A monoclonal antibody against human homocysteine-induced endoplasmic reticulum protein (Herp)

A useful tool for evaluating endoplasmic reticulum stress

Yumiko Oka, Yasuhiko Hirabayashi, Tomonori Ishii, Reiko Takahashi, Takeshi Sasaki

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)431-437
Number of pages7
JournalTohoku Journal of Experimental Medicine
Volume212
Issue number4
DOIs
Publication statusPublished - Jul 27 2007

Fingerprint

Endoplasmic Reticulum Stress
Staphylococcal Protein A
Homocysteine
Endoplasmic Reticulum
Monoclonal Antibodies
Proteins
HeLa Cells
Immunosorbents
lysyl-aspartyl-glutamyl-leucine
Assays
Western Blotting
Enzyme-Linked Immunosorbent Assay
Membranes
Thapsigargin
Mercaptoethanol
Hyperhomocysteinemia
Endothelial cells
Enzymes
Hybridomas
Fluorescence Microscopy

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

A monoclonal antibody against human homocysteine-induced endoplasmic reticulum protein (Herp) : A useful tool for evaluating endoplasmic reticulum stress. / Oka, Yumiko; Hirabayashi, Yasuhiko; Ishii, Tomonori; Takahashi, Reiko; Sasaki, Takeshi.

In: Tohoku Journal of Experimental Medicine, Vol. 212, No. 4, 27.07.2007, p. 431-437.

Research output: Contribution to journalArticle

Oka, Yumiko ; Hirabayashi, Yasuhiko ; Ishii, Tomonori ; Takahashi, Reiko ; Sasaki, Takeshi. / A monoclonal antibody against human homocysteine-induced endoplasmic reticulum protein (Herp) : A useful tool for evaluating endoplasmic reticulum stress. In: Tohoku Journal of Experimental Medicine. 2007 ; Vol. 212, No. 4. pp. 431-437.
@article{7d4f8860e9bb4553abe66e3ee932d7ee,
title = "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.",
author = "Yumiko Oka and Yasuhiko Hirabayashi and Tomonori Ishii and Reiko Takahashi and Takeshi Sasaki",
year = "2007",
month = "7",
day = "27",
doi = "10.1620/tjem.212.431",
language = "English",
volume = "212",
pages = "431--437",
journal = "Tohoku Journal of Experimental Medicine",
issn = "0040-8727",
publisher = "Tohoku University Medical Press",
number = "4",

}

TY - JOUR

T1 - A monoclonal antibody against human homocysteine-induced endoplasmic reticulum protein (Herp)

T2 - A useful tool for evaluating endoplasmic reticulum stress

AU - Oka, Yumiko

AU - Hirabayashi, Yasuhiko

AU - Ishii, Tomonori

AU - Takahashi, Reiko

AU - Sasaki, Takeshi

PY - 2007/7/27

Y1 - 2007/7/27

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=38449123788&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=38449123788&partnerID=8YFLogxK

U2 - 10.1620/tjem.212.431

DO - 10.1620/tjem.212.431

M3 - Article

VL - 212

SP - 431

EP - 437

JO - Tohoku Journal of Experimental Medicine

JF - Tohoku Journal of Experimental Medicine

SN - 0040-8727

IS - 4

ER -