| BackgroundReceptor for advanced glycation end products(RAGE)is a newly identified member of the immunoglobulin superfaniily of cell-surface molecules. Human RAGE (hRAGE) consists of 404 amino acid residues, including a sinal peptide of 22 amino acids and an extracellular domain of 320 amino acids. The latter domain is also named soluble RAGE(sRAGE). sRAGE has two N-linked glycosylation sites locating near the amino terminus (at amino acid positions 25 and 81 of the mature protein), which lie in a V-like domain. There are also two C-like domains in this region. These structures are important to the molecular stability and the function of identifying ligands. Besides, there is a transmembrance region of 21 hydrophobic amino acids followed by a highly charged intracellular domain of 41 amino acids. As a new member of the immunoglobulin superfamily of cell-surface molecules, it shares significant homology with MUC 18, nerve cell adhesion molecule(NCAM)and the cytoplasmic domain of CD20. It has been demonstrated that RAGE isnot a specifical receptor for advanced glycation end products(AGE) which has been recognized firstly as a ligand of RAGE. In fact, RAGE recognizes a ligand family whose member include amphoterin, amyloid-?peptide, extracellular newly identified RAGE-binding protein(EN-RAGE) and high mobility group protein Bl(HMGBl). The interaction of these ligands and RAGE plays important role in the pathogenesis of many diseases, such as diabetic mellitus, dialysis-related amyloidosis(DRA)and Alzheimer's disease(AD).AGE accumulates during the process of normal aging in the plasma and tissues, but to an accelerated degree in patients with diabetes, DRA and AD. A central means by which AGE are believed to impart their pathogenic effects is via interaction with its cellular receptors; the best-characterized of these is RAGE. RAGE is present at low levels on a range of cell types, including endothelial cells, monocytes, mesangial cells, vascular smooth muscle cells and neurocytes. However, at sites of accumulated RAGE ligands, there is a marked increase in RAGE-expressing cells with functional receptor up-regulation. One consequence of AGE-RAGE interaction is the generation of enhanced cellular oxidant stress, a means by which cell signaling pathways may be activated, thereby resulting in altered cellular phenotype and cellular dysfunction.The discovery of RAGE and development of reagents to block its interaction with AGE should provide insights into the role of this ligand-receptor interaction in the pathogenesis of diseases. Anti-RAGE antibody and sRAGE are generally accepted blocking reagents.11sRAGE is the excellular domain of RAGE, which can bind with AGE specifically but not mediate the biological effects. Foreign scholars have successfully cloned the extracellular domain of RAGE and prepared anti-RAGE antibody, but these products are not available commercially. There is no report about cloning the extracellular domain of RAGE in China up to now, so that hold up further investigation on RAGE. The aims of this project are: (l)cloning and expressing the extracellular and cytoplasmic domains of RAGE, and identify their biological functions; (2)examining the effect of AGE-RAGE interaction on the synthesis of IL-8 in endothelial cells; (3)investigating the role of ./V-link glycans on the interaction of AGE-RAGE; (4)screening potential proteins which could interact with RAGE in cytoplasm.Methods/. DNA Cloning, Protein Expression and Purification of the Extracellular and Cytoplamic Domains of RAGE(1) Subcloning method was performed to construct the expression vectors of the extracellular and cytoplamic domains of human RAGE (hRAGE-E and hRAGE-C). The extracellular domain was recombined with pET-14b vector with a His-tag, and the cytoplamic domain was recombined with pGEX-KG vector with a GST-tag.(2) hRAGE-E fusion protein was induced by 1 mmol/L IPTG and purified with Ni-NTA resin with a denature procedure, then large12amount of this fusion protein was renatured.
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