Joint Research on KDN-Glycoconjugates in Mammalian Cells

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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