Parent J B, Yeo T K, Yeo K T, Olden K
Mol Cell Biochem. 1986 Nov-Dec;72(1-2):21-33. doi: 10.1007/BF00230633.
To investigate our earlier hypothesis that carbohydrates play a regulatory role in the intracellular transport of secretory glycoproteins, we used 1-deoxynojirimycin (DNJ), and inhibitor of glucosidase I and II of the rough endoplasmic reticulum (RER), to modify the structure of N-linked glycan moieties of secretory glycoproteins of human hepatoma (Hep G2) cells in culture. Using a pulse-chase protocol, we found that treatment of Hep G2 cultures with 1.25 mM DNJ markedly reduced the rate of secretion of alpha 1-protease inhibitor, ceruloplasmin, and alpha 2-macroglobulin, but had no effect on the export of fibronectin, alpha-fetoprotein and transferrin, nor on albumin which lacks carbohydrate. For example, 50% of newly synthesized alpha 1-protease inhibitor, the glycoprotein most dramatically affected, was secreted by 27 min in control cultures versus 110 min in DNJ-treated cultures. Percoll gradient cell fractionation analyses revealed that DNJ inhibited transport of the affected secretory glycoproteins in the RER segment of the ER/Golgi pathway. For example, 50% of newly synthesized alpha 1-protease inhibitor was lost from the RER fraction by 10 min in untreated cells, but 70 min was required for the transport of a similar amount of protein in DNJ-treated cells. DNJ treatment also inhibited the rate at which the N-linked glycan moieties of the affected glycoproteins became resistant to endo H in the Golgi. Since the glycan moiety of secreted forms of the affected glycoproteins were fully processed to the complex structure, suggesting escape from DNJ inhibition, we concluded that removal of terminal glucose residues from the glycan chain of secretory glycoproteins is required for their transport from the RER to the Golgi. We suggest that the oligosaccharide moieties on alpha 1-protease inhibitor, ceruloplasmin and alpha 2-macroglobulin form part of the binding site for a receptor which regulates transport of these glycoproteins.
为了验证我们早期提出的假说，即碳水化合物在分泌性糖蛋白的细胞内运输中起调节作用，我们使用了1-脱氧野尻霉素（DNJ），一种粗面内质网（RER）中葡糖苷酶I和II的抑制剂，来改变培养的人肝癌（Hep G2）细胞分泌性糖蛋白的N-连接聚糖部分的结构。采用脉冲追踪实验方案，我们发现用1.25 mM DNJ处理Hep G2培养物可显著降低α1-蛋白酶抑制剂、铜蓝蛋白和α2-巨球蛋白的分泌速率，但对纤连蛋白、甲胎蛋白和转铁蛋白的输出没有影响，对不含碳水化合物的白蛋白也没有影响。例如，受影响最显著的糖蛋白——新合成的α1-蛋白酶抑制剂，在对照培养物中27分钟时有50%分泌，而在DNJ处理的培养物中则为110分钟。Percoll梯度细胞分级分离分析表明，DNJ抑制了内质网/高尔基体途径的RER段中受影响的分泌性糖蛋白的运输。例如，在未处理的细胞中，10分钟时新合成的α1-蛋白酶抑制剂有50%从RER组分中消失，但在DNJ处理的细胞中，运输等量蛋白质需要70分钟。DNJ处理还抑制了受影响糖蛋白的N-连接聚糖部分在高尔基体中对内切糖苷酶H产生抗性的速率。由于受影响糖蛋白分泌形式的聚糖部分已完全加工成复杂结构，表明已逃脱DNJ的抑制，我们得出结论，分泌性糖蛋白聚糖链上末端葡萄糖残基的去除是其从RER运输到高尔基体所必需的。我们认为，α1-蛋白酶抑制剂、铜蓝蛋白和α2-巨球蛋白上的寡糖部分构成了调节这些糖蛋白运输的受体结合位点的一部分。
