The role of N-linked glycosylation on the assembly and secretion of VLDL

载脂蛋白B （apoB）是一种巨大的糖蛋白，对超低密度脂蛋白（VLDL）的组装至关重要。在这项研究中，我们研究了载脂蛋白ob的n -链糖基化在VLDL的组装和分泌中的作用。tunicamycin （TM）处理大鼠肝细胞降低了apoB100的合成和apoB100和apoB48的分泌。转染的McA-RH7777细胞中，重组人载脂蛋白ob变体（即apoB17、B29、B34、B42和B48，分别代表载脂蛋白ob的氨基末端17%、29%等）的分泌也不同程度地减少。由于载脂蛋白ob的氨基末端17%对VLDL组装至关重要，我们测试了B17中单糖基化位点（Asn^<158>）的功能。Asn^<158>- gln取代降低了apoB17的分泌，但对apoB37和apoB48的分泌影响不大。相比之下，所有（五个）糖基化位点的突变减少了apoB50的分泌，其水平与TM处理的野生型相似。此外，诱变显著减少突变的apoB50作为VLDL的分泌，尽管它对作为致密脂蛋白的apoB50分泌的影响相对较小。同样，用TM处理原代大鼠肝细胞可减少apoB48作为VLDL的分泌。在这些细胞中，TM减少了载脂蛋白ob从微粒体膜到管腔的运动，从而减少了管腔中VLDL的出现。因此，我们认为载脂蛋白ob的n -链糖基化在VLDL的组装和分泌中起重要作用。

Apolipoprotein B (apoB) is the huge glycoprotein that is essential for the assembly of very low density lipoproteins (VLDL). In this study, we investigated the role of N-linked glycosylation of apoB in the assembly and secretion of VLDL.Treatment of rat hepatocytes with tunicamycin (TM) decreased synthesis of apoB100 and secretion of apoB100 and apoB48. Secretion of recombinant human apoB variants (i.e. apoB17, B29, B34, B42, and B48 representing the amino-terminal 17%, 29% and so on of apoB) from transfected McA-RH7777 cells was also decreased by TM to different degrees. Since the amino-terminal 17% of apoB is essential for VLDL assembly, we tested the function of the singly glycosylation site (Asn^<158>) within B17. Asn^<158>-to-Gln substitution decreased secretion of apoB17 but had little effect on apoB37 and apoB48 secretion. In contrast, mutagenesis of all (five) glycosylation sites decreased the secretion of apoB50 at levels similar to that of their wild type counterparts treated with TM.Furthermore, the mutagenesis markedly diminished the secretion of mutated apoB50 as VLDL, although it had relatively little effect on apoB50 secretion as dense lipoproteins. Similarly, treatment of primary rat hepatocytes with TM decreased secretion of apoB48 as VLDL.In these cells, TM decreased the movement of apoB from the microsomal membranes to the lumen and thereby diminished the appearance of VLDL in the lumen. Thus, we conclude that N-linked glycosylation of apoB plays an important role in the assembly and secretion of VLDL.