| Budget Amount *help |
¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1998: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Vitronectin (VN) is a multifunctional glycoprotein mainly existing in animal serum and extracellular matrix, In this study, brain VN was isolated and characterized for the first time as the basis of the functional studies of tissue VN.
1 Isolation of brain VN Immunoblot analysis of porcine brain extract showed that approximately 60 and 40 kDa protein bands on SOS-PAGE reacted with anti-VN antibody. Unlike serum VN, brain VN was found to bind with heparin-column under non-denaturing conditions. The 62 kDa protein fraction was obtained from the brain extract using DEAE-HPLC and found to react with anti-porcine serum VN.HRP-lectin binding assay indicated that 62 kDa protein was mainly O- glycosylated although porcine serum VNwas mainly N-glycosylated. The 62kDa protein bound to HRP-heparin and weakly to beta-endorphin on western blotting.
On the other hand, cDNA cloning was performed from a cDNA expression library derived from Alzheimer's diseased patient brain, by the method of plaque-hybridization with HRP-anti-human VN antibody. Positive 4 clones were obtained from 8 x 10^4 clones.
2 Lipid Binding Activities of Vitronectin It was found that plasma vitronectin bound to various sulfated lipids and acidic phospholipids but not gangliosides by the use of ELISA and affinity chromatography. Only the activated open form of vitronectin bound to sulfatide, while vitronectin bound to cholesterol 3-sulfate regardless of its conformational state.
3 Identification of Binding Domains for Biological Ligands Each binding domain for lipids, beta-endorphin, type-1 collagen, and heparin was determined by ELISA using recombinant domains and deletion mutants of human vitronectin expressed as fusion proteins linked to glutathione S-transferase in E.coli. Both recombinant proteins containing Hi or H2 domains were found to have sulfatide-binding activity. The ligand binding activities found in this work would provide a clue for functional studies and purification of tissue VN.
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