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Identification of Chondroitin Sulfate Glucuronyltransferase as Chondroitin Synthase-3 Involved in Chondroitin Polymerization

2008; Elsevier BV; Volume: 283; Issue: 17 Linguagem: Inglês

10.1074/jbc.m707549200

ISSN

1083-351X

Autores

Tomomi Izumikawa, Toshiyasu Koike, Shoko Shiozawa, Kazuyuki Sugahara, Jun’ichi Tamura, Hiroshi Kitagawa,

Tópico(s)

Polysaccharides Composition and Applications

Resumo

Recently, we demonstrated that chondroitin polymerization is achieved by any two combinations of human chondroitin synthase-1 (ChSy-1), ChSy-2 (chondroitin sulfate synthase 3, CSS3), and chondroitin-polymerizing factor (ChPF). Although an additional ChSy family member, called chondroitin sulfate glucuronyltransferase (CSGlcA-T), has been identified, its involvement in chondroitin polymerization remains unclear because it possesses only glucuronyltransferase II activity responsible for the elongation of chondroitin sulfate (CS) chains. Herein, we report that CSGlcA-T exhibits polymerization activity on α-thrombomodulin bearing the truncated linkage region tetrasaccharide through its interaction with ChSy-1, ChSy-2 (CSS3), or ChPF, and the chain length of chondroitin formed by the co-expressed proteins in various combinations is different. In addition, ChSy family members co-expressed in various combinations exhibited distinct but overlapping acceptor substrate specificities toward the two synthetic acceptor substrates, GlcUAβ1–3Galβ1-O-naphthalenemethanol and GlcUAβ1–3Galβ1-O-C2H4NH-benzyloxycarbonyl, both of which share the disaccharide sequence with the glycosaminoglycan-protein linkage region tetrasaccharide. Moreover, overexpression of CSGlcA-T increased the amount of CS in HeLa cells, whereas the RNA interference of CSGlcA-T resulted in a reduction of the amount of CS in the cells. Furthermore, the analysis using the CSGlcA-T mutant that lacks any glycosyltransferase activity but interacts with other ChSy family members showed that the glycosyltransferase activity of CSGlcA-T plays an important role in chondroitin polymerization. Overall, these results suggest that chondroitin polymerization is achieved by multiple combinations of ChSy-1, ChSy-2, CSGlcA-T, and ChPF and that each combination may play a unique role in the biosynthesis of CS. Based on these results, we renamed CSGlcA-T chondroitin synthase-3 (ChSy-3). Recently, we demonstrated that chondroitin polymerization is achieved by any two combinations of human chondroitin synthase-1 (ChSy-1), ChSy-2 (chondroitin sulfate synthase 3, CSS3), and chondroitin-polymerizing factor (ChPF). Although an additional ChSy family member, called chondroitin sulfate glucuronyltransferase (CSGlcA-T), has been identified, its involvement in chondroitin polymerization remains unclear because it possesses only glucuronyltransferase II activity responsible for the elongation of chondroitin sulfate (CS) chains. Herein, we report that CSGlcA-T exhibits polymerization activity on α-thrombomodulin bearing the truncated linkage region tetrasaccharide through its interaction with ChSy-1, ChSy-2 (CSS3), or ChPF, and the chain length of chondroitin formed by the co-expressed proteins in various combinations is different. In addition, ChSy family members co-expressed in various combinations exhibited distinct but overlapping acceptor substrate specificities toward the two synthetic acceptor substrates, GlcUAβ1–3Galβ1-O-naphthalenemethanol and GlcUAβ1–3Galβ1-O-C2H4NH-benzyloxycarbonyl, both of which share the disaccharide sequence with the glycosaminoglycan-protein linkage region tetrasaccharide. Moreover, overexpression of CSGlcA-T increased the amount of CS in HeLa cells, whereas the RNA interference of CSGlcA-T resulted in a reduction of the amount of CS in the cells. Furthermore, the analysis using the CSGlcA-T mutant that lacks any glycosyltransferase activity but interacts with other ChSy family members showed that the glycosyltransferase activity of CSGlcA-T plays an important role in chondroitin polymerization. Overall, these results suggest that chondroitin polymerization is achieved by multiple combinations of ChSy-1, ChSy-2, CSGlcA-T, and ChPF and that each combination may play a unique role in the biosynthesis of CS. Based on these results, we renamed CSGlcA-T chondroitin synthase-3 (ChSy-3). Chondroitin sulfates (CSs) 3The abbreviations used are: CS, chondroitin sulfate; ChSy, chondroitin synthase; GalNAcT-II, β1,4-N-acetylgalactosaminyltransferase II; GlcAT-II, β1,3-glucuronyltransferase II; GalNAcT-I, β1,4-N-acetylgalactosaminyltransferase I; TM, thrombomodulin; Cbz, benzyloxycarbonyl; NM, naphthalenemethanol; MES, 2-(N-morpholino)ethanesulfonic acid; GFP, green fluorescent protein; GlcUA, d-glucuronic acid; siRNA, small interfering RNA; NTA, nitrilotriacetic acid; HPLC, high pressure liquid chromatography; GFP, green fluorescent protein; EGFP, enhanced GFP; ER, endoplasmic reticulum. are universally ubiquitous molecules distributed on cell surfaces and in extracellular matrices (1Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1680) Google Scholar, 2Esko J.D. Selleck S.B. Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1254) Google Scholar, 3Prydz K. Dalen K.T. J. Cell Sci. 2000; 113: 193-205Crossref PubMed Google Scholar, 4Sugahara K. Kitagawa H. Curr. Opin. Struct. Biol. 2000; 10: 518-527Crossref PubMed Scopus (356) Google Scholar). CS is a linear, sulfated polysaccharide composed of repeating disaccharide units consisting of alternating uronic acid (GlcUA) and GalNAc residues and synthesized as a proteoglycan bound to specific Ser residues in the core protein (1Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1680) Google Scholar, 2Esko J.D. Selleck S.B. Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1254) Google Scholar, 3Prydz K. Dalen K.T. J. Cell Sci. 2000; 113: 193-205Crossref PubMed Google Scholar, 4Sugahara K. Kitagawa H. Curr. Opin. Struct. Biol. 2000; 10: 518-527Crossref PubMed Scopus (356) Google Scholar). Compelling evidence has shown that CS-proteoglycans play crucial roles in a number of physiological phenomena, such as cell adhesion, morphogenesis, neural network formation, and cell division (5Perrimon N. Bernfield M. Nature. 2000; 404: 725-728Crossref PubMed Scopus (662) Google Scholar, 6Sugahara K. Mikami T. Uyama T. Mizuguchi S. Nomura K. Kitagawa H. Curr. Opin. Struct. Biol. 2003; 13: 612-620Crossref PubMed Scopus (600) Google Scholar). Therefore, an understanding of CS synthesis and its regulatory mechanism underlying diverse CS functions is essential. The biosynthesis of CS is initiated by the addition of Xyl to specific serine residues in the core protein, followed by the sequential addition of two Gal residues and a GlcUA residue, forming the tetrasaccharide linkage structure GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser. Each transferring reaction is catalyzed by the corresponding glycosyltransferase. Then chondroitin polymerization with alternating GalNAc and GlcUA takes place, forming the repeating disaccharide region. Then a number of sulfotransferases modify the chondroitin backbone with sulfate at specific positions, resulting in the structural diversity of CS (7Kusche-Gullberg M. Kjellén L. Curr. Opin. Struct. Biol. 2003; 13: 605-611Crossref PubMed Scopus (245) Google Scholar). To date, six homologous glycosyltransferases, all of which are probably responsible for CS biosynthesis, have been cloned. We and others have revealed four chondroitin-synthesizing enzymes: chondroitin synthase-1 (ChSy-1), chondroitin synthase-2 (ChSy-2)/chondroitin sulfate synthase-3 (CSS3), and chondroitin GalNAc transferases 1 and 2 (8Kitagawa H. Uyama T. Sugahara K. J. Biol. Chem. 2001; 276: 38721-38726Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 9Uyama T. Kitagawa H. Tamura J. Sugahara K. J. Biol. Chem. 2002; 277: 8841-8846Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 10Gotoh 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, 11Sato 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 (88) Google Scholar, 12Uyama T. Kitagawa H. Tanaka J. Tamura J. Ogawa T. Sugahara K. J. Biol. Chem. 2003; 278: 3072-3078Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). ChSy-1 and ChSy-2 show dual glycosyltransferase activities of GlcUA transferase II (GlcAT-II) and GalNAc transferase II (GalNAcT-II), which are responsible for synthesizing the repeating disaccharide units of CS, whereas chondroitin GalNAc transferases 1 and 2 catalyze chain initiation and elongation, exhibiting activities of N-acetylgalactosaminyltransferase I (GalNAcT-I) and GalNAcT-II (9Uyama T. Kitagawa H. Tamura J. Sugahara K. J. Biol. Chem. 2002; 277: 8841-8846Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 10Gotoh 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, 11Sato 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 (88) Google Scholar, 12Uyama T. Kitagawa H. Tanaka J. Tamura J. Ogawa T. Sugahara K. J. Biol. Chem. 2003; 278: 3072-3078Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In addition, chondroitin sulfate GlcUA transferase (CSGlcA-T) has been identified by others (13Gotoh 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 (65) Google Scholar). Previously, we revealed that chondroitin polymerization could be demonstrated in vitro when ChSy-1 was co-expressed with chondroitin-polymerizing factor (ChPF), which shows a weak yet significant homology to ChSy-1 (14Kitagawa H. Izumikawa T. Uyama T. Sugahara K. J. Biol. Chem. 2003; 278: 23666-23671Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 15Yada T. Gotoh M. Sato T. Shionyu M. Go M. Kaseyama H. Iwasaki H. Kikuchi N. Kwon Y.-D. Togayachi A. Kudo T. Watanabe H. Kimata K. Narimatsu H. J. Biol. Chem. 2003; 278: 30235-30247Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Although ChPF has little glycosyltransferase activity, co-expression of ChPF and ChSy-1 resulted in a marked augmentation of not only the glycosyltransferase activity but also the polymerase activity of ChSy-1 (14Kitagawa H. Izumikawa T. Uyama T. Sugahara K. J. Biol. Chem. 2003; 278: 23666-23671Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). In addition, co-expressed ChSy-2 (CSS3) and ChSy-1 or ChSy-2 (CSS3) and ChPF showed chondroitin polymerase activities (16Izumikawa T. Uyama T. Okuura Y. Sugahara K. Kitagawa H. Biochem. J. 2007; 403: 545-552Crossref PubMed Scopus (87) Google Scholar). Thus, chondroitin polymerization was achieved by any two combinations of ChSy-1, ChSy-2 (CSS3), and ChPF. Although an additional ChSy family member, called CSGlcA-T, has been identified, the involvement of CSGlcA-T in chondroitin polymerization remains unclear, because it possesses only glucuronyltransferase II activity responsible for the elongation of CS chains (13Gotoh 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 (65) Google Scholar). Herein, we report that CSGlcA-T exhibits polymerization activity onto α-thrombomodulin bearing the truncated linkage region tetrasaccharide through its interaction with ChSy-1, ChSy-2 (CSS3), or ChPF. Based on these results, we renamed CSGlcA-T chondroitin synthase-3 (ChSy-3). Materials—UDP-[U-14C]GlcUA (285.2 mCi/mmol) and UDP-[3H]GalNAc (10 Ci/mmol) were purchased from PerkinElmer Life Sciences. Unlabeled UDP-GlcUA and UDP-GalNAc were obtained from Sigma. Chondroitin (a chemically desulfated derivative of whale cartilage chondroitin sulfate A) and Arthrobacter aurescens chondroitinase ABC (EC 4.2.2.4) were purchased from Seikagaku Corp. (Tokyo, Japan). Purified α-thrombomodulin (α-TM) (17Nawa K. Sakano K. Fujiwara H. Sato Y. Sugiyama N. Teruuchi T. Iwamoto M. Marumoto Y. Biochem. Biophys. Res. Commun. 1990; 171: 729-737Crossref PubMed Scopus (57) Google Scholar) was provided by the research institute, Dai-ichi Pharmaceutical Co. (Tokyo, Japan), and contained a linkage region tetrasaccharide, GlcUAβ1–3Galβ1–3Galβ1–4Xyl (18Nadanaka S. Kitagawa H. Sugahara K. J. Biol. Chem. 1998; 273: 33728-33734Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). GlcUAβ1–3Galβ1-O-naphthalenemethanol (NM) and GlcUAβ1–3Galβ1-O-C2H4NH-Cbz were chemically synthesized (19Izumikawa T. Kitagawa H. Mizuguchi S. Nomura K.H. Nomura K. Tamura J. Gengyo-Ando K. Mitani S. Sugahara K. J. Biol. Chem. 2004; 279: 53755-53761Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Superdex™ Peptide HR10/30 and Superdex™ 200 10/300 GL columns were obtained from Amersham Biosciences. Construction of a Soluble Form of CSGlcA-T (ChSy-3)—The cDNA fragment of a truncated form of CSGlcA-T (ChSy-3), lacking the first 57 N-terminal amino acids, was amplified by PCR with KIAA1402 cDNA obtained from the Kazusa DNA Research Institute (Chiba, Japan) as a template using a 5′-primer (5′-GAAGATCTAGAGCTCGGCTAGACCAAAG-3′) containing an in-frame BglII site and a 3′-primer (5′-GAAGATCTCCATCTTGCCTTGCCCTTCC-3′) containing a BglII site located 72 bp downstream of the stop codon. PCR was carried out with KOD-Plus DNA polymerase (TOYOBO, Tokyo) for 30 cycles at 94 °C for 30 s, 58 °C for 30 s, and 68 °C for 150 s in 5% (v/v) dimethyl sulfoxide. The PCR fragment was subcloned into the BamHI site of pGIR201protA (20Kitagawa H. Paulson J.C. J. Biol. Chem. 1994; 269: 1394-1401Abstract Full Text PDF PubMed Google Scholar), resulting in the fusion of the insulin signal sequence and the protein A sequence present in the vector, as described previously (8Kitagawa H. Uyama T. Sugahara K. J. Biol. Chem. 2001; 276: 38721-38726Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 14Kitagawa H. Izumikawa T. Uyama T. Sugahara K. J. Biol. Chem. 2003; 278: 23666-23671Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 16Izumikawa T. Uyama T. Okuura Y. Sugahara K. Kitagawa H. Biochem. J. 2007; 403: 545-552Crossref PubMed Scopus (87) Google Scholar). The nucleotide sequence of the amplified cDNA was determined in a 377 DNA sequencer (PE Applied Biosystems). Soluble forms of ChSy-1, ChSy-2, and ChPF were constructed previously (8Kitagawa H. Uyama T. Sugahara K. J. Biol. Chem. 2001; 276: 38721-38726Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 14Kitagawa H. Izumikawa T. Uyama T. Sugahara K. J. Biol. Chem. 2003; 278: 23666-23671Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 16Izumikawa T. Uyama T. Okuura Y. Sugahara K. Kitagawa H. Biochem. J. 2007; 403: 545-552Crossref PubMed Scopus (87) Google Scholar). Site-directed Mutagenesis—A two-stage PCR mutagenesis method was used to construct CSGlcA-T (ChSy-3) mutant. Two separate PCRs were performed to generate two overlapping gene fragments using the soluble form of CSGlcA-T (ChSy-3) cDNA as a template. In the first PCR, the sense 5′-primer described above and the antisense internal mutagenic primer listed below were used: D184A 5′-GCACATATGTGGCATCCTGCATGATG-3′ (the mutated nucleotide is underlined). In the second round of PCR, the sense internal mutagenic primer (complementary to the antisense internal mutagenic primer) and the antisense 3′-primer described above were used. These two PCR products were gel-purified and then used as a template for a third PCR containing the sense 5′-primer and the antisense 3′-primer described above. The final PCR fragment was subcloned into the BamHI site of pGIR201protA (20Kitagawa H. Paulson J.C. J. Biol. Chem. 1994; 269: 1394-1401Abstract Full Text PDF PubMed Google Scholar). The nucleotide sequence of the amplified cDNA was determined in a 377 DNA sequencer (PE Applied Biosystems). Expression of a Soluble Form of CSGlcA-T (ChSy-3) and Enzyme Assays—The expression plasmid (6.0 μg) was transfected into COS-1 cells on 100-mm plates using FuGENE™ 6 (Roche Applied Science) according to the manufacturer's instructions. For co-transfection experiments, the CSGlcA-T (ChSy-3) and ChSy-1, ChSy-2, or ChPF expression plasmids (3.0 μg each) were co-transfected into COS-1 cells on 100-mm plates using FuGENE 6, as above. Two days after transfection, 1 ml of the culture medium was collected and incubated with 10 μl of IgG-Sepharose (Amersham Biosciences) for 1 h at 4 °C. The beads recovered by centrifugation were washed with and then resuspended in the assay buffer and tested for GalNAcT and GlcUA transferase activities, as described below. To quantify the protein absorbed onto IgG-Sepharose beads, the bound protein was eluted with 1 m acetic acid and then quantified using the BCA Protein Assay Reagent (enhanced protocol; Pierce). Assays for GalNAcT-II and GlcAT-II were carried out using chondroitin as an acceptor and UDP-GalNAc or UDP-GlcUA as a sugar donor, respectively, as described previously (21Kitagawa H. Tsutsumi K. Ujikawa M. Goto F. Tamura J. Neumann K.W. Ogawa T. Sugahara K. Glycobiology. 1997; 7: 531-537Crossref PubMed Scopus (56) Google Scholar, 22Kitagawa H. Ujikawa M. Tsutsumi K. Tamura J. Neumann K.W. Ogawa T. Sugahara K. Glycobiology. 1997; 7: 905-911Crossref PubMed Scopus (39) Google Scholar). Polymerization reactions using α-TM, GlcUAβ1–3Galβ1-O-NM, or GlcUAβ1–3Galβ1-O-C2H4NH-Cbz as acceptors were coincubated in reaction mixtures containing the following constituents in a total volume of 20 μl: 1 nmol of α-TM, 100 nmol of GlcUAβ1–3Galβ1-O-NM or 100 nmol of GlcUAβ1–3Galβ1-O-C2H4NH-Cbz, 0.25 mm UDP-[3H]Gal-NAc (5.28 × 105 dpm), 0.25 mm UDP-GlcUA, 100 mm MES buffer, pH 6.5 or 5.8, 10 mm MnCl2, and 10 μl of the resuspended beads. The mixtures were incubated at 37 °C overnight. Characterization of the Enzyme Reaction Products—Products of polymerization reactions on α-TM were isolated by gel filtration on a Superdex peptide column with 0.2 m NH4HCO3 as the eluent. The [3H]GalNAc-labeled oligosaccharide chains were released from α-TM by alkaline reduction treatment using 1.0 m NaBH4, 0.05 m NaOH and then exhaustively digested with chondroitinase ABC using 50 mIU of the enzyme for 1 h, as described previously (23Sugahara K. Shigeno K. Masuda M. Fujii N. Kurosaka A. Takeda K. Carbohydr. Res. 1994; 255: 145-163Crossref PubMed Scopus (82) Google Scholar). An aliquot of the enzyme digest was subjected to gel filtration on a Superdex peptide column, as described above. To determine the size of reaction products, the remaining aliquot was subjected to gel filtration on a Superdex 200 column with 0.2 m NH4HCO3 as the eluent. Calibration of the Superdex 200 column was performed using a series of commercial polysaccharides of known size. Pull-down Assays—The cDNA fragment of a truncated form of CSGlcA-T (ChSy-3), lacking the first 57 N-terminal amino acids of putative CSGlcA-T (ChSy-3), was amplified using a 5′-primer (5′-CGGAATTCAGAGCTCGGCTAGACCAAAG-3′) containing an in-frame EcoRI site and a 3′-primer (5′-CGGAATTCCCATCTTGCCTTGCCCTTCC-3′) containing an EcoRI site. The cDNA fragment of a truncated form of ChSy-2, lacking the first 129 N-terminal amino acids of ChSy-2, was also amplified using a 5′-primer (5′-GCTCTAGAGGCTGCCGGTCCGGGCAG-3′) containing an in-frame XbaI site and a 3′-primer (5′-GCTCTAGACAATCTTAAAGGAGTCCTATGTA-3′) containing an XbaI site. Each DNA fragment was inserted into a pcDNA3Ins-His expression vector, resulting in the fusion of the protein with the insulin signal sequence and His6 sequence present in the vector. Combinations of these constructs and the protein A-tagged expression vectors were transfected into COS-1 cells on 100-mm plates using FuGENE™ 6 (Roche Applied Science) according to the manufacturer's instructions. Two days after transfection, 1 ml of the culture medium was collected and incubated with 10 μl of Ni2+-NTA-agarose (Qiagen) overnight at 4 °C. The beads recovered by centrifugation were washed with TBS buffer containing Tween 20 three times and subjected to SDS-PAGE (7% gel), and proteins were transferred to a polyvinylidene difluoride membrane. The membrane, after blocking in PBS containing 2% skim milk and 0.1% Tween 20, was incubated with IgG antibody and then treated with anti-mouse IgG conjugated with horseradish peroxidase (Amersham Biosciences). Proteins bound to the antibody were visualized with an ECL advance kit (Amersham Biosciences). Subcellular Localization—The cDNA fragment encoding CSGlcA-T (ChSy-3) was amplified using a 5′-primer (5′-CGGAATTCCTGGCAGGGCCTACCACC-3′) containing an EcoRI site and a 3′-primer (5′-CGGAATTCTGCTATTGGCCTGCTCCT-3′) containing an EcoRI site. The cDNA fragment encoding ChSy-1 was amplified using a 5′-primer (5′-CCCTCGAGGGAGCGGCGCGGGCATG-3′) containing an XhoI site and a 3′-primer (5′-CCCTCGAGGGCTGTCCTCACTGAGCCA-3′) containing an in-frame XhoI site. The cDNA fragment encoding ChSy-2 was amplified using a 5′-primer (5′-CCCTCGAGGCGACAGCCCAGCGAGCGTCCG-3′) containing an XhoI site and a 3′-primer (5′-CGGAATTCGAGTTCGATTGTACCTGACACC-3′) containing an in-frame EcoRI site. The cDNA fragment encoding ChPF was amplified using a 5′-primer (5′-CCCTCGAGACTCCTCTGGCTGCTCTGG-3′) containing an XhoI site and a 3′-primer (5′-CGGAATTCTGCTGTTGCCCTGCTCCT-3′) containing an in-frame EcoRI site. PCR was carried out with KOD-Plus DNA polymerase (TOYOBO) for 30 cycles at 94 °C for 30 s, 53 °C for 42 s, and 68 °C for 180 s in 5% (v/v) dimethyl sulfoxide. Each PCR fragment was subcloned into the pEGFP-N1 or pDsRed-Monomer-N1 expression vector (Clontech). The Golgi marker vector (pEGFP-Golgi) was constructed using the pECFP-Golgi vector (Clontech) that harbors a sequence encoding the N-terminal 81 amino acids of human β1–4-galactosyltransferase (24Watzele G. Berger E.G. Nucleic Acids Res. 1990; 18: 7174Crossref PubMed Scopus (29) Google Scholar). This region of human β1–4-galactosyltransferase contains the membrane-anchoring signal peptide that targets the fusion protein to the trans-medial region of the Golgi apparatus (25Llopis J. McCaffery J.M. Miyawaki A. Farquhar M.G. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6803-6808Crossref PubMed Scopus (930) Google Scholar, 26Yamaguchi N. Fukuda M.N. J. Biol. Chem. 1995; 270: 12170-12176Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 27Gleeson P.A. Teasdale R.D. Burke J. Glycoconj. J. 1994; 11: 381-394Crossref PubMed Scopus (47) Google Scholar). The region from pECFP-Golgi was digested with NheI and BamHI and subcloned into pEGFP-N1. In addition, the ER marker vector (pDsRed2-ER) was obtained from Clontech. Combinations of GFP-tagged and DsRed-Monomer-tagged expression vectors (3.0 μg each) were transfected into HeLa cells on glass bottom dishes (Matsunami Glass) using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. Fluorescent images were obtained using a laser-scanning confocal microscope, FLUOVIEW (Olympus, Tokyo, Japan). Northern Blot Analysis—A commercial human 12-Lane Multiple Tissue Northern blot (Clontech) membrane was used for the analysis. The membrane was probed with a gel-purified, radiolabeled (>1 × 109 cpm/μg), 871-bp CSGlcA-T (ChSy-3)-specific fragment corresponding to nucleotides 3031–3903 of the CSGlcA-T cDNA (GenBank™ accession number AB095612). Establishment of an Expression Vector for CSGlcA-T (ChSy-3) and Preparation of Cells That Stably Overexpress CSGlcA-T (ChSy-3)—The cDNA fragment encoding CSGlcA-T (ChSy-3) was amplified from KIAA1402 cDNA as a template using a 5′-primer (5′-CGGAATTCCTGGCAGGGCCTACCACC-3′) containing an EcoRI site and a 3′-primer (5′-CGGAATTCCCATCTTGCCTTGCCCTTCC-3′) containing an EcoRI site. PCR was carried out with KOD-Plus DNA polymerase (TOYOBO) for 30 cycles at 94 °C for 30 s, 53 °C for 42 s, and 68 °C for 180 s in 5% (v/v) dimethyl sulfoxide. The PCR fragments were subcloned into the EcoRI site of the pCMV expression vector (Invitrogen). The nucleotide sequence of the amplified cDNA was determined in a 377 DNA sequencer (PE Applied Biosystems). The expression plasmid (6.7 μg) was transfected into HeLa cells on 100-mm plates using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. Transfectants were cultured in the presence of 1,000 μg/ml G418. Then resultant colonies were picked up and propagated for experiments. RNA Interference of the CSGlcA-T (ChSy-3) Gene—A 25-mer double-stranded RNA composed of sense 5′-GGCUUACAGUGAAAUAGAACAACUGAG-3′ and antisense 5′-CAGUUGUUCUAUUUCACUGUAAGCCAU-3′ sequences and of sense 5′-UCGGCUAGACCAAAGUGAUGAAGACAG-3′ and antisense 5′-GUCUUCAUCACUUUGGUCUAGCCGAAU-3′ sequences for CSGlcA-T (ChSy-3) was designed and purchased from iGENE (Tsukuba, Japan). Silencing, scrambled RNA composed of sequences with no homology to known human sequences (iGENE) was used as a control. The HeLa cells were transfected with 10 nm small interfering RNA (siRNA) using TransIT-TKO transfection reagent (Takara, Otsu, Japan). Quantitative Real Time Reverse Transcription-PCR—Total RNA was extracted from HeLa cells using a QuickPrep total RNA extraction kit (Amersham Biosciences). The cDNA was synthesized from ∼1 μg of total RNA using Moloney murine leukemia virus reverse transcriptase (Promega) and an oligo(dT)20-M4 adaptor primer (Takara). Primer sequences used were as follows: CSGlcA-T (ChSy-3), a forward primer 5′-GCTCGGCTAGACCAAAG-3′ and a reverse primer 5′-TGTAGCTCGGGAGGTCA-3′; and glyceraldehyde-3-phosphate dehydrogenase, a forward primer 5′-ATGGGTGTGAACCATGAGAAGTA-3′ and a reverse primer 5′-GGCAGTGATGGCATGGAC-3′. Quantitative real time reverse transcription-PCR was performed using a FastStart DNA Master plus SYBR Green I (Roche Applied Science) in a LightCycler ST300 (Roche Applied Science). The expression level of CSGlcA-T (ChSy-3) mRNA was normalized to that of the glyceraldehyde-3-phosphate dehydrogenase transcript. Derivatization of Glycosaminoglycans from HeLa Cells Using a Fluorophore, 2-Aminobenzamide—Cells were homogenized in acetone and air-dried. The dried materials were digested with heat-pretreated (60 °C for 30 min) actinase E in 200 μl of 0.1 m borate-sodium, pH 8.0, containing 10 mm calcium acetate at 60 °C for 24 h. Following incubation, each sample was treated with trichloroacetic acid, and the resultant precipitate was removed by centrifugation. The soluble fraction was extracted with ether. The aqueous phase was neutralized with 1.0 m sodium carbonate and adjusted to contain 80% ethanol. The resultant precipitate was dissolved in 50 mm pyridine acetate and subjected to gel filtration on a PD-10 column using 50 mm pyridine acetate as an eluent. The flow-through fractions were collected and evaporated to dryness. The dried sample was subsequently dissolved in water. Digestion with chondroitinase ABC (5 mIU) was conducted as described previously at 37 °C for 1 h in a total volume of 10 μl (28Sugahara K. Ohkita Y. Shibata Y. Yoshida K. Ikegami A. J. Biol. Chem. 1995; 270: 7204-7212Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Reactions were terminated by boiling for 1 min. Each digest was derivatized with 2-aminobenzamide and then analyzed by HPLC, as reported previously (29Kinoshita A. Sugahara K. Anal. Biochem. 1999; 269: 367-378Crossref PubMed Scopus (191) Google Scholar). Glycosyltransferase Activity of CSGlcA-T (ChSy-3)—Recent studies revealed that co-expression of any two of ChSy-1, ChSy-2 (CSS3), and ChPF augmented glycosyltransferase activities when compared with ChSy-1 or ChSy-2 expressed alone (14Kitagawa H. Izumikawa T. Uyama T. Sugahara K. J. Biol. Chem. 2003; 278: 23666-23671Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 16Izumikawa T. Uyama T. Okuura Y. Sugahara K. Kitagawa H. Biochem. J. 2007; 403: 545-552Crossref PubMed Scopus (87) Google Scholar). These findings prompted us to investigate whether the co-expression of an additional ChSy family member, CSGlcA-T (ChSy-3), despite having only GlcAT-II activity responsible for the elongation of CS chains (13Gotoh 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 (65) Google Scholar), with ChSy-1, ChSy-2 (CSS3), or ChPF might augment the glycosyltransferase activities. Hence, co-expression of CSGlcA-T (ChSy-3) with ChSy-1, ChSy-2 (CSS3), or ChPF was carried out. To facilitate the functional analysis of CSGlcA-T (ChSy-3), a soluble form of CSGlcA-T (ChSy-3) was generated by replacing the first 57 amino acids of the protein with a cleavable insulin signal sequence and a protein A IgG-binding domain, as described under "Experimental Procedures." Then the soluble protein was expressed in COS-1 cells as a recombinant protein fused with the protein A IgG-binding domain. The fusion protein secreted into the medium was adsorbed onto IgG-Sepharose beads for purification to eliminate endogenous glycosyltransferases, and then the protein-bound beads were used as an enzyme source. When CSGlcA-T (ChSy-3) bound to beads was evaluated for glycosyltransferase activities using chondroitin as an acceptor and either UDP-GalNAc or UDP-GlcUA as a donor substrate, weak GlcAT-II and GalNAcT-II activities were detected (Table 1). These results were distinct from the findings of Gotoh et al. (13Got

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