Piha M, Lindstedt L, Kovanen P T
Wihuri Research Institute, Helsinki, Finland.
Biochemistry. 1995 Aug 15;34(32):10120-9. doi: 10.1021/bi00032a004.
During atherogenesis, lipid droplets appear in the extracellular space of the arterial intima. We previously observed generation of lipid droplets on the surface of exocytosed mast cell granules when granule neutral proteases degraded the granule-bound LDL particles and the particles became unstable and fused [Kovanen, P.T., & Kokkonen, J.O. (1991) J. Biol. Chem. 266, 4430-4436]. We have now extended our studies to the fluid phase and examined the effects of several proteases (trypsin, alpha-chymotrypsin, Pronase, plasmin, kallikrein, and thrombin) all known for their ability to cleave the apolipoprotein B-100 component (apoB-100) of LDL. The fused LDL particles were separated from unfused particles by gel filtration or by density gradient ultracentrifugation. Proteolytic degradation of LDL with trypsin, alpha-chymotrypsin, or Pronase led to fragmentation of apoB-100 and release of the fragments from the LDL particles and triggered particle fusion. In contrast, proteolytic degradation of LDL with plasmin, kallikrein, or thrombin, which also led to fragmentation of apoB-100 but not to release of fragments, did not trigger particle fusion. With advancing degradation of apoB-100, particles having progressively lower densities and larger sizes were generated. Thus, after incubation for 24 h with alpha-chymotrypsin (apoB-100:alpha-chymotrypsin mass ratio 10:1) 40% of the apoB-100 was degraded and about 30% of the LDL particles had fused and reached diameters of up to 70 nm and densities ranging from 1.020 to < 1.005 g/mL. When the proteolyzed LDL particles, both unfused and fused, were incubated with macrophages, only those particles that had undergone fusion were ingested and converted into intracellular cholesteryl ester droplets. Thus proteolysis of LDL with release of apoB-100 fragments renders the particles sufficiently unstable to fuse and thus to become liable to ingestion by macrophages. Since the fused LDL particles resemble the extracellular lipid droplets in the atherosclerotic arterial intima and generate foam cells in vitro, these findings support the idea that proteolytic fusion of LDL is an atherogenic process.
在动脉粥样硬化形成过程中，脂滴出现在动脉内膜的细胞外空间。我们之前观察到，当颗粒中性蛋白酶降解颗粒结合的低密度脂蛋白（LDL）颗粒，使其变得不稳定并融合时，在胞吐的肥大细胞颗粒表面会产生脂滴[科瓦宁，P.T.，& 科科宁，J.O.（1991年）《生物化学杂志》266卷，4430 - 4436页]。我们现在将研究扩展到液相，并研究了几种蛋白酶（胰蛋白酶、α - 糜蛋白酶、链霉蛋白酶、纤溶酶、激肽释放酶和凝血酶）的作用，这些蛋白酶都以能够切割LDL的载脂蛋白B - 100成分（apoB - 100）而闻名。通过凝胶过滤或密度梯度超速离心将融合的LDL颗粒与未融合的颗粒分离。用胰蛋白酶、α - 糜蛋白酶或链霉蛋白酶对LDL进行蛋白水解降解会导致apoB - 100片段化，并使片段从LDL颗粒中释放出来，从而引发颗粒融合。相比之下，用纤溶酶、激肽释放酶或凝血酶对LDL进行蛋白水解降解，虽然也会导致apoB - 100片段化，但不会使片段释放，不会引发颗粒融合。随着apoB - 100降解程度的加深，会产生密度逐渐降低、尺寸逐渐增大的颗粒。因此，用α - 糜蛋白酶（apoB - 100与α - 糜蛋白酶质量比为10:1）孵育24小时后，40%的apoB - 100被降解，约30%的LDL颗粒发生融合，直径达到70纳米，密度范围为1.020至<1.005克/毫升。当将未融合和已融合的经蛋白水解的LDL颗粒与巨噬细胞一起孵育时，只有那些发生融合的颗粒会被摄取并转化为细胞内胆固醇酯滴。因此，LDL的蛋白水解并释放apoB - 100片段会使颗粒变得足够不稳定从而发生融合，进而易于被巨噬细胞摄取。由于融合的LDL颗粒类似于动脉粥样硬化动脉内膜中的细胞外脂滴，并在体外产生泡沫细胞，这些发现支持了LDL的蛋白水解融合是一个动脉粥样硬化形成过程的观点。
