Biology of Proteolytic Derivatives of Lp(a)

Lp(a) 蛋白水解衍生物的生物学

• 批准号: 7201615
• 负责人: Angelo M Scanu
• 金额: $ 36.74万
• 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/7201615
• 关键词: Area; Biology; C-terminal; Cardiovascular system; Carotid Arteries; Carotid Artery Plaques; Chemicals; Cleaved cell; Cultured Cells; DNA; Endarterectomy; Exhibits; Genetic Polymorphism; Heterogeneity; Human; In Vitro; Inflammatory; Interleukin-8; Kringles; Lasers; Lesion; Light; Link; Lipoprotein (a); Low-Density Lipoproteins; Lysine; Mass Spectrum Analysis; Metalloproteases; Methods; Monoclonal Antibodies; Nature; Numbers; Pathogenicity; Phospholipids; Play; Predisposition; Production; Property; Protease Domain; Proteins; Proteolysis; Proteomics; Purpose; Recombinants; Reporting; Role; Structure; Techniques; Variant; adduct; apolipoprotein Lp(a+); base; cytokine; macrophage; size

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 apoB100 以 1:1 摩尔比与载脂蛋白（a），apo（a）（一种高度糖基化的多环结构）共价连接。 apo(a) 的特点是其异质性，这是由于其大小多态性取决于 2 型三环重复的数量和对蛋白水解切割的敏感性。对裂解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 端区域（我们称为 F2）中包含的生物活性微结构域（片段）组成。到目前为止，F2 中含有 KIV 的微域的生物活性已得到证实。然而，我们最近表明，F2 中的 KV 蛋白酶结构域 PD 能够刺激培养的人巨噬细胞中白细胞介素 8 的产生，并将这种效应归因于 KV 中与氧化磷脂 ox-PL 连接的赖氨酸残基，并且还表明这些加合物可以被单克隆抗体 EO6 识别。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，我们将继续对不含 PD 的含有 KV 的重组体进行研究，以通过质谱技术明确鉴定与 ox-PL 连接有关的赖氨酸残基以及后者的化学性质。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的apo(a)离散片段。我们还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，对病变人类颈动脉的动脉内膜切除段的富含巨噬细胞的区域进行激光显微切割。拟议研究的结果应有助于阐明 ox-PL 加合物是否在 Lp(a) 的心血管致病性中发挥作用。