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Cloning, Golgi Localization, and Enzyme Activity of the Full-length Heparin/Heparan Sulfate-Glucuronic Acid C5-epimerase

2001; Elsevier BV; Volume: 276; Issue: 24 Linguagem: Inglês

10.1074/jbc.m100880200

1083-351X

Brett E. Crawford, Sara K. Olson, Jeffrey D. Esko, MariaA.S. Pinhal,

Fibroblast Growth Factor Research

While studying the cellular localization and activity of enzymes involved in heparan sulfate biosynthesis, we discovered that the published sequence for the glucuronic acid C5-epimerase responsible for the interconversion ofd-glucuronic acid and l-iduronic acid residues encodes a truncated protein. Genome analysis and 5′-rapid amplification of cDNA ends was used to clone the full-length cDNA from a mouse mastocytoma cell line. The extended cDNA encodes for an additional 174 amino acids at the amino terminus of the protein. The murine sequence is 95% identical to the human epimerase identified from genomic sequences and fits with the general size and structure of the gene from Drosophila melanogasterand Caenorhabditis elegans. Full-length epimerase is predicted to have a type II transmembrane topology with a 17-amino acid transmembrane domain and an 11-amino acid cytoplasmic tail. An assay with increased sensitivity was devised that detects enzyme activity in extracts prepared from cultured cells and in recombinant proteins. Unlike other enzymes involved in glycosaminoglycan biosynthesis, the addition of a c-myctag or green fluorescent protein to the highly conserved COOH-terminal portion of the protein inhibits its activity. The amino-terminally truncated epimerase does not localize to any cellular compartment, whereas the full-length enzyme is in the Golgi, where heparan sulfate synthesis is thought to occur. While studying the cellular localization and activity of enzymes involved in heparan sulfate biosynthesis, we discovered that the published sequence for the glucuronic acid C5-epimerase responsible for the interconversion ofd-glucuronic acid and l-iduronic acid residues encodes a truncated protein. Genome analysis and 5′-rapid amplification of cDNA ends was used to clone the full-length cDNA from a mouse mastocytoma cell line. The extended cDNA encodes for an additional 174 amino acids at the amino terminus of the protein. The murine sequence is 95% identical to the human epimerase identified from genomic sequences and fits with the general size and structure of the gene from Drosophila melanogasterand Caenorhabditis elegans. Full-length epimerase is predicted to have a type II transmembrane topology with a 17-amino acid transmembrane domain and an 11-amino acid cytoplasmic tail. An assay with increased sensitivity was devised that detects enzyme activity in extracts prepared from cultured cells and in recombinant proteins. Unlike other enzymes involved in glycosaminoglycan biosynthesis, the addition of a c-myctag or green fluorescent protein to the highly conserved COOH-terminal portion of the protein inhibits its activity. The amino-terminally truncated epimerase does not localize to any cellular compartment, whereas the full-length enzyme is in the Golgi, where heparan sulfate synthesis is thought to occur. l-iduronic acid Chinese hamster ovary green fluorescent protein polymerase chain reaction yellow fluorescent protein untranslated region phosphate-buffered saline bovine serum albumin piperazine-N,N′-bis(2-ethanesulfonic acid) 3-(N-morpholino)propanesulfonic acid 2-(N-morpholino)ethanesulfonic acid Heparan sulfate proteoglycans are located on the cell surface and in the extracellular matrix, where they play important roles in cell adhesion, differentiation, and growth in vitro and in vivo (1Lindahl U. Kusche-Gullberg M. Kjellén L. J. Biol. Chem. 1998; 273: 24979-24982Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 2Selleck S.B. Trends Genet. 2000; 16: 206-212Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 3Park P.W. Reizes O. Bernfield M. J. Biol. Chem. 2000; 275: 29923-29926Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). To a large extent, these biological activities depend on the heparan sulfate chains attached to the core protein. Heparan sulfate, a type of glycosaminoglycan, initially assembles by the copolymerization of N-acetyl-d-glucosamine (GlcNAc) and d-glucuronic acid (GlcA). The backbone then undergoes extensive modification initiated by theN-deacetylation and N-sulfation of subsets of GlcNAc residues. Subsequently, d-GlcA residues adjacent to the N-sulfated sugars are converted tol-IdoUA1 by a C5-epimerase and are sulfated at C-2 by a specific sulfotransferase. The glucosamine units also can be sulfated at C-6 and to a lesser extent at C-3. The blocklike arrangement of the modified residues confers specific binding properties to the chains for protein ligands, which in turn facilitate various biological activities. Many of the enzymes involved in heparan sulfate and heparin formation seem to be members of multienzyme gene families. Two exceptions are the C5-epimerase that interconverts d-GlcA andl-IdoUA and the 2-O-sulfotransferase that adds sulfate to C-2 of IdoUA residues and to a lesser extent GlcA residues. The C5-epimerase has been partially purified from mouse mastocytoma (4Malmström A. Rodén L. Feingold D.S. Jacobsson I. Bäckström G. Lindahl U. J. Biol. Chem. 1980; 255: 3878-3883Abstract Full Text PDF PubMed Google Scholar) and purified to homogeneity from bovine liver (5Campbell P. Hannesson H.H. Sandbäck D. Rodén L. Lindahl U. Li J. J. Biol. Chem. 1994; 269: 26953-26958Abstract Full Text PDF PubMed Google Scholar). A bovine cDNA for the epimerase has been cloned as well (6Li 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). Kinetic studies have clarified the substrate specificity of the epimerase, showing that the enzyme will react with both d-GlcA (forward reaction) andl-IdoUA (reverse reaction) when these residues are located toward the reducing side of N-sulfated glucosamine residues, but it will not react with uronic acids that are O-sulfated or that are adjacent to O-sulfated glucosamine residues (7Jacobsson I. Bäckström G. Höök M. Lindahl U. Feingold D.S. Malmström A. Rodén L. J. Biol. Chem. 1979; 254: 2975-2982Abstract Full Text PDF PubMed Google Scholar,8Jacobsson I. Lindahl U. Jensen J.W. Rodén L. Prihar H. Feingold D.S. J. Biol. Chem. 1984; 259: 1056-1063Abstract Full Text PDF PubMed Google Scholar). This specificity is consistent with the overall order of modification, suggesting that epimerization begins to occur after GlcNAc N-deacetylation and N-sulfation but before glucosamine residues undergo 6-O-sulfation and 3-O-sulfation (7Jacobsson I. Bäckström G. Höök M. Lindahl U. Feingold D.S. Malmström A. Rodén L. J. Biol. Chem. 1979; 254: 2975-2982Abstract Full Text PDF PubMed Google Scholar, 9Lindahl U. Jacobsson I. Höök M. Bäckström G. Feingold D.S. Biochem. Biophys. Res. Commun. 1976; 70: 492-499Crossref PubMed Scopus (28) Google Scholar). The fact that the epimerase seems to be represented only once in vertebrate and invertebrate genomes suggests that the extent of uronic acid epimerization depends on the level of enzyme expression and production of the N-sulfated tracts. In an attempt to study the cellular localization and potential interaction of the epimerase with other enzymes in the pathway, we discovered that the published bovine sequence encodes a truncated protein. 2M. A. S. Pinhal, B. Smith, J. Aikawa, K. Kimata, and J. D. Esko, unpublished results. This report provides the full-length sequence from mouse and human, an improved set of conditions for assaying the epimerase in cell extracts, and a demonstration that the enzyme is localized to the Golgi in vertebrate cells. Chinese hamster ovary cells (CHO-K1) were obtained from the American Type Culture Collection (CCL-61, Manassa, VA). MST cells were derived from the Furth murine mastocytoma (10Montgomery R.I. Lidholt K. Flay N.W. Liang J. Vertel B. Lindahl U. Esko J.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11327-11331Crossref PubMed Scopus (28) Google Scholar). CHO cells were grown in Ham's F-12 medium (Life Technologies, Inc.), and MST cells were grown in RPMI 1640 medium. Both media were supplemented with 10% (v/v) fetal bovine serum (Hyclone Laboratories), 100 μg/ml streptomycin sulfate, and 100 units/ml penicillin G. The cells were cultured at 37 °C under an atmosphere of 5% CO2 in air at 100% relative humidity. A murine epimerase cDNA fragment corresponding to the published bovine sequence (GenBankTM accession number AF003927) was cloned from an MST cDNA library using PCR, the forward primer 5′-ATGTCCTTTGAAGGCTACAATGTGG-3′, and the reverse primer 5′-CTAGTTGTGCTTTGCCCGGCTGCC-3′, which anneal with the first and last 24 bases of the partial bovine sequence (6Li 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). The PCR product was blunt-end cloned into pGEM (Promega) for sequencing. Subsequently, the primers XhoEpi5′ (5′-CCCCGGCTCGAGGCCGCCATGTCCTTTGAAGCCTACAATG-3′) and BamEpi3′ (5′-CTGGATCCTAGTTGTGCTTTGCCCGG-3′) were used to amplify the cDNA from the pGEM epimerase clone for transfer into pCDNA3.1 (Invitrogen) using the XhoI and BamHI sites. To generate the YFP-tagged truncated epimerase, the primers XhoEpi5′ and 3′Epi-GFPBam (5′-CTGGATCCCCGTTGTGCTTTGCCCGG-3′) were used to amplify the epimerase cDNA, which was cloned into the XhoI andBamHI sites of pEYFP-N1. The cDNA containing the full-length epimerase was cloned using a primer designed to the proposed 5′ end deduced from the human genomic DNA sequence (GenBankTM accession number AC026992). This primer, 5EpiXhoI (5′-CTCGAGCCATGCGTTGCTTGGCAGCTCGG-3′), was used with an internal reverse primer, 3EpiBam (5′-GGATCCGAGATTCCATGCCGCGCTCGTACAAG-3′), to amplify the 5′ 900 base pairs of the cDNA from the murine MST cDNA library. The cDNA encoding the full-length epimerase was then constructed by digesting the truncated epimerase in pCDNA3.1 with XhoI and HindIII and then by inserting the extended 5′ end amplified by PCR. The GFP-tagged full-length epimerase was generated by amplifying the full coding region from pCDNA3.1 with the primers 5EpiXhoI and 3′Epi-GFPBam and by cloning into the XhoI andBamHI sites of pEGFP-N1. The coding sequence was verified by directly sequencing PCR products from three independent amplifications from the MST cDNA library. All PCR amplifications were done using Vent DNA polymerase (New England Biolabs), and clones were sequenced on an ABI 373 DNA sequencer using dye terminator cycle sequencing. 5′-Rapid amplification of cDNA ends was performed according to the manufacturer's instructions (CLONTECH) with mRNA isolated from MST cells. Two gene-specific primers were used based on the murine sequence for the epimerase described above, 3EpiBam (5′-GGATCCGAGATTCCATGCCGCGCTCGTACAAG-3′) and BC15 (5′-ACATTGGTGGATCTAGACTT-3′). Analysis of five independent clones, each with the same 5′ end, yielded a consensus sequence. The sequence of the 3′ end of the murine coding sequence was determined by amplifying the 3′ end from an MST cDNA library using BC11 (5′-GGAGACCACAGAAAAGAATC-3′) and BC42 (5′-GGAGACCACAGAAAAGAATC-3′). BC11 was designed to anneal between nucleotides 1164 and 1183 of the mouse epimerase cDNA, whereas BC42 was designed to anneal to the 3′-untranslated region (UTR) and was designed based on nucleotides 1832–1855 of the partial human sequence (GenBankTMaccession number AB020643). The PCR product was cloned, and three independent isolates were sequenced. All three clones contained two silent changes from the published human sequence. The GenBankTM accession numbers for the murine cDNA and encoded protein are AF325532 and AAG42004, respectively. Epimerase constructs were generated with COOH-terminal c-myc and GFP tags by subcloning into pCDNA3.1 containing myc/His6 and pEGFP (CLONTECH), respectively. An amino-terminal c-myc-tagged clone was generated by annealing the oligonucleotides BC46 (5′-ATGTCTAGAGAACAAAAACTCATCTCAGAAGAGGATCTGTCTAGAGCA-3′) and BC47 (5′-TGCTCTAGACAGATCCTCTTCTGAGATGAGTTTTTGTTCTCTAGACAT-3′), which codes for a myc tag. The oligonucleotides were boiled for 1 min, cooled on ice, phosphorylated with polynucleotide kinase (New England Biolabs), and cloned in-frame into theEcoRV site in the polylinker region of pcDNA3.1 containing the full-length epimerase. The clone used in these experiments actually contained two Myc tags in a tandem repeat at the amino terminus. CHO cells were transiently transfected with 2 μg of plasmid DNA using LipofectAMINE according to the manufacturer's directions (Life Technologies, Inc.). Cells were grown on 24-well glass microscope slides and were processed for enzyme localization studies 24–36 h after transfection. After the cells were fixed for 1 h with 2% paraformaldehyde in 75 mm phosphate buffer, pH 7.5, they were rinsed several times with phosphate-buffered saline (PBS) (11Dulbecco R. Vogt M. J. Exp. Med. 1954; 99: 167-182Crossref PubMed Scopus (2004) Google Scholar). Cells were then permeabilized with 1% Triton X-100 (v/v) and 0.1% bovine serum albumin (BSA) (w/v) in PBS. The primary antibody, mouse anti-Myc (Invitrogen) monoclonal antibody, and rabbit polyclonal anti-α-mannosidase II antiserum (a gift from Marilyn G. Farquhar, University of California, San Diego) were diluted 1:400 in PBS with 1% BSA and incubated for 1 h with the fixed cells. To remove the unbound primary antibody, the cells were washed several times for 30 min with PBS containing 0.1% BSA. The samples were then incubated for 1 h with the secondary antibody, anti-rabbit Cy5 (Qccuvate Chemicals, NY) or anti-mouse-TRITC (Sigma), diluted 1:200 in PBS containing 1% BSA. After several washes, the cells were mounted with Vectashield containing 4′,6-diamidino-2-phenylindole for nuclear staining (Vector Laboratories). The photomicrographs shown in Fig. 5,A–D, were captured with a Photometrics charge-coupled device mounted on a Nikon microscope adapted to a DeltaVision (Applied Precision, Inc.) deconvolution imaging system. The data sets were deconvolved and analyzed using SoftWorx software (Applied Precision, Inc.) on a Silicon Graphics Octane work station. The photomicrograph shown in Fig. 5 E was captured with a Hamamatsu C5810 three-color chilled charge-coupled device camera mounted on a Zeiss Axiophot (×100 lens) microscope. Normal and transfected cells were washed twice with cold PBS and once with cold 0.25 m sucrose in 20 mm Tris, pH 7.4, and were then scraped with a rubber policeman into 100 μl of cold buffer containing 0.25 msucrose, 20 mm Tris-HCl, pH 7.4, 20 μmphenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, and 0.5 μg/ml pepstatin. Cells were lysed by sonication with a microtip sonicator, and the protein concentration was quantitated with the Bradford assay (Bio-Rad) using BSA as the standard. The extracts were stable when stored at −20 °C. The epimerase substrate consisted of modifiedN-acetylheparosan and was prepared as described (12Hagner-McWhirter Å. Hannesson H.H. Campbell P. Westley J. Rodén L. Lindahl U. Li J.P. Glycobiology. 2000; 10: 159-171Crossref PubMed Scopus (43) Google Scholar). Briefly, Escherichia coli K5 capsular polysaccharide was labeled in vivo withd-[5-3H]glucose (PerkinElmer Life Sciences) and purified from the growth medium. The GlcNAc residues in the labeled polysaccharide were N-deacetylated to near completion with anhydrous hydrazine and hydrazine sulfate (Sigma) and wereN-sulfated with trimethylamine sulfur trioxide complex (Sigma). The concentration of N-acetylheparosan was determined by a carbazol assay for uronic acids (13Bitter T. Muir H.M. Anal. Biochem. 1962; 4: 330-334Crossref PubMed Scopus (5217) Google Scholar), which yielded a radiospecific activity of 76 cpm/pmol GlcA (43 Ci/mol). Detection of epimerase activity was based on the release of3H from [5-3H]GlcA units in the polysaccharide and recovery as 3H2O (12Hagner-McWhirter Å. Hannesson H.H. Campbell P. Westley J. Rodén L. Lindahl U. Li J.P. Glycobiology. 2000; 10: 159-171Crossref PubMed Scopus (43) Google Scholar). Initial assays were set up according to the published reaction conditions ("original"), which contained 50 mm HEPES, 15 mm EDTA, 100 mm KCl, and 0.015% Triton X-100, pH 7.4 (12Hagner-McWhirter Å. Hannesson H.H. Campbell P. Westley J. Rodén L. Lindahl U. Li J.P. Glycobiology. 2000; 10: 159-171Crossref PubMed Scopus (43) Google Scholar). Protein, substrate, and various ancillary factors were adjusted to maximize the activity detected in normal and transfected cells. The "revised" assay consisted of 25 mm HEPES, pH 7.0, 0.1% Triton X-100, 300 pmol of3H-sulfated heparosan substrate, and 2 μg of cell protein in a total volume of 20 μl. Some assays contained 40 mmCaCl2, but divalent cations were later found not to be required. The reactions were incubated for 2 h at 37 °C and halted by the addition of 50 μl of cold 50 mm sodium acetate buffer, pH 4.0, containing 50 mm LiCl. The sample and a 100-μl rinse of the tube with buffer (25 mm HEPES, pH 7.0, and 0.1% Triton X-100) were transferred to a 0.4-ml column of DEAE-Sephacel (Amersham Pharmacia Biotech) that was equilibrated with the same buffer. The column was washed with 0.9 ml of assay buffer, and the 3H2O recovered in the flow-through fractions was counted by liquid scintillation spectrometry using Ultima Gold (Packard Instrument Co.). A reagent blank containing everything except a source of enzyme was included as a control. This yielded values of ∼200 cpm, which were subtracted from the experimental values that ranged from 300 to 3000 counts. All assays were done in duplicate with comparable results from three or more independent experiments. Cells were harvested, and 25 μg of protein for each sample was analyzed by SDS-polyacrylamide gel electrophoresis on a 10% gel. The samples were transferred to a nitrocellulose membrane using the Bio-Rad Mini Protean II system. The membrane was blocked at 4 °C overnight with 4% BSA in Tris-buffered saline (10 mm Tris, pH 8.0, and 150 mm NaCl) with 0.1% Tween 20 (TBST). Mouse anti-GFP (CLONTECH) and mouse anti-Myc (Invitrogen) were diluted 1:1000 and 1:5000 in TBST, respectively, and incubated for 1 h with the membrane at room temperature with shaking. The membrane was washed three times with TBST before the application of the secondary antibody, goat anti-mouse horseradish peroxidase (Bio-Rad) diluted 1:3000 in TBST. The membrane was washed six times with TBST and developed with SuperSignal West Pico chemiluminescent substrate (Pierce). The GlcA C5-epimerase was previously purified from murine mastocytoma and bovine liver (4Malmström A. Rodén L. Feingold D.S. Jacobsson I. Bäckström G. Lindahl U. J. Biol. Chem. 1980; 255: 3878-3883Abstract Full Text PDF PubMed Google Scholar, 5Campbell P. Hannesson H.H. Sandbäck D. Rodén L. Lindahl U. Li J. J. Biol. Chem. 1994; 269: 26953-26958Abstract Full Text PDF PubMed Google Scholar). Sequencing the purified protein yielded proline as the amino-terminal amino acid, suggesting that the protein had been proteolytically cleaved during the purification process (6Li 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). Several internal peptides also were generated by controlled proteolysis, and the peptide sequences were used to design oligonucleotides for screening a bovine lung cDNA library. A cDNA sequence was obtained that was 3085 base pairs and contained an open reading frame corresponding to a 444-amino acid protein from the first in-frame ATG codon. The deduced amino acid sequence predicted a 49,905-Da protein with a potential transmembrane domain located near the amino terminus. Expression of this clone in the baculovirus system yielded the expected activity (6Li 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). Further analysis of the epimerase sequence suggested that it was incomplete. First, computer-aided analysis using PSORT did not indicate the predicted transmembrane domain and in fact suggested that the protein was most likely soluble (14Nakai K. Kanehisa M. Genomics. 1992; 14: 897-911Crossref PubMed Scopus (1368) Google Scholar). As described below, this was confirmed in localization studies with GFP-tagged constructs. Second, alignment of the published bovine epimerase sequence with other orthologs in the GenBankTM data base suggested that the bovine sequence was potentially missing a large domain from the amino terminus (Fig. 1). A BLAST search revealed a human genomic DNA clone (GenBankTM accession number AC026992) containing an extended open reading frame that more closely matched the size of the epimerase orthologs found inCaenorhabditis elegans and Drosophila melanogaster. In addition, a partial human cDNA also was identified in the sequence data bases (GenBankTM accession number AB020643). Based on this information, we cloned an extended cDNA from mouse mastocytoma mRNA using primers based on the human sequence. This fragment was further extended using 5′-rapid amplification of cDNA ends. The additional sequence added 753 nucleotides including 231 nucleotides in a 5′-UTR and 522 coding nucleotides. The 5′-UTR contains an in-frame termination codon (TGA) 21 base pairs upstream of the new initiation codon, suggesting that the cDNA encodes the full-length epimerase (Fig. 2). The context of the new start codon conforms to an "adequate" Kozak sequence (AATatgC, consensus RNNatgY, where R = A or G and Y = T or C) (15Kozak M. Mamm. Genome. 1996; 7: 563-574Crossref PubMed Scopus (757) Google Scholar), whereas the previously suggested start codon lacks A or G at −3 (tttatgt). Previous studies reported a 3′-UTR sequence of ∼1.6 kilobases (6Li 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). Because the mRNA was found to be ∼5 kilobases, an additional untranslated sequence of ∼1.4 kilobases apparently exists, but its location is unknown. The revised sequence adds 174 amino acids to the amino terminus of the previous sequence of the bovine epimerase for a total of 618 amino acids. The full-length epimerase predicts a protein of 70,099 Da with a relatively basic isoelectric point (pI = 8.95). The protein contains a stretch of 17 hydrophobic residues located 11 amino acids from the amino terminus. As expected, PSORT predicts this as a transmembrane domain, and therefore the protein would most likely have a type II transmembrane topology like other enzymes involved in polysaccharide biosynthesis. The full-length clone contains three potential N-linked glycosylation sites at residues 225, 304, and 394, consistent with previous studies suggesting that the protein contained one or more Asn-linked chains. Using the extended sequence to perform a BLAST search of the GenBankTM data bases did not reveal additional homologs, suggesting that there may be only one heparin/heparan sulfate C5-epimerase (16Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60174) Google Scholar). A comparison of C. elegans, D. melanogaster, mouse, bovine, and human epimerase sequences indicated weak homology in the amino-terminal domain (residues 1–171) followed by a region of high identity (62%, residues 172–223). However, neither of these regions seems critical for catalytic activity because the purified protein from bovine liver was truncated at residue 248 (6Li 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). The COOH-terminal domain was also highly conserved across phylogeny (60% identity, residues 497–618), suggesting that this may represent an important functional part of the protein. Expression studies of the truncated and full-length epimerase were undertaken to determine whether the additional 174 amino acids had any effect on catalysis. Initial attempts to assay the basal enzyme activity in cell extracts prepared from CHO and MST cells met with limited success. Expression of the truncated or full-length enzyme gave variable results, suggesting that the assay originally described for the bovine enzyme might not be optimal for cultured cells or recombinant enzymes expressed in cultured cells (4Malmström A. Rodén L. Feingold D.S. Jacobsson I. Bäckström G. Lindahl U. J. Biol. Chem. 1980; 255: 3878-3883Abstract Full Text PDF PubMed Google Scholar, 5Campbell P. Hannesson H.H. Sandbäck D. Rodén L. Lindahl U. Li J. J. Biol. Chem. 1994; 269: 26953-26958Abstract Full Text PDF PubMed Google Scholar). Variation of each component improved the activity additively. Monovalent salts were inhibitory in contrast to previous findings (Fig.3 A) (5Campbell P. Hannesson H.H. Sandbäck D. Rodén L. Lindahl U. Li J. J. Biol. Chem. 1994; 269: 26953-26958Abstract Full Text PDF PubMed Google Scholar, 7Jacobsson I. Bäckström G. Höök M. Lindahl U. Feingold D.S. Malmström A. Rodén L. J. Biol. Chem. 1979; 254: 2975-2982Abstract Full Text PDF PubMed Google Scholar). Divalent cations, such as Ca2+ or Mg2+, and EDTA (up to 40 mm) had no effect (4Malmström A. Rodén L. Feingold D.S. Jacobsson I. Bäckström G. Lindahl U. J. Biol. Chem. 1980; 255: 3878-3883Abstract Full Text PDF PubMed Google Scholar, 7Jacobsson I. Bäckström G. Höök M. Lindahl U. Feingold D.S. Malmström A. Rodén L. J. Biol. Chem. 1979; 254: 2975-2982Abstract Full Text PDF PubMed Google Scholar). The activity was highly dependent on detergent, even in sonicated extracts, with maximal effects obtained with 0.1% Triton X-100 (Fig. 3 B). However, other detergents inhibited the reaction, suggesting that the effect was not merely because of solubilization of the protein from membranes. The pH optimum was ∼7.0, which is in general agreement with previous findings (Fig.3 C) (4Malmström A. Rodén L. Feingold D.S. Jacobsson I. Bäckström G. Lindahl U. J. Biol. Chem. 1980; 255: 3878-3883Abstract Full Text PDF PubMed Google Scholar, 5Campbell P. Hannesson H.H. Sandbäck D. Rodén L. Lindahl U. Li J. J. Biol. Chem. 1994; 269: 26953-26958Abstract Full Text PDF PubMed Google Scholar, 7Jacobsson I. Bäckström G. Höök M. Lindahl U. Feingold D.S. Malmström A. Rodén L. J. Biol. Chem. 1979; 254: 2975-2982Abstract Full Text PDF PubMed Google Scholar), but the activity showed marked sensitivity to the type of buffer (Fig. 3 D). HEPES was found to be optimal. Under the revised conditions, the reaction was proportional with time for over 2 h and with protein concentration in the range of 1–30 μg. The K m of the enzyme for theN-deacetylated/N-sulfated heparosan was estimated to be 25 μm GlcA equivalents (∼500 pmol of GlcA/assay). With 300 pmol of GlcA/assay, a 4.5-fold increase in epimerase activity in MST cell extracts and a 6.5-fold enhancement in CHO cell extracts were observed compared with the original conditions (Fig.3 E). At lower concentrations of substrate, the difference was even more dramatic (data not shown). Transient transfection of CHO cells revealed 4-fold greater activity associated with the full-length protein compared with the truncated enzyme in the revised assay (Fig. 4). Increasing the substrate 10-fold did not enhance the rate of reaction for either recombinant enzyme (data not shown). These findings indicated that the natural amino terminus was not a prerequisite to detect activity, which is consistent with previous findings showing that the truncated protein purified from liver and mastocytoma had substantial activity (4Malmström A. Rodén L. Feingold D.S. Jacobsson I. Bäckström G. Lindahl U. J. Biol. Chem. 1980; 255: 3878-3883Abstract Full Text PDF PubMed Google Scholar, 5Campbell P. Hannesson H.H. Sandbäck D. Rodén L. Lindahl U. Li J. J. Biol. Chem. 1994; 269: 26953-26958Abstract Full Text PDF PubMed Google Scholar, 12Hagner-McWhirter Å. Hannesson H.H. Campbell P. Westley J. Rodén L. Lindahl U. Li J.P. Glycobiology. 2000; 10: 159-171Crossref PubMed Scopus (43) Google Scholar, 17Hagner-McWhirter Å. Lindahl U. Li J.P. Biochem. J. 2000; 347: 69-75Crossref PubMed Google Scholar). Extracts prepared from cells transfected with epimerase containing a COOH-terminal GFP or YFP tag were analyzed by Western blotting with an anti-GFP monoclonal antibody. As shown in the inset of Fig. 4, the tagged full-length protein was present at higher levels than the truncated enzyme. Both forms were engineered into a near perfect Kozak sequence in the expression vector, suggesting that their expression was similar. Thus, we believe that the lower amount of the truncated enzyme was caused by decreased stability. As shown below, the truncated enzyme was also mislocalized, which may add to its instability. Thus, the amino-terminal domain does not seem to enhance the intrinsic activity of the enzyme. Fusing c-myc or GFP to the COOH terminus resulted in a dramatic reduction of enzyme activity (<1 pmol/min/mg), but when a c-myc tag was placed at the amino terminus, enzyme activity was normal (23 pmol/min/mg versus 26 pmol/min/mg, respectively). These findings suggested that the highly conserved COOH terminus plays an important role in binding, conformation, or catalysis. Recent investigations into the catalytic mechanism of the C5-epimerase implicated two polyprotic bases in the proton exchanges at C-5 (17Hagner-McWhirter Å. Lindahl U. Li J.P. Biochem. J. 2000; 347: 69-75Crossref PubMed Google Scholar) that are possibly mediated by two lysine residues. Interestingly, two lysine residues (amino acids 547 and 616) in the COOH-terminal domain of the epimerase are highly conserved across phylogeny (Fig. 1, asterisks). Adding GFP to the COOH terminus of other enzymes involved in heparin/heparan sulfate biosynthesis does not result in loss of activity (18McCormick C. Duncan G. Goutsos K.T. Tufaro F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 668-673Crossref PubMed Scopus (373) Google Scholar).2 To study the intracellular localization of epimerase, cDNAs encoding the truncated and full-length enzymes were fused to GFP or c-mycand expressed in CHO cells. Full-length epimerase was located in a juxtanuclear position, co-localizing with the Golgi marker, α-mannosidase II (Fig. 5,A–C). This localization was observed with tags on either the C or amino terminus, indicating that the location of the tag did not interfere with subcellular localization signals in the protein (Fig. 5 E). When the truncated epimerase was expressed, it behaved as a soluble protein exhibiting diffuse cytoplasmic staining (Fig. 5 D). This is not an unexpected result given that the protein lacks a signal peptide. The mislocalization of the truncated enzyme may act to destabilize its structure and activity (Fig. 4, inset). Future studies of the epimerase will be greatly expedited by having the full-length sequence. Interestingly, very little information is available about the function of IdoUA in the biological activity of heparin and heparan sulfate. In general, it is assumed that the greater conformational flexibility of IdoUA will enhance the binding opportunities for heparin and heparan sulfate (19Mulloy B. Forster M.J. Glycobiology. 2000; 10: 1147-1156Crossref PubMed Scopus (272) Google Scholar). The best studied example is the interaction of antithrombin with a heparin pentasaccharide, in which a critical IdoUA residue located to the reducing side of a central 3-O-sulfated glucosomine unit confers high affinity binding to antithrombin (20Atha D.H. Lormeau J.C. Petitou M. Rosenberg R.D. Choay J. Biochemistry. 1985; 24: 6723-6729Crossref PubMed Scopus (189) Google Scholar). Fibroblast growth factor-2 also apparently requires at least one IdoUA unit for binding and activation (21Maccarana M. Casu B. Lindahl U. J. Biol. Chem. 1993; 268: 23898-23905Abstract Full Text PDF PubMed Google Scholar, 22Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. J. Biol. Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar). In the former case, the addition of the 2-O-sulfate group to the IdoUA residue seems to be dispensable (20Atha D.H. Lormeau J.C. Petitou M. Rosenberg R.D. Choay J. Biochemistry. 1985; 24: 6723-6729Crossref PubMed Scopus (189) Google Scholar, 23Bjork I. Lindahl U. Mol. Cell. Biochem. 1982; 48: 161-182Crossref PubMed Scopus (364) Google Scholar), whereas in the latter it is essential for binding (24Bai X.M. Esko J.D. J. Biol. Chem. 1996; 271: 17711-17717Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 25Pellegrini L. Burke D.F. Von Delft F. Mulloy B. Blundell T.L. Nature. 2000; 407: 1029-1034Crossref PubMed Scopus (630) Google Scholar, 26Schlessinger J. Plotnikov A.N. Ibrahimi O.A. Eliseenkova A.V. Yeh B.K. Yayon A. Linhardt R.J. Mohammadi M. Mol. Cell. 2000; 6: 743-750Abstract Full Text Full Text PDF PubMed Scopus (973) Google Scholar). These findings suggest that in some cases the IdoUA may play a direct role in binding to the ligand, whereas in others it may simply serve as a scaffold for placement of a critical sulfate residue. In both cases, the epimerase plays an essential role in creating the preferred binding site for the ligand. With full-length recombinant enzyme now available, it should be possible to engineer binding sites in isolated oligosaccharides and to explore the function of epimerasein vivo by creating mutants in cells and model organisms. We thank James Feramisco and Brian Smith from the Digital Imaging Shared Resource at the University of California, San Diego Cancer Center for their help in the deconvolution microscopy.