A monoclonal antibody against human homocysteine-induced endoplasmic reticulum protein (Herp): a useful tool for evaluating endoplasmic reticulum stress.
A monoclonal antibody against human homocysteine-induced endoplasmic reticulum protein (Herp): a useful tool for evaluating endoplasmic reticulum stress.
复制标题
DOI：
10.1620/tjem.212.431
复制
发表时间：
2007-08
期刊：
影响因子：
--
通讯作者：
Y. Oka;Y. Hirabayashi;T. Ishii;R. Takahashi;Takeshi Sasaki
中科院分区：
文献类型：
--
作者：
Y. Oka;Y. Hirabayashi;T. Ishii;R. Takahashi;Takeshi Sasaki
高同型半胱氨酸血症是血管损伤的危险因素之一。同型半胱氨酸诱导血管内皮细胞的内质网（ER）应激，随后产生同型半胱氨酸诱导的ER蛋白（Herp）。Herp被认为具有对抗ER应激和抑制细胞凋亡的保护作用，但其具体机制尚不清楚。为了精确检测Herp蛋白，我们建立了一个产生抗人Herp单克隆抗体（mAb）的鼠杂交瘤克隆，命名为HT 2。经酶联免疫吸附试验（ELISA）和Western blot分析证实HT 2 mAb与Herp特异性结合。在ELISA中，HT 2 mAb能够以剂量依赖的方式与Herp结合，并且其结合被重组Herp中断。在Western印迹分析中，对应于Herp的54-kDa条带用HT 2 mAb在未处理的HeLa细胞的膜部分中检测到，并且其表达在已经用同型半胱氨酸、毒胡萝卜素或2-巯基乙醇处理的ER应激HeLa细胞中显著增加。重要的是，在用印迹膜孵育之前，通过用重组Herp吸收HT 2 mAb消除信号。免疫荧光显微镜显示，HT 2单抗染色ER应激的HeLa细胞的核周细胞质，这与识别ER的抗KDEL（Lys-Asp-Glu-Leu）单抗的染色模式相似。相反，未处理的HeLa细胞用HT 2 mAb弱染色。因此，HT 2 mAb可用于Herp的定量和/或定性检测，并可用于研究Herp在各种病理状态下的作用。
Hyperhomocysteinemia has been reported as one of the risk factors for vascular damage. Homocysteine induces endoplasmic reticulum (ER) stress in vascular endothelial cells, which is followed by production of homocysteine-induced ER protein (Herp). Herp has been thought to have a protective role against ER stress and inhibition of apoptosis, but the details are still obscure. To detect Herp protein precisely, we established a murine hybridoma clone producing an anti-human Herp monoclonal antibody (mAb), named HT2. The specific binding of HT2 mAb to Herp was confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. In ELISA, HT2 mAb was able to bind to Herp in a dose-dependent manner, and its binding was interrupted by recombinant Herp. In Western blot analysis, a 54-kDa band corresponding to Herp was detected with HT2 mAb in the membrane fraction of untreated HeLa cells, and its expression was remarkably increased in ER-stressed HeLa cells that had been treated with homocysteine, thapsigargin, or 2-mercaptoethanol. Importantly, the signal was eliminated by absorption of HT2 mAb with recombinant Herp prior to incubation with the blotted membrane. Immunofluorescence microscopy revealed that HT2 mAb stained the perinuclear cytoplasm of ER-stressed HeLa cells, which was similar to the staining pattern with anti-KDEL (Lys-Asp-Glu-Leu) mAb that recognizes the ER. In contrast, untreated HeLa cells were weakly stained with HT2 mAb. Thus, the HT2 mAb is useful in the quantitative and/or qualitative detection of Herp and to study the role of Herp at a variety of pathological states.