Regulation of chondroitin sulfate biosynthesis by specific sulfation: acceptor specificity of serum β-GalNAc transferase revealed by structurally defined oligosaccharides

Kitagawa, Hiroshi; Tsutsumi, Kae; Ujikawa, Miho; Goto, Fumitaka; Tamura, Jun’ichi; Neumann, Karoline T.; Ogawa, Tomoya; Sugahara, Kazuyuki

doi:10.1093/glycob/7.4.531

Cited by 59 publications

(51 citation statements)

References 22 publications

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“…The key step in producing the low molecular weight TM is the first GalNAc transfer reaction to the proteoglycan linkage tetrasaccharide regulated by sulfation at 3C of GlcA, which leads to regulation of the cell-surface anticoagulant potential. This is compatible with the finding that GalNAc transferase catalyzing chondroitin chain elongation cannot transfer GalNAc to 3-O-sulfated GlcA (30). This sulfation may be accomplished either with a similar sulfotransferase, as reported for the detection of SO 4 -3GlcA␤1-4Xyl␤-4-methylumbelliferone from the substrate, 4-methylumbelliferyl ␤-Xyl (31), or with the sulfotransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope (32,33).…”

Section: Discussionsupporting

confidence: 86%

Novel Proteoglycan Linkage Tetrasaccharides of Human Urinary Soluble Thrombomodulin, SO4-3GlcAβ1–3Galβ1–3(±Siaα2–6)Galβ1–4Xyl

Wakabayashi¹,

Natsuka²,

Mega³

et al. 1999

Journal of Biological Chemistry

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O-linked sugar chains with xylose as a reducing end linked to human urinary soluble thrombomodulin were studied. Sugar chains were liberated by hydrazinolysis followed by N-acetylation and tagged with 2-aminopyridine. Two fractions containing pyridylaminated Xyl as a reducing end were collected. Their structures were determined by partial acid hydrolysis, two-dimensional sugar mapping combined with exoglycosidase digestions, methylation analysis, mass spectrometry, and NMR as SO 4 -3GlcA␤1-3Gal␤1-3(؎Sia␣2-6)Gal␤1-4Xyl. These sugar chains could bind to an HNK-1 monoclonal antibody. This is believed to be the first example of a proteoglycan linkage tetrasaccharide with glucuronic acid 3-sulfate and sialic acid.

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Section: Discussionsupporting

confidence: 86%

Novel Proteoglycan Linkage Tetrasaccharides of Human Urinary Soluble Thrombomodulin, SO4-3GlcAβ1–3Galβ1–3(±Siaα2–6)Galβ1–4Xyl

Wakabayashi¹,

Natsuka²,

Mega³

et al. 1999

Journal of Biological Chemistry

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“…*The values represent the averages of two independent experiments. † GlcA␤1-4GlcNAc␣1-(4GlcA␤1-4GlcNAc␣1-)n. ‡ GlcNAc␣1-(4GlcA␤1-4GlcNAc␣1-)n. § A mixture of GlcA␤1-(3GalNAc␤1-4GlcA␤1-)n and GalNAc␤1-(4GlcA␤1-3GalNAc␤1-)n. Under the conditions, the chondroitin-synthesizing enzyme system (11,35,36) as a positive control showed marked activities of CS-GlcAT-II and GalNAcT-II. ¶ GlcA␤1-3GalNAc.…”

Section: Extl3 Protein Harbors Both Glcnact-i and Glcnact-ii Activitimentioning

confidence: 96%

“…For GlcNAc transferase assays, the reaction mixture of a total volume of 20 l contained 10 l of the resuspended beads, 100 mM 2-(N-morpholino)ethanesulfonic acid-NaOH (pH 6.5), 10 mM MgCl 2 , 1 mM ATP, GlcA␤1-3Gal␤1-O-C 2 H 4 NHCbz (250 nmol), or tetrasaccharide-serine (1 nmol) for GlcNAcT-I assay (31) or N-acetylheparosan oligosaccharides with the nonreducing terminal GlcA (20 g) for GlcNAcT-II assay (32) as acceptors, and 250 M UDP-[ 3 H]GlcNAc (about 1.1 ϫ 10 6 dpm). Enzyme assays for GlcAT-I (33), HS-GlcAT-II (32), and ␣-GalNAc transferase (34), ␤-GalNAc transferase for CS chain elongation (35), and GlcA transferase for CS chain elongation (CS-GlcAT-II) (8, 36) were carried out as described previously.…”

Section: Expression Of the Soluble Forms Of Extl1 And Extl3 In Cos-1 mentioning

confidence: 99%

“…Neither GlcA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser nor GlcA␤1-3Gal␤1-O-C 2 H 4 NHCbz, a substrate for GlcNAcT-I (31), was used as an acceptor. Because mammalian chondroitin-synthesizing enzymes have not been cloned, ␤-GalNAc and GlcA transferase activities (35,36) were measured by using chondroitin as an acceptor and UDP-GalNAc or UDP-GlcA as a donor substrate, but no activity was detected. N-Acetylchondrosine, GlcA␤1-3GalNAc, which is an acceptor substrate for the unique ␣-GalNAc transferase activity of EXTL2 (26,34), did not serve as an acceptor either.…”

Section: Extl1 Is a Likely ␣14-glcnac Transferase For The Synthesis mentioning

confidence: 99%

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Human tumor suppressor EXT gene family members EXTL1 and EXTL3 encode α1,4- N -acetylglucosaminyltransferases that likely are involved in heparan sulfate/ heparin biosynthesis

Kim

Kitagawa

Tamura

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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156

144

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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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“…7 Chondroitin beta1,4-N-acetylgalactosaminyltransferase-1 (ChGn-1) is involved in an important step for the synthesis of CSPGs. 8 Chondroitin sulfate chains consist of repeating disaccharide units of N-acetylgalactosamine (GalNAc) and glucuronic acid, which are sulfated at either the C6 or C4 position of GalNAc. The integrity of the chondroitin sulfate chain is maintained by elongation (biosynthesis) of the chain, which is catalyzed by ChGn-1, ChGn-2 and sulfotransferases.…”

Section: Introductionmentioning

confidence: 99%

Chondroitin beta-1,4-N-acetylgalactosaminyltransferase-1 missense mutations are associated with neuropathies

et al. 2010

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Chondroitin sulfate proteoglycans (CSPGs) in the peripheral nervous system likely participate as regulatory molecules in the process of axonal degeneration and regeneration. We investigated the chondroitin beta1,4-N-acetylgalactosaminyltransferase-1 (ChGn-1) gene in 114 patients affected with neuropathies including Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, hereditary motor and sensory neuropathy (HMSN) and unknown etiology. The controls were 196 patients with other neurological diseases. We found novel missense mutations in two patients with neuropathy (Bell's palsy, unknown HMSN) in exons 5 (H234R) and 10 (M509R), respectively. None of the patients with other neurological diseases had either of these mutations. We then synthesized the two soluble forms of ChGn-1, containing each of the above mutations. Each of the soluble mutants was expressed in COS-1 cells and the mutant proteins were purified. The purified mutant proteins were used for western blotting analysis using an anti-ChGn-1 antibody and evaluated for glycosyltransferase activities. Although the expression of the ChGn-1 mutant proteins was confirmed by western blotting, they exhibited no N-acetylgalactosamineT-II activities. It is possible that these mutations are associated with the pathogenetic mechanisms of the peripheral neuropathies.

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Regulation of chondroitin sulfate biosynthesis by specific sulfation: acceptor specificity of serum β-GalNAc transferase revealed by structurally defined oligosaccharides

Cited by 59 publications

References 22 publications

Novel Proteoglycan Linkage Tetrasaccharides of Human Urinary Soluble Thrombomodulin, SO4-3GlcAβ1–3Galβ1–3(±Siaα2–6)Galβ1–4Xyl

Novel Proteoglycan Linkage Tetrasaccharides of Human Urinary Soluble Thrombomodulin, SO4-3GlcAβ1–3Galβ1–3(±Siaα2–6)Galβ1–4Xyl

Human tumor suppressor EXT gene family members EXTL1 and EXTL3 encode α1,4- N -acetylglucosaminyltransferases that likely are involved in heparan sulfate/ heparin biosynthesis

Chondroitin beta-1,4-N-acetylgalactosaminyltransferase-1 missense mutations are associated with neuropathies

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