A monoclonal antibody against human homocysteine-induced endoplasmic reticulum protein (Herp): A useful tool for evaluating endoplasmic reticulum stress

Abstract

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 cyloplasm 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. © 2007 Tohoku University Medical Press.