Joint Research on KDN-Glycoconjugates in Mammalian Cells

我们的研究目的是通过揭示KDN-糖缀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得到的结果。KDN-糖缀合物在哺乳动物中的发生和分布：(1)我们建立了一种灵敏的KDN化学检测方法，包括用荧光染料标记释放的KDN和用高效液相色谱鉴定标记的KDN。这种方法使我们能够证明KDN存在于猪、大鼠和人的各种细胞和组织中。(2)单克隆抗体mAb的免疫特异性。以一系列寡核苷酸与磷脂偶联为抗原，采用ELISA法精确测定kdn8kdn。(3)我们纯化了能够特异性切割KDN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酶切可以消除KDN表位的免疫染色性，从而为KDN表位的鉴定提供了很好的控制。(4)利用上述两种探针(mAb；kdn8kdn和KDNase)，我们成功地证明了在大鼠的各种细胞和组织中存在oligoKDN结构。有趣的是，oligoKDN结构在肌肉、肾脏、肺和脑中的表达受到发育调节。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kdn8kdn阳性成分。其中一些仅在胎儿中表达，而在正常发育的成人中不表达，这表明oligoKDN是肿瘤发育碳水化合物抗原（如聚唾液酸）的一个成员。kdn -糖缀合物表达的调控机制。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，我们发现KDN-9-磷酸合成酶是形成KDN单糖的关键酶。该酶催化man -6-磷酸与磷酸烯醇丙酮酸缩合生成knd -9-磷酸。kdn -糖缀合物的发育表达和酶活性将是重要的阐明。少

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