Becker Lev, Nesheim Michael E, Koschinsky Marlys L
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Biochemistry. 2006 Aug 15;45(32):9919-28. doi: 10.1021/bi060283t.
The assembly of lipoprotein(a) (Lp(a)) particles occurs via a two-step mechanism in which noncovalent interactions between apolipoprotein(a) (apo(a)) and the apolipoproteinB-100 component of low density lipoprotein precede the formation of a single disulfide bond. Although we have previously demonstrated that the rate constant for the covalent step of Lp(a) assembly can be enhanced by altering the conformational status of apo(a), the resultant rates of covalent Lp(a) particle formation measured in vitro are relatively slow. The large excess of Lp(a) (over apo(a)) observed in vivo can be accounted for by a preferential clearance of apo(a) over Lp(a) and/or a sufficiently high rate of covalent Lp(a) assembly. In the present study, we report that cultured human hepatoma cells secrete an oxidase activity that dramatically enhances the rate of covalent Lp(a) assembly. This activity is likely possessed by a protein because it is heat-sensitive and is retained in the concentrate following ultrafiltration through a 5 kDa cutoff filter. However, a small molecule cofactor for the activity is suggested by the observation that the activity is lost upon dialysis. Plots of Lp(a) assembly rate versus input apo(a) concentration gave rectangular hyperbolae; the reaction displayed an unusual dependence on the concentration of apoB-100, with increasing concentrations of apoB-100 resulting in slower rates of Lp(a) assembly at low concentrations of apo(a), an effect that was alleviated by higher apo(a) concentrations. Interestingly, V(max(app))/K(m(app)) ratios were insensitive to apoB-100 concentration, which is diagnostic of a ping-pong reaction mechanism. In this way, the putative Lp(a) oxidase may be functionally analogous to protein disulfide isomerase, which exhibits a similar mechanism during the catalysis of disulfide bond formation during protein folding, although we have ruled out a role for this enzyme in Lp(a) assembly.
脂蛋白(a)[Lp(a)]颗粒的组装通过两步机制进行，其中载脂蛋白(a)[apo(a)]与低密度脂蛋白的载脂蛋白B-100成分之间的非共价相互作用先于单个二硫键的形成。尽管我们之前已经证明，通过改变apo(a)的构象状态可以提高Lp(a)组装共价步骤的速率常数，但体外测定的共价Lp(a)颗粒形成的最终速率相对较慢。体内观察到的Lp(a)（相对于apo(a)）大量过剩，可以通过apo(a)相对于Lp(a)的优先清除和/或共价Lp(a)组装的足够高的速率来解释。在本研究中，我们报告培养的人肝癌细胞分泌一种氧化酶活性，该活性显著提高共价Lp(a)组装的速率。这种活性可能由一种蛋白质拥有，因为它对热敏感，并且在通过5 kDa截留滤器超滤后的浓缩物中保留。然而，透析后活性丧失的观察结果提示存在一种小分子辅因子。Lp(a)组装速率与输入apo(a)浓度的关系图呈矩形双曲线；该反应对apoB-100浓度表现出异常依赖性，在低浓度apo(a)时，随着apoB-100浓度增加，Lp(a)组装速率减慢，而在较高apo(a)浓度时这种效应得到缓解。有趣的是，V(max(app))/K(m(app))比值对apoB-100浓度不敏感，这是乒乓反应机制的特征。通过这种方式，推测的Lp(a)氧化酶在功能上可能类似于蛋白质二硫键异构酶，后者在蛋白质折叠过程中二硫键形成的催化过程中表现出类似的机制，尽管我们已经排除了该酶在Lp(a)组装中的作用。
