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Vitronectin expressed in porcine brain and characterization of lipid binding activities related to tissue vitronectins.

玻连蛋白在猪脑中的表达以及与组织玻连蛋白相关的脂质结合活性的表征。

基本信息

批准号：
09680585
负责人：
OGAWA Haruko
金额：
$ 0.83万
依托单位：
GRADUATE SCHOOL OF HUMANITIES AND SCIENCES OCHANOMIZU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

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.

玻连蛋白（Vitronectin，VN）是一种多功能糖蛋白，主要存在于动物血清和细胞外基质中，本研究首次分离并鉴定了猪脑VN，为研究组织VN的功能奠定了基础。1猪脑VN的分离猪脑提取液免疫印迹分析表明，在SOS-PAGE上约60和40 kDa的蛋白条带与抗VN抗体反应。与血清VN不同，脑VN被发现在非变性条件下与肝素柱结合。用DEAE-HPLC从脑提取物中分离出62 kDa蛋白组分，发现其与抗猪血清VN反应，HRP-凝集素结合试验表明62 kDa蛋白主要是O-糖基化的，而猪血清VN主要是N-糖基化的。另一方面，利用HRP-抗人VN抗体噬斑杂交法，从阿尔茨海默病患者脑组织cDNA表达文库中克隆了该蛋白的cDNA。从8 × 10^4个克隆中获得了4个阳性克隆。2玻连蛋白的脂质结合活性通过ELISA和亲和层析发现，血浆玻连蛋白与各种硫酸化脂质和酸性磷脂结合，但不与神经节苷脂结合。只有激活的开放形式的玻连蛋白结合硫脂，而玻连蛋白结合胆固醇3-硫酸酯，而不管其构象状态。3生物配体的结合域的鉴定脂质，β-内啡肽，1型胶原蛋白和肝素的每个结合域通过ELISA测定，使用重组域和缺失突变体的人玻连蛋白表达为融合蛋白连接谷胱甘肽S-转移酶在大肠杆菌中。发现含有H1或H2结构域的两种重组蛋白都具有硫酸酯结合活性。本工作中发现的配体结合活性将为组织VN的功能研究和纯化提供线索。