|Budget Amount *help
¥4,080,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2007: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2006: ¥2,000,000 (Direct Cost: ¥2,000,000)
RAGE (receptor for advanced glycation endproducts) is a cell surface receptor that bind several ligands, such as AGE (advanced glycation endproducts), HMGB-1 (high mobility group box-I protein), amyloid beta. The intracellular signals evoked by RAGE-ligand interaction were thought to play pivotal roles in development of various diseases. However, the mechanism of generation of intracellular signals by RAGE was poorly understood.
This study shed lights on several new aspects of RAGE signaling mechanism as follows.
I. Recombinant wild-type, de-N-glycosylation and G82S RAGE proteins were produced in COS-7 cells, purified and assayed for ligand-binding abilities. De-N-glycosylation at N81 and G82S mutation decreased Kd for glycolaldehyde-derived AGE to three orders of magnitude lower levels compared with wild-type. AGE-induced upregulation of VEGF mRNA was significantly augmented in endothelial cell-derived ECV304 cells expressing de-N-glycosylated and G82S RAGE when compared with wild-type
2. RAGE molecules of living cell surface were labeled by fluorescent dye-conjugated monoclonal antibody that binds to extracellular domain of RAGE (mAbl3G4), and the dynamic state of RAGE was analyzed by confocal fluorescence laser microscopy. The results indicated that RAGE molecules were constitutively internalized, and pinocytotic mechanism was, at least partly, involved in RAGE internalization. The rate of RAGE internalization was not altered by addition of RAGE ligand.
3. RAGE molecules of living cell surface were labeled by mixture of mAbl3G4 conjugated with Alexa 488 and mAbl3G4 with Alexa 594, which are a pair of donor and acceptor for FRET (fluorescence resonance energy transfer), to detect oligomerization of RAGE molecule by FRET. The results indicated that the RAGE molecule oligomerized upon binding its ligand.
4. Low molecular weight RAGE ligands such as low molecular weight heparin, which are speculated not to induce the RAGE oligomerization, did not evoke RAGE-mediated intracellular signal, and inhibited the signaling by high molecular weight ligands.
These new findings strongly suggested that the oligomerization is the first step of the signal generation, and revealed the principle of the development of RAGE antagonists that should be promising candidates of the preventive medicine for RAGE-related diseases. Less