|Budget Amount *help
¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1991 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1990 : ¥1,400,000 (Direct Cost : ¥1,400,000)
1. -Identification of furin involved in the processing of proproteins
1) Primary structure of furin deduced from the cDNA : The cDNA of furin was isolated from human and rat liver cDNA libraries. The cDNA sequences indicate that the human and rat furins consist of 794 and 793 amino acids, respectively. The primary structure of each furin shows high homology to that of the yeast Kex2 protease, a prohormone-processing enzyme, and contains three potential sites for glycosylation and a transmenbrane domain at a COOH-terminal region.
2) Intracellular localization and processing activity of furin : The furin cDNA was transfected into COS-1 cells. Immunoelectron-microscopical observations revealed that furin is localized in the Golgi complex. When albumin or complement C3 was expressed by transfection of each cDNA, each proform was completely processed into a mature form by furin co-expressed in the same cells. These results indicate that furin, localized in the Golgi complex, is an endoproteas
e involved in the processing of proforms of secretory proteins.
2. Biosynthesis, processing and transport of membrane proteins
1) GPI-anchored proteins : We have established that the ectoenzyme alkaline phosphatase (ALP) is initially synthesized as a proform having a hydrophobic sequence at the COOH terminus, which is immediately cleaved and replaced by a glycosyl-phosphatidylinositol (GPI) anchor, and the GPI-anchored form is transported to the cell surface. When the biosynthesis of GPI was completely blocked by 2-fluoro-2-deoxyglucose, the newly synthesized ALP remained in the endoplasmic reticulum as the proform, which was then gradually converted to a soluble form by proteolytic removal of the COOH-terminal segment, followed by its secretion. The results indicate that the GPI anchoring is prerequisite for ALP to be transported to the cell surface.
2) An enzyme defect due to proteolytic degradation in the endoplasmic reticulum (ER) : The ectoenzyme dipeptidyl peptidase IV (DPPIV) was found to be defective in a rat substrain. Cloning and sequencing of the DPPIV cDNA identified a point mutation which leads to substitution of Gly^<633>->Arg at the active site sequence of DPPIV. In addition, the mutant DPPIV was found to undergo rapid degradation in the ER without being transported to the cell surface. Thus, it is concluded that the rapid degradation of the mutant DPPIV in the ER results, in the enzyme defect in the affected rat. Less