Hussain M M, Zanni E E, Kelly M, Zannis V I
Section of Molecular Genetics, Boston University Medical School, MA.
Biochim Biophys Acta. 1989 Jan 23;1001(1):90-101. doi: 10.1016/0005-2760(89)90311-1.
We have studied apolipoprotein synthesis, intracellular modification and secretion by primary adult rat hepatocyte cultures using continuous pulse or pulse chase labeling with [35S]methionine, immunoprecipitation and two-dimensional isoelectric focusing/polyacrylamide gel electrophoresis. The flotation properties of the newly secreted apolipoproteins were studied by discontinuous density gradient ultracentrifugation and one- and two-dimensional polyacrylamide gel electrophoresis. These studies showed that rat hepatocyte apoE is modified intracellularly to produce minor isoproteins that differ in size and charge. One of these minor isoproteins represents a monosialated apoE form (apoE3s1). Similarly, apoCIII is modified intracellularly to produce a disialated apoCIII form (apoCIIIs2), whereas newly synthesized apoA-I and apoA-IV are not glycosylated and overlap on two-dimensional gels with the proapoA-I and the plasma apoA-IV form, respectively. Both unmodified and modified apolipoproteins are secreted into the medium. Separation of secreted apolipoproteins by density gradient ultracentrifugation has shown that 50% of apoE, 80% of apoA-I, and more than 90% of apoA-IV and apoCIII are secreted in a lipid-poor form, whereas apoB-100 and apoB-48 are 100% associated with lipids. ApoB-100 floats in the VLDL and IDL regions, whereas apoB-48 is found in all lipoprotein fractions. ApoE and small amounts of apoA-I, apoA-IV and apoCIII float in the HDL region. Small amounts of apoE and apoCIII are also found in the VLDL and IDL regions, and apoE in the LDL region. Ultracentrifugation of nascent lipoproteins in the presence of rat serum promoted flotation of apoA-I and apoA-IV in the HDL fraction and resulted in increased flotation and distribution of apoE and apoCs in VLDL, IDL and LDL regions. These observations are consistent with the hypothesis that intracellular assembly of lipoproteins involves apoB-48 and apoB-100 forms, whereas a large portion of apoA-I, apoCIII and apoA-IV can be secreted in a lipid-poor form, which associates extracellularly with preexisting lipoproteins.
我们利用[35S]甲硫氨酸连续脉冲或脉冲追踪标记、免疫沉淀以及二维等电聚焦/聚丙烯酰胺凝胶电泳，对原代成年大鼠肝细胞培养物中的载脂蛋白合成、细胞内修饰和分泌进行了研究。通过不连续密度梯度超速离心以及一维和二维聚丙烯酰胺凝胶电泳，研究了新分泌载脂蛋白的漂浮特性。这些研究表明，大鼠肝细胞载脂蛋白E在细胞内被修饰，产生大小和电荷不同的次要同型蛋白。其中一种次要同型蛋白代表单唾液酸化的载脂蛋白E形式（apoE3s1）。同样，载脂蛋白CIII在细胞内被修饰，产生双唾液酸化的载脂蛋白CIII形式（apoCIIIs2），而新合成的载脂蛋白A-I和载脂蛋白A-IV不进行糖基化，在二维凝胶上分别与前载脂蛋白A-I和血浆载脂蛋白A-IV形式重叠。未修饰和修饰的载脂蛋白均分泌到培养基中。通过密度梯度超速离心分离分泌的载脂蛋白表明，50%的载脂蛋白E、80%的载脂蛋白A-I以及超过90%的载脂蛋白A-IV和载脂蛋白CIII以脂质贫乏的形式分泌，而载脂蛋白B-100和载脂蛋白B-48则100%与脂质结合。载脂蛋白B-100漂浮在极低密度脂蛋白（VLDL）和中间密度脂蛋白（IDL）区域，而载脂蛋白B-48存在于所有脂蛋白组分中。载脂蛋白E以及少量的载脂蛋白A-I、载脂蛋白A-IV和载脂蛋白CIII漂浮在高密度脂蛋白（HDL）区域。在VLDL和IDL区域也发现少量的载脂蛋白E和载脂蛋白CIII，在低密度脂蛋白（LDL）区域发现载脂蛋白E。在大鼠血清存在的情况下对新生脂蛋白进行超速离心，促进载脂蛋白A-I和载脂蛋白A-IV在HDL组分中的漂浮，并导致载脂蛋白E和载脂蛋白C在VLDL、IDL和LDL区域的漂浮和分布增加。这些观察结果与以下假设一致：脂蛋白的细胞内组装涉及载脂蛋白B-48和载脂蛋白B-100形式，而很大一部分载脂蛋白A-I、载脂蛋白CIII和载脂蛋白A-IV可以以脂质贫乏的形式分泌，在细胞外与预先存在的脂蛋白结合。
