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• W1992048265 abstract "The murine gene for the glucuronyl C5-epimerase involved in heparan sulfate biosynthesis was cloned, using a previously isolated bovine lung cDNA fragment (Li, J.-P., Hagner-McWhirter, Å., Kjellén, L., Palgi, J., Jalkanen, M., and Lindahl, U. (1997)J. Biol. Chem. 272, 28158–28163) as probe. The ∼11-kilobase pair mouse gene contains 3 exons from the first ATG to stop codon and is localized to chromosome 9. Southern analysis of the genomic DNA and chromosome mapping suggested the occurrence of a single epimerase gene. Based on the genomic sequence, a mouse liver cDNA was isolated that encodes a 618-amino acid residue protein, thus extending by 174 N-terminal residues the sequence deduced from the (incomplete) bovine cDNA. Comparison of murine, bovine, and human epimerase cDNA structures indicated 96–99% identity at the amino acid level. A cDNA identical to the mouse liver species was demonstrated in mouse mast cells committed to heparin biosynthesis. These findings suggest that the iduronic acid residues in heparin and heparan sulfate, despite different structural contexts, are generated by the same C5-epimerase enzyme. The catalytic activity of the recombinant full-length mouse liver epimerase, expressed in insect cells, was found to be >2 orders of magnitude higher than that of the previously cloned, smaller bovine recombinant protein. The ∼52-kDa, similarly highly active, enzyme originally purified from bovine liver (Campbell, P., Hannesson, H. H., Sandbäck, D., Rodén, L., Lindahl, U., and Li, J.-P. (1994) J. Biol. Chem.269, 26953–26958) was found to be associated with an ∼22-kDa peptide generated by a single proteolytic cleavage of the full-sized protein. The murine gene for the glucuronyl C5-epimerase involved in heparan sulfate biosynthesis was cloned, using a previously isolated bovine lung cDNA fragment (Li, J.-P., Hagner-McWhirter, Å., Kjellén, L., Palgi, J., Jalkanen, M., and Lindahl, U. (1997)J. Biol. Chem. 272, 28158–28163) as probe. The ∼11-kilobase pair mouse gene contains 3 exons from the first ATG to stop codon and is localized to chromosome 9. Southern analysis of the genomic DNA and chromosome mapping suggested the occurrence of a single epimerase gene. Based on the genomic sequence, a mouse liver cDNA was isolated that encodes a 618-amino acid residue protein, thus extending by 174 N-terminal residues the sequence deduced from the (incomplete) bovine cDNA. Comparison of murine, bovine, and human epimerase cDNA structures indicated 96–99% identity at the amino acid level. A cDNA identical to the mouse liver species was demonstrated in mouse mast cells committed to heparin biosynthesis. These findings suggest that the iduronic acid residues in heparin and heparan sulfate, despite different structural contexts, are generated by the same C5-epimerase enzyme. The catalytic activity of the recombinant full-length mouse liver epimerase, expressed in insect cells, was found to be >2 orders of magnitude higher than that of the previously cloned, smaller bovine recombinant protein. The ∼52-kDa, similarly highly active, enzyme originally purified from bovine liver (Campbell, P., Hannesson, H. H., Sandbäck, D., Rodén, L., Lindahl, U., and Li, J.-P. (1994) J. Biol. Chem.269, 26953–26958) was found to be associated with an ∼22-kDa peptide generated by a single proteolytic cleavage of the full-sized protein. heparan sulfate d-glucuronic acid l-iduronic acid glycosaminoglycan fluorescence in situhybridization 4′,6-diamidino-2-phenylindole 2,5-anhydromannitol (formed by reduction of terminal 2,5-anhydromannose residues with NaBH4) polyacrylamide gel electrophoresis kilobase pair fetal calf serum base pair open reading frame polymerase chain reaction reverse transcriptase-PCR N-deacetylase/N-sulfotransferase Heparin and heparan sulfate (HS)1 are both linear, sulfated glycosaminoglycans based on a common carbohydrate backbone of alternating d-glucosamine (GlcN) and hexuronic acid (d-glucuronic (GlcUA) orl-iduronic acid (IdoUA)) units. Both polymer types are synthesized as proteoglycans. Heparin occurs in connective tissue-type mast cells, whereas HS has a ubiquitous distribution and is produced by almost all mammalian cells. The biosynthesis of heparin and HS follows a common pathway that involves: (i) formation of a GlcUA-Gal-Gal-Xyl-Ser carbohydrate-protein linkage region; (ii) assembly of repeating -GlcUA-GlcNAc- disaccharide units; and (iii) modification of the [GlcUA-GlcNAc]n polymers (1Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (397) Google Scholar, 2Lindahl U. Kusche-Gullberg M. Kjellén L. J. Biol. Chem. 1998; 273: 24979-24982Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). The latter process includes N-deacetylation andN-sulfation of GlcNAc units, C5-epimerization of GlcUA to IdoUA units, and O-sulfation at various sites of the sugar residues. Heparin is highly modified, heavily N- andO-sulfated and with more IdoUA than GlcUA units. Generally, a predominant proportion of heparin chains consists of trisulfated -IdoUA(2-OSO3)-GlcNSO3(6-OSO3)- units. By comparison, HS has a more variable and heterogeneous structure, with highly modified, less modified, and unmodified sequences arranged in domain-type fashion (3Lyon M. Gallagher J.T. Matrix Biol. 1998; 17: 485-493Crossref PubMed Scopus (113) Google Scholar, 4Casu, B., and Lindahl, U. (2001) Adv. Carbohydr. Chem. Biochem., in press.Google Scholar). The IdoUA units occur in essentially two types of domains, composed of contiguousN-sulfated, and of alternating N-sulfated andN-acetylated disaccharide units, respectively (5Maccarana M. Sakura Y. Tawada A. Yoshida K. Lindahl U. J. Biol. Chem. 1996; 271: 17804-17810Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). The sequence variability of the chains is believed to reflect the functional role of HS glycosaminoglycans (GAGs) in specific interactions with different proteins (1Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (397) Google Scholar, 2Lindahl U. Kusche-Gullberg M. Kjellén L. J. Biol. Chem. 1998; 273: 24979-24982Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 3Lyon M. Gallagher J.T. Matrix Biol. 1998; 17: 485-493Crossref PubMed Scopus (113) Google Scholar, 4Casu, B., and Lindahl, U. (2001) Adv. Carbohydr. Chem. Biochem., in press.Google Scholar, 6Bernfield M. Kokenyesi R. Kato M. Hinkes M. Spring J. Gallo R. Lose E. Annu. Rev. Cell Biol. 1992; 8: 333-364Crossref PubMed Scopus (970) Google Scholar). IdoUA residues generally appear to promote protein binding due to their conformational flexibility and have been identified as invariable constituents of protein-binding HS domains (4Casu, B., and Lindahl, U. (2001) Adv. Carbohydr. Chem. Biochem., in press.Google Scholar). The reaction catalyzed by the C5-epimerase therefore is crucial for many biological functions of heparin and HS. The mechanisms in control of the structural diversity of heparin and, in particular, of HS are only partly understood but clearly rely on the substrate specificities of the enzymes involved (2Lindahl U. Kusche-Gullberg M. Kjellén L. J. Biol. Chem. 1998; 273: 24979-24982Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 4Casu, B., and Lindahl, U. (2001) Adv. Carbohydr. Chem. Biochem., in press.Google Scholar). The enzymes required to synthesize a HS chain have all been cloned. Notably, several of these proteins, including species committed to polymer modification, occur in multiple forms that are encoded by different genes. Some of these species have been shown to differ with regard to kinetic properties and/or substrate specificity from the respective homologous forms (7Eriksson I. Sandbäck D. Ek B. Lindahl U. Kjellén L. J. Biol. Chem. 1994; 269: 10438-10443Abstract Full Text PDF PubMed Google Scholar, 8Shworak N.W. Liu J. Petros L.M. Zhang L. Kobayashi M. Copeland N.G. Jenkins N.A. Rosenberg R.D. J. Biol. Chem. 1999; 274: 5170-5184Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 9Habuchi H. Tanaka M. Habuchi O. Yoshida K. Suzuki H. Ban K. Kimata K. J. Biol. Chem. 2000; 275: 2859-2868Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 10Aikawa J. Grobe K. Tsujimoto M. Esko J.D. J. Biol. Chem. 2001; 276: 5876-5882Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The IdoUA units of heparin and HS chains occur in widely different structural contexts, which range from the minimally sulfated -GlcNSO3-IdoUA-GlcNAc- sequence found in HS molecules to the extensively sulfated -GlcNSO3(6-OSO3)-IdoUA(2-OSO3)-GlcNSO3(6-OSO3)- structure typical of heparin (4Casu, B., and Lindahl, U. (2001) Adv. Carbohydr. Chem. Biochem., in press.Google Scholar). These findings raise the question as to whether the C5-epimerase, similar to other enzymes in the same biosynthetic process, also occurs in genetically distinct forms. More specifically, is the same epimerase committed to the formation of heparin, in the mast cell, and to HS, in other cells? Following the isolation of the GlcUA C5-epimerase from bovine liver, the corresponding cDNA was cloned from bovine lung, and a recombinant protein with significant catalytic activity was expressed in insect cells (11Li J.-P. Hagner-McWhirter Å. Kjellén L. Palgi J. Jalkanen M. Lindahl U. J. Biol. Chem. 1997; 272: 28158-28163Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In the present study we have addressed the question of genetic polymorphism by cloning the murine C5-epimerase gene. Only one form of the gene was found. Moreover, we have cloned the C5-epimerase cDNA from mouse liver that generates HS, and from mouse mastocytoma cells that produce heparin. The results strongly suggest that the same enzyme protein is implicated in both biosynthetic processes. Finally, analysis of the epimerase gene structure indicated that the bovine cDNA previously cloned (11Li J.-P. Hagner-McWhirter Å. Kjellén L. Palgi J. Jalkanen M. Lindahl U. J. Biol. Chem. 1997; 272: 28158-28163Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) was incomplete, as ∼28% of the 5′-terminal coding region was missing. This conclusion was verified by cloning and expression of the full-length protein, with the catalytic activity much higher than that of the previously expressed truncated form. A mouse genomic library (Lambda FX-II from Stratagene) was screened with a 1407-bp DNA probe from the bovine epimerase coding sequence, labeled with [32P]dCTP (PerkinElmer Life Sciences). Approximately 2 × 106 phages were plated at a density of 250,000 plaques per 20 × 20-cm plate, and duplicate nylon filters were prepared from each plate. Hybridization was performed at 60 °C in 5× Denhardt's hybridization solution, containing 100 μg of salmon sperm DNA/ml. The final washes were in 0.1× SSC (1× SSC is 150 mm NaCl, 15 mm sodium citrate, pH 7.0) containing 0.1% SDS. Plaques that produced positive signals on both replicas were selected for second and third round screening. Five positive clones were found. Two of the clones were digested, clone 5a with EcoRI and clone 64 withSacI, and the resultant fragments were cloned into pUC 119 and BlueScript vectors, respectively. The insert-containing plasmids were purified using the Qiagen plasmid kit and subsequently sequenced. Nucleotide sequencing was performed using the BigDye terminator method and an ABI Prism 310 Genetic Analyzer according to the instructions of the manufacturer (PerkinElmer Life Sciences). Exon/intron boundaries were determined by primer walking sequencing on both strands of the subclones. The size of introns was estimated by agarose gel electrophoresis. The exons were sequenced from both strands. Southern blot analysis was performed according to Sambrook et al. (12Sambrook J. Fritsch E.F. Maniatis T. Harbor C.S. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 7.18-7.26Google Scholar). Mouse genomic DNA (20 μg), prepared from liver using an Easy Prep kit (Amersham Pharmacia Biotech), was digested with restriction enzymes, and the products were separated by electrophoresis on an 0.8% agarose gel. After separation, the gel was treated with 0.5 m NaOH for 30 min and neutralized in 0.5 m Tris-HCl buffer, pH 7.4, containing 1.5 m NaCl. The DNA fragments were transferred onto a Hybond-N+ nylon transfer membrane (Amersham Pharmacia Biotech). A fragment of an ∼800-bp intron DNA from genomic clone 5a was labeled with [32P]dCTP using Klenow enzyme from Roche Molecular Biochemicals and applied as probe. Hybridization was performed at 65 °C in ExpressHyb hybridization solution (CLONTECH) for 1 h followed by washing with 0.5× SSC containing 0.5% SDS at room temperature twice for 20 min. The membrane was exposed to a x-ray film for 3 days. Mouse lymphocytes were isolated from normal spleen and cultured at 37 °C in RPMI 1640 medium supplemented with 15% fetal calf serum (FCS), 3 μg/ml concanavalin A, 10 μg/ml lipopolysaccharide, and 5 × 10−5m mercaptoethanol for 44 h. Human lymphocytes were isolated from blood and cultured in α-minimal essential medium supplemented with 10% FCS and phytohemagglutinin at 37 °C for 68–72 h. Both lymphocyte cultures were synchronized by treatment with bromodeoxyuridine (0.18 mg/ml). The synchronized cells were washed three times with serum-free medium and recultured at 37 °C for 4–6 h in α-minimal essential medium with thymidine (2.5 μg/ml). The cells were harvested, and chromosome slides were made by hypotonic treatment, fixation, and air drying. The full-length mouse epimerase cDNA was biotinylated with dATP using Life Technologies, Inc., BioNick labeling kit (15 °C, 1 h) (13Heng H.H. Squire J. Tsui L.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9509-9513Crossref PubMed Scopus (521) Google Scholar). The procedure for fluorescence in situ hybridization (FISH) detection was as described (13Heng H.H. Squire J. Tsui L.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9509-9513Crossref PubMed Scopus (521) Google Scholar, 14Heng H.H. Tsui L.C. Chromosoma. 1993; 102: 325-332Crossref PubMed Scopus (431) Google Scholar). Briefly, chromosome slides were baked at 55 °C for 1 h and were then treated with RNase A, denatured in 70% formamide in 2× SSC for 2 min at 70 °C, and finally dehydrated with ethanol. The probe was denatured at 75 °C for 5 min in a hybridization mixture consisting of 50% formamide and 10% dextran sulfate. The slides were pre-hybridized for 15 min at 37 °C, and the probe was loaded on the denatured slides. After overnight hybridization, slides were washed, and signals were detected as well as amplified using the published method (13Heng H.H. Squire J. Tsui L.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9509-9513Crossref PubMed Scopus (521) Google Scholar). FISH signals and the 4′-6-diamidino-2-phenylindole (DAPI) banding patterns were recorded by separate photographs, and the assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with DAPI-banded chromosomes (14Heng H.H. Tsui L.C. Chromosoma. 1993; 102: 325-332Crossref PubMed Scopus (431) Google Scholar). Mouse multiple tissue Northern blot was purchased from CLONTECH. A DNA probe of 837 bp from the C-terminal end region of bovine cDNA clone was labeled with [32P]dCTP by use of Klenow enzyme. The hybridization was carried out in ExpressHyb hybridization solution at 60 °C for 1 h, and the blot was then washed with 0.1× SSC containing 0.5% SDS at 60 °C. The membrane was exposed to a Kodak x-ray film for 2 days. Primers for cloning of mouse cDNA were designed based on the nucleotide sequence obtained by sequencing the exons of the genomic clones. The sense primer corresponds to bp 1–26 of the open reading frame (5′-ATGCGTTGTTTGGCAGCTCGGGTCAA). The antisense primer corresponds to the 3′-end of the coding sequence (bp 1829–1854), excluding the stop codon (5′-GTTGTGCTTTGCCCTACTGCCTTTAA). PCR was performed using a mouse liver QUICK-CloneTM cDNA (CLONTECH) as template under the following conditions: 1 cycle of 94 °C for 1 min, 30 cycles each of 94 °C for 30 s, 60 °C for 45 s, and 72 °C for 1 min, and a final extension at 72 °C for 10 min. Mouse mastocytoma mast cells (denoted “mast cells” in this paper), established in culture after passage through an ascites stage (15Jacobsson K.-G. Riesenfeld J. Lindahl U. J. Biol. Chem. 1985; 260: 12154-12159Abstract Full Text PDF PubMed Google Scholar), were grown in Dulbecco's modified Eagle's medium containing 50 μg/ml penicillin, 50 μg/ml streptomycin, and 10% FCS until confluent. Total RNA was extracted from one flask (75 ml) of cultured mast cells according to the LiCl/urea/SDS procedure of Sambrooket al. (12Sambrook J. Fritsch E.F. Maniatis T. Harbor C.S. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 7.18-7.26Google Scholar). About 1 μg of total RNA was used as template for RT-PCR. The single strand cDNA was prepared in a volume of 20 μl with 1st Strand cDNA Synthesis Kit for RT-PCR (Roche Molecular Biochemicals). A portion (5 μl) of the RT-PCR reaction mixture was used for amplification of C5-epimerase cDNA using the primer pair described above under the same conditions. For cloning of the 5′-terminal portion of bovine C5-epimerase cDNA, PCR was performed using a bovine lung gt10 cDNA library (CLONTECH) as template under the conditions described above. The sense primer is 5′-ATGCGTTGTTTGGCAGCTCGGGTCAA, corresponding to bp 1–26 of the open reading frame of mouse cDNA. The antisense primer is 5′-GCAGCCCTTGGGCACAGTCCAGTCATTGGGCTTGC corresponding to bp 287–321 of the bovine cDNA reported earlier (11Li J.-P. Hagner-McWhirter Å. Kjellén L. Palgi J. Jalkanen M. Lindahl U. J. Biol. Chem. 1997; 272: 28158-28163Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). All PCR products were directly cloned into a TOPOTM-TA Cloning vector (Invitrogen) according to the protocol provided by the manufacturer and were subsequently sequenced. The sequencing was carried out as described for the genomic DNA. Based on the cDNA sequence of the mouse C5-epimerase, new primers were designed and used for generation of a fragment without transmembrane domain for recombinant expression. PCR was performed by using the full-length mouse C5-epimerase cDNA as a template under the conditions described above. The resulting fragment was inserted into a modified (by introducing EGT signal peptide, FLAG epitope, enterokinase cleavage site, and His tag at the 5′-end) pIZ/V5 expression vector (Invitrogen) and subsequently sequenced. The expression construct was introduced into Sf9 insect cells, and the cells were cultured according to the manufacturer's instructions. The medium was collected and analyzed for epimerase activity. Medium containing recombinant epimerase (100 ml) was applied to a 20-ml column ofO-desulfated heparin immobilized on Sepharose (16Campbell P. Hannesson H.H. Sandbäck D. Rodén L. Lindahl U. Li J.-P. J. Biol. Chem. 1994; 269: 26953-26958Abstract Full Text PDF PubMed Google Scholar), equilibrated in 50 mm HEPES buffer, pH 7.4, 100 mm KCl, 15 mm EDTA. After extensive washing with the same buffer, the bound material was eluted with the same HEPES buffer containing 300 mm KCl, 15 mm EDTA. The eluted material was concentrated by centrifugation in an Ultrafree-MC 10,000 filter unit (Millipore). Bovine liver C5-epimerase was extracted and purified to homogeneity as described (16Campbell P. Hannesson H.H. Sandbäck D. Rodén L. Lindahl U. Li J.-P. J. Biol. Chem. 1994; 269: 26953-26958Abstract Full Text PDF PubMed Google Scholar). Peptide sequences were determined as described (11Li J.-P. Hagner-McWhirter Å. Kjellén L. Palgi J. Jalkanen M. Lindahl U. J. Biol. Chem. 1997; 272: 28158-28163Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Purified enzyme preparations were analyzed by SDS-PAGE (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). After electrophoresis, gels were stained with silver and documented. Epimerase assay was based on the release of 3H (recovered as3H2O) from a C5-3H-labeled polysaccharide substrate, as described (11Li J.-P. Hagner-McWhirter Å. Kjellén L. Palgi J. Jalkanen M. Lindahl U. J. Biol. Chem. 1997; 272: 28158-28163Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). For determination of enzyme activity, tissues were freshly dissected from a (A/Sn × Leaden) F1 mouse that had been inoculated previously with Furth mastocytoma cells in a hind leg (18Furth J. Hagen P. Hirsch E.I. Proc. Soc. Exp. Biol. Med. 1957; 95: 824-828Crossref PubMed Scopus (50) Google Scholar). The tissues were immediately homogenized in 10 volumes of 50 mm HEPES, pH 7.4, containing 100 mm KCl, 15 mm EDTA, 1% Triton X-100, and protease inhibitors (16Campbell P. Hannesson H.H. Sandbäck D. Rodén L. Lindahl U. Li J.-P. J. Biol. Chem. 1994; 269: 26953-26958Abstract Full Text PDF PubMed Google Scholar). Lysates were shaken at 4 °C for 30 min and centrifuged. The supernatants were collected and assayed for enzyme activity (11Li J.-P. Hagner-McWhirter Å. Kjellén L. Palgi J. Jalkanen M. Lindahl U. J. Biol. Chem. 1997; 272: 28158-28163Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and for total protein (19Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Controls were performed to ascertain that all determinations of epimerase activity in tissue extracts fell within the linear range of the assay. Enzymatic conversion of GlcUA to IdoUA units was directly demonstrated using purified recombinant mouse C5-epimerase (∼50 ng of protein in 100 μl of incubation mixture) and a metabolically [1-3H]glucose-labeled [4GlcUAβ1–4GlcNSO3α1-]n polysaccharide substrate, as described (11Li J.-P. Hagner-McWhirter Å. Kjellén L. Palgi J. Jalkanen M. Lindahl U. J. Biol. Chem. 1997; 272: 28158-28163Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Briefly, the partially epimerized polysaccharide was subjected to deaminative cleavage with nitrous acid (pH 1.5 reaction) followed by reduction, and the resultant labeled GlcUA-aManR (glucuronyl-2,5-anhydromannitol) and IdoUA-aManR (iduronyl-2,5-anhydromannitol) disaccharides were separated by paper chromatography and quantified by scintillation counting. Mastocytoma cells established from the Furth tumor (15Jacobsson K.-G. Riesenfeld J. Lindahl U. J. Biol. Chem. 1985; 260: 12154-12159Abstract Full Text PDF PubMed Google Scholar) were cultured in RPMI 1640 medium (Life Technologies, Inc.) containing 50 μg/ml penicillin, 50 μg/ml streptomycin, and 10% FCS. To label the cells, 50 μCi of Na35SO4 (Amersham Pharmacia Biotech) was added per ml of culture medium. After incubation for 24 h, the medium was collected, and the cells were lysed in 50 mm HEPES, pH 7.4, containing 0.1 m KCl and 1% Triton X-100. The cell lysate was kept on ice for 30 min and centrifuged. The supernatant and medium were pooled and treated with 0.5 m NaOH at 4 °C overnight and subsequently neutralized. The sample, containing released GAG chains, was diluted and applied to a DEAE-Sephacel column (4 ml) equilibrated in 0.05m NaAc, pH 4.0, containing 0.05 m NaCl. Labeled GAGs were eluted with a gradient of 0.05–1.5 m NaCl in the same buffer. The fractions containing labeled GAGs were pooled and concentrated. Analytical chromatography on DEAE-Sephacel was performed using the same buffer and salt gradient but in a high pressure liquid chromatography system. Treatments of GAGs with chondroitinase ABC (20Lindahl B. Eriksson L. Lindahl U. Biochem. J. 1995; 306: 177-184Crossref PubMed Scopus (98) Google Scholar) or with nitrous acid at pH 1.5 (21Shively J.E. Conrad H.E. Biochemistry. 1976; 15: 3932-3942Crossref PubMed Scopus (667) Google Scholar) were carried out as described. Unlabeled heparin and chondroitin sulfate standards were detected by the carbazole reaction for hexuronic acid (22Bitter T. Muir H.M. Anal. Biochem. 1962; 4: 330-334Crossref PubMed Scopus (5218) Google Scholar). Screening a genomic library from mouse liver with the previously described32P-labeled bovine epimerase cDNA (11Li J.-P. Hagner-McWhirter Å. Kjellén L. Palgi J. Jalkanen M. Lindahl U. J. Biol. Chem. 1997; 272: 28158-28163Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) as a probe yielded five positive clones that were purified and further characterized. The two largest clones, 5a and 64, were 16–18 kb in size, whereas the remaining three ranged 8–12 kb. All clones showed sequence overlap with at least three of the other clones (Fig.1 A). Digestion of clone 5a with EcoRI released seven fragments that were subsequently cloned into pUC119. Clone 64 was cleaved with SacI, and the resultant 3 major fragments were cloned into BlueScript. Analysis of exon-intron organization revealed that the C5-epimerase is encoded by only 3 exons, of which the largest one (exon 3) encodes more than 50% of the protein (Fig. 1 B). The genomic sequences defining the exon/intron boundaries (splice sites) follow the gt/ag consensus rule (Table I). The precise match between the open reading frame (ORF) sequence in the exons and the cDNA (data not shown) suggests that the identified genomic clone represents the functional gene for the C5-epimerase.Table IIntron-exon boundary sequences of the mouse C5-epimerase geneExon no.5′-Splice acceptor and 5′-end sequenceExon size3′-End sequence and 3′-splice donorIntron sizebpbp1>586GTTGAAGgtaggt>15002ttatagGTGTGCCA246TGCTCCAGgtgagt>70003ccacagAAACCAGT>1025 Open table in a new tab In order to address whether the C5-epimerase is encoded by a single gene in the mouse genome, we performed Southern blot hybridization of genomic DNA that had been digested with restriction enzymes as indicated in Fig.2. The probe used was an intron fragment (indicated by the asterisk in Fig. 1 A) generated from clone 5a by digestion with EcoRI. This ∼800-bp probe detected a single band of the same size in the sample cleaved withEcoRI and single bands of the expected sizes (estimated based on restriction enzyme mapping) upon cleavage withHindIII (∼3.5 kb), NcoI (∼5 kb),SacI (∼6.5 kb), and PstI (∼8 kb). By using the full-length mouse epimerase cDNA as probe, a single chromosomal locus was detected by FISH mapping in both mouse and human chromosomes. The FISH detection efficiency with this probe was 85% on mouse chromosomes and 63% on human chromosomes. The mouse epimerase is localized to chromosome number 9 (Fig.3 A) and the human epimerase to chromosome number 15 (Fig. 3 B), in accordance with the homology relationship between human and mouse genomes. The loci were further defined based on the combined information from 10 photos each. The epimerase probe thus was mapped to mouse chromosome 9, region C and D (Fig. 3 C), and to human chromosome 15, region q23-q24 (Fig. 3 D). These findings along with the Southern analysis indicate that the C5-epimerase is encoded by a single gene. Northern analysis of a mouse multiple tissue Northern mRNA blot by hybridization with a 32P-labeled cDNA probe corresponding to the C-terminal region of bovine C5-epimerase revealed a single transcript of ∼5 kb in all tissues examined. The highest expression level was seen in liver, whereas small amounts of transcript were seen in spleen (Fig.4 A). Yet epimerase activity in spleen was similar to that in kidney or lung (Fig. 4 B). Although we have no explanation to this discrepancy, we note that two other enzymes involved in the biosynthesis of HS,N-deacetylase/N-sulfotransferase 1 and 2-O-sulfotransferase, showed similar low levels of mRNA expression in spleen (23Kusche-Gullberg M. Eriksson I. Sandbäck Pikas D. Kjellén L. J. Biol. Chem. 1998; 273: 11902-11907Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 24Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M. (2001) Biochemistry, in press.Google Scholar). Mouse mastocytoma showed high epimerase activity (Fig. 4 B) and contained the same ∼5-kb epimerase transcript (11Li J.-P. Hagner-McWhirter Å. Kjellén L. Palgi J. Jalkanen M. Lindahl U. J. Biol. Chem. 1997; 272: 28158-28163Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) as the other murine tissues. Previous studies in our laboratory led to the isolation of a highly active ∼52-kDa epimerase from bovine liver (16Campbell P. Hannesson H.H. Sandbäck D. Rodén L. Lindahl U. Li J.-P. J. Biol. Chem. 1994; 269: 26953-26958Abstract Full Text PDF PubMed Google Scholar) and to the subsequent cloning of a bovine lung cDNA (11Li J.-P. Hagner-McWhirter Å. Kjellén L. Palgi J. Jalkanen M. Lindahl U. J. Biol. Chem. 1997; 272: 28158-28163Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). This cDNA encoded a protein consisting of 444 amino acid residues. N-terminal analysis of the purified liver enzyme showed that it lacked 73 amino acid residues predicted from the cDNA and thus represented a truncated form. Yet the recombinant protein, expressed in insect cells, had a catalytic activity much lower than that of the purified liver enzyme. Although this discrepancy could have several reasons, we noted that also the cloned protein could be incomplete, since the 5′-end of the cDNA open reading frame was not defined with certainty. With the gene structure for the epimerase at hand it became possible to reassess this question, by renewed cDNA isolation based on the genomic sequence information. Appropriate oligonucleotide primers were therefore designed based on the genomic sequences, as described under “Experimental Procedures”, and used for PCR amplification using mouse liver cDNA as template. Agarose gel electrophoresis of the PCR product revealed one strong band of ∼2 kb in size," @default.
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• W1992048265 date "2001-01-01" @default.
• W1992048265 modified "2023-10-17" @default.
• W1992048265 title "Characterization of the d-Glucuronyl C5-epimerase Involved in the Biosynthesis of Heparin and Heparan Sulfate" @default.
• W1992048265 cites W1515314521 @default.
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• W1992048265 cites W1971540677 @default.
• W1992048265 cites W1976962628 @default.
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• W1992048265 cites W1993745855 @default.
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• W1992048265 cites W2013582082 @default.
• W1992048265 cites W2024559068 @default.
• W1992048265 cites W2027880172 @default.
• W1992048265 cites W2029055264 @default.
• W1992048265 cites W2039644166 @default.
• W1992048265 cites W2041420763 @default.
• W1992048265 cites W2043484559 @default.
• W1992048265 cites W2044804476 @default.
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• W1992048265 cites W2046899925 @default.
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• W1992048265 cites W2113266466 @default.
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• W1992048265 cites W2141843510 @default.
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• W1992048265 cites W2160835895 @default.
• W1992048265 cites W2323311778 @default.
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