The putative tumor suppressors EXT1 and EXT2 form a stable complex that accumulates in the Golgi apparatus and catalyzes the synthesis of heparan sulfate.
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Hereditary multiple exostoses, a dominantly inherited genetic disorder characterized by multiple cartilaginous tumors, is caused by mutations in members of the EXT gene family, EXT1 or EXT2. The proteins encoded by these genes, EXT1 and EXT2, are endoplasmic reticulum-localized type II transmembrane glycoproteins that possess or are tightly associated with glycosyltransferase activities involved in the polymerization of heparan sulfate. Here, by testing a cell line with a specific defect in EXT1 in in vivo and in vitro assays, we show that EXT2 does not harbor significant glycosyltransferase activity in the absence of EXT1. Instead, it appears that EXT1 and EXT2 form a hetero-oligomeric complex in vivo that leads to the accumulation of both proteins in the Golgi apparatus. Remarkably, the Golgi-localized EXT1/EXT2 complex possesses substantially higher glycosyltransferase activity than EXT1 or EXT2 alone, which suggests that the complex represents the biologically relevant form of the enzyme(s). These findings provide a rationale to explain how inherited mutations in either of the two EXT genes can cause loss of activity, resulting in hereditary multiple exostoses. (+info)
Synthesis of hyaluronan of distinctly different chain length is regulated by differential expression of Xhas1 and 2 during early development of Xenopus laevis.
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The localization of hyaluronan has been determined in tailbud stage embryos of Xenopus laevis using a neurocan-alkaline phosphatase fusion protein. This polysaccharide was located between the germ layers and enriched in mesenchyme, the lumen of the neural tube, the embryonic gut, the hepatic cavity and the heart. A full-length cDNA for a hyaluronan synthase, Xhas2 has been cloned. The expression pattern of Xhas1 and 2 is closely similar to the distribution of hyaluronan in the embryo. Xhas1 produces hyaluronan with a molecular mass of around 40-200 kDa, while the product formed by Xhas2 has a molecular mass above 1 million Da. (+info)
Preparation of oligomeric beta-glycosides from cellulose and hemicellulosic polysaccharides via the glycosyl transferase activity of a trichoderma reesei cellulase.
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Oligoglycosyl (allyl, 2,3-dihydroxypropyl, ethyl, 2-hydroxyethyl, and methyl) beta-glycosides were generated by endo -transglycosylation reactions catalyzed by commercially available Trichoderma reesei cellulase. A polymeric donor substrate (xyloglucan or cellulose) was incubated with the enzyme in an aqueous solution containing 20% of the acceptor alcohol (allyl alcohol, glycerol, ethanol, ethylene glycol, and methanol, respectively). The products of these reactions included oligomeric alkyl beta-glycosides and reducing oligosaccharides. The high yield of alkyl beta-glycosides may be explained by the resistance of the xyloglucan beta-glycosides to cellulase-mediated hydrolysis. The resistance of the oligoxyloglucan beta-glycosides to endo glucanase catalyzed hydrolysis supports the hypothesis that productive binding of the glycan substrate depends on its interaction with enzyme subsites on both sides of the cleavage point, leading to distortion of the ring geometry of the residue whose glycosidic bond is cleaved. Oligoxyloglucan beta-glycosides were purified by a combination of gel-permeation and reversed-phase HPLC and were structurally characterized by MS and NMR spectroscopy. These results demonstrate that novel oligosaccharide beta-glycosides can be efficiently produced by enzyme-catalyzed fragmentation/transglycosylation reactions starting with a polysaccharide donor substrate. This class of reactions may represent a convenient source of beta-glycosides to be used as synthons for the rapid synthesis of complex glycans. (+info)
Osteogenic protein 1 stimulates cells-associated matrix assembly by normal human articular chondrocytes: up-regulation of hyaluronan synthase, CD44, and aggrecan.
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OBJECTIVE: To determine the effects of osteogenic protein 1 (OP-1) on hyaluronan (HA), CD44, and aggrecan biosynthesis as well as the contribution of these molecules in promoting matrix assembly by human articular chondrocytes. METHODS: Normal human chondrocytes were cultured with or without OP-1 treatment. Changes in the relative expression of messenger RNA (mRNA) for HA synthases 2 and 3 (HAS-2 and HAS-3), CD44, and aggrecan were determined by competitive quantitative reverse transcriptase-polymerase chain reaction. Accumulation of HA was characterized by indirect staining, CD44 by flow cytometry, and aggrecan biosynthesis by 35SO4 incorporation. RESULTS: OP-1 stimulated the expression of HAS-2, CD44, and aggrecan mRNA in a time-dependent manner, resulting in increased expression of HA, CD44, and aggrecan. Prominent increases in HA-rich cell-associated matrices were also observed. CONCLUSION: OP-1 stimulates not only the synthesis of matrix macromolecules such as aggrecan, but also the synthesis of other molecules required for matrix retention, namely, HA and CD44. (+info)
Inhibition of hyaluronan synthesis by vesnarinone in cultured human myofibroblasts.
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Hyaluronan (HA), which is a major component of the extracellular matrix (ECM), is regulated during myofibroproliferative responses to numerous forms of inflammatory stimuli. It is a key factor involved in cellular migration and adherence. The development of a potent and non-toxic inhibitor of HA synthesis would open up a new avenue for the treatment of fibrocontractive diseases such as pulmonary fibrosis and liver cirrhosis. In this study, the effects of vesnarinone (OPC-8212: 3,4-dihydro-6-[4-(3, 4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone) on the secretion of HA in human myofibroblast cell lines (MRC-5 and LI90 cells, referred to as pulmonary and hepatic myofibroblasts, respectively) were examined. Vesnarinone specifically and dose-dependently inhibited HA secretion by myofibroblasts up-regulated by fetal calf serum (FCS). The treatment of vesnarinone did not modify the phenotype of myofibroblast cells in culture. Vesnarinone also potently inhibited the HA secretion by the two myofibroblast cell lines up-regulated by transforming growth factor-beta1 (TGF-beta1) or tumor necrosis factor-alpha (TNF-alpha). The addition of vesnarinone to myofibroblasts resulted in a significant decrease of HA synthase (HAS) activity, with or without the addition of FCS or either cytokine. These findings suggest that vesnarinone inhibits the secretion of HA in myofibroblasts by specifically suppressing HAS activity, and may therefore prove useful for the treatment of chronic inflammation and tissue fibrosis. (+info)
Two new tailoring enzymes, a glycosyltransferase and an oxygenase, involved in biosynthesis of the angucycline antibiotic urdamycin A in Streptomyces fradiae Tu2717.
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Urdamycin A, the principal product of Streptomyces fradiae Tu2717, is an angucycline-type antibiotic and anticancer agent containing C-glycosidically linked D-olivose. To extend knowledge of the biosynthesis of urdamycin A the authors have cloned further parts of the urdamycin biosynthetic gene cluster. Three new ORFs (urdK, urdJ and urdO) were identified on a 3.35 kb fragment, and seven new ORFs (urdL, urdM, urdJ2, urdZl, urdGT2, urdG and urdH) on an 8.05 kb fragment. The deduced products of these genes show similarities to transporters (urdJ and urdJ2), regulatory genes (urdK), reductases (urdO), cyclases (urdL) and deoxysugar biosynthetic genes (urdG, urdH and urdZ1). The product of urdM shows striking sequence similarity to oxygenases (N-terminal sequence) as well as reductases (C-terminal sequence), and the deduced amino acid sequence of urdGT2 resembles those of glycosyltransferases. To determine the function of urdM and urdGT2, targeted gene inactivation experiments were performed. The resulting urdM deletion mutant strains accumulated predominantly rabelomycin, indicating that UrdM is involved in oxygenation at position 12b of urdamycin A. A mutant in which urdGT2 had been deleted produced urdamycin I, urdamycin J and urdamycin K instead of urdamycin A. Urdamycins I, J and K are tetracyclic angucyclinones lacking a C-C connected deoxysugar moiety. Therefore UrdGT2 must catalyse the earliest glycosyltransfer step in the urdamycin biosynthetic pathway, the C-glycosyltransfer of one NDP-D-olivose. (+info)
Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-mhz (1)h and (13)c NMR analysis.
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We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen, Campylobacter jejuni OH4384, which has been associated with Guillain-Barre syndrome. We first cloned a gene encoding an alpha-2, 3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a beta-1, 4-N-acetylgalactosaminyl-transferase (cgtA), a beta-1, 3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked alpha-2,3- to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of (3)J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two related C. jejuni strains that express shorter ganglioside mimics in their LOS. (+info)
The accumulation of Man(6)GlcNAc(2)-PP-dolichol in the Saccharomyces cerevisiae Deltaalg9 mutant reveals a regulatory role for the Alg3p alpha1,3-Man middle-arm addition in downstream oligosaccharide-lipid and glycoprotein glycan processing.
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N-Glycans in nearly all eukaryotes are derived by transfer of a precursor Glc(3)Man(9)GlcNAc(2) from dolichol (Dol) to consensus Asn residues in nascent proteins in the endoplasmic reticulum. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide-lipid properly, and the alg9 mutant, accumulates Man(6)GlcNAc(2)-PP-Dol. High-field (1)H NMR and methylation analyses of Man(6)GlcNAc(2) released with peptide-N-glycosidase F from invertase secreted by Deltaalg9 yeast showed its structure to be Manalpha1,2Manalpha1,2Manalpha1, 3(Manalpha1,3Manalpha1,6)-Manbeta1,4GlcNAcbeta1, 4GlcNAcalpha/beta, confirming the addition of the alpha1,3-linked Man to Man(5)GlcNAc(2)-PP-Dol prior to the addition of the final upper-arm alpha1,6-linked Man. This Man(6)GlcNAc(2) is the endoglycosidase H-sensitive product of the Alg3p step. The Deltaalg9 Hex(7-10)GlcNAc(2) elongation intermediates were released from invertase and similarly analyzed. When compared with alg3 sec18 and wild-type core mannans, Deltaalg9 N-glycans reveal a regulatory role for the Alg3p-dependent alpha1,3-linked Man in subsequent oligosaccharide-lipid and glycoprotein glycan maturation. The presence of this Man appears to provide structural information potentiating the downstream action of the endoplasmic reticulum glucosyltransferases Alg6p, Alg8p and Alg10p, glucosidases Gls1p and Gls2p, and the Golgi Och1p outerchain alpha1,6-Man branch-initiating mannosyltransferase. (+info)