Biosynthesis of heparin/heparan sulphate: mechanism of epimerization of glucuronyl C-5

Hagner-McWhirter, Åsa; Lindahl, Ulf; Li, Jinping

doi:10.1042/bj3470069

Cited by 44 publications

(43 citation statements)

References 32 publications

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“…Divalent The triangle indicates the putative initiating Met residue in the original bovine sequence (6). The asterisks indicate conserved lysine residues in the COOH terminus that may be involved in catalysis (17).…”

Section: Resultsmentioning

confidence: 99%

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

Crawford

Olson

Esko

et al. 2001

Journal of Biological Chemistry

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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 of D-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 melanogaster and 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-myc tag 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 fulllength enzyme is in the Golgi, where heparan sulfate synthesis is thought to occur.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 (1-3). 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 the N-deacetylation and N-sulfation of subsets of GlcNAc residues. Subsequently, D-GlcA residues adjacent to the N-sulfated sugars are converted to L-IdoUA 1 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 and L-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 (4) and purified to homogeneity from bovine liver (5). A bovine cDNA for the epimerase has been cloned as well (6). Kinetic studies have clarified the substrate specificity of the epimerase, showing that the enzyme will react with both D...

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Section: Resultsmentioning

confidence: 99%

“…ings showing that the truncated protein purified from liver and mastocytoma had substantial activity (4,5,12,17). 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.…”

Section: Resultsmentioning

confidence: 99%

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

Crawford

Olson

Esko

et al. 2001

Journal of Biological Chemistry

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“…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- [5-3 H]glucose, followed by N-deacetylation and N-sulfation of the resultant radiolabeled polysaccharide (21,22).…”

Section: Methodsmentioning

confidence: 99%

Biosynthesis of Dermatan Sulfate

Maccarana

Olander

Malmström

et al. 2006

Journal of Biological Chemistry

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138

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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 ϳ43,000-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)5 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 (1-5). 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 outgrowthpromoting activity mediated by rare structures in cerebral CS/DS (6 -9). 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 flexibili...

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“…Fractions of 1 ml were collected and analyzed for radioactivity. O-Sulfated disaccharides were first isolated by preparative high voltage paper electrophoresis at pH 5.3 (0.083 M pyridine, 0.05 M acetic acid) and were then identified by paper chromatography (ethyl acetate/acetic acid/H 2 O, 3:1:1 by volume) (10 3 and 30 ϫ 10 3 dpm 3 H, respectively) obtained by Bio-Gel P-10 chromatography were digested with 10.4 units of ␤-glucuronidase in 200 l of 0.05 M sodium acetate, pH 5, at 37°C overnight. Test incubations with appropriate disaccharide standards ascertained that the enzyme preparation contained ␤-glucuronidase but no ␣-iduronidase activity (not shown).…”

Section: Materials-d-[u-mentioning

confidence: 99%

“…1). Incubation with epimerase of an appropriate polysaccharide substrate containing C5-3 H-labeled hexuronic acid residues will release the label from GlcA as well as IdoA units; conversely, incubation of unlabeled substrate in 3 H 2 O leads to incorporation of label into both epimers (10). Is epimerization reversible also during HS biosynthesis in an intact cell?…”

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confidence: 99%