|Budget Amount *help
¥2,500,000 (Direct Cost : ¥2,500,000)
Fiscal Year 1996 : ¥2,500,000 (Direct Cost : ¥2,500,000)
Oligosaccharides modify the structure, dynamics and physical properties of the proteins to which they are covalently bound through nitrogen to aspargine or oxygen to serine or threonine. After the breakthrough of cDNA cloning, the primary structure is deduced from the sequence of nucleotides in the cDNA encoding the protein. From the derived sequence, certain properties of the protein can be predicted including potential glycosilation sites for N-linked glycans. Oligosaccharide structure and function, however, can not be deduced from the nucleotide sequence. In this research project, we have studied on the biological activity of several glycoproteins including Leukemia lnhibitory Factor (LIF), Placental Lactogen (PL) and Chorionic Gonadotropin (CG) using techniques including molecular cloning, site-directed mutagenesis and organic synthesis of oligosaccharides. We have cloned the cDNA which encodes LIF in the rat. LIF is a member of a cytokine superfamily and has diverse functions in the various tissues and cells. Of seven possible N-glycosilation sites, six sites are glycosilated at positions of 9,34,63,73,96 and 116. These oligosaccharides are involved in the biological function when the activity was evaluated in the DA-1a cell proliferation system. PL-lm is a member of the placental prolactin family and has two possible N-glycosilation sites (79 and 128). The mutants of PLs of which substituting the Asn 79 and Asn 128 with Gln showed significant decrease of Nb2 cell proliferation. Equine CG (eCG) is a unique member of gonadotropin, because it possesses both LH and FSH activities. The eCG mutant which lack N-glycan of a-subunit (Asn56) showed complete loss of LH-like activity but showed potent FSH-like activity. eCG has also O-glycans at the C-teminal portion of b-subunit. The mutant which lack O-linked oligosaccharides was also shoed potent FSH like activity. Thus, we demonstrated the biological importance of oligosaccharides in the glycoproteins.