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

在微生物中，存在动植物中没有的N-和O-连接的糖链。它们的生物学功能和合成还没有被阐明。本项目对新型O-连接糖链的结构进行了测定，并对N-连接低聚糖中与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)从黑曲霉α-Glu-cosidase中分离得到5种O-连接糖链，经单糖分析、糖外糖苷酶处理、MS、1D-和2D-核磁共振鉴定为：I)甘露糖，II)具有α-1，2-键的甘露二糖，III)分支型葡萄糖甘露糖，IV)两种分枝(IV-a)和线形(IV-b)结构的甘露三糖。糖链III和IV-b是新发现的糖链。(2)利用黑曲霉α-葡萄糖苷酶制备半乳糖转移酶的底物和产物。由于黑曲霉细胞提取液中UDP-GALF合成酶和GALF转移酶活性较低，因此对其培养条件进行了研究。在发酵液中添加麦芽糖或淀粉可提高这两种酶的活性，并诱导淀粉酶(分泌蛋白)的产生。当细胞被破坏时，观察到活性的丧失。结果表明，洗涤剂对GALF转移酶有稳定作用。经多次层析，得到纯度较高的转移酶制剂，在电泳法中未见单一条带。目前正在进行纯化，以确定氨基酸序列并分离酶基因。

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.