Bradley W A, Hwang S L, Karlin J B, Lin A H, Prasad S C, Gotto A M, Gianturco S H
J Biol Chem. 1984 Dec 10;259(23):14728-35.
Using thrombin and trypsin as probes, we determined: first, that low-density lipoprotein (LDL) receptor binding determinants switch from apolipoprotein (apo) E to apo-B within the very-low-density lipoprotein (VLDL) Sf 20-60 region of the metabolic cascade from VLDL1 (Sf 100-400) of hypertriglyceridemic (HTG) human subjects to LDL. Second, two different conformations of apo-E exist in HTG-VLDL Sf greater than 60, one accessible (greater than or equal to 1 mol/mol of particle) and one inaccessible (1-2 mol/mol) to both thrombin and the LDL receptor; normal VLDL (Sf greater than 60) have only the inaccessible conformation and therefore do not bind to the LDL receptor. Third, thrombin degrades apo-B into large fragments, three of which have electrophoretic mobilities similar to B-48, B-74, and B-26; this, however, has no effect on apo-B-mediated receptor binding. Fibroblast studies showed that thrombin could abolish receptor uptake of HTG-VLDL1 and HTG-VLDL2 (Sf 60-100), had little or no effect on HTG-VLDL3 (Sf 20-60), and no effect on uptake of intermediate-density lipoprotein (IDL) or LDL. Trypsin abolished the binding of HTG-VLDL1 and HTG-VLDL2, reduced that of HTG-VLDL3, but had little to no effect on IDL or LDL binding. Immunochemical techniques revealed that thrombin cleaved some apo-E into the E-22 and E-12 fragments; after trypsin treatment no apo-E was detected in any HTG-lipoprotein. Normal VLDL subclasses contained less apo-E than the corresponding HTG-VLDL subclasses and it was not cleaved by thrombin. Apo-B immunoreactivities of VLDL subclasses were not significantly changed after treatment with thrombin, although thrombin cleaved some of the B-100 of each VLDL subclass, and all apo-B in IDL and LDL, into 4-6 major large fragments. Trypsin converted all of the apo-B of each lipoprotein into smaller fragments (Mr less than 100,000). We conclude that apo-E of the thrombin-accessible conformation mediates uptake of HTG-VLDL1 and HTG-VLDL2 but that apo-B alone is sufficient to mediate receptor binding of IDL and LDL; the switch from apo-E to apo-B as the primary or sufficient binding determinant occurs within the VLDL3 (Sf 20-60) region of the metabolic cascade, where receptor binding first appears in VLDL subclasses from normal subjects.
首先，在高甘油三酯血症（HTG）患者的极低密度脂蛋白（VLDL）从VLDL1（Sf 100 - 400）到低密度脂蛋白（LDL）的代谢级联反应的极低密度脂蛋白（VLDL）Sf 20 - 60区域内，低密度脂蛋白（LDL）受体结合决定簇从载脂蛋白（apo）E转换为apo - B。其次，在HTG - VLDL Sf大于60中存在两种不同构象的apo - E，一种对凝血酶和LDL受体均可及（≥1摩尔/摩尔颗粒），另一种不可及（1 - 2摩尔/摩尔）；正常VLDL（Sf大于60）仅具有不可及构象，因此不与LDL受体结合。第三，凝血酶将apo - B降解为大片段，其中三个片段的电泳迁移率与B - 48、B - 74和B - 26相似；然而，这对apo - B介导的受体结合没有影响。成纤维细胞研究表明，凝血酶可消除HTG - VLDL1和HTG - VLDL2（Sf 60 - 100）的受体摄取，对HTG - VLDL3（Sf 20 - 60）影响很小或无影响，对中间密度脂蛋白（IDL）或LDL的摄取无影响。胰蛋白酶消除了HTG - VLDL1和HTG - VLDL2的结合，降低了HTG - VLDL3的结合，但对IDL或LDL结合影响很小或无影响。免疫化学技术显示，凝血酶将一些apo - E切割成E - 22和E - 12片段；胰蛋白酶处理后，在任何HTG - 脂蛋白中均未检测到apo - E。正常VLDL亚类所含apo - E比相应的HTG - VLDL亚类少，且不被凝血酶切割。尽管凝血酶将每个VLDL亚类的一些B - 100以及IDL和LDL中的所有apo - B切割成4 - 6个主要大片段，但VLDL亚类的apo - B免疫反应性在凝血酶处理后无显著变化。胰蛋白酶将每种脂蛋白的所有apo - B转化为较小片段（分子量小于100,000）。我们得出结论，可及构象的apo - E介导HTG - VLDL1和HTG - VLDL2的摄取，但单独的apo - B足以介导IDL和LDL的受体结合；作为主要或充分结合决定簇从apo - E到apo - B的转换发生在代谢级联反应的VLDL3（Sf 20 - 60）区域，在这里正常受试者的VLDL亚类中首次出现受体结合。
