| Project/Area Number |
09671097
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Hematology
|
|---|
| Research Institution | Tokyo Medical and Dental University |
|---|
Principal Investigator |
HIROSAWA Shinsaku Medical Department, Associate Professor, 医学部, 助教授 (50143574)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
YAMAMOTO Koh Medical Department, Assistant, 医学部, 助手 (00281717)
|
|---|
| Project Period (FY) |
1997 – 1998
|
| Project Status |
Completed (Fiscal Year 1998)
|
| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
|
|---|
| Keywords | alpha_2-plasmin inhibitor / mutant protein / endoplasmic reticulum / impaired secretion / プロテアーゼ / α2-プラスミンインヒビター |
|---|
| Research Abstract |
We have previously shown that deficiency of alpha_2-plasmin inhibitor (alpha_2Pl) is caused by a frame-shift mutation of one base citidine, leading to a replacement of normal 12 residues with 178 amino acids. The mutant alpha_2Pl, designated as alpha_2Pl Nara, is retained in the endoplasmic reticulum (ER).
In this study, we analyzed degradation mechanism of retained mutant protein using ER and also tried to characterize proteases involved in ist degradation.
Normal and mutant alpha_2Pl cDNA were subcloned into a plasmid pBlueScript under T7 promoter. And mRNAs were synthesized with T7 promoter. Using synthesized mRNA, ^<35>S-methionine, other amino acids mixture, and ER, alpha_2Pl proteins were translated with in vitro translation of rabbit reticulocyte lysate. Synthesized proteins showed cleavage of a signal peptide and glycosilation at 4 sites. While normal alpha_2Pl showed no change during incubation, preferential degradation of mutant alpha_2Pl molecules in an endoplasmic reticulum was demonstrated. Furthermore, in-vitro analysis using stable cell lines expressing mutant alpha_2Pl suggested that mutant protein bind transiently to calnexin. But we could not demonstrate any involvement of ubiquitin for the degradation of mutant alpha_2Pl proteins.
|
