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Functional Domain of Marine Invertebrate Lectin

海洋无脊椎动物凝集素的功能域

基本信息

批准号：
01560229
负责人：
MURAMOTO Koji
金额：
$ 1.15万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

The amino acid sequences of multiple galactose-binding lectins isolated from the coelomic fluid of the acorn barnacle, Megabalanus rosa have been determined. 140K lectin was composed of identical subunits (Mr 22K) which were cross-linked by interchain disulfide bonds. The subunit was a glycoprotein on 173 amino acids. 64K lectin was composed of four identical subunits (Mr 16K) of 138 amino acids. The subunits were crossーlinked by disulfide bonds to form dimers at the C-terminal segments. They had a domain which was homologous with the carbohydrate-recognition domain of the calcium-dependent (C-type) animal lectins. The denaturation by urea and other agents of the lectins was followed by high-performance size exclusion chromatography and agglutinating activity against rabbit erythrocytes. The subunit structure of 64K lectin was highly stable either in 6M urea and 50% isopropanol, sustainig the activity, whereas 140K lectin easily denatured.A photoactivatable D-galactosamine derivative was prepared for the photoaffinity labeling of the lectins to localize carbohydrate binding sites. The lectins were also subjected to various chemical modification in order to ascertain the amino acid residues responsible for its carbohydrate-binding property. Modification of lysine and tyrosine did not affect their activities. However, modification of tryptophan, arginine and histidine led to complete losses of the activities.Upon treatment with cyanogen bromide, 64K lectin yielded two fragments, a small one including interchain disulfide bonds and a large one corresponding to the carbohydrate-recognition domain, by gel filtration. The isolated domain was demonstrated to retain the carbohydrate binding activity by affinity chromatography on a HCI treated Sepharose 4B column.The above results indicate that marine invertebrate lectins can be used as a functional domain to construct various kinds of multifunctional proteins.

从橡子藤壶Megabalanus rosa的体腔液中分离的多种半乳糖结合凝集素的氨基酸序列已经确定。140 K凝集素由相同的亚基（Mr 22 K）组成，这些亚基通过链间二硫键交联。该亚基是由173个氨基酸组成的糖蛋白。64 K凝集素由4个相同的亚基（Mr 16 K）组成，由138个氨基酸组成。亚基通过二硫键交联，在C-末端片段形成二聚体。它们具有与钙依赖性（C型）动物凝集素的碳水化合物识别结构域同源的结构域。用尿素和其他试剂对凝集素进行变性，然后进行高效体积排阻层析和对兔红细胞的凝集活性。64 K凝集素在6 M尿素和50%异丙醇中的亚基结构稳定性高，保持活性，而140 K凝集素则易变性，制备了一种可光活化的D-半乳糖胺衍生物，用于光亲和标记凝集素的糖结合位点。凝集素也进行了各种化学修饰，以确定负责其碳水化合物结合特性的氨基酸残基。赖氨酸和酪氨酸的修饰不影响其活性。经溴化氰处理后，64 K凝集素经凝胶过滤得到两个片段，一个小片段含有链间二硫键，另一个大片段对应于糖识别结构域。经HCl处理的Sepharose 4 B柱亲和层析证明，分离的结构域保留了糖结合活性，表明海洋无脊椎动物凝集素可作为功能结构域构建多种功能蛋白。