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

Abstract

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...

Heparin and Heparan Sulfate Biosynthesis

“…Orthologs have been found in C. elegans and Drosophila (79). The full-length enzyme is located in the Golgi apparatus (79). During enzyme purification, the truncated form was tightly associated with a 22-kDa fragment derived from the N-terminus of the full length form encoded by the same gene.…”

“…During enzyme purification, the truncated form was tightly associated with a 22-kDa fragment derived from the N-terminus of the full length form encoded by the same gene. This fragment may be needed for Golgi localization since the truncation leads to mislocation in the cytosol (79) or it may have a regulatory function since the full-length form shows 4-fold greater activity compared with the truncated enzyme. Although the inversion of configuration at C5 is reversible, O-sulfation at C2 of the uronic acid or at C6 of one or both of the neighboring glucosamine residues appears to preclude C5-epimerization to IdoA or back epimerization to GlcA (2).…”

“…Uronyl C5-epimerase interconverts GlcA and IdoA in heparin and HS and has been purified to homogeneity from bovine liver in a truncated soluble form of 52 kDa (77). A cDNA encoding the full-length type-II transmembrane protein has been cloned from mice and humans (78,79). The enzyme does not modify chondroitin nor does the C5-epimerase for DS biosynthesis modify heparin/HS precursor polysaccharides.…”

Decreased expression of human D‐glucuronyl C5‐epimerase in breast cancer

“…It is highly homologous to murine and bovine epimerase which have been cloned earlier. [12][13][14] GLCE is responsible for GlcUA to IdoUA epimerization only in HS but not in DS, for which a specific DS epimerase was discovered. 15 Thus, GLCE could be involved in any processes where appropriate HS structure is important.…”

Oligosaccharide Library-based Assessment of Heparan Sulfate 6-O-Sulfotransferase Substrate Specificity

“…Along with polymerization the chains undergo a series of modification steps that involve five distinct enzyme families; (i) N-deacetylase/N-sulfotransferase removes the N-acetyl group from GlcNAc units and replaces it with an N-sulfate group (GlcNS); (ii) GlcUA C5 epimerase converts GlcUA to its C5 epimer L-iduronate (IdoUA); (iii) 2-O-sulfotransferase adds 2-O-sulfate groups to IdoUA and GlcUA residues; (iv) 6-O-sulfotransferase (6-OST), and (v) 3-O-sulfotransferase (3-OST) transfer O-sulfate groups to C6 and C3, respectively, of GlcNAc or GlcNS units (2)(3)(4). The N-deacetylase/N-sulfotransferase (5-8), the 6-OST (9), and the 3-OST (10 -12) families each contain several members, whereas only one GlcUA C5 epimerase (13,14) and one hexuronate 2-Osulfotransferase (15, 16) have been described. During enzymatic HS modification, only parts of the available target units are modified.…”