| 1. |
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| 2. |
- Kitagawa, H, et al.
(författare)
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N-acetylgalactosamine (GalNAc) transfer to the common carbohydrate-protein linkage region of sulfated glycosaminoglycans : identification of UDP-GaINAc:chondro oligosaccharide aNacetylgalactosaminyltransferase in fetal bovine serum
- 1995
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Ingår i: The Journal of Biological Chemsitry. - 0021-9258. ; 270:38, s. 22190-22195
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Tidskriftsartikel (refereegranskat)abstract
- During the course of a study to elucidate the role ofmodification of the common polysaccharide-protein linkagestructure, GlcAb1–3Galb1–3Galb1–4Xylb1-O-Ser, inbiosynthetic sorting mechanisms of the different sulfatedglycosaminoglycan chains, a novel N-acetylgalactosamine(GalNAc) transferase was discovered in fetalbovine serum. The enzyme catalyzed the transfer of[3H]GalNAc from UDP-[3H]GalNAc to linkage tetrasaccharideand hexasaccharide serines synthesized chemicallyand to various regular oligosaccharides containingterminal D-glucuronic acid (GlcA), which were preparedfrom chondroitin and chondroitin sulfate using testicularhyaluronidase digestion. The labeled products obtainedwith the linkage tetra- and hexasaccharideserines and with the tetrasaccharide (GlcAb1–3GalNAc)2were resistant to digestion with chondroitinase AC-IIand b-N-acetylhexosaminidase but sensitive to a-Nacetylgalactosaminidasedigestion, indicating that theenzyme is an a-N-acetylgalactosaminyltransferase. Thisfinding is in contrast to that of Rohrmann et al. (Rohrmann,K., Niemann, R., and Buddecke, E. (1985) Eur. J.Biochem., 148, 463–469), who reported that a correspondingproduct was susceptible to digestion with b-Nacetylhexosaminidase.The presence of a sulfate groupat C4 of the penultimate GalNAc or Gal units markedlyinhibited the transfer of GalNAc to the terminal GlcA,while a sulfate group at C6 of the GalNAc had little effecton the transfer. Moreover, a slight but significant transferof [3H]GalNAc was observed to an oligosaccharidecontaining terminal 2-O-sulfated GlcA as acceptor,whereas no incorporation was detected into oligosaccharidescontaining terminal unsaturated or 3-O-sulfatedGlcA units. These results suggest that this novelserum enzyme is a UDP-GalNAc:chondro-oligosaccharidea1–3- or 1–4-N-acetylgalactosaminyltransferase.
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| 3. |
- Senay, Clair, et al.
(författare)
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The EXT1/EXT2 tumor suppressors : catalytic activities and role in heparan sulfate biosynthesis
- 2000
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Ingår i: EMBO Reports. - : EMBO. - 1469-221X .- 1469-3178. ; 1:3, s. 282-286
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Tidskriftsartikel (refereegranskat)abstract
- The D-glucuronyltransferase and N-acetyl-D-glucosaminyltransferase reactions in heparan sulfate biosynthesis have been associated with two genes, EXT1 and EXT2, which are also implicated in the inherited bone disorder, multiple exostoses. Since the cell systems used to express recombinant EXT proteins synthesize endogenous heparan sulfate, and the EXT proteins tend to associate, it has not been possible to define the functional roles of the individual protein species. We therefore expressed EXT1 and EXT2 in yeast, which does not synthesize heparan sulfate. The recombinant EXT1 and EXT2 were both found to catalyze both glycosyltransferase reactions in vitro. Coexpression of the two proteins, but not mixing of separately expressed recombinant EXT1 and EXT2, yields hetero-oligomeric complexes in yeast and mammalian cells, with augmented glycosyltransferase activities. This stimulation does not depend on the membrane-bound state of the proteins.
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| 4. |
- Griffiths, G, et al.
(författare)
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Characterization of the glycosyltransferase enzyme from the Escherichia coli K5 capsule gene cluster and identification and characterization of the glucuronyl active site.
- 1998
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 273:19, s. 11752-7
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial capsular polysaccharides play an important role in virulence and survival. The Escherichia coli K5 capsule consists of a repeat structure of -4)GlcA-beta(1,4)-GlcNAc alpha(1-, identical to N-acetylheparosan. A 60-kDa protein, KfiC, has been identified as a bifunctional glycosyltransferase, responsible for the alternating alpha and beta addition of each UDP-sugar to the nonreducing end of the polysaccharide chain. Using hydrophobic cluster analysis, a conserved secondary structure motif characteristic of beta-glycosyltransferases was identified along with two highly conserved aspartic acid residues at positions 301 and 352 within the KfiC protein. Site-directed mutagenesis was used to identify catalytically active amino acids within domain A of the KfiC protein. The conserved aspartic acid residues at 301 and 352 were shown to be critical for the beta addition of UDP-GlcA (uridine diphosphoglucuronic acid) to defined nonreducing end oligosaccharide acceptors, suggesting that these conserved aspartic acid residues are catalytically important for beta-glycosyltransferase activity. A deleted derivative of the kfiC gene was generated, which encoded for a truncated KfiC (kfiC') protein. This protein lacked 139 amino acids at the C terminus. This enzyme had no UDP-GlcA transferase activity but still retained UDP-GlcNAc transferase activity, indicating that two separate active sites are present within the KfiC protein.
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| 5. |
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| 6. |
- Lidholt, Kerstin, et al.
(författare)
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Assessment of glycosaminoglycan-protein linkage tetrasaccharides as acceptors for GalNAc- and GlcNAc-transferases from mouse mastocytoma.
- 1997
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Ingår i: Glycoconjugate Journal. - 0282-0080 .- 1573-4986. ; 14:6, s. 737-742
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Tidskriftsartikel (refereegranskat)abstract
- Two glycosaminoglycan-protein linkage tetrasaccharide-serine compounds, GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and GlcA beta 1-3Gal(4-O-sulfate)beta 1-3Gal beta 1-4Xyl beta 1-O-Ser, were tested as hexosamine accepters, using UDP-[H-3]GlcNAc and UDP-[H-3]GalNAc as sugar donors, and solubilized mouse mastocytoma microsomes as enzyme source. The nonsulfated Ser-tetrasaccharide was found to function as an acceptor for a GalNAc residue, whereas the Ser-tetrasaccharide containing a sulfated galactose unit was inactive. Characterization of the radio-labelled product by digestion with alpha-N-acetylgalactosaminidase and beta-N-acetylhexosaminidase revealed that the [H-3]GalNAc unit was alpha-linked, as in the product previously synthesized using serum enzymes, and not beta-linked as found in the chondroitin sulfate polymer. Heparan sulfate/heparin biosynthesis could not be primed by either of the two linkage Ser-tetrasaccharides, since no transfer of [H-3]GlcNAc from UDP-[H-3]GlcNAc could be detected. By contrast, transfer of a [H-3]GlcNAc unit to a [GlcA beta 1-4GlcNAca1-4](2)-GlcA beta 1-4-aMan hexasaccharide acceptor used to assay the GlcNAc transferase involved in chain elongation, was readily detected. These results are in agreement with the recent proposal that two different N-acetylglucosaminyl transferases catalyse the biosynthesis of heparan sulfate. Although the mastocytoma system contains both the heparan sulfate/heparin and chondroitin sulfate biosynthetic enzymes the Ser-tetrasaccharides do not seem to fulfil the requirements to serve as accepters for the first HexNAc transfer reactions involved in the formation of these polysaccharides.
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| 7. |
- Lidholt, Kerstin, et al.
(författare)
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Biosynthesis of heparin : Modulation of polysaccharide chain length in a cell-free system
- 1988
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 254:2, s. 571-578
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Tidskriftsartikel (refereegranskat)abstract
- The formation of heparin-precursor polysaccharide (N-acetylheparosan) was studied with a mouse mastocytoma microsomal fraction. Incubation of this fraction with UDP-[3H]GlcA and UDP-GlcNAc yielded labelled macromolecules that could be depolymerized, apparently to single polysaccharide chains, by alkali treatment, and thus were assumed to be proteoglycans. Label from UDP-[3H]GlcA (approx. 3 microM) is transiently incorporated into microsomal polysaccharide even in the absence of added UDP-GlcNAc, probably owing to the presence of endogenous sugar nucleotide. When the concentration of exogenous UDP-GlcNAc was increased to 25 microM the rate of incorporation of 3H increased and proteoglycans carrying polysaccharide chains with an Mr of approx. 110,000 were produced. Increasing the UDP-GlcNAc concentration to 5 mM led to an approx. 4-fold decrease in the rate of 3H incorporation and a decrease in the Mr of the resulting polysaccharide chains to approx. 6000 (predominant component). When both UDP-GlcA and UDP-GlcNAc were present at high concentrations (5 mM) the rate of polymerization and the polysaccharide chain size were again increased. The results suggest that the inhibition of polymerization observed at grossly different concentrations of the two sugar nucleotides, UDP-GlcA and UDP-GlcNAc, may be due either to interference with the transport of one of these precursors across the Golgi membrane or to competitive inhibition of one of the glycosyltransferases. The maximal rate of chain elongation obtained, under the conditions employed, was about 40 disaccharide units/min. The final length of the polysaccharide chains was directly related to the rate of the polymerization reaction.
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| 8. |
- Lind, T, et al.
(författare)
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Biosynthesis of heparin/heparan sulfate. Identification of a 70-kDa protein catalyzing both the D-glucuronosyl- and the N-acetyl-D-glucosaminyltransferase reactions.
- 1993
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 268:28, s. 20705-8
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Tidskriftsartikel (refereegranskat)abstract
- The D-glucuronosyl- (GlcA) and N-acetyl-D-glucosaminyl- (GlcNAc) transferase reactions involved in heparin/heparan sulfate biosynthesis were assayed, measuring transfer of radiolabeled GlcA or GlcNAc monosaccharide units from the corresponding UDP-sugars to the appropriate oligosaccharide acceptors. The assays were applied to enzyme purification from bovine serum. The two activities remained inseparable through a series of different chromatographic steps, resulting in approximately -2000-fold purification. Further purification was achieved by chromatofocusing, which showed an isoelectric point of pH approximately -7.0, similar for both activities. SDS-polyacrylamide gel electrophoresis (PAGE) of subfractions from the chromatofocusing procedure revealed an approximately 70-kDa protein in amounts reflecting enzyme activity. SDS-PAGE followed by extraction of gel segments and renaturation of proteins showed that the GlcA- and GlcNAc-transferase activities were both recovered from the same single segment, corresponding to the 70-kDa component. It is proposed that the two glycosyltransferase reactions are catalyzed by the same Golgi enzyme (see also Lidholt, K., Weinke, J. L., Kiser, C. S., Lugemwa, F. N., Bame, K. J., Cheifetz, S., Massagué, J., Lindahl, U., and Esko, J. D. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2267-2271).
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| 9. |
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| 10. |
- Tsuchida, Kazunori, et al.
(författare)
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Purification and characterization of fetal bovine serum beta-N-acetyl-D-galactosaminyltransferase and beta-D-glucuronyltransferase involved in chondroitin sulfate biosynthesis
- 1999
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 264:2, s. 461-467
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Tidskriftsartikel (refereegranskat)abstract
- beta-N-Acetylgalactosaminyltransferase II and beta-glucuronyltransferase II, involved in chondroitin sulfate biosynthesis, transfer an N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) residue, respectively, through beta-linkages to an acceptor chondroitin oligosaccharide derived from the repeating disaccharide region of chondroitin sulfate. They were copurified from fetal bovine serum approximately 2500-fold and 850-fold, respectively, by sequential chromatographies on Red A-agarose, phenyl-Sepharose, S-Sepharose and wheat germ agglutinin-agarose. Identical and inseparable chromatographic profiles of both glycosyltransferase activities obtained through the above chromatographic steps and gel filtration suggest that the purified enzyme activities are tightly coupled, which could imply a single enzyme with dual transferase activities; beta-N-acetylgalactosaminyltransferase and beta-glucuronyltransferase, reminiscent of the heparan sulfate polymerase reaction. However, when a polymerization reaction was performed in vitro with the purified serum enzyme preparation under the polymerization conditions recently developed for the chondroitin-synthesizing system, derived from human melanoma cells, each monosaccharide transfer took place, but no polymerization occurred. These results may suggest that the purified serum enzyme preparation contains both beta-N-acetylgalactosaminyltransferase II and beta-glucuronyltransferase II activities on a single polypeptide or on the respective polypeptides forming an enzyme complex, but is different from that obtained from melanoma cells in that it transfers a single GalNAc or GlcA residue but does not polymerize chondroitin.
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