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

微生物中有动物和植物中没有的N-和o -链糖链。它们的生物学功能和合成尚未阐明。本项目对新型o -连接糖链进行了结构测定，并对n -连接低聚糖中与α-半乳糖呋喃基结构（Galf）形成有关的酶，UDP-Galf合成酶和Galf转移酶进行了分析。(1)对从黑曲霉α-葡萄糖苷酶中化学分离得到的5种o键糖链进行纯化，通过单糖分析、外置糖苷酶处理、质谱、1D- nmr和2D-NMR确定了以下结构：1)甘露糖，2)具有α-1,2-连锁的甘露糖，3)支链型甘露糖，4)支链型（iv-a）和线性（iv-b）两种甘露糖。iii和iv-b糖链是新发现的。(2)以A为原料制备半胱氨酸转移酶的底物和产物。尼日尔α-gluco-sidase。结果表明：A细胞提取物中udp -半胱氨酸合成酶和半胱氨酸转移酶活性明显降低。尼日尔低，考察了栽培条件。在培养液中添加麦芽糖或淀粉可通过诱导淀粉酶（分泌蛋白）提高这两种酶的活性。当细胞被破坏时，观察到活动的丧失。结果表明，该洗涤剂对半胱氨酸转移酶具有稳定作用。经多次层析，获得了纯度高、电泳分析无单带的转移酶制剂。现在正在进行纯化，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.