Biology of Proteolytic Derivatives of Lp(a)

Lp(a) 蛋白水解衍生物的生物学

• 批准号: 7024471
• 负责人: Angelo M Scanu
• 金额: $ 37.84万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7024471
• 关键词: adduct; apolipoproteins; atherosclerotic plaque; blood lipoprotein metabolism; carotid artery; clinical research; cytokine; endopeptidases; human subject; human tissue; inflammation; laser capture microdissection; low density lipoprotein; macrophage; mass spectrometry; oxidized lipid; phospholipids; plasmapheresis; protein structure function; proteolysis; recombinant proteins; tissue /cell culture

DESCRIPTION (provided by applicant): Lipoprotein(a), Lp(a), is an LDL variant, having as a protein moiety apoB100 linked covalently on a 1:1 molar basis to apolipoprotein(a), apo(a), a highly glycosylated multikringle structure. Hallmarks of apo(a) are its heterogeneity due to its size polymorphism dependent on the number of kringle type 2 repeats and susceptibility to proteolytic cleavage. Studies in vitro and in cell culture on cleaved products and recombinants have provided evidence that apo(a) is made of bioactive microdomains (fragments) contained in the C-terminal region that we call F2. Thus far, the bioactivity in F2 has been demonstrated for KIV-containing microdomains. However, we have recently shown that the KV-protease domain, PD, in F2 is able to stimulate the production of interleukin-8 in cultured human macrophages and assigned this effect to lysine residues in KV linked to oxidized phospholipids, ox-PL and also shown that these adducts are recognized by the monoclonal antibody, EO6. On the basis of these observations we will explore the hypothesis that KV-PD contributes to the athero-thrombogenic properties of Lp(a). To this effect, we will pursue the studies on KV-containing recombinants, with no PD, to unequivocally identify the lys residues involved in linkage with ox-PLs and the chemical nature of the latter by mass spectroscopy techniques. We have recently reported discrete fragments of apo(a) in extracts of human carotid plaques in a microenvironment rich in pro-inflammatory cytokines and metalloproteinases. We have also immunological evidence that these fragments contain ox-PL adducts. Thus, we wish to better define the properties of these fragments and compare them with those exhibited by the products generated in vitro by both apo(a) proteolysis and recombinant techniques. For this purpose, we will use immunochemical methods, proteomic and DNA array of laser-microdissected macrophage-rich areas of endarterectomy segments of lesioned human carotid arteries. The results of the proposed studies should shed light on whether ox-PL adducts play a role in the cardiovascular pathogenicity of Lp(a).

描述（由申请人提供）：脂蛋白（a），Lp（a），是一种LDL变体，其蛋白质部分apoB 100以1：1摩尔比共价连接到载脂蛋白（a），apo（a），一种高度糖基化的多三环结构。载脂蛋白（a）的特征是其异质性，这是由于其大小多态性依赖于kringle 2型重复序列的数量和对蛋白水解切割的敏感性。对切割产物和重组体的体外和细胞培养研究提供了证据，证明apo（a）是由C-末端区域（我们称之为F2）中含有的生物活性微区（片段）组成的。到目前为止，F2中的生物活性已被证明是含有KIV的微域。然而，我们最近已经表明，KV-蛋白酶结构域，PD，在F2能够刺激白细胞介素-8的生产在培养的人巨噬细胞，并分配这种效果的赖氨酸残基KV连接到氧化磷脂，ox-PL，也表明这些加合物被单克隆抗体，EO 6。在这些观察的基础上，我们将探讨KV-PD有助于Lp（a）的动脉粥样硬化血栓形成特性的假设。为此，我们将继续研究含有KV的重组体，没有PD，明确确定参与连接ox-PL的赖氨酸残基和后者的化学性质，通过质谱技术。我们最近报道了在富含促炎细胞因子和金属蛋白酶的微环境中人颈动脉斑块提取物中apo（a）的离散片段。我们也有免疫学证据表明，这些片段含有ox-PL加合物。因此，我们希望更好地定义这些片段的特性，并将它们与通过apo（a）蛋白水解和重组技术在体外产生的产物所表现出的特性进行比较。为此，我们将使用免疫化学方法，蛋白质组学和DNA阵列的激光显微切割的巨噬细胞丰富的地区的动脉内膜切除术段的病变的人颈动脉。这些研究结果将有助于阐明ox-PL加合物是否在Lp（a）的心血管致病性中发挥作用。