Molecular Mechanism of Novel N-Linked Sugar Chain Having α-Galactofuranosyl Structure

具有α-呋喃半乳糖基结构的新型N-连接糖链的分子机制

• 批准号: 10660068
• 负责人: KIMURA Atsuo
• 金额: $ 2.3万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660068/
• 关键词: N-Linked Sugar Chain; O-Linked Sugar Chain

In microorganisms there are the N- and O-linked sugar chains which are not found in animals and plants. Their biological function and synthesis have not been elucidated. In this project the structural determination of novel O-linked sugar chains was done, and the enzymes concerned with the formation of α-galactofuranosyl structure (Galf) in N-linked oligosaccharide, UDP-Galf synthetase and Galf transferase, were analyzed. (1) Five kinds of O-linked sugar chains, which were separated chemically from Aspergillus niger α-glu-cosidase, were purified, and the following structures were determined by monosaccharide analysis, exo-glycosidase treatment, and MS, 1D- and 2D-NMR: I) mannose, ii) mannobiose having α-1,2-linkage, iii) glucosylmannobiose of branched type, iv) two mannotrioses of branched (iv-a) and linear (iv-b) structures. Sugar chains of iii and iv-b were novel ones. (2) The substrate and product for Galf transferase were prepared from A .niger α-gluco-sidase. Since the activities of UDP-Galf synthetase and Galf transferase in cell extract of A .niger were low, the cultivation conditions were examined. The addition of maltose or starch to culture broth increased the both enzyme activities with induction of amylases (secretory proteins). When disruption of cells, the loss of activities was observed. It was found that the detergent stabilized the Galf transferase. The transferase preparation of high purity, which did not give a single band in electophoretic analysis, was obtained after several chromatographies. The purification is now doing to figure out the amino acid sequence and to separate the enzyme gene.

在微生物中存在动物和植物中没有的N-和O-连接的糖链。它们的生物学功能和合成尚未阐明。本课题对新型O-连接糖链进行了结构测定，并对N-连接寡糖中与α-呋喃半乳糖基结构（Galf）形成有关的酶--UDP-Galf合成酶和Galf转移酶进行了分析。(1)从尼日尔α-葡萄糖苷酶中分离出5种O-连接糖链，经单糖分析、外切糖苷酶处理、MS、1D-NMR和2D-NMR鉴定其结构为：I）甘露糖，ii）具有α-1，2-键的甘露二糖，iii）支链型葡糖基甘露二糖，iv）支链（iv-a）和直链（iv-b）结构的两种甘露三糖。iii和iv-b的糖链是新的糖链。(2)以尼日尔α-葡萄糖苷酶为底物制备半乳糖醛酸转移酶的底物和产物。由于尼日尔曲霉细胞提取物中UDP-Galf合成酶和Galf转移酶的活性较低，因此对培养条件进行了考察。在发酵液中添加麦芽糖或淀粉可提高两种酶的活性，并诱导淀粉酶（分泌蛋白）的产生。当细胞破裂时，观察到活性的丧失。发现去污剂稳定Galf转移酶。经多次层析后，得到了电泳不出现单一条带的高纯度转移酶制剂。目前正在进行纯化，以确定氨基酸序列并分离酶基因。

项目成果

H. Mori, T. Kobayashi, T. Tonokawa, A. Tatematsu, H. Matsui, A.Kimura & S. Chiba: "Molecular Cloning of an α-Amylase CDNA from Germinatipg Cotyledons of Kidney Bean (Phaseolus vulgaris L. cv Toramame)"Oyo Toshitsu Kagaku. 45-3. 261-267 (1998)

H. Mori、T. Kobayashi、T. Tonokawa、A. Tatematsu、H. Matsui、A. Kimura 和 S. Chiba：“从芸豆 (Phaseolus vulgaris L. cv Toramame) Germinatipg 子叶中 α-淀粉酶 CDNA 的分子克隆）“大洋俊科学。45-3.261-267（1998）

永坂 曜介: "Corticium rolfsiiのグルコアミラーゼに関する研究"応用糖質科学. 46(2). 169-178 (1999)

Yosuke Nagasaka：“Corticium rolfsii 的葡糖淀粉酶的研究”，《应用糖科学》46(2) (1999)。

K.Nozaki: "Amylases and Branching Enzyme of Developing Kidney Been Seeds on Native Electrophoretic Gel"J.Apple.Glycosci. 45(2). 117-122 (1998)

K.Nozaki：“在天然电泳凝胶上开发肾种子的淀粉​​酶和分支酶”J.Apple.Glycosci。

木村淳夫: "澱粉粒の酵素分解に見い出された生成物の"固定化現象""BRAINテクノニュース. 72(3). 27-29 (1999)

Atsuo Kimura：“淀粉颗粒酶促降解中发现的产品“固定现象””BRAIN Techno News 72(3) (1999)。

S.Igaki: "Catalytic Reaction Mechanism on α-Secondary Kinetic Isotope Effects in Hydrolytic Cleavage of α-Glucosidic Linkage by α-Glycosidases" Oyo Toshitsu Kagaku(J.Appl.Glycosci.). 45・3. 269-274 (1998)

S.Igaki：“α-糖苷酶水解α-糖苷键的α-二级动力学同位素效应的催化反应机制”Oyo Toshitsu Kagaku（J.Appl.Glycosci.）45·3（1998）。