The tumor suppressors EXT1 and EXT2 are associated with hereditary multiple exostoses and encode bifunctional glycosyltransferases essential for chain polymerization of heparan sulfate (HS) and its analog, heparin (Hep). Three highly homologous EXT-like genes, EXTL1-EXTL3, have been cloned, and EXTL2 is an ␣1,4-GlcNAc transferase I, the key enzyme that initiates the HS͞Hep synthesis. In the present study, truncated forms of EXTL1 and EXTL3, lacking the putative NH 2-terminal transmembrane and cytoplasmic domains, were transiently expressed in COS-1 cells and found to harbor ␣-GlcNAc transferase activity. EXTL3 used not only N-acetylheparosan oligosaccharides that represent growing HS chains but also GlcA␤1-3Gal␤1-O-C 2H4NH-benzyloxycarbonyl (Cbz), a synthetic substrate for ␣-GlcNAc transferase I that determines and initiates HS͞Hep synthesis. In contrast, EXTL1 used only the former acceptor. Neither EXTL1 nor EXTL3 showed any glucuronyltransferase activity as examined with N-acetylheparosan oligosaccharides. Heparitinase I digestion of each transferase-reaction product showed that GlcNAc had been transferred exclusively through an ␣1,4-configuration. Hence, EXTL3 most likely is involved in both chain initiation and elongation, whereas EXTL1 possibly is involved only in the chain elongation of HS and, maybe, Hep as well. Thus, their acceptor specificities of the five family members are overlapping but distinct from each other, except for EXT1 and EXT2 with the same specificity. It now has been clarified that all of the five cloned human EXT gene family proteins harbor glycosyltransferase activities, which probably contribute to the synthesis of HS and Hep.

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The relationship between the metabolites of glycyrrhizin (18beta-glycyrrhetinic acid-3-O-beta-D-glucuronopyranosyl-(1-->2)-beta-D-glucuronide, GL) and their biological activities was investigated. By human intestinal microflora, GL was metabolized to 18beta-glycyrrhetinic acid (GA) as a main product and to 18beta-glycyrrhetinic acid-3-O-beta-D-glucuronide (GAMG) as a minor product. The former reaction was catalyzed by Eubacterium L-8 and the latter was by Streptococcus LJ-22. Among GL and its metabolites, GA and GAMG had more potent in vitro anti-platelet aggregation activity than GL. GA also showed the most potent cytotoxicity against tumor cell lines and the potent inhibitory activity on rotavirus infection as well as growth of Helicobacter pylori. GAMG, the minor metabolite of GL, was the sweetest.

The Drosophila melanogaster genome contains three putative glucuronyltransferases homologous to human GlcAT-I and GlcAT-P. These enzymes are predicted to be ␤1,3-glucuronyltransferases involved in the synthesis of the glycosaminoglycan (GAG)-protein linkage region of proteoglycans and the HNK-1 carbohydrate epitope of glycoproteins, respectively. The genes encode active enzymes, which we have designated DmGlcAT-I, DmGlcAT-BSI, and DmGlcAT-BSII (where BS stands for "broad specificity"). Protein A-tagged truncated soluble forms of all three enzymes efficiently transfer GlcUA from UDP-GlcUA to the linkage region trisaccharide Gal␤1-3Gal␤1-4Xyl. Strikingly, DmGlcAT-I has specificity for Gal␤1-3Gal␤1-4Xyl, whereas DmGlcAT-BSI and DmGlcAT-BSII act on a wide array of substrates with non-reducing terminal ␤1,3-and ␤1,4-linked Gal residues. Their highest activities are obtained with asialoorosomucoid with a terminal Gal␤1-4GlcNAc sequence, indicating their possible involvement in the synthesis of the HNK-1 epitope in addition to the GAG-protein linkage region. Gal␤1-3GlcNAc and Gal␤1-3GalNAc, disaccharide structures widely found in N-and O-glycans of glycoproteins and glycolipids, also serve as acceptors for DmGlcAT-BSI and -BSII. Transcripts of all three enzymes are ubiquitously expressed throughout the developmental stages and in adult tissues of Drosophila. Thus, all three glucuronyltransferases are likely involved in the synthesis of the GAG-protein linkage region in Drosophila, and DmGlcAT-BSI and -BSII appear to be involved in various GlcUA transfer reactions for the synthesis of proteoglycans, glycoproteins, and glycolipids. This activity distinguishes these glucuronyltransferases from their mammalian homologs GlcAT-P and GlcAT-D (or -S). Sequence alignment of the Drosophila glucuronyltransferases with homologs in human, rat, and Caenorhabditis elegans demonstrates the conservation of a majority of the critical amino acid residues in the active sites of the three Drosophila enzymes. Proteoglycans (PGs)1 play an essential role in a variety of biological processes such as cell-cell adhesion, cell proliferation, and tissue morphogenesis (1, 2). PGs consist of a core protein and sulfated glycosaminoglycan (GAG) side chains. PGs can be classified into three groups based on the nature of their GAGs: heparan sulfate (HS)-type PGs, chondroitin sulfate (CS)-type PGs, and keratan sulfate PGs. There is increasing evidence that uniquely sulfated domain structures of GAG side chains are critically involved in various functions of these PGs (3-5), and defective synthesis of GAGs causes aberrant morphology and even embryonic lethality during development (6).In biosynthesis, HS or CS linear chains are differentially assembled on the common linkage region tetrasaccharide Glc-UA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-, which takes place on a specific serine residue in a given core protein (7). Transfer of either ␣1,4-GlcNAc or ␤1,4-GalNAc to the tetrasaccharide linkage region terminus mediated by the GAG-specific hexosaminyltransferases determin...

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Heparan, the common unsulfated precursor of heparan sulfate (HS) and heparin, is synthesized on the glycosaminoglycan-protein linkage region tetrasaccharide GlcUA-Gal-Gal-Xyl attached to the respective core proteins presumably by HS co-polymerases encoded by EXT1 and EXT2, the genetic defects of which result in hereditary multiple exostoses in humans. Although both EXT1 and EXT2 exhibit GlcNAc transferase and GlcUA transferase activities required for the HS synthesis, no HS chain polymerization has been demonstrated in vitro using recombinant enzymes. Here we report in vitro HS polymerization. Recombinant soluble enzymes expressed by co-transfection of EXT1 and EXT2 synthesized heparan polymers with average molecular weights greater than 1.7 ؋ 10 5 using UDP-[ 3 H]GlcNAc and UDPGlcUA as donors on the recombinant glypican-1 core protein and also on the synthetic linkage region analog GlcUA-Gal-O-C 2 H 4 NH-benzyloxycarbonyl. Moreover, in our in vitro polymerization system, a part time proteoglycan, ␣-thrombomodulin, that is normally modified with chondroitin sulfate served as a polymerization primer for heparan chain. In contrast, no polymerization was achieved with a mixture of individually expressed EXT1 and EXT2 or with acceptor substrates such as N-acetylheparosan oligosaccharides or the linkage region tetrasaccharide-Ser, which are devoid of a hydrophobic aglycon, suggesting the critical requirement of core protein moieties in addition to the interaction between EXT1 and EXT2 for HS polymerization. Heparan sulfate (HS)1 proteoglycans found at cell surfaces and in extracellular matrices play vital functions in many biological processes such as cell proliferation, migration, differentiation, recognition, adhesion, and tissue morphogenesis in the animal kingdom, and the HS glycosaminoglycan moiety, a structural analog of heparin, is essential for these functions (1). HS has recently attracted much attention since it plays critical roles during development, especially in the major signaling pathways such as those of fibroblast growth factor, Wnt, and Hedgehog (2, 3). HS is synthesized to have highly heterogeneous structures with various sizes and sulfated patterns, which are spatially and temporally regulated. Therefore, understanding of HS synthesis and its regulatory mechanisms underlying diverse HS functions is essential.Heparan, the HS chain backbone, is synthesized by glycosyltransferases encoded by EXT1 and EXT2 (4) in the EXT (exostosin) gene family, which were first identified as causative genes of a genetic bone disorder, hereditary multiple exostoses (5, 6), and were subsequently demonstrated to function as tumor suppressor genes (7-9). Later it was reported that both EXT1 and EXT2 encode bifunctional glycosyltransferases with N-acetylglucosaminyltransferase II (GlcNAcT-II) and glucuronyltransferase II activities required for chain elongation of heparan chains consisting of -(4GlcUA␤1-4GlcNAc␣1) n -, and they were suggested to be HS co-polymerases, although no direct evidence was presented for chain po...

A novel type of heparinase (heparin lyase, no EC number) has been purified from Bacteroides stercoris HJ-15, isolated from human intestine, which produces three kinds of heparinases. The enzyme was purified to apparent homogeneity by a combination of QAE-cellulose, DEAE-cellulose, CM-Sephadex C-50, hydroxyapatite, and HiTrap SP chromatographies with a final specific activity of 19.5 mmol/min/mg. It showed optimal activity at pH 7.2 and 45 degrees C and the presence of 300 mM KCl greatly enhanced its activity. The purified enzyme activity was inhibited by Cu(2+), Pb(2+), and some agents that modify histidine and cysteine residues, and activated by reducing agents such as dithiothreitol and 2-mercaptoethanol. This purified Bacteroides heparinase is an eliminase that shows its greatest activity on bovine intestinal heparan sulfate, and to a lesser extent on porcine intestinal heparan sulfate and heparin. This enzyme does not act on acharan sulfate but de-O-sulfated acharan sulfate and N-sulfoacharan sulfate were found to be poor substrates. The substrate specificity of this enzyme is similar to that of Flavobacterial heparinase II. However, an internal amino acid sequence of the purified Bacteroides heparinase shows significant (73%) homology to Flavobacterial heparinase III and only 43% homology to Flavobacterial heparinase II. These findings suggest that the Bacteroidal heparinase is a novel enzyme degrading GAGs.