Edelstein C, Mandala M, Pfaffinger D, Scanu A M
Department of Medicine, University of Chicago, Illinois 60637, USA.
Biochemistry. 1995 Dec 19;34(50):16483-92. doi: 10.1021/bi00050a032.
We previously observed that rhesus monkey lipoprotein(a) [Lp(a)], is lysine-binding defective (Lys-) and attributed this deficiency to the presence of Arg72 in the lysine-binding site (LBS) of kringle IV-10 of apolipoprotein(a) [apo(a)] [Scanu, A.M., Miles, L.A., Fless, G.M., Pfaffinger, D., Eisenbart, J., Jackson, E., Hoover-Plow, J.L., Brunck, T., & Plow, E.F. (1993) J. Clin. Invest. 91, 283-291]. We also identified human mutants having Arg72 instead of Trp72 (wild type) in the LBS of kringle IV-10 [Scanu, A M., Pfaffinger, D., lEE, J.C., & Hinman, J. (1994) Biochim. Biophys. Acta 1227, 41-45]. Unique to the human mutant phenotype were the very low levels of plasma Lp(a), suggesting structural differences between human and rhesus apo(a) and a possible divergent mode of Lp(a) assembly. In order to explore the possibility of a relationship between apo(a) LBS and Lp(a) assembly, we developed a novel method for isolating wild-type and mutant apo(a) phenotypes in a free form by subjecting each parent Lp(a) to mild reductive conditions using 2 mM dithioerythritol (DTE) and 100 mM of the lysine analogue, epsilon-aminocaproic acid (EACA). The application of this method to the study of wild-type and mutant apo(a) species showed that regardless of the source of Lp(a), i.e., positive lysine binding (Lys+) or negative lysine binding (Lys-), all of the isolated free apo(a)s were Lys+. Moreover, incubation of free apo(a)s with their autologous human or rhesus low-density lipoproteins (LDL) generated Lp(a) complexes which were structurally and functionally indistinguishable from their parent native Lp(a). In each instance, the reassembly process was inhibited by the presence of either EACA or proline. These two reagents had a minimal effect on either Lp(a) or reassembled Lp(a) [RLp(a)]. Free apo(a) bound to apoB100 of very low density lipoproteins (VLDL) to form a triglyceride-rich Lp(a). These results show that (1) both human and rhesus Lp(a) are amenable to dissassembly and reassembly, (2) the presence of Arg72 in the LBS of kringle IV-10 is not involved, at least directly, in this process, (3) its cleavage from apoB100 opens up in apo(a) a domain that is both EACA and proline sensitive and involved in Lp(a) assembly, and (4) the apoB100 of VLDL is also competent to bind apo(a). Our observations also suggest that the difference in plasma Lp(a) levels between the rhesus and the human mutant, both having Arg72 in the LBS of apo(a) kringle IV-10, is not related to the assembly process, but more likely to a divergence in production/secretion rates between the two apo(a) phenotypes.
我们之前观察到，恒河猴脂蛋白(a)[Lp(a)]存在赖氨酸结合缺陷（Lys-），并将这种缺陷归因于载脂蛋白(a)[apo(a)]kringle IV-10赖氨酸结合位点（LBS）中存在的精氨酸72 [斯坎努，A.M., 迈尔斯，L.A., 弗莱斯，G.M., 普法芬格，D., 艾森巴特，J., 杰克逊，E., 胡佛-普洛，J.L., 布伦克，T., & 普洛，E.F. (1993) 《临床研究杂志》91, 283 - 291]。我们还鉴定出在kringle IV-10的LBS中具有精氨酸72而非色氨酸72（野生型）的人类突变体[斯坎努，A.M., 普法芬格，D., 李，J.C., & 欣曼，J. (1994) 《生物化学与生物物理学报》1227, 41 - 45]。人类突变体表型的独特之处在于血浆Lp(a)水平极低，这表明人类和恒河猴apo(a)之间存在结构差异以及Lp(a)组装可能存在不同模式。为了探究apo(a) LBS与Lp(a)组装之间关系的可能性，我们开发了一种新方法，通过使用2 mM二硫苏糖醇（DTE）和100 mM赖氨酸类似物ε-氨基己酸（EACA）对每个亲本Lp(a)进行温和还原条件处理，以游离形式分离野生型和突变型apo(a)表型。将该方法应用于野生型和突变型apo(a)物种的研究表明，无论Lp(a)的来源如何，即正赖氨酸结合（Lys+）或负赖氨酸结合（Lys-），所有分离出的游离apo(a)都是Lys+。此外，将游离apo(a)与其自身的人类或恒河猴低密度脂蛋白（LDL）孵育会产生Lp(a)复合物，这些复合物在结构和功能上与其亲本天然Lp(a)无法区分。在每种情况下，EACA或脯氨酸的存在都会抑制重新组装过程。这两种试剂对Lp(a)或重新组装的Lp(a)[RLp(a)]影响极小。游离apo(a)与极低密度脂蛋白（VLDL）的apoB100结合形成富含甘油三酯的Lp(a)。这些结果表明：(1)人类和恒河猴Lp(a)都适合进行分解和重新组装；(2)kringle IV-10的LBS中精氨酸72的存在至少直接不参与此过程；(3)其从apoB100的切割在apo(a)中打开了一个对EACA和脯氨酸敏感且参与Lp(a)组装的结构域；(4)VLDL的apoB100也能够结合apo(a)。我们的观察结果还表明，在apo(a) kringle IV-10的LBS中都具有精氨酸72的恒河猴和人类突变体之间血浆Lp(a)水平的差异与组装过程无关，而更可能与两种apo(a)表型的产生/分泌速率差异有关。
