|Budget Amount *help
¥3,900,000 (Direct Cost : ¥3,900,000)
Fiscal Year 1999 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1998 : ¥2,400,000 (Direct Cost : ¥2,400,000)
Glycosyltransferases in the Golgi apparatus are type-II membrane proteins which consist of an N-terminal cytoplasmic region, a trans membrane region, a stem region and a c-terminal catalytic region facing to lumen of Golgi. In order to elucidate the molecular mechanisms underlying Golgi retention of glycosyl-transferases, we carried out the following two experiments.
(1) Molecular behavior of mutant Lewis enzymes in vivo : The expression of type-1 Lewis antigens on erythrocytes and in digestive organs is determined by a Lewis type α(1, 3/1, 4)fucosyltransferase (Lewis enzyme) encoded by the FUT3 gene (Lewis gene) . We have classified the Lewis alleles in the Japanese population into four types, the wild-type allele (Le) and three mutated alleles, le1, le2, and le3. We carried out an extensive study on the biological properties of the three mutant Lewis enzymes using native tissues. The missense mutation, Leu20 to Arg, in the transmembrane domain reduces retention of the le3 enzyme in th
e Golgi membrane resulting in an apparent reduction of enzyme activity as revealed by the lack of Lewis antigen synthesis. So the le3 enzyme can not synthesize Lewis-active glycolipids, which result in the Lewis antigen-negative phenotype of erythrocytes.
(2) Cloning and identification of genes encoding proteins interacting with glycosyltransferases : The two-hybrid system is an in vivo yeast-based system that identifies interaction between two proteins by reconstituting active transcription factor dimmers. cDNAs have been prepared from human non-cancerous right hemi-colon tissues obtained as a surgical sample and a cDNA library constructed in the GAL4 activation domain vector pPC86. We have reported that Lewis type α1, 3/1, 4 fucosyltransferase (FUT3) , H enzyme, Se enzyme, α2,3sialyltransferases (ST3Gal I and ST3Gal IV) and β1,4galactosyl-transferase I(β1,4Galt I) are all expressed in the normal right hemi-colon. Their genes have been cloned in frame with the GAL4 sequence encoding the DNA-binding domain into pDBLeu as the "bait". We carried out screening and obtained clones as the candidate molecules interacting with glycosyltransferases. Less