 × 600 mm) with 0.2 m NH4HCO3 at a flow rate of 2 ml per min, and absorbance was monitored at 225 nm. The peak fractions were pooled, and desalted on the PD10 desalting column with distilled water as an eluate. The uronic acid contents were then determined by the Bitter-Muir's method using d-glucuronic acid as a standard (30Bitter T. Muir H.M. Anal. Biochem. 1962; 4: 330-334Crossref PubMed Scopus (5205) Google Scholar). The desalted solution was lyophilized and redissolved in distilled water at a concentration of 1 mm for each oligosaccharide. Glycosyltransferase Assays—The glycosyltransferase activities were investigated with radioactive forms of UDP-GlcUA, UDP-GalNAc, UDP-GlcNAc, UDP-Gal, and various acceptor saccharide substrates, including polymer chondroitin, 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 GlcAT-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 6.2, and 10 mm MnCl2 in a total volume of 30 μl. The reaction mixtures were incubated at 37 °C for 1 h with mixing, and the reaction was stopped by boiling for 5 min, and then radiolabeled products were separated from free UDP-[3H]GalNAc or UDP-[14C]GlcUA by gel filtration using Superdex™ peptide HR10/30 column (10 × 300 mm) with 0.2 m NaCl as an eluant or HiLoad 16/60 Superdex 30-pg column (16 × 600 mm) with 0.2 m NH4HCO3 as an eluate (28Gotoh 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 (64) Google Scholar). The labeled products recovered were quantified by liquid scintillation counting. For the acceptor substrates of oligosaccharide with an aromatic residue (methoxyphenyl- or benzyl-) at the reducing terminus, 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) (28Gotoh 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 (64) 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. In repetitions of the experiments when different batches of the enzyme were used, an aliquot was first analyzed by SDS-PAGE and Western blotting using anti-FLAG BioM2 antibody with FLAG-BAP as a standard to obtain a comparable amount of enzyme. Identification of the Enzyme Reaction Products—Each product from the GlcAT-II reaction using chondroitin or CS11 and 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 lyophilized. In order to identify the linkage, the dried sample (about 20 pmol of radiolabeled material) from GlcAT-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 for 1 h or 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. For confirmation of the linkage structure, we determined whether the product could serve as an acceptor for Escherichia coli strain K4 chondroitin polymerase, which synthesizes chondroitin, and the resultant products could be digested with chondroitinase ACII completely (31Ninomiya 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 (88) Google Scholar). Briefly, the 20 pmol of radiolabeled materials was lyophilized and served as substrate for K4 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. This was followed by boiling 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 lyophilized. The dried sample (about 20 pmol of radiolabeled material) from the E. coli strain K4 chondroitin polymerase reaction 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 for 1 h. The enzyme digests were analyzed again using the same Superdex Peptide HR10/30 column as 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 for 1 h or 100 milliunits of α-N-acetylgalactosaminidase in a total volume of 100 μlof50mm sodium citrate buffer, pH 4.5, at 37 °C overnight. The enzyme digests were analyzed again using the same Superdex Peptide HR10/30 column as described above. Quantitative Analysis of the CSS2 Transcript in Human Tissues by Real Time PCR—For quantification of CSS2 transcripts, we employed the real time PCR method, as described in detail previously (32Iwai 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 (138) 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 the probe for CSS2 were as follows: the forward primer, 5′-GCTGAACTGGAACGCACGTA-3′, and the reverse primer, 5′-CGGGATGGTGCTGGAATAC-3′, and the probe, 5′-AGATCCAGGAGTTACAGTGG-3′, with a minor groove binder. The primer sets and the probes for CSGlcAT and CSS1 were as follows: the forward primer for CSGlcAT, 5′-GTGAAATAGAACAACTGCAGGCTC-3′, and the reverse primer for CSGlcAT, 5′-GAGAAGGTGTGCTGCTCTGTGA-3′, the probe for CSGlcAT, 5′-CGGAACCTGACCGTGC-3′, with a minor groove binder; 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′ with a minor groove binder. PCR products were continuously measured with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). The relative amounts of their transcripts were normalized to the amount of GAPDH transcript in the same cDNA. Comparison of the C-terminal Domain Structure between CSS2 and CSGlcAT by Homology Modeling—The molecular structures of the C-terminal halves of CSS2 (C-CSS2; amino acid residues 474–775) and CSGlcAT (C-CSGlcAT; amino acid residues 455–772) were estimated from the model of the C-terminal half of CSS1 (C-CSS1; amino acid residues 500–802) because the amino acid sequences of C-CSS2 and C-CSGlcAT are far diverged from other glycosyltransferases for which the three-dimensional structures are known. From the consensus result of three threading methods, 3D-PSSM (www.sbg.bio.ic.uk/~3dpssm/), FUGUE (www-cryst.bioc.cam.ac.uk/~fugue/prfsearch.html), and GenTHREADER (bioinf.cs.ucl.ac.uk/psiform.html), we chose bovine β4-galactosyltransferase (β4GalT-1) as a template for homology modeling of C-CSS1, because β4GalT-1 obtained the best score by 3D-PSSM and FUGUE and the second highest score by GenTHREADER. Furthermore, CSS1 has been classified into the same glycosyltransferase family (Family GT7) with β4GalT-1 by CAZy (afmb.cnrs-mrs.fr/~cazy/CAZY/index.html), in which CSGalNAcT-1 and CSGalNAcT-2 are also the same family members. With the pairwise alignment of C-CSS1 and β4GalT-1 derived from 3D-PSSM, homology modeling of C-CSS1 was performed using FAMS (33Ogata K. Umeyama H. J. Mol. Graphics Model. 2000; 18 (305–256): 258-272Crossref PubMed Scopus (86) Google Scholar). Then the amino acid sequences of C-CSS1, C-CSS2, C-CSGlcAT, CSGalNAcT-1 (amino acid residues 228–532), and CSGalNAcT-2 (amino acid residues 237–542) were aligned using ClustalW (34Thompson J.D. Higgins D.G.
Referência(s)