Biosynthesis of Dermatan Sulfate
2006; Elsevier BV; Volume: 281; Issue: 17 Linguagem: Inglês
10.1074/jbc.m513373200
ISSN1083-351X
AutoresMarco Maccarana, Benny Olander, Johan Malmström, Kerstin Tiedemann, Ruedi Aebersold, Ulf Lindahl, Jin‐Ping Li, Anders Malmström,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoWe identified the gene encoding chondroitin-glucuronate C5-epimerase (EC 5.1.3.19) that converts d-glucuronic acid to l-iduronic acid residues in dermatan sulfate biosynthesis. The enzyme was solubilized from bovine spleen, and an ∼43,000-fold purified preparation containing a major 89-kDa candidate component was subjected to mass spectrometry analysis of tryptic peptides. SART2 (squamous cell carcinoma antigen recognized by T cell 2), a protein with unknown function highly expressed in cancer cells and tissues, was identified by 18 peptides covering 26% of the sequence. Transient expression of cDNA resulted in a 22-fold increase in epimerase activity in 293HEK cell lysate. Moreover, overexpressing cells produced dermatan sulfate chains with 20% of iduronic acid-containing disaccharide units, as compared with 5% for mock-transfected cells. The iduronic acid residues were preferentially clustered in blocks, as in naturally occurring dermatan sulfate. Given the discovered identity, we propose to rename SART2 (Nakao, M., Shichijo, S., Imaizumi, T., Inoue, Y., Matsunaga, K., Yamada, A., Kikuchi, M., Tsuda, N., Ohta, K., Takamori, S., Yamana, H., Fujita, H., and Itoh, K. (2000) J. Immunol. 164, 2565–2574) with a functional designation, chondroitin-glucuronate C5-epimerase (or DS epimerase). DS epimerase activity is ubiquitously present in normal tissues, although with marked quantitative differences. It is highly homologous to part of the NCAG1 protein, encoded by the C18orf4 gene, genetically linked to bipolar disorder. NCAG1 also contains a putative chondroitin sulfate sulfotransferase domain and thus may be involved in dermatan sulfate biosynthesis. The functional relation between dermatan sulfate and cancer is unknown but may involve known iduronic acid-dependent interactions with growth factors, selectins, cytokines, or coagulation inhibitors. We identified the gene encoding chondroitin-glucuronate C5-epimerase (EC 5.1.3.19) that converts d-glucuronic acid to l-iduronic acid residues in dermatan sulfate biosynthesis. The enzyme was solubilized from bovine spleen, and an ∼43,000-fold purified preparation containing a major 89-kDa candidate component was subjected to mass spectrometry analysis of tryptic peptides. SART2 (squamous cell carcinoma antigen recognized by T cell 2), a protein with unknown function highly expressed in cancer cells and tissues, was identified by 18 peptides covering 26% of the sequence. Transient expression of cDNA resulted in a 22-fold increase in epimerase activity in 293HEK cell lysate. Moreover, overexpressing cells produced dermatan sulfate chains with 20% of iduronic acid-containing disaccharide units, as compared with 5% for mock-transfected cells. The iduronic acid residues were preferentially clustered in blocks, as in naturally occurring dermatan sulfate. Given the discovered identity, we propose to rename SART2 (Nakao, M., Shichijo, S., Imaizumi, T., Inoue, Y., Matsunaga, K., Yamada, A., Kikuchi, M., Tsuda, N., Ohta, K., Takamori, S., Yamana, H., Fujita, H., and Itoh, K. (2000) J. Immunol. 164, 2565–2574) with a functional designation, chondroitin-glucuronate C5-epimerase (or DS epimerase). DS epimerase activity is ubiquitously present in normal tissues, although with marked quantitative differences. It is highly homologous to part of the NCAG1 protein, encoded by the C18orf4 gene, genetically linked to bipolar disorder. NCAG1 also contains a putative chondroitin sulfate sulfotransferase domain and thus may be involved in dermatan sulfate biosynthesis. The functional relation between dermatan sulfate and cancer is unknown but may involve known iduronic acid-dependent interactions with growth factors, selectins, cytokines, or coagulation inhibitors. Proteoglycans consist of glycosaminoglycan (GAG) 5The abbreviations used are: GAG, glycosaminoglycan; aTalR, 2,5-anhydro-d-talitol (formed by the reduction of anhydrotalose units, obtained by deamination of galactosamine residues); CS, chondroitin sulfate; dK4, defructosylated K4; DS, dermatan sulfate; HexA, hexuronic acid; HS, heparan sulfate; IdoUA, l-iduronic acid; LC, liquid chromatography; MS, mass spectrometry; SART2, squamous cell carcinoma antigen recognized by T cell 2; MES, 4-morpholineethanesulfonic acid; BSA, bovine serum albumin; DTT, dithiothreitol; FBS, fetal bovine serum; ConA, concanavalin A; PAPS, adenosine 3′-phosphate,5′-phosphosulfate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. chains covalently linked to core proteins. The GAGs play important roles in mediating biological functions of proteoglycans, mainly due to their ability to interact with a variety of proteins (1Iozzo R.V. Nat. Rev. Mol. Cell Biol. 2005; 6: 646-656Crossref PubMed Scopus (399) Google Scholar, 2Perrimon N. Bernfield M. Semin. Cell Dev. Biol. 2001; 12: 65-67Crossref PubMed Scopus (98) Google Scholar, 3Handel T.M. Johnson Z. Crown S.E. Lau E.K. Proudfoot A.E. Annu. Rev. Biochem. 2005; 74: 385-410Crossref PubMed Scopus (432) Google Scholar, 4Kinsella M.G. Bressler S.L. Wight T.N. Crit. Rev. Eukaryotic Gene Expression. 2004; 14: 203-234Crossref PubMed Scopus (140) Google Scholar, 5Esko J.D. Lindahl U. J. Clin. Investig. 2001; 108: 169-173Crossref PubMed Scopus (788) Google Scholar). Chondroitin sulfate (CS)/dermatan sulfate (DS) proteoglycans carry GAGs composed of alternating units of GalNAc and GlcA, or IdoUA in the case of DS. The chondroitin/CS/DS family has been ascribed a variety of physiological/developmental effects that range from control of basic cellular processes such as cell division in Caenorhabditis elegans, scaffold functions in various types of connective tissue, to highly cell type-specific effects, as exemplified by the neurite outgrowth-promoting activity mediated by rare structures in cerebral CS/DS (6Bao X. Muramatsu T. Sugahara K. J. Biol. Chem. 2005; 280: 35318-35328Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 7Clement A.M. Nadanaka S. Masayama K. Mandl C. Sugahara K. Faissner A. J. Biol. Chem. 1998; 273: 28444-28453Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 8Sugahara K. Mikami T. Uyama T. Mizuguchi S. Nomura K. Kitagawa H. Curr. Opin. Struct. Biol. 2003; 13: 612-620Crossref PubMed Scopus (595) Google Scholar, 9Trowbridge J.M. Gallo R.L. Glycobiology. 2002; 12: R117-R125Crossref PubMed Google Scholar). Protein ligands targeted by CS/DS include growth factors, modulators of blood coagulation, selectins, and chemokines. It is important to define the CS/DS structures involved in selective protein binding and to understand how they are generated. The IdoUA-containing domains of DS chains are of particular significance in this regard, because IdoUA residues endow conformational flexibility to the polymer, which is believed to facilitate protein interactions (10Ferro D.R. Provasoli A. Ragazzi M. Casu B. Torri G. Bossennec V. Perly B. Sinay P. Petitou M. Choay J. Carbohydr. Res. 1990; 195: 157-167Crossref PubMed Scopus (215) Google Scholar). Biosynthesis of CS/DS involves initial formation of a precursor polysaccharide composed of alternating GlcA and GalNAc residues, which subsequently undergoes a series of modification reactions (11Silbert J.E. Sugumaran G. IUBMB Life. 2002; 54: 177-186Crossref PubMed Scopus (257) Google Scholar). Our previous studies established that the generation of IdoUA units in DS (12Malmstrom A. Fransson L.A. J. Biol. Chem. 1975; 250: 3419-3425Abstract Full Text PDF PubMed Google Scholar, 13Malmstrom A. J. Biol. Chem. 1984; 259: 161-165Abstract Full Text PDF PubMed Google Scholar), as well as in heparin/heparan sulfate (14Hook M. Lindahl U. Backstrom G. Malmstrom A. Fransson L. J. Biol. Chem. 1974; 249: 3908-3915Abstract Full Text PDF PubMed Google Scholar), occurs by C5-epimerization of a portion of the GlcA residues previously incorporated into the polysaccharide chain. Moreover, CS/DS polysaccharides are O-sulfated at C2 of GlcA and IdoUA and C4 and/or C6 of GalNAc (15Kusche-Gullberg M. Kjellen L. Curr. Opin. Struct. Biol. 2003; 13: 605-611Crossref PubMed Scopus (243) Google Scholar). Notably, IdoUA units are consistently found adjacent to 4-O-sulfated GalNAc residues. The extent of these modifications varies between tissues and seems to be influenced by the core protein structure (16Seidler D.G. Breuer E. Grande-Allen K.J. Hascall V.C. Kresse H. J. Biol. Chem. 2002; 277: 42409-42416Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In addition, both epimerization and sulfation can be affected by growth factors (17Tiedemann K. Olander B. Eklund E. Todorova L. Bengtsson M. Maccarana M. Westergren-Thorsson G. Malmstrom A. Glycobiology. 2005; 15: 1277-1285Crossref PubMed Scopus (50) Google Scholar). The enzymes involved in the biosynthesis of heparin/heparan sulfate and CS/DS have been cloned, all except the chondroitin-glucuronate C5-epimerase required for IdoUA formation in DS (in the following denoted DS epimerase). In this study, we therefore purified this enzyme ∼43,000-fold from bovine spleen microsomes and identified by mass spectrometry a candidate protein, SART2 (squamous cell carcinoma antigen recognized by T cells 2) that had previously been cloned but not assigned any specific function. Transgenic expression of this protein in mammalian cell lines yielded a product with DS epimerase activity, capable of inducing IdoUA formation in exogenous chondroitin substrate. Moreover, cells transfected with DS epimerase synthesized dermatan sulfate chains with increased IdoUA content compared with mock-transfected control cells. AH-Sepharose, Sephadex G-25, Red-Sepharose, and ConA-Sepharose gels, Mono-Q 5/5, Superose 12 HR 10/30, Mono-Q PC 1.6/5 (operated with Smart System), Superdex Peptide 10/300 GL, and PD-10 columns were obtained from Amersham Biosciences, as was d-[1-14C]glucose (57 mCi/mmol). d-[5-3H]Glucose (20 Ci/mmol) was from PerkinElmer Life Sciences, and inorganic carrier-free [35S]sulfate was from PerkinElmer Life Sciences. Chondroitinase ABC, AC-I, and B were from Seikagaku. Cell lines were from ATCC. Cell culture reagents were from Invitrogen. DNA oligonucleotides were synthesized by DNA Technology A/S, Denmark. Other chemicals were of reagent grade and were obtained from various commercial sources. K4 polysaccharide was a gift from P. Oreste (Italfarmaco, Milan, Italy). After defructosylation (18Rodriguez M.L. Jann B. Jann K. Eur. J. Biochem. 1988; 177: 117-124Crossref PubMed Scopus (106) Google Scholar), it was coupled to AH-Sepharose by a carbodiimide-mediated procedure (19Malmstrom A. Roden L. Feingold D.S. Jacobsson I. Backstrom G. Lindahl U. J. Biol. Chem. 1980; 255: 3878-3883Abstract Full Text PDF PubMed Google Scholar). Protease inhibitors were obtained from Sigma and applied at a final concentration of 1 mm phenylmethylsulfonyl fluoride and 1 μg/ml each of aprotinin, leupeptin, and pepstatin. Metabolically 3H- or 14C-labeled K4 polysaccharides (DS epimerase substrates) were prepared by growing a K4-producing Escherichia coli strain in medium containing d-[5-3H]glucose or d-[1-14C]glucose, as described previously (20Hannesson H.H. Hagner-McWhirter A. Tiedemann K. Lindahl U. Malmstrom A. Biochem. J. 1996; 313: 589-596Crossref PubMed Scopus (42) Google Scholar). The resulting labeled K4 polysaccharides were defructosylated (dK4). HS epimerase substrate was generated in an analogous manner by incubating E. coli K5 bacteria with d-[5-3H]glucose, followed by N-deacetylation and N-sulfation of the resultant radiolabeled polysaccharide (21Hagner-McWhirter A. Lindahl U. Li J. Biochem. J. 2000; 347: 69-75Crossref PubMed Google Scholar, 22Hagner-McWhirter A. Li J.P. Oscarson S. Lindahl U. J. Biol. Chem. 2004; 279: 14631-14638Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). DS epimerase was assayed by its ability to release 3H-labeled water from chondroitin substrate containing [3H]GlcA residues, essentially as described (23Malmstrom A. Aberg L. Biochem. J. 1982; 201: 489-493Crossref PubMed Scopus (34) Google Scholar), but with some modifications based on preliminary kinetics analysis of semipurified enzyme (data not shown). Enzyme samples, containing 3 mg of BSA as carrier unless otherwise stated, were desalted at 4 °C on Sephadex G-25 columns (0.7 × 3 cm), equilibrated with desalting buffer (20 mm MES (pH 5.5 at 37 °C), 10% glycerol, 0.5 mm EDTA, 0.1% Triton X-100, 1 mm DTT, protease inhibitors). Incubations were performed in a 100-μl final volume of 0.8× desalting buffer, 0.5 mg of BSA, 2 mm MnCl2, 0.5% Nonidet P-40, and 30,000 dpm [5-3H]dK4 or [1-14C]dK4 (∼200 μm HexA for both substrates). Incubations were done at 37 °C for 2–14 h and stopped by boiling for 5 min, and samples were centrifuged. For quantitation of [3H]water, 90 μl of the incubations were transferred to a distillation tube containing 200 μl of (unlabeled) water. After distillation (24Backstrom G. Hook M. Lindahl U. Feingold D.S. Malmstrom A. Roden L. Jacobsson I. J. Biol. Chem. 1979; 254: 2975-2982Abstract Full Text PDF PubMed Google Scholar), 200μl of the distillate was analyzed by scintillation counting. The assay is linear up to 3000 dpm of released 3H. Protein was estimated by the Bradford assay (Bio-Rad), using BSA as standard. HexA content was determined by the carbazole reaction (25Bitter T. Muir H.M. Anal. Biochem. 1962; 4: 330-334Crossref PubMed Scopus (5205) Google Scholar). Bovine spleen was obtained fresh from the local slaughterhouse and was processed immediately. All procedures were carried out at 4 °C, and all buffers contained 1 mm DTT as reducing agent. Step 1. Microsomal Preparation and Extraction—On day 1, three spleens were freed from surrounding fat tissue, cut into 1–2-cm cubes, washed twice with cold distilled water, and placed in homogenization buffer (20 mm MES, pH 6.5, 250 mm sucrose, 5 mm EDTA, protease inhibitors). Batches of 350 × g of tissue were first homogenized without any added buffer in a food processor and were then rehomogenized three times after step additions of 350, 500, and 500 ml of buffer. The homogenate was centrifuged for 15 min at 8,000 × g. The resulting supernatant was centrifuged at 38,400 × g for 45 min. The 38,400 × g pellet was extracted with 90 ml of solubilization buffer (20 mm MES, pH 6.5, 150 mm NaCl, 1 mm EDTA, 1% glycerol, 1% Triton X-100, protease inhibitors). The combined extracted material from 11 such preparations, in all corresponding to 3.9 kg of tissue, was subjected to two strokes with a 200-ml Potter device and centrifuged at 38,400 × g for 45 min. The supernatant was collected. On day 2, the final supernatant from day 1 was further centrifuged at 125,000 × g for 40 min. Approximately half of the resulting supernatant was clear and was collected, whereas the other half was turbid and was diluted 1:2 with solubilization buffer and recentrifuged, and the resulting clear supernatant was collected. Step 2. Red-Sepharose—The microsomal extract was applied at a flow rate of 1.5 ml/min to a Red-Sepharose column (5 × 13 cm). After application, the flow rate increased to 8 ml/min. The column was washed with 50 bed volumes of 10 mm MES, pH 6.5, 1 mm EDTA, 1% glycerol, 150 mm NaCl, 0.1% Triton X-100; further washed with 4 bed volumes of 10 mm MES, pH 6.5, 1 mm EDTA, 10% glycerol (Buffer A) containing 150 mm NaCl; and finally eluted with 4 liters of 10 mm MES, pH 6.5, 0.1 mm EDTA, 10% glycerol, 2 m NaCl. Step 3. ConA-Sepharose—The eluted material from Step 2 was adjusted to 1 mm MgCl2, 1 mm CaCl2, 0.1% Triton X-100 and applied to a column of ConA-Sepharose (2.6 × 6 cm; gel not reutilized) run at 2 ml/min. The gel was washed with 30 bed volumes of 10 mm MES, pH 6.5, 0.1 mm EDTA, 10% glycerol, 1 mm MnCl2, 1 mm CaCl2, 0.1% Triton X-100 (Buffer B), 0.5 m NaCl, and then with 6 bed volumes of Buffer B, 20 mm NaCl. To save elution buffer (Buffer B, 20 mm NaCl, 0.5 m methyl-α-d-mannoside) and increase recovery, five 90-ml elutions were performed, each involving about 12 cycles each of 30 min of buffer recirculation followed by 30 min without recirculation. Step 4. dK4-Sepharose—The eluate from Step 3 was applied to a dK4-Sepharose column (2.6 × 3 cm) at 2 ml/min. The column was washed with 10 bed volumes of Buffer A, 20 mm NaCl, 10 mm CHAPS, further washed with 10 bed volumes of Buffer A, 20 mm NaCl, and finally eluted with Buffer A, 150 mm NaCl. Fractions with epimerase activity were pooled, dialyzed versus Buffer A, 10 mm NaCl, and concentrated by applying them to a Mono-Q 5/5 column, run at 0.5 ml/min. After application, the column was inverted and eluted at a flow rate of 0.1 ml/min with Buffer A, 1 m NaCl. Step 5. Superose 12—The 1-ml concentrated material from Step 4 was injected to a Superose 12 column, which was subsequently eluted at 0.05 ml/min with Buffer A, 150 mm NaCl. Fractions of ∼0.3 ml were collected (Fig. 2A) and analyzed for epimerase activity. Active fractions were pooled, diluted with Buffer A to a final NaCl concentration of 20 mm, and concentrated on a Mono-Q PC 1.6/5 column, operated as described above. Step 6. Superose 12—The 0.2-ml concentrated material from Step 5 was injected to a second Superose 12 column, operated as above. Step 7. Red-Sepharose—The most active fraction from the previous step was diluted with Buffer A to 100 mm NaCl; CHAPS was added to final 10 mm concentration, and the sample was batch-incubated with 50 μl of fresh Red-Sepharose gel. After incubation for 2 h, the gel was washed five times with 500 μl of Buffer A, 100 mm NaCl, 10 mm CHAPS, further washed five times with 1 ml of the same solution without CHAPS, and finally eluted with 10 × 150 μl of Buffer A, 2 m NaCl. The ∼0.6 million-fold purified analytical sample shown in Fig. 2B, lane 2, was prepared as above, with one modification; an initial elution step with Buffer A, 1 m NaCl preceded the final elution with Buffer A, 2 m NaCl. The final eluate contained ∼10% of the epimerase activity incubated with the Red-Sepharose gel. Material from the last purification step was precipitated with trichloroacetic acid, and the pellet was resuspended in reducing Laemmli buffer. SDS-PAGE was carried out on NuPAGE pre-made 10% acrylamide gels (Invitrogen) that were stained with Brilliant Blue G Colloidal staining solution (Sigma). Gel bands of interest were cut out, and the proteins were digested with trypsin (Promega) as described (26Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7822) Google Scholar). Electrospray ionization-based LC-MS/MS analyses were carried out using an Agilent 1100 series instrumentation (Agilent Technologies, Palo Alto, CA) on a 75-μm × 10.5-cm fused silica microcapillary reversed phase column (5-μm Magic C18 beads; Michrom Bioresources). The column was eluted at 200 nl/min using a gradient of 5–35% acetonitrile in 0.1% formic acid, over a 50-min period as described (27Lee H. Yi E.C. Wen B. Reily T.P. Pohl L. Nelson S. Aebersold R. Goodlett D.R. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2004; 803: 101-110Crossref PubMed Scopus (33) Google Scholar). LC-MS/MS was performed using an LTQ ion trap (Thermofinnigan, San Jose, CA) with an electrospray voltage of 2 kV. The instrument was set up to perform one MS scan (400–1600 Da) followed by three MS/MS analyses in a data-dependent mode with an intensity threshold of 15,000 counts. The repeat count was set to 3, the repeat duration to 30 s, and the exclusion duration was 60 s; and the exclusion list size was 100. Data base search was carried out using SEQUEST (28Yates III, J.R. Eng J.K. McCormack A.L. Schieltz D. Anal. Chem. 1995; 67: 1426-1436Crossref PubMed Scopus (1110) Google Scholar), and the search results were further analyzed by peptide and protein prophet as described previously (29Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3897) Google Scholar, 30Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3635) Google Scholar). The identified peptides and corresponding proteins were stored and analyzed in a MySQL data model, which was used to accommodate further analysis of the data (31Malmstrom L. Marko-Varga G. Westergren-Thorsson G. Laurell T. Malmstrom J. BMC Bioinformatics,. 2006; (in press)PubMed Google Scholar). SART2 human cDNA clone (IMAGE number 5272885) was obtained from RZPD, Germany, and subcloned into pcDNA 3.1/myc-His vector (Invitrogen) using XhoI and AgeI restriction sites (underlined in the primers) introduced by PCR using primers 5′-GATCCTCGAGATGAGGACTCACACACGGGG-3′ and 5′-GATCACCGGTACACTGTGATTGGGAACAAGA-3′, respectively. The insert was confirmed by sequencing. Ligation into the expression vector resulted in a construct with SART2 in-frame with a C-terminal His6 tag. CHO-K1 cells, maintained in F12-K medium, 10% FBS, HFL-1 cells and 293HEK cells, both maintained in minimum Eagle's medium, 10% FBS, were grown in 6-well plates and transiently transfected with pcDNA-His or pcDNA SART2-His plasmid, using Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer's instruction. After 48 h, cells were washed with phosphate-buffered saline and lysed in 20 mm MES, pH 6.5, 150 mm NaCl, 10% glycerol, 2 mm DTT, 1 mm EDTA, 1% Triton X-100, protease inhibitors. After 30 min at 4 °C, cell lysate was centrifuged for 1 h at 20,000 × g, and 200 μl of the supernatant was desalted (without carrier BSA added prior to desalting), followed by determination of protein content and enzyme activity. DA strain rats, 6 weeks old, were sacrificed, and the organs were put in ∼3-fold excess (v/w) lysis buffer (see above for buffer composition and lysate preparation). The predominantly muscular tissues heart, uterus, and skeletal muscle were ground and further homogenized by three Potter strokes. For the remaining soft tissues the use of the Potter homogenizer alone was adequate. Two strategies were adopted to analyze the reaction products. In one experimental set, samples of 15,000 dpm of [1-14C]dK4, recovered after incubation with cellular lysate as described in Fig. 4A, were digested with chondroitinase AC-I, and the split products were analyzed by gel chromatography, as described below. Alternatively, the overall composition of enzyme-incubated [1-14C]-labeled dK4 was determined by analysis of labeled HexA-aTalR disaccharides, generated by N-deacetylation followed by deaminative cleavage and reduction of the products essentially as described (20Hannesson H.H. Hagner-McWhirter A. Tiedemann K. Lindahl U. Malmstrom A. Biochem. J. 1996; 313: 589-596Crossref PubMed Scopus (42) Google Scholar). Briefly, samples were subjected to hydrazinolysis (64% hydrazine, 36% H2O, 1% hydrazine sulfate) at 100 °C for 16 h, and the product was reisolated on a PD-10 column eluted with water and then treated with HNO2 at pH 3.9. The resultant disaccharides, representing 64% of the total radioactivity, were reduced with NaBH4 and recovered by gel chromatography on a Superdex Peptide column, eluted at 0.3 ml/min with 0.2 m NH4HCO3. The labeled disaccharides were separated by paper chromatography (20Hannesson H.H. Hagner-McWhirter A. Tiedemann K. Lindahl U. Malmstrom A. Biochem. J. 1996; 313: 589-596Crossref PubMed Scopus (42) Google Scholar). Transient transfection of 293HEK cells, grown in 75-cm2 flasks was performed as described above, with pcDNA-His or pcDNA SART2-His plasmid. After transfection, cell were grown for 24 h in ordinary minimum Eagle's medium, washed once in sulfur-deprived Dulbecco's modified Eagle's medium, maintained for 2 h in 10 ml of sulfur-deprived Dulbecco's modified Eagle's medium, 10% FBS, and finally 100 μCi/ml 35SO4 was added. After an additional 24-h culture period, medium was collected and applied to a 2-ml DE52 column, equilibrated with 50 mm acetate, pH 5.5, 0.1% Triton X-100, 6 m urea. The column was washed with 30 bed volumes of equilibration buffer and then with 5 volumes of water, 5 volumes of 0.2 m NH4HCO3, and finally eluted with 10 volumes of 2 m NH4HCO3. Ten μg of cold DS were added, and the samples were lyophilized and then subjected to alkaline β-elimination (50 mm KOH, 1 m NaBH4, 45 °C for 16 h). GAG chains were reisolated using the DE52 column, operated as described above. The GAG chains were deaminated at pH 1.5 (32Bienkowski M.J. Conrad H.E. J. Biol. Chem. 1985; 260: 356-365Abstract Full Text PDF PubMed Google Scholar), and intact CS/DS chains were recovered after gel filtration. 35S-Labeled CS/DS chains (15,000 dpm) were digested for 4 h at 37 °C in 200 μl of 0.1 m Tris-HCl/sodium acetate, pH 7.3, with 36 milliunits of chondroitinase ABC or 10 milliunits of chondroitinase AC-I. Chondroitinase B digestions were performed with 8 milliunits of enzyme in 200 μl of 20 mm Tris-HCl/sodium acetate, pH 7.3, 50 mm NaCl, 4 mm CaCl2, 0.01% BSA. Digests were heated at 100 °C for 2 min, mixed with 100 μg of heparin, and applied to a Superdex Peptide column, operated at 0.3 ml/min in 0.2 m NH4HCO3. Purification of DS Epimerase—Initial data base searches for clues to the DS epimerase coding sequence based on similarity with the HS or alginate C5-epimerases were unsuccessful, and we therefore decided to isolate the DS epimerase protein. Screening various rat tissues for enzyme activity pointed to spleen as the richest source of DS epimerase (Fig. 1). A survey of bovine tissues gave similar results (data not shown), and bovine spleen was therefore selected as starting material for purification of DS epimerase. The overall purification process (see "Experimental Procedures") is summarized in Table 1. Solubilized microsomes were first applied to Red-Sepharose gel, yielding a 10-fold purification with excellent recovery. A ConA-Sepharose column efficiently removed contaminants but allowed only 30–40% recovery of epimerase activity. dK4-Sepharose provided consistent 3-fold purification, with high recovery. After concentration, the partially purified enzyme was applied to a Superose 12 column. Most of the protein emerged as high molecular weight complexes, but the epimerase fraction was more retarded and relatively homogeneous in size, being eluted 4-fold and purified with the peak of activity at the position of a 67-kDa marker (Fig. 2A). Size fractionation was refined by reapplication of the active pool to the same column, thus yielding a sharp peak with 80% of the eluted activity in two effluent fractions, again with a 4-fold purification. The most active fraction was incubated, batchwise, with a small amount of Red-Sepharose gel. Most of the activity was recovered and 14-fold further purified. The complexity of this sample, purified altogether ∼43,000-fold, was assessed on SDS-PAGE (Fig. 2B, lane 1). An 89-kDa band was considered of particular interest, because the (Colloidal Blue) staining intensity of this band in the eluted fractions from Superose 12 and Red-Sepharose correlated with epimerase activity (data not shown). Furthermore, by modifying the elution conditions of the last purification on Red-Sepharose (see "Experimental Procedures"), ∼0.6 millionfold purified active material was obtained, at the expense of recovery. Silver staining after SDS-PAGE showed the 89-kDa band as a major component (Fig. 2B, lane 2).TABLE 1Purification of DS epimeraseTotal proteinTotal activityRecoverySpecific activityPurificationmgdpm/h × 10-6%dpm/h/mg × 10-3-foldMicrosomal preparation (step 1)12,1029210081Red-Sepharose (step 2)1,03377847510ConA-Sepharose (step 3)54283052068dK4-Sepharose (step 4)10.9 (6.5)aValues are given in parentheses after concentration.18 (12)aValues are given in parentheses after concentration.19 (13)aValues are given in parentheses after concentration.1,650220Superose 12 (step 5)1.53 (0.32)aValues are given in parentheses after concentration.9.3 (6.6)aValues are given in parentheses after concentration.10 (7)aValues are given in parentheses after concentration.6,080800Superose 12 (step 6)0.0671.82.026,9003,500Red-Sepharose (step 7)∼0.004bEvaluation was from Colloidal Blue staining after SDS-PAGE.1.31.4∼330,000∼43,000Red-Sepharose (step 7), analytical samplecSee purification, "Step 7" under "Experimental Procedures."∼1 ngdEvaluation was from silver staining after SDS-PAGE.0.005NAeNA indicates not applicable.∼5,000,000∼600,000a Values are given in parentheses after concentration.b Evaluation was from Colloidal Blue staining after SDS-PAGE.c See purification, "Step 7" under "Experimental Procedures."d Evaluation was from silver staining after SDS-PAGE.e NA indicates not applicable. Open table in a new tab Gene Identification—Seven SDS-PAGE Colloidal Blue-stained bands (Fig. 2B, lane 1) were trypsinized and subjected to electrospray ionization-based LC-MS/MS analysis. Data base search of the results against the NCBI nonredundant (NRP.nci.fasta.20041115) data base yielded 56 proteins identified with more than one peptide matched and with a protein prophet probability of 1.0 (29Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3897) Google Scholar, 30Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3635) Google Scholar). Several proteins were identified in more than one band, decreasing the number of unique proteins identified to 33. The following four proteins were identified from the 89-kDa gel band, believed to contain the DS epimerase: human SART2 (UPTR:Q9UL01; 6 peptides), lactotransferrin (gi.7428768; 11 peptides), hu-k4 (Q92853; 2 peptides), and pld3 protein (O35405; 2 peptides). The seven LC-MS/MS runs were then searched against a data base generated from the merged output from two genome-wide gene-finder programs, GenScan and GeneId and protein sequences from Bos taurus at NCBI (www.ncbi.nlm.nih.gov). Again, the 89-kDa band yielded four proteins with more than one pept
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