The objective of this research project was to establish methods for enzymatic synthesis of neo-glycoconjugates containing new sialic acid (deaminoneuraminic acid, KDN) residues with the aim of utilization of such neo-KDN-glycoconjugates as new bioactive and/or organic materials. The following results were obtained :
1. Search for KDN-specific glycosyltransferases and glycosidases-(1) Two KDN-transferases from rainbow trout ovary and sperm were found to be useful for neo-KDN-glycoconjugate synthesis ; -(2) Glycosidase highly specific for KDN residue was found and purified from a soil bacterium, and used for first identification of KDN-glycoconjugates in mammalian cells and tissues ;
2. Establishment of methods for enzymatic synthesis of neo-KDN-glycoconjugates was achieved. Three synthetic processes using bacterial and animal enzymes were involved : synthesis of monosaccharide KDN from mannose and pyruvate with N-acylneuraminate lyase, synthesis of CMP-KDN from CTP and KDN with CMP-KDN synthetase, and incorporation of KDN residues into various glycoconjugates from CMP-KDN catalyzed by KDN-transferases. Using this method, glycoproteins, glycolipids, and oligosaccharides were able to be prepared in sufficient amounts for testing the biological and chemical properties. The enzymes involved in the latter two processes were discovered and prepared from rainbow trout by us.
3. Neo-KDN-transferrin, prepared according to the methods described in 2, was examined for ligand activity to a plant lectin (SSA), which is known to be specific to NeuAc residue, revealing that SSA recognized KDN residue as well.
4. For the purpose of in vivo synthesis of KDN-glycoconjugates, key enzyme(s) which determines KDN-glycoconjugate synthesis was searched for, and involvement of KDN 9-phosphate synthetase was suggested.