Biology of Proteolytic Derivatives of Lp(a)

描述（由申请人提供）：脂蛋白(a), Lp(a)，是一种LDL变体，其蛋白质片段apoB100以1:1的摩尔比例与载脂蛋白(a)，载脂蛋白(a)共价连接，是一种高度糖基化的多分子结构。载脂蛋白(a)的特征是其异质性，这是由于其大小多态性取决于kringle 2型重复序列的数量和对蛋白水解裂解的敏感性。对裂解产物和重组体的体外和细胞培养研究表明，载脂蛋白(a)是由c端F2区域的生物活性微域（片段）组成的。到目前为止，F2中的生物活性已被证实为含有kiv的微结构域。然而，我们最近发现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，并将这种作用定位于KV中与氧化磷脂ox-PL相连的赖氨酸残基，并表明这些加合物可被单克隆抗体EO6识别。在这些观察的基础上，我们将探讨KV-PD有助于Lp的动脉粥样硬化-血栓形成特性的假设(a)。为此，我们将继续研究不含PD的含kv重组体，通过质谱技术明确识别与ox-PLs连接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载脂蛋白(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性质，并将它们与体外载脂蛋白(a)蛋白水解和重组技术产生的产物进行比较。为此，我们将使用免疫化学方法，蛋白质组学和DNA阵列对病变人颈动脉内膜切除术段的富含巨噬细胞的激光微解剖区域进行检测。拟议的研究结果应该阐明ox-PL加合物是否在Lp的心血管致病性中起作用(a)。

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).