Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa 277-8562 Chiba, Japan.
Glycobiology. 2012 Dec;22(12):1709-20. doi: 10.1093/glycob/cws114. Epub 2012 Jul 20.
Newly synthesized glycoproteins destined for secretion are transported from the endoplasmic reticulum (ER), through the Golgi and toward the cell surface. In this secretion pathway, several intracellular ER- or Golgi-resident transmembrane proteins serve as cargo receptors. ER-Golgi intermediate compartment (ERGIC)-53, VIP36 and VIPL, which have an L-type lectin domain within the luminal portion, participate in the vectorial transport of glycoproteins via sugar-protein interactions. To understand the nature of these receptors, monoclonal antibodies were generated against human ERGIC-53, VIP36 and VIPL using 293T cells expressing these receptors on cell surfaces. These cells were used to immunize rats and for screening antibody-producing clones. Flow cytometric analysis and immunoprecipitation studies showed that the obtained monoclonal antibodies bound specifically to the corresponding cargo receptors. Immunostaining of HeLa cells using the monoclonal antibodies showed that the localization of ERGIC-53 changed from relatively broad distribution in both the ER and the Golgi under normal conditions to a compact distribution in the Golgi under ER stress conditions. This redistribution was also observed by the overexpression of ERGIC-53 and abrogated by co-expression with VIPL but not VIP36. Real-time polymerase chain reaction revealed that ERGIC-53 along with several chaperone proteins was up-regulated after tunicamycin treatment; however, the expression of VIPL was unchanged. Furthermore, ERGIC-53 co-precipitated with VIPL but not VIP36, indicating that ERGIC-53 may interact with VIPL in the ER, which may regulate the localization of ERGIC-53 inside cells. Taken together, these observations provide new insights into the regulation of these cargo receptors and the quality control of glycoproteins within cells.
新合成的分泌型糖蛋白从内质网（ER）经高尔基氏体（Golgi）向细胞表面运输。在这条分泌途径中，几种细胞内的内质网或高尔基体内的跨膜蛋白作为货物受体。内质网-高尔基体中间池（ERGIC）-53、VIP36 和 VIPL，在腔部分内具有 L 型凝集素结构域，通过糖蛋白相互作用参与糖蛋白的定向运输。为了了解这些受体的性质，使用在细胞表面表达这些受体的 293T 细胞，针对人 ERGIC-53、VIP36 和 VIPL 生成了单克隆抗体。这些细胞被用于免疫大鼠并筛选产生抗体的克隆。流式细胞分析和免疫沉淀研究表明，获得的单克隆抗体特异性结合相应的货物受体。使用单克隆抗体对 HeLa 细胞进行免疫染色显示，在 ER 应激条件下，ERGIC-53 的定位从正常条件下在 ER 和高尔基体中相对广泛的分布转变为高尔基体中的紧凑分布。这种重分布也可以通过 ERGIC-53 的过表达观察到，并通过与 VIPL 共表达而不是 VIP36 消除。实时聚合酶链反应显示，在衣霉素处理后，ERGIC-53 与几种伴侣蛋白一起上调；然而，VIPL 的表达没有变化。此外，ERGIC-53 与 VIPL 共沉淀，但与 VIP36 不沉淀，表明 ERGIC-53 可能在 ER 中与 VIPL 相互作用，这可能调节 ERGIC-53 在细胞内的定位。总之，这些观察结果为这些货物受体的调节和细胞内糖蛋白的质量控制提供了新的见解。
