Paananen K, Saarinen J, Annila A, Kovanen P T
Wihuri Research Institute, Helsinki, Finland.
J Biol Chem. 1995 May 19;270(20):12257-62. doi: 10.1074/jbc.270.20.12257.
Lipid droplets resembling those seen in the extracellular space of the arterial intima were generated in vitro when granule proteases of rat serosal mast cells degraded the apolipoprotein B-100 (apoB-100) component of granule-bound low density lipoprotein (LDL), and the particles fused on the granule surface (Paanenen, K., and Kovanen, P. T. (1994) J. Biol. Chem. 269, 2023-2031). Moreover, the binding of the fused particles to the heparin proteoglycan component of the granules was found to be strengthened. We have now treated LDL particles with alpha-chymotrypsin and examined the strength with which the proteolytically modified LDL binds to human aortic proteoglycans on an affinity column. We found that chymotryptic degradation of the LDL particles triggered particle fusion. The higher the degree of proteolytic degradation, the higher were the degree of fusion and the strength of binding to the aortic proteoglycans. Separation of the proteolyzed particles by size exclusion chromatography into two fractions, unfused and fused particles, and analysis of their binding strengths revealed that not only the fused but also the unfused proteolyzed particles bound more tightly to the proteoglycans than did the native LDL particles. To investigate the mechanism underlying this increase in binding strength, we attached [13C]dimethyl groups to the lysines and used NMR spectroscopy to quantify the active lysine residues of apoB-100, which are thought to be located in basic areas of apoB-100 and involved in binding of LDL to proteoglycans. Analysis of the 13C-labeled particles showed that, despite loss of apoB-100 fragments from the particles, the number of active lysine residues in the unfused proteolyzed particles had not decreased. In the fused proteolyzed particles, the number of active lysine residues was markedly increased. Thus, proteolytic fusion appears to increase the number of basic domains of apoB-100, which would explain the observed increase in the strength of binding of the modified LDL particles to arterial proteoglycans. Since the fused particles resemble the small lipid droplets found in the atherosclerotic arterial intima, this LDL modification offers a plausible mechanism for the focal accumulation of lipid droplets in the extracellular proteoglycan matrix during atherogenesis.
当大鼠浆膜肥大细胞的颗粒蛋白酶降解颗粒结合的低密度脂蛋白（LDL）的载脂蛋白B - 100（apoB - 100）成分时，体外产生了类似于在动脉内膜细胞外空间中看到的脂滴，并且这些颗粒在颗粒表面融合（帕内嫩，K.，和科瓦宁，P. T.（1994年）《生物化学杂志》269，2023 - 2031）。此外，发现融合颗粒与颗粒的肝素蛋白聚糖成分的结合得到加强。我们现在用α - 胰凝乳蛋白酶处理LDL颗粒，并在亲和柱上检测经蛋白水解修饰的LDL与人主动脉蛋白聚糖结合的强度。我们发现LDL颗粒的胰凝乳蛋白酶降解引发了颗粒融合。蛋白水解降解程度越高，融合程度和与主动脉蛋白聚糖的结合强度就越高。通过尺寸排阻色谱将蛋白水解颗粒分离为未融合和融合颗粒两个部分，并分析它们的结合强度，结果显示不仅融合的而且未融合的蛋白水解颗粒都比天然LDL颗粒更紧密地结合到蛋白聚糖上。为了研究这种结合强度增加的潜在机制，我们将[13C]二甲基基团连接到赖氨酸上，并使用核磁共振光谱法对apoB - 100的活性赖氨酸残基进行定量，这些残基被认为位于apoB - 100的碱性区域并参与LDL与蛋白聚糖的结合。对13C标记颗粒的分析表明，尽管颗粒中apoB - 100片段有所丢失，但未融合的蛋白水解颗粒中活性赖氨酸残基的数量并未减少。在融合的蛋白水解颗粒中，活性赖氨酸残基的数量显著增加。因此，蛋白水解融合似乎增加了apoB - 100的碱性结构域数量，这可以解释观察到的修饰LDL颗粒与动脉蛋白聚糖结合强度的增加。由于融合颗粒类似于在动脉粥样硬化动脉内膜中发现的小脂滴，这种LDL修饰为动脉粥样硬化形成过程中细胞外蛋白聚糖基质中脂滴的局部积累提供了一种合理的机制。
