제8 혈액응고인자와 저밀도 지질 단백질 수용체관련 단백질의 상호작용 연구

Abstract

Low density lipoprotein receptor-related protein (LRP) is a receptor responsible for the clearance of proteases including factor VIII (FVIII) from blood. The binding sites on FVIII for LRP are within its A2, A3, and C2 domains, and these sites interact with clusters II and IV of LRP. Binding of FVIII to LRP might serve to both regulate and clear FVIII. However, given its critical role in endocytosis and cholesterol homeostasis, inhibiting LRP itself has not been considered as a strategy to increase the half-life of FVIII. A structure-based understanding at the atomic level of the ligand binding interfaces of FVIII is critical in designing recombinant FVIII with altered binding properties and in developing drugs that will inhibit FVIII-ligand interactions. We successfully cloned, overexpressed, and purified complementary-type repeats double (CRD) domains (CR3-4, CR4-5, CR5-6, CR6-7, CR7-8, CR8-9, CR21-22, CR22-23, CR23-24, CR24-25, CR25-26, CR26-27, CR28-29, CR29-30) and cluster II, IV of LRP in E. coli. The CRD domains were purified about 4 mg of protein per liter of culture, while cluster II and IV were purified about 5 mg of protein per liter of culture. The interaction between the purified protein and FVIII was examined using dot blot and SPR method. The interaction between FVIII and CRD domains (CR3-4, CR4-5, CR24-25, CR25-26) was confirmed as a result of dot blot. And the interaction between FVIII and CRD domains (CR3-4, CR4-5, CR21-22, CR23-24, CR24-25, CR26-27, CR28-29, CR29-30) was confirmed as a result of SPR method. CR4-5 showed the strongest affinity with SPR. This result can be used as a basic material for structural analysis of LRP and FVIII complex.