Biology of Proteolytic Derivatives of Lp(a)

说明书(申请人提供)：脂蛋白(A)，Lp(A)，是一种低密度脂蛋白变异体，具有以1：1摩尔的方式共价连接到载脂蛋白(A)，apo(A)的蛋白质部分apoB100，apo(A)是高度糖基化的多环结构。载脂蛋白(A)的特点是它的异质性，因为它的大小、多态依赖于kringle类型2重复的数量以及对蛋白水解性切割的敏感性。对切割产物和重组体的体外和细胞培养研究表明，apo(A)是由C-末端区域包含的生物活性微域(片段)组成的，我们称之为F2。到目前为止，在F2中已经证明了含有KIV的微域的生物活性。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产生IL-8，并将这种作用归因于KV中与氧化磷脂相连的赖氨酸残基OX-PL，还表明这些加合物可被单抗EO6识别。在这些观察的基础上，我们将探索KV-PD与Lp(A)的动脉粥样硬化-血栓形成特性有关的假设。为此，我们将继续对不含PD的KV重组体进行研究，以明确地鉴定与ox-pls连锁的赖氨酸残基，并利用质谱学技术确定后者的化学性质。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，并将它们与通过apo(A)蛋白分解和重组技术在体外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阵列对受损的人颈动脉内膜切除节段的巨噬细胞丰富区域进行激光显微解剖。建议的研究结果应该有助于阐明OX-PL加合物是否在Lp(A)的心血管致病中起作用。

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).