van Vlijmen B J, van Dijk K W, van't Hof H B, van Gorp P J, van der Zee A, van der Boom H, Breuer M L, Hofker M H, Havekes L M
TNO Prevention and Health, Gaubius Laboratory, 2301 CE Leiden, The Netherlands.
J Biol Chem. 1996 Nov 29;271(48):30595-602. doi: 10.1074/jbc.271.48.30595.
Apolipoprotein E2(Arg-158 --> Cys) (APOE2) transgenic mice were generated and compared to the previously generated apolipoprotein E3-Leiden (APOE3-Leiden) transgenic mice to study the variable expression of hyperlipoproteinemia associated with these two APOE variants. In the presence of the endogenous mouse Apoe gene, the expression of the APOE3-Leiden gene resulted in slightly elevated levels of serum cholesterol as compared with control mice (2.7 +/- 0. 5 versus 2.1 +/- 0.2 mmol/liter, respectively), whereas the expression of the APOE2(Arg-158 --> Cys) gene did not affect serum cholesterol levels, even after high/fat cholesterol feeding. The extreme cholesterol level usually found in apoE-deficient mice (Apoe-/- mice; 23.6 +/- 5.0 mmol/liter) could be rescued by introducing the APOE3-Leiden gene (APOE3-Leiden.Apoe-/-; 3.6 +/- 1. 5 mmol/liter), whereas the expression of the APOE2(Arg-158 --> Cys) gene in Apoe-/- mice minimally reduced serum cholesterol levels (APOE2.Apoe-/-; 16.6 +/- 2.9 mmol/liter). In vivo very low density lipoprotein (VLDL) turnover studies revealed that APOE2.Apoe-/- VLDL and APOE3-Leiden.Apoe-/- VLDL display strongly reduced fractional catabolic rates as compared with control mouse VLDL (4.0 and 6.1 versus 22.1 pools/h). In vitro low density lipoprotein (LDL) receptor binding studies using HepG2 and J774 cells showed that APOE2. Apoe-/- VLDL is completely defective in binding to the LDL receptor, whereas APOE3-Leiden.Apoe-/- VLDL still displayed a considerable binding activity to the LDL receptor. After transfection of APOE2.Apoe-/- and APOE3-Leiden.Apoe-/- mice with adenovirus carrying the gene for the receptor-associated protein (AdCMV-RAP), serum lipid levels strongly increased (15.3 to 42.8 and 1.4 to 15.3 mmol/liter for cholesterol and 5.0 to 35.7 and 0.3 to 20. 7 mmol/liter for triglycerides, respectively). This indicates that RAP-sensitive receptors, possibly the LDL receptor-related protein (LRP), mediate the plasma clearance of both APOE2.Apoe-/- and APOE3-Leiden. Apoe-/- VLDL. We conclude that in vivo the APOE2 variant is completely defective in LDL receptor binding but not in binding to LRP, whereas for the APOE3-Leiden mutant both LRP and LDL receptor binding activity are only mildly affected. As a consequence of this difference, APOE2.Apoe-/- develop more severe hypercholesterolemia than APOE3-Leiden.Apoe-/- mice.
构建了载脂蛋白E2（精氨酸158→半胱氨酸）（APOE2）转基因小鼠，并与先前构建的载脂蛋白E3-莱顿（APOE3-莱顿）转基因小鼠进行比较，以研究与这两种APOE变体相关的高脂蛋白血症的可变表达。在内源性小鼠Apoe基因存在的情况下，与对照小鼠相比，APOE3-莱顿基因的表达导致血清胆固醇水平略有升高（分别为2.7±0.5与2.1±0.2 mmol/升），而APOE2（精氨酸158→半胱氨酸）基因的表达即使在高胆固醇/高脂肪喂养后也不影响血清胆固醇水平。通常在载脂蛋白E缺陷小鼠（Apoe-/-小鼠；23.6±5.0 mmol/升）中发现的极高胆固醇水平可通过引入APOE3-莱顿基因得到挽救（APOE3-莱顿.Apoe-/-；3.6±1.5 mmol/升），而APOE2（精氨酸158→半胱氨酸）基因在Apoe-/-小鼠中的表达仅使血清胆固醇水平略有降低（APOE2.Apoe-/-；16.6±2.9 mmol/升）。体内极低密度脂蛋白（VLDL）周转研究表明，与对照小鼠VLDL相比，APOE2.Apoe-/- VLDL和APOE3-莱顿.Apoe-/- VLDL的分数分解代谢率显著降低（分别为4.0和6.1与22.1池/小时）。使用HepG2和J774细胞进行的体外低密度脂蛋白（LDL）受体结合研究表明，APOE2.Apoe-/- VLDL与LDL受体结合完全缺陷，而APOE3-莱顿.Apoe-/- VLDL对LDL受体仍显示出相当大的结合活性。用携带受体相关蛋白基因的腺病毒（AdCMV-RAP）转染APOE2.Apoe-/-和APOE3-莱顿.Apoe-/-小鼠后，血清脂质水平大幅升高（胆固醇分别从15.3升至42.8和从1.4升至15.3 mmol/升，甘油三酯分别从5.0升至35.7和从0.3升至20.7 mmol/升）。这表明对RAP敏感的受体，可能是低密度脂蛋白受体相关蛋白（LRP），介导了APOE2.Apoe-/-和APOE3-莱顿.Apoe-/- VLDL的血浆清除。我们得出结论，在体内，APOE2变体在LDL受体结合方面完全缺陷，但在与LRP结合方面无缺陷，而对于APOE3-莱顿突变体，LRP和LDL受体结合活性仅受到轻微影响。由于这种差异，APOE2.Apoe-/-比APOE3-莱顿.Apoe-/-小鼠发生更严重的高胆固醇血症。
