Biosynthesis and biological roles of specific sulfation pattern of chondroitin sulfate

硫酸软骨素特定硫酸化模式的生物合成和生物学作用

• 批准号: 18570125
• 负责人: HABUCHI Osami
• 金额: $ 2.63万
• 依托单位: Aichi University of Education
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18570125/
• 关键词: chondroitin sulfate; sulfotransferases; gene knockout; chondroitin sulfate E; C6ST; GalNAc4S-6ST; N-glycan; bone marow derived mast cells; GalNAc4S-6ST; Tryptase; Thrombin; イカ軟骨

1. We generated GalNAc4S-6ST KO mice. GalNAc4S-65T-/- mice lost synthetic activity of CS-E completely in every tissues. Bone marrow derived mast cells derived from the GalNAc4S-6ST-/- mice could not synthesize CS-E and showed decreased activity of tryptase.2. We established stable clone expressing GalNAc4S-6ST from rat chondrosarcoma cell line. This clone synthesized and secreted proteoglycan containing CS-E. Chain length of CS produced by the clone was much shorter than the length of CS produced by the parent cells, suggesting that formation of GalNAc (4,6-SO4) residues by GalNAc4S-6ST may inhibit chain elongation.3. We established a new method to synthesize a unique nonrreducing terminal sequence GalNAc(4,6-SO_4)-GlcA(2SO_4)-GalNAc(6SO_4), using 2OST and GalNAc4S-6ST from GalNAc(4SO_4)-GlcA-GalNAc(6SO_4).4.We synthesized various C6ST-1 mutants devoid of N-glycosylation sites by the site-directed mutagenesis, and showed that N-glycans attached to C6ST-1 contributed to production of active enzyme, intracellular localization and substrate specificity of C6ST-I.5.We cloned squid GalNAc4S-63T cDNA from the squid cartilage and prepared the recombinant enzyme. CS-E synthesized by the recombinant GalNAc4S-6ST inhibit thrombin in the antithrombin III-independent manner

1.我们产生GalNAc 4S-6ST KO小鼠。GalNAc 4S-65 T-/-小鼠各组织CS-E合成活性完全丧失。GalNAc 4S-6ST-/-小鼠骨髓来源的肥大细胞不能合成CS-E，类胰蛋白酶活性降低.我们从大鼠软骨肉瘤细胞系中建立了稳定表达GalNAc 4S-6ST的克隆。该克隆合成并分泌含有CS-E的蛋白聚糖。克隆产生的CS链长远短于亲本细胞产生的CS链长，表明GalNAc 4S-6ST形成的GalNAc（4，6-SO 4）残基可能抑制了CS链的延长.我们建立了一种新的合成非还原性末端序列GalNAc的方法（4，6-SO_4）-GlcA（2SO_4）-GalNAc（6SO_4）的2 OST和GalNAc（4S-6ST）。4.通过定点突变的方法合成了C6 ST-1缺失N-糖基化位点的突变体，从鱿鱼软骨中克隆了鱿鱼GalNAc 4S-63 T的cDNA，并制备了重组酶。由重组GalNAc 4S-6ST合成的CS-E以抗凝血酶III非依赖性方式抑制凝血酶

项目成果

Knockout Mice and Proteoglycans in

敲除小鼠和蛋白多糖

• 期刊: COMPREHENSIVE GLYCOSCIENCE.(Kamerling, J. P. et. al. eds.) 3,4,10
• 作者: Kimata;K. Habuchi;O. Habuchi;H. Watanabe;H
• 通讯作者: H

Synthesis of chondroitin sulfate containing an E-D tetrasaccharide structure by the recombinant squid sulfotransferase

重组鱿鱼磺基转移酶合成含E-D四糖结构的硫酸软骨素

• 发表时间: 2007
• 作者: T. Yamaguchi;S. Ohtake;K. Kimata;O. Habuchi
• 通讯作者: O. Habuchi

Enzymatic synthesis of highly sulfated chondroitin sulfate that mimics thrombomoduline

模拟血栓调节素的高度硫酸化硫酸软骨素的酶法合成

• 发表时间: 2007
• 作者: N. Kusakabe;S. Ohtake;T. Yamaguchi;H. Habuchi;O. Habuchi
• 通讯作者: O. Habuchi

The utility of formalin-fixed paraffin-embedded tissue blocks for quantitative analysis of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase mRNA expressed by colorectal cancer cells

福尔马林固定石蜡包埋组织块定量分析结直肠癌细胞表达的 N-乙酰半乳糖胺 4-硫酸 6-O-磺基转移酶 mRNA

• 发表时间: 2007
• 期刊: Acta Histochem. Cytochem 40
• 作者: Ito;Y.;Watanabe;M.;Nishizawa;T.;Omachi;T.;Kobayashi;T.;Habuchi;O.;and Nakayama
• 通讯作者: and Nakayama

Molecular cloning of squid N-acetylgalactosamine 4-sulfate 6-0-sulfotransferase and synthesis of a unique chondroitin sulfate containing E-D hybrid tetrasaccharide structure by the recombinant enzyme

鱿鱼N-乙酰半乳糖胺4-硫酸6-0-磺基转移酶的分子克隆及重组酶合成独特的含E-D杂合四糖结构的硫酸软骨素

• 发表时间: 2007
• 期刊: Glycobiology 17
• 作者: Yamaguchi;T.;Ohtake;S.;Kimata;K.;Habuchi;O
• 通讯作者: O