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Joint Research on KDN-Glycoconjugates in Mammalian Cells

KDN-糖复合物在哺乳动物细胞中的联合研究

基本信息

批准号：
07044228
负责人：
KITAGIMA Ken
金额：
$ 2.3万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1995
资助国家：
日本
起止时间：
1995 至无数据
项目状态：
已结题

项目摘要

An objective of our research was to elucidate biological significance of KDN glycans by revealing the developmental expression and biosynthetic pathway of KDN-glycoconjugates. The followings are the results obtained under our research project.1. Occurrence and distribution of KDN-glycoconjugates in mammal : (1) We developed a sensitive method for chemical detection of KDN,consisting of labeling of liberated KDN with a fluorescent dye and identification of labeled KDN on HPLC.This method enabled us to show the presence of KDN in various cells and tissues of pig, rat, and human. (2) Immunospecificity of monoclonal antibody mAb.kdn8kdn was precisely determined by ELISA using a series of oligoKDN conjugated with phospholipid as antigens. (3) We purified bacterial KDNase which could specifically cleave ketosidic linkages of KDN to utilize the enzyme for immunodetection of the KDN epitopes, because disappearance of the immunostainability by the digestion with KDNase would give an excellent c … More ontrol for the identification of KDN epitopes. (4) Using the above two probes (mAb.kdn8kdn and KDNase), we successfully demonstrated the presence of oligoKDN structure in various cells and tissues of rat. Interestingly, the expression of oligoKDN structure in muscle, kidney, lung, and brain was developmentally regulated.2. Searching for KDN-glycoconjugates in tumor cells. We detected a kdn8kdn-positive component in several kinds of tumor cells. Some of them were expressed only in fetus, but not in adult, under normal development, thus indicating that oligoKDN constituted a member of oncodevelopmental carbohydrate antigens, such as polysialic acid.3. Regulatory mechanism for expression of KDN-glycoconjugates. We have already demonstrated the importance of the formation of CMP-KDN as a donor substrate for a KDN-transferase. Here we showed the identification of KDN-9-phosphate synthetase as a key enzyme responsible for formation of KDN monosaccharide. The enzyme catalyzed the condensation of Man-6-phosphate with phosphoenolpyruvic acid to form KND-9-phosphate. Developmental expression of KDN-glycoconjugates and the enzyime activity would be important to be elucidated. Less

本研究的目的是通过揭示KDN-糖复合物的发育表达和生物合成途径来阐明KDN聚糖的生物学意义。以下是我们在研究项目中获得的结果。哺乳动物体内KDN-糖复合物的存在和分布：（1）建立了一种灵敏的KDN的化学检测方法，即用荧光染料标记KDN，并在HPLC上鉴定标记的KDN，这种方法使我们能够显示KDN在猪、大鼠和人的各种细胞和组织中的存在。(2)以磷脂偶联的oligokDN为抗原，用ELISA法测定了单克隆抗体mAb.kdn8kdn的免疫特异性。(3)我们纯化了能特异性切割KDN的酮苷键的细菌KDNase，利用该酶进行KDN表位的免疫检测，因为用KDNase消化后免疫标记的消失将提供良好的检测结果。 ...更多信息用于鉴定KDN表位的对照。(4)利用上述两种探针（mAb.kdn8kdn和KDNase），我们成功地证明了oligoKDN结构在大鼠各种细胞和组织中的存在。有趣的是，寡KDN结构在肌肉、肾脏、肺和脑中的表达受到发育调控.在肿瘤细胞中寻找KDN-糖缀合物。我们在几种肿瘤细胞中检测到了kdn 8 kdn阳性成分。在正常发育过程中，有些基因仅在胎儿中表达，而在成人中不表达，提示oligoKDN是肿瘤发育糖抗原的一员，如聚唾液酸. KDN-糖缀合物表达的调节机制。我们已经证明了形成CMP-KDN作为KDN转移酶的供体底物的重要性。在这里，我们表明KDN-9-磷酸合成酶的鉴定作为一个关键酶负责形成KDN单糖。该酶催化Man-6-磷酸与磷酸烯醇式丙酮酸缩合形成KND-9-磷酸。发育中的表达和酶的活性将是重要的是要阐明的KDN-糖复合物。少