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Kazuyuki Sugahara - One of the best experts on this subject based on the ideXlab platform.
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mutations in biosynthetic enzymes for the protein linker region of chondroitin dermatan heparan sulfate cause skeletal and skin dysplasias
BioMed Research International, 2015Co-Authors: Shuji Mizumoto, Shuhei Yamada, Kazuyuki SugaharaAbstract:Glycosaminoglycans, including chondroitin, dermatan, and heparan sulfate, have various roles in a wide range of biological events such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Their polysaccharides covalently attach to the serine residues on specific core proteins through the common linker region Tetrasaccharide, -xylose-galactose-galactose-glucuronic acid, which is produced through the stepwise addition of respective monosaccharides by four distinct glycosyltransferases. Mutations in the human genes encoding the glycosyltransferases responsible for the biosynthesis of the linker region Tetrasaccharide cause a number of genetic disorders, called glycosaminoglycan linkeropathies, including Desbuquois dysplasia type 2, spondyloepimetaphyseal dysplasia, Ehlers-Danlos syndrome, and Larsen syndrome. This review focused on recent studies on genetic diseases caused by defects in the biosynthesis of the common linker region Tetrasaccharide.
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structural determination of five novel Tetrasaccharides containing 3 o sulfated d glucuronic acid and two rare oligosaccharides containing a β d glucose branch isolated from squid cartilage chondroitin sulfate e
Biochemistry, 2004Co-Authors: Akiko Kinoshitatoyoda, Shuhei Yamada, Stuart M Haslam, Kayhooi Khoo, Makiko Sugiura, Howard R Morris, A Dell, Kazuyuki SugaharaAbstract:Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAβ1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel Tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing d-Glc. Their structures were determined by fast atom bombardment mass spectrometry and 1H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel Tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but...
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structural determination of five novel Tetrasaccharides containing 3 o sulfated d glucuronic acid and two rare oligosaccharides containing a beta d glucose branch isolated from squid cartilage chondroitin sulfate e
Biochemistry, 2004Co-Authors: Akiko Kinoshitatoyoda, Shuhei Yamada, Stuart M Haslam, Kayhooi Khoo, Makiko Sugiura, Howard R Morris, And Anne Dell, Kazuyuki SugaharaAbstract:Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAbeta1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel Tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing D-Glc. Their structures were determined by fast atom bombardment mass spectrometry and (1)H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel Tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but rather interspersed in the CS-E polysaccahride chains, being preferentially located in the highly sulfated sequences. The predominant structure on the nearest nonreducing side of a GlcA(3-O-sulfate) residue was GalNAc(4-O-sulfate) (80%), whereas that on the reducing side was GalNAc(4,6-O-disulfate) (59%). The structural variety in the vicinity of the GlcA(3-O-sulfate) residue might represent the substrate specificity of the unidentified chondroitin GlcA 3-O-sulfotransferase. The results also revealed a trisaccharide and a pentasaccahride sequence, both of which contained a beta-d-Glc branch at the C6 position of the constituent GalNAc residue. Approximately 5 mol % of all disaccharide units were substituted by Glc in the CS-E preparation used.
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novel Tetrasaccharides isolated from squid cartilage chondroitin sulfate e contain unusual sulfated disaccharide units glca 3 o sulfate beta1 3galnac 6 o sulfate or glca 3 o sulfate beta1 3galnac
Journal of Biological Chemistry, 1997Co-Authors: Akiko Kinoshita, Shuhei Yamada, Stuart M Haslam, Howard R Morris, A Dell, Kazuyuki SugaharaAbstract:Abstract We previously isolated novel Tetrasaccharides containing 3-O-sulfated glucuronic acid from king crab cartilage chondroitin sulfate K and demonstrated that the disaccharide units containing 3-O-sulfated glucuronic acid were decomposed by chondroitinase ABC digestion (Sugahara, K., Tanaka, Y., Yamada, S., Seno, N., Kitagawa, H., Haslam, S. M., Morris, H. R., and Dell, A. (1996) J. Biol. Chem. 271, 26745–26754). The findings indicated the necessity to re-evaluate the disaccharide compositions of chondroitin sulfate preparations purified from other biological sources and analyzed using the above enzyme. In this study, to evaluate squid cartilage chondroitin sulfate E a series of even-numbered oligosaccharides were isolated after exhaustive digestion with sheep testicular hyaluronidase and subsequent fractionation by gel chromatography. The Tetrasaccharide fraction was subfractionated by high performance liquid chromatography on an amine-bound silica column. Systematic structural analysis of five major fractions, h,l, m, n, and q, by fast atom bombardment mass spectrometry, enzymatic digestions in conjunction with capillary electrophoresis, and 500-MHz 1H NMR spectroscopy revealed one disulfated, three trisulfated, and one tetrasulfated Tetrasaccharide structure: fraction h, GlcAβ1–3GalNAc(4S)β1–4GlcAβ1–3GalNAc(4S); fractionl, GlcA(3S)β1–3GalNAc(6S)β1–4GlcAβ1–3GalNAc(4S); fractionm, GlcA(3S)β1–3GalNAc(4S)β1–4GlcAβ1–3GalNAc(4S); fractionn, GlcAβ1–3GalNAc(4S,6S)β1–4GlcAβ1–3GalNAc(4S); and fraction q, GlcA(3S)β1–3GalNAc(4S,6S)β1–4GlcAβ1–3GalNAc(4S), where 3S, 4S, and 6S represent 3-O-, 4-O- and 6-O-sulfate, respectively. The structures found in fractions h and m as well as the unsaturated counterpart of that found in fraction n have been reported, whereas those in fractions l and q are novel in that they contained unusual disulfated and trisulfated disaccharide units where GlcA(3S) is directly linked to GalNAc(6S) and GalNAc(4S,6S), respectively. These novel Tetrasaccharide sequences are distinct from those found in other chondroitin sulfate isoforms and may play key roles in the biological functions and activities of chondroitin sulfate E not only from squid cartilage but also from mammalian cells and tissues.
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preparation of a series of sulfated Tetrasaccharides from shark cartilage chondroitin sulfate d using testicular hyaluronidase and structure determination by 500 mhz1h nmr spectroscopy
Glycoconjugate Journal, 1996Co-Authors: Kazuyuki Sugahara, Yukako Tanaka, Shuhei YamadaAbstract:Six Tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz1H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the Tetrasaccharides. One fraction was found to contain two Tetrasaccharide components. All the seven Tetrasaccharides shared the common core structure GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcAβ1-3GalNAc(4-sulfate) and/or GlcAβ1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated Tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)β1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcAβ1-3GalNAc(4-or 6-sulfate). These sulfated Tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum α-N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.
Shuhei Yamada - One of the best experts on this subject based on the ideXlab platform.
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mutations in biosynthetic enzymes for the protein linker region of chondroitin dermatan heparan sulfate cause skeletal and skin dysplasias
BioMed Research International, 2015Co-Authors: Shuji Mizumoto, Shuhei Yamada, Kazuyuki SugaharaAbstract:Glycosaminoglycans, including chondroitin, dermatan, and heparan sulfate, have various roles in a wide range of biological events such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Their polysaccharides covalently attach to the serine residues on specific core proteins through the common linker region Tetrasaccharide, -xylose-galactose-galactose-glucuronic acid, which is produced through the stepwise addition of respective monosaccharides by four distinct glycosyltransferases. Mutations in the human genes encoding the glycosyltransferases responsible for the biosynthesis of the linker region Tetrasaccharide cause a number of genetic disorders, called glycosaminoglycan linkeropathies, including Desbuquois dysplasia type 2, spondyloepimetaphyseal dysplasia, Ehlers-Danlos syndrome, and Larsen syndrome. This review focused on recent studies on genetic diseases caused by defects in the biosynthesis of the common linker region Tetrasaccharide.
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structural determination of five novel Tetrasaccharides containing 3 o sulfated d glucuronic acid and two rare oligosaccharides containing a β d glucose branch isolated from squid cartilage chondroitin sulfate e
Biochemistry, 2004Co-Authors: Akiko Kinoshitatoyoda, Shuhei Yamada, Stuart M Haslam, Kayhooi Khoo, Makiko Sugiura, Howard R Morris, A Dell, Kazuyuki SugaharaAbstract:Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAβ1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel Tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing d-Glc. Their structures were determined by fast atom bombardment mass spectrometry and 1H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel Tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but...
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structural determination of five novel Tetrasaccharides containing 3 o sulfated d glucuronic acid and two rare oligosaccharides containing a beta d glucose branch isolated from squid cartilage chondroitin sulfate e
Biochemistry, 2004Co-Authors: Akiko Kinoshitatoyoda, Shuhei Yamada, Stuart M Haslam, Kayhooi Khoo, Makiko Sugiura, Howard R Morris, And Anne Dell, Kazuyuki SugaharaAbstract:Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAbeta1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel Tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing D-Glc. Their structures were determined by fast atom bombardment mass spectrometry and (1)H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel Tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but rather interspersed in the CS-E polysaccahride chains, being preferentially located in the highly sulfated sequences. The predominant structure on the nearest nonreducing side of a GlcA(3-O-sulfate) residue was GalNAc(4-O-sulfate) (80%), whereas that on the reducing side was GalNAc(4,6-O-disulfate) (59%). The structural variety in the vicinity of the GlcA(3-O-sulfate) residue might represent the substrate specificity of the unidentified chondroitin GlcA 3-O-sulfotransferase. The results also revealed a trisaccharide and a pentasaccahride sequence, both of which contained a beta-d-Glc branch at the C6 position of the constituent GalNAc residue. Approximately 5 mol % of all disaccharide units were substituted by Glc in the CS-E preparation used.
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novel Tetrasaccharides isolated from squid cartilage chondroitin sulfate e contain unusual sulfated disaccharide units glca 3 o sulfate beta1 3galnac 6 o sulfate or glca 3 o sulfate beta1 3galnac
Journal of Biological Chemistry, 1997Co-Authors: Akiko Kinoshita, Shuhei Yamada, Stuart M Haslam, Howard R Morris, A Dell, Kazuyuki SugaharaAbstract:Abstract We previously isolated novel Tetrasaccharides containing 3-O-sulfated glucuronic acid from king crab cartilage chondroitin sulfate K and demonstrated that the disaccharide units containing 3-O-sulfated glucuronic acid were decomposed by chondroitinase ABC digestion (Sugahara, K., Tanaka, Y., Yamada, S., Seno, N., Kitagawa, H., Haslam, S. M., Morris, H. R., and Dell, A. (1996) J. Biol. Chem. 271, 26745–26754). The findings indicated the necessity to re-evaluate the disaccharide compositions of chondroitin sulfate preparations purified from other biological sources and analyzed using the above enzyme. In this study, to evaluate squid cartilage chondroitin sulfate E a series of even-numbered oligosaccharides were isolated after exhaustive digestion with sheep testicular hyaluronidase and subsequent fractionation by gel chromatography. The Tetrasaccharide fraction was subfractionated by high performance liquid chromatography on an amine-bound silica column. Systematic structural analysis of five major fractions, h,l, m, n, and q, by fast atom bombardment mass spectrometry, enzymatic digestions in conjunction with capillary electrophoresis, and 500-MHz 1H NMR spectroscopy revealed one disulfated, three trisulfated, and one tetrasulfated Tetrasaccharide structure: fraction h, GlcAβ1–3GalNAc(4S)β1–4GlcAβ1–3GalNAc(4S); fractionl, GlcA(3S)β1–3GalNAc(6S)β1–4GlcAβ1–3GalNAc(4S); fractionm, GlcA(3S)β1–3GalNAc(4S)β1–4GlcAβ1–3GalNAc(4S); fractionn, GlcAβ1–3GalNAc(4S,6S)β1–4GlcAβ1–3GalNAc(4S); and fraction q, GlcA(3S)β1–3GalNAc(4S,6S)β1–4GlcAβ1–3GalNAc(4S), where 3S, 4S, and 6S represent 3-O-, 4-O- and 6-O-sulfate, respectively. The structures found in fractions h and m as well as the unsaturated counterpart of that found in fraction n have been reported, whereas those in fractions l and q are novel in that they contained unusual disulfated and trisulfated disaccharide units where GlcA(3S) is directly linked to GalNAc(6S) and GalNAc(4S,6S), respectively. These novel Tetrasaccharide sequences are distinct from those found in other chondroitin sulfate isoforms and may play key roles in the biological functions and activities of chondroitin sulfate E not only from squid cartilage but also from mammalian cells and tissues.
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preparation of a series of sulfated Tetrasaccharides from shark cartilage chondroitin sulfate d using testicular hyaluronidase and structure determination by 500 mhz1h nmr spectroscopy
Glycoconjugate Journal, 1996Co-Authors: Kazuyuki Sugahara, Yukako Tanaka, Shuhei YamadaAbstract:Six Tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz1H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the Tetrasaccharides. One fraction was found to contain two Tetrasaccharide components. All the seven Tetrasaccharides shared the common core structure GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcAβ1-3GalNAc(4-sulfate) and/or GlcAβ1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated Tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)β1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcAβ1-3GalNAc(4-or 6-sulfate). These sulfated Tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum α-N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.
Howard R Morris - One of the best experts on this subject based on the ideXlab platform.
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structural determination of five novel Tetrasaccharides containing 3 o sulfated d glucuronic acid and two rare oligosaccharides containing a β d glucose branch isolated from squid cartilage chondroitin sulfate e
Biochemistry, 2004Co-Authors: Akiko Kinoshitatoyoda, Shuhei Yamada, Stuart M Haslam, Kayhooi Khoo, Makiko Sugiura, Howard R Morris, A Dell, Kazuyuki SugaharaAbstract:Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAβ1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel Tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing d-Glc. Their structures were determined by fast atom bombardment mass spectrometry and 1H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel Tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but...
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structural determination of five novel Tetrasaccharides containing 3 o sulfated d glucuronic acid and two rare oligosaccharides containing a beta d glucose branch isolated from squid cartilage chondroitin sulfate e
Biochemistry, 2004Co-Authors: Akiko Kinoshitatoyoda, Shuhei Yamada, Stuart M Haslam, Kayhooi Khoo, Makiko Sugiura, Howard R Morris, And Anne Dell, Kazuyuki SugaharaAbstract:Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAbeta1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel Tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing D-Glc. Their structures were determined by fast atom bombardment mass spectrometry and (1)H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel Tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but rather interspersed in the CS-E polysaccahride chains, being preferentially located in the highly sulfated sequences. The predominant structure on the nearest nonreducing side of a GlcA(3-O-sulfate) residue was GalNAc(4-O-sulfate) (80%), whereas that on the reducing side was GalNAc(4,6-O-disulfate) (59%). The structural variety in the vicinity of the GlcA(3-O-sulfate) residue might represent the substrate specificity of the unidentified chondroitin GlcA 3-O-sulfotransferase. The results also revealed a trisaccharide and a pentasaccahride sequence, both of which contained a beta-d-Glc branch at the C6 position of the constituent GalNAc residue. Approximately 5 mol % of all disaccharide units were substituted by Glc in the CS-E preparation used.
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novel Tetrasaccharides isolated from squid cartilage chondroitin sulfate e contain unusual sulfated disaccharide units glca 3 o sulfate beta1 3galnac 6 o sulfate or glca 3 o sulfate beta1 3galnac
Journal of Biological Chemistry, 1997Co-Authors: Akiko Kinoshita, Shuhei Yamada, Stuart M Haslam, Howard R Morris, A Dell, Kazuyuki SugaharaAbstract:Abstract We previously isolated novel Tetrasaccharides containing 3-O-sulfated glucuronic acid from king crab cartilage chondroitin sulfate K and demonstrated that the disaccharide units containing 3-O-sulfated glucuronic acid were decomposed by chondroitinase ABC digestion (Sugahara, K., Tanaka, Y., Yamada, S., Seno, N., Kitagawa, H., Haslam, S. M., Morris, H. R., and Dell, A. (1996) J. Biol. Chem. 271, 26745–26754). The findings indicated the necessity to re-evaluate the disaccharide compositions of chondroitin sulfate preparations purified from other biological sources and analyzed using the above enzyme. In this study, to evaluate squid cartilage chondroitin sulfate E a series of even-numbered oligosaccharides were isolated after exhaustive digestion with sheep testicular hyaluronidase and subsequent fractionation by gel chromatography. The Tetrasaccharide fraction was subfractionated by high performance liquid chromatography on an amine-bound silica column. Systematic structural analysis of five major fractions, h,l, m, n, and q, by fast atom bombardment mass spectrometry, enzymatic digestions in conjunction with capillary electrophoresis, and 500-MHz 1H NMR spectroscopy revealed one disulfated, three trisulfated, and one tetrasulfated Tetrasaccharide structure: fraction h, GlcAβ1–3GalNAc(4S)β1–4GlcAβ1–3GalNAc(4S); fractionl, GlcA(3S)β1–3GalNAc(6S)β1–4GlcAβ1–3GalNAc(4S); fractionm, GlcA(3S)β1–3GalNAc(4S)β1–4GlcAβ1–3GalNAc(4S); fractionn, GlcAβ1–3GalNAc(4S,6S)β1–4GlcAβ1–3GalNAc(4S); and fraction q, GlcA(3S)β1–3GalNAc(4S,6S)β1–4GlcAβ1–3GalNAc(4S), where 3S, 4S, and 6S represent 3-O-, 4-O- and 6-O-sulfate, respectively. The structures found in fractions h and m as well as the unsaturated counterpart of that found in fraction n have been reported, whereas those in fractions l and q are novel in that they contained unusual disulfated and trisulfated disaccharide units where GlcA(3S) is directly linked to GalNAc(6S) and GalNAc(4S,6S), respectively. These novel Tetrasaccharide sequences are distinct from those found in other chondroitin sulfate isoforms and may play key roles in the biological functions and activities of chondroitin sulfate E not only from squid cartilage but also from mammalian cells and tissues.
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structural studies on the bacterial lyase resistant Tetrasaccharides derived from the antithrombin iii binding site of porcine intestinal heparin
Journal of Biological Chemistry, 1993Co-Authors: Shuhei Yamada, Kazuyuki Sugahara, Keiichi Yoshida, Kayhooi Khoo, Makiko Sugiura, Howard R Morris, A DellAbstract:Abstract Three discrete Tetrasaccharide structures which are resistant to Flavobacterium heparinase and heparitinases I and II were isolated from porcine intestinal heparin after exhaustive digestion with a mixture of all the above enzymes, and the tri-, tetra-, and penta-sulfated structures were determined by negative ion mode fast atom bombardment mass spectrometry and 500-MHz 1H NMR analysis as delta 4,5GlcA beta 1-4GlcNAc (6-sulfate)alpha 1-4GlcA beta 1-4GlcN(N,3-disulfate), delta 4,5 GlcA beta 1-4GlcNAc(6-sulfate)alpha 1-4GlcA beta 1-4GlcN (N,3,6-trisulfate), and delta 4,5GlcA beta 1-4GlcN (N,6-disulfate)alpha 1-4GlcA beta 1-4GlcN(N,3,6-trisulfate). The three components share the 3-O-sulfated reducing GlcN and the 6-O-sulfated internal GlcN, indicating that they are structural variants derived from the nonreducing portion of the minimal pentasaccharide sequence required for binding to antithrombin III. Isolation of the pentasulfated component has never been reported. Their unexpected resistance to heparitinases I and II indicates that 3-O-sulfation of the reducing GlcN contributes to the resistant nature of these Tetrasaccharides to the enzymes. The present study demonstrates that the nonreducing trisaccharide portion of the structural variants of the antithrombin III-binding pentasaccharide sequence can be isolated in Tetrasaccharides resistant to heparinase/heparitinases I and II, while the rest of the repeating region is degraded into disaccharide units. The lyase treatment is applicable to evaluation of heparin/heparan sulfate preparations in terms of the presence or absence of the specific structure containing the 3-O-sulfated GlcN representing biosynthetic precursors, intermediates or final products of the binding site.
Malcolm Lyon - One of the best experts on this subject based on the ideXlab platform.
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the binding properties of minimal oligosaccharides reveal a common heparan sulfate dermatan sulfate binding site in hepatocyte growth factor scatter factor that can accommodate a wide variety of sulfation patterns
Journal of Biological Chemistry, 2009Co-Authors: Jon A Deakin, John T Gallagher, Barbel S Blaum, Dusan Uhrin, Malcolm LyonAbstract:Abstract Heparan sulfate (HS)/heparin and dermatan sulfate (DS) both bind with high affinity to hepatocyte growth factor/scatter factor (HGF/SF) and function as necessary co-factors in vitro. How both these two structurally distinct glycosaminoglycans (GAGs) are recognized has remained unclear. We have now reconciled this issue using a panel of minimal tri- and Tetrasaccharide sequences of variable but well defined sulfation patterns in combination with further development of the gel mobility shift assay to allow simultaneous comparisons of relative protein affinities/selectivities for different oligosaccharides. From this approach it would seem that a minimum binding sequence is a disulfated trisaccharide comprised of an internal iduronate flanked by monosulfated hexosamine residues and that additional sulfation further enhances affinity. However, the similarity in recognition of HS/heparin and DS seems to arise primarily from a lack of any apparent positional requirement for sulfation. Thus, isomers of HS/heparin Tetrasaccharides containing only two sulfates irrespective of whether they are purely N-, 2-O-, or 6-O-sulfates bind with equivalent apparent affinity as a disulfated DS Tetrasaccharide. In addition, the NMR chemical shifts induced in NK1 (the truncated variant of HGF/SF comprised of the N-terminal and first Kringle domains) by titration with either heparin or DS oligosaccharides strongly indicate that both bind to essentially the same site. Together, these observations reveal an unexpected degree of flexibility in the GAG-HGF/SF interface, allowing a single binding site in the protein to accommodate iduronate-containing sequences of variable sulfation pattern and/or density from different GAGs.
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the interactions of hepatocyte growth factor scatter factor and its nk1 and nk2 variants with glycosaminoglycans using a modified gel mobility shift assay elucidation of the minimal size of binding and activatory oligosaccharides
Journal of Biological Chemistry, 2004Co-Authors: Malcolm Lyon, Ermanno Gherardi, Jon A Deakin, Daniel Lietha, John T GallagherAbstract:Full-length hepatocyte growth factor/scatter factor interacts with both heparan and dermatan sulfates and is critically dependent upon them as cofactors for activation of the tyrosine kinase receptor Met. Two C-terminally truncated variants (NK1 and NK2) of this growth factor also occur naturally. Their glycosaminoglycan binding properties are not clear. We have undertaken a comparative study of the heparan/dermatan sulfate binding characteristics of all three proteins. This has entailed the development of a modified gel mobility shift assay, utilizing fluorescence end-tagged oligosaccharides, that is also widely applicable to the analysis of many glycosaminoglycan-protein interactions. Using this we have shown that all three hepatocyte growth factor/scatter factor variants share identical heparan/dermatan sulfate binding properties and that both glycosaminoglycans occupy the same binding site. The minimal size of the oligosaccharide that binds with high affinity in all cases is a Tetrasaccharide from heparan sulfate but a hexasaccharide from dermatan sulfate. These findings demonstrate that functional glycosaminoglycan binding is restricted to a binding site situated solely within the small N-terminal domain. The same minimal size fractions are also able to promote hepatocyte growth factor/scatter factor-mediated activation of Met and consequent downstream signaling in the glycosaminoglycan-deficient Chinese hamster ovary pgsA-745 cells. A covalent complex of heparan sulfate Tetrasaccharide with monovalent growth factor is also active. The binding and activity of Tetrasaccharides put constraints upon the possible interactions and molecular geometry within the ternary signaling complex.
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fibroblast growth factor 2 binds to small heparin derived oligosaccharides and stimulates a sustained phosphorylation of p42 44 mitogen activated protein kinase and proliferation of rat mammary fibroblasts
Biochemical Journal, 2002Co-Authors: Maryse Delehedde, Malcolm Lyon, John T Gallagher, Philip S Rudland, David G FernigAbstract:We examine the relationship between the chain length of heparin-derived oligosaccharides, fibroblast growth factor (FGF)-2 binding kinetics and the ability of the oligosaccharides to allow FGF-2-induced proliferation of chlorate-treated rat mammary fibroblasts. First, using an optical biosensor, we show that FGF-2 did not bind disaccharides, but definitively bound to Tetrasaccharides. As the chain length increased from Tetrasaccharide to octasaccharide, there was a substantial increase in k(ass) (564000 M(-1) x s(-1) to 2000000 M(-1) x s(-1), respectively) and affinity (K(d) 77 nM to 11 nM, respectively) for FGF-2. From decasaccharides and longer, the k(ass) and affinity for FGF-2 was reduced substantially (tetradecasaccharide k(ass) 470000 M(-1) x s(-1), K(d) 30 nM). In chlorate-treated, and hence sulphated, glycosaminoglycan-deficient cells, FGF-2 alone or in the presence of disaccharides did not stimulate DNA synthesis and it only elicited an early transient dual phosphorylation of p42/44 mitogen-activated protein kinase (MAPK). In the same cells FGF-2 in the presence of Tetrasaccharides and longer oligosaccharides was able to restore DNA synthesis and enable the sustained dual phosphorylation of p42/44(MAPK). However, the oligosaccharides from Tetrasaccharides to octasaccharides were less potent in proliferation assays than deca- and longer oligosaccharides. Therefore, there was no correlation between the binding parameters and the potency of the oligosaccharides in DNA synthesis assays. These results demonstrate that Tetrasaccharides are able to bind FGF-2 and enable FGF-2 to stimulate cell proliferation, which sets important boundary conditions for models of the FGF-2-heparan sulphate-FGF receptor complex.
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hepatocyte growth factor scatter factor binds to small heparin derived oligosaccharides and stimulates the proliferation of human hacat keratinocytes
Journal of Biological Chemistry, 2002Co-Authors: Maryse Delehedde, Malcolm Lyon, Rishma Vidyasagar, Timothy J Mcdonnell, David G FernigAbstract:Abstract Hepatocyte growth factor/scatter factor (HGF/SF) acts via a dual receptor system consisting of the MET tyrosine kinase receptor and heparan sulfate or dermatan sulfate proteoglycans. In optical biosensor binding assays, competition by oligosaccharides for binding of HGF/SF to immobilized heparin showed that disaccharides failed to compete, whereas Tetrasaccharides inhibited HGF/SF binding (IC50 8 μg/ml). The inhibitory potency of the oligosaccharides increased as their length increased by successive disaccharide units, to reach a maximum (IC50 1 μg/ml) at degree of polymerization (dp) 10. In binding assays, HGF/SF was found to bind directly to oligosaccharides as small as dp 4, and the binding parameters were similar for oligosaccharides of dp 4–14 (k a 2.2–45.3 × 106 m −1 s−1, k d0.033–0.039 s−1, and K d 9–16 nm). In human keratinocytes, HGF/SF stimulated DNA synthesis, and this was dependent on a sustained phosphorylation of p42/44MAPK. In chlorate-treated and hence sulfated glycosaminoglycan-deficient HaCaT cells, the stimulation of DNA synthesis by HGF/SF was almost abolished. Heparin-derived oligosaccharides from dp 2 to dp 24 were added together with HGF/SF to chlorate-treated cells to determine the minimum size of oligosaccharides able to restore HGF/SF activity. At restricted concentrations of oligosaccharides (4 ng/ml), HGF/SF required decasaccharides, whereas at higher concentrations (100 ng/ml) even Tetrasaccharides were able to partly restore DNA synthesis. The results suggest that HGF/SF binds to a Tetrasaccharide and that although this is sufficient to enable the stimulation of DNA synthesis, longer oligosaccharides are more efficient, perhaps by virtue of their ability to bind more easily other molecules.
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structural determination of five novel Tetrasaccharides containing 3 o sulfated d glucuronic acid and two rare oligosaccharides containing a β d glucose branch isolated from squid cartilage chondroitin sulfate e
Biochemistry, 2004Co-Authors: Akiko Kinoshitatoyoda, Shuhei Yamada, Stuart M Haslam, Kayhooi Khoo, Makiko Sugiura, Howard R Morris, A Dell, Kazuyuki SugaharaAbstract:Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAβ1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel Tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing d-Glc. Their structures were determined by fast atom bombardment mass spectrometry and 1H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel Tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but...
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novel Tetrasaccharides isolated from squid cartilage chondroitin sulfate e contain unusual sulfated disaccharide units glca 3 o sulfate beta1 3galnac 6 o sulfate or glca 3 o sulfate beta1 3galnac
Journal of Biological Chemistry, 1997Co-Authors: Akiko Kinoshita, Shuhei Yamada, Stuart M Haslam, Howard R Morris, A Dell, Kazuyuki SugaharaAbstract:Abstract We previously isolated novel Tetrasaccharides containing 3-O-sulfated glucuronic acid from king crab cartilage chondroitin sulfate K and demonstrated that the disaccharide units containing 3-O-sulfated glucuronic acid were decomposed by chondroitinase ABC digestion (Sugahara, K., Tanaka, Y., Yamada, S., Seno, N., Kitagawa, H., Haslam, S. M., Morris, H. R., and Dell, A. (1996) J. Biol. Chem. 271, 26745–26754). The findings indicated the necessity to re-evaluate the disaccharide compositions of chondroitin sulfate preparations purified from other biological sources and analyzed using the above enzyme. In this study, to evaluate squid cartilage chondroitin sulfate E a series of even-numbered oligosaccharides were isolated after exhaustive digestion with sheep testicular hyaluronidase and subsequent fractionation by gel chromatography. The Tetrasaccharide fraction was subfractionated by high performance liquid chromatography on an amine-bound silica column. Systematic structural analysis of five major fractions, h,l, m, n, and q, by fast atom bombardment mass spectrometry, enzymatic digestions in conjunction with capillary electrophoresis, and 500-MHz 1H NMR spectroscopy revealed one disulfated, three trisulfated, and one tetrasulfated Tetrasaccharide structure: fraction h, GlcAβ1–3GalNAc(4S)β1–4GlcAβ1–3GalNAc(4S); fractionl, GlcA(3S)β1–3GalNAc(6S)β1–4GlcAβ1–3GalNAc(4S); fractionm, GlcA(3S)β1–3GalNAc(4S)β1–4GlcAβ1–3GalNAc(4S); fractionn, GlcAβ1–3GalNAc(4S,6S)β1–4GlcAβ1–3GalNAc(4S); and fraction q, GlcA(3S)β1–3GalNAc(4S,6S)β1–4GlcAβ1–3GalNAc(4S), where 3S, 4S, and 6S represent 3-O-, 4-O- and 6-O-sulfate, respectively. The structures found in fractions h and m as well as the unsaturated counterpart of that found in fraction n have been reported, whereas those in fractions l and q are novel in that they contained unusual disulfated and trisulfated disaccharide units where GlcA(3S) is directly linked to GalNAc(6S) and GalNAc(4S,6S), respectively. These novel Tetrasaccharide sequences are distinct from those found in other chondroitin sulfate isoforms and may play key roles in the biological functions and activities of chondroitin sulfate E not only from squid cartilage but also from mammalian cells and tissues.
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structural studies on the bacterial lyase resistant Tetrasaccharides derived from the antithrombin iii binding site of porcine intestinal heparin
Journal of Biological Chemistry, 1993Co-Authors: Shuhei Yamada, Kazuyuki Sugahara, Keiichi Yoshida, Kayhooi Khoo, Makiko Sugiura, Howard R Morris, A DellAbstract:Abstract Three discrete Tetrasaccharide structures which are resistant to Flavobacterium heparinase and heparitinases I and II were isolated from porcine intestinal heparin after exhaustive digestion with a mixture of all the above enzymes, and the tri-, tetra-, and penta-sulfated structures were determined by negative ion mode fast atom bombardment mass spectrometry and 500-MHz 1H NMR analysis as delta 4,5GlcA beta 1-4GlcNAc (6-sulfate)alpha 1-4GlcA beta 1-4GlcN(N,3-disulfate), delta 4,5 GlcA beta 1-4GlcNAc(6-sulfate)alpha 1-4GlcA beta 1-4GlcN (N,3,6-trisulfate), and delta 4,5GlcA beta 1-4GlcN (N,6-disulfate)alpha 1-4GlcA beta 1-4GlcN(N,3,6-trisulfate). The three components share the 3-O-sulfated reducing GlcN and the 6-O-sulfated internal GlcN, indicating that they are structural variants derived from the nonreducing portion of the minimal pentasaccharide sequence required for binding to antithrombin III. Isolation of the pentasulfated component has never been reported. Their unexpected resistance to heparitinases I and II indicates that 3-O-sulfation of the reducing GlcN contributes to the resistant nature of these Tetrasaccharides to the enzymes. The present study demonstrates that the nonreducing trisaccharide portion of the structural variants of the antithrombin III-binding pentasaccharide sequence can be isolated in Tetrasaccharides resistant to heparinase/heparitinases I and II, while the rest of the repeating region is degraded into disaccharide units. The lyase treatment is applicable to evaluation of heparin/heparan sulfate preparations in terms of the presence or absence of the specific structure containing the 3-O-sulfated GlcN representing biosynthetic precursors, intermediates or final products of the binding site.
