Thrift R N, Drisko J, Dueland S, Trawick J D, Davis R A
Atherosclerosis Research Center, University of Colorado Health Sciences Center, Denver 80262.
Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9161-5. doi: 10.1073/pnas.89.19.9161.
To explore the process of lipoprotein assembly, plasmids encoding truncated forms of apolipoprotein B (apoB) were transfected into Chinese hamster ovary (CHO) fibroblasts. (One, encoding apoB53, the N-terminal 53% of apoB100, can direct the assembly and secretion of lipoproteins when expressed in hepatoma cells, while the other, encoding the shorter apoB15, does not direct lipoprotein assembly.) Expression of apoB15 in CHO cells resulted in the accumulation of apoB15 protein in both medium and cells. In contrast, apoB was not detectable in medium or within CHO cells transfected with the plasmid encoding apoB53, despite the expression of apoB53 mRNA. ApoB53 did accumulate within transfected cells incubated with the thiol protease inhibitor N-acetylleucylleucylnorleucinal (ALLN), suggesting that it is synthesized but completely degraded in the absence of the inhibitor. ApoB53 was not secreted despite its presence within ALLN-treated cells. Essentially all the apoB53 that accumulated in microsomes from ALLN-treated cells was associated with the membrane and was susceptible to degradation by exogenous trypsin, indicating exposure on the cytoplasmic face of the membrane. Thus, translocation of apoB53 across the endoplasmic reticulum membrane is blocked. However, the apoB53 bound to concanavalin A, suggesting that it is glycosylated and therefore partly exposed to the lumen as well. ApoB requires a unique process, not expressed in CHO fibroblasts, for its complete translocation and entrance into the secretory pathway. This process might account for the inability of abetalipoproteinemic patients to secrete apoB.
为了探究脂蛋白组装的过程，将编码截短形式载脂蛋白B（apoB）的质粒转染到中国仓鼠卵巢（CHO）成纤维细胞中。（一种编码apoB53，即apoB100的N端53%，在肝癌细胞中表达时可指导脂蛋白的组装和分泌，而另一种编码较短的apoB15，则不能指导脂蛋白组装。）apoB15在CHO细胞中的表达导致apoB15蛋白在培养基和细胞中都有积累。相比之下，在用编码apoB53的质粒转染的CHO细胞的培养基或细胞内未检测到apoB，尽管有apoB53 mRNA的表达。在用巯基蛋白酶抑制剂N - 乙酰亮氨酰 - 亮氨酰 - 正亮氨酸（ALLN）处理的转染细胞中，apoB53确实有积累，这表明它已被合成，但在没有抑制剂的情况下会完全降解。尽管apoB53存在于经ALLN处理的细胞中，但并未分泌。基本上，在经ALLN处理的细胞的微粒体中积累的所有apoB53都与膜结合，并且易被外源性胰蛋白酶降解，这表明它暴露在膜的细胞质面上。因此，apoB53跨内质网膜的转运受阻。然而，apoB53与伴刀豆球蛋白A结合，这表明它已被糖基化，因此也部分暴露于内质网腔中。apoB需要一个在CHO成纤维细胞中未表达的独特过程，才能完成其转运并进入分泌途径。这个过程可能解释了无β脂蛋白血症患者无法分泌apoB的原因。
