ARTICLE |
Correspondence to: Hiroyoshi Komatsu, Dept. of Immunology, School of Allied Health Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan. E-mail: hkomatsu@ahs.kitasato-u.ac.jp
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Summary |
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This is the first report to describe the successful detection of human gastrointestinal glutathione peroxidase in normal tissues by Western blotting and immunohistochemical staining techniques. Four hybridoma clones producing monoclonal antibodies (MAbs) against the human gastrointestinal glutathione peroxidase were established from mice immunized with a gastrointestinal glutathione peroxidase-derived peptide. The MAbs did not crossreact with other members of the glutathione peroxidase family, be it cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, or extracellular glutathione peroxidase. Although the MAbs were found to react with a 24-kD protein in a Western blotting assay using gastric carcinoma cell extracts as antigen, they did not react with a B-lymphoblastoid cell extract. Immunohistochemical staining showed gastrointestinal glutathione peroxidase localized in the cytoplasm and in the nucleus of gastric carcinoma cells. Moreover, gastrointestinal glutathione peroxidase was detected in tissue extracts of human stomach, small intestine, large intestine, liver, and gallbladder by Western blotting, and its localization was immunohistochemically confirmed in the mucosal epithelia of the basal area of gastric pits and intestinal crypts.
(J Histochem Cytochem 49:759766, 2001)
Key Words: gastrointestinal glutathione, peroxidase, monoclonal antibody, immunohistochemical staining, Western blotting, gastric carcinoma cell
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Introduction |
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THE GLUTATHIONE PEROXIDASE FAMILY (GPx) are selenoenzymes that catalyze the reduction of hydrogen peroxide, organic hydrogen peroxides, or lipid peroxides in the presence of glutathione and function to detoxify peroxides in the cell (
In addition to these three isozymes, a novel selenium-dependent GPx was characterized by expressing a cDNA isolated from human hepatoma HepG2 cells. The mRNA was expressed in gastrointestinal tissues but not in other human tissues such as kidney, heart, lung, placenta, or uterus. Therefore, the new GPx was designated gastrointestinal GPx (giGPx, GPx2) (
However, the localization of giGPx protein in human tissues and its biological characteristics, other than its enzymatic activity, remain unknown. As a first step to resolve these questions, we attempted to establish mouse hybridomas that produce a monoclonal antibody (MAb) against human giGPx. In this article, we describe the characterization of MAbs against human giGPx, from mice immunized with a giGPx-derived peptide. Moreover, we demonstrate human giGPx protein expression in stomach, small intestine, large intestine, liver and gallbladder by Western blotting and immunohistochemical staining techniques using the human giGPx MAbs.
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Materials and Methods |
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Cells
KATO-III (
giGPx mRNA expression in KATO-III, OCUM-1, and TAB089 cells was assayed by RT-PCR. giGPx mRNA expression was detected in KATO-III and OCUM-1 cells, but not in TAB089 cells (data not shown).
Synthesis of Human giGPx-derived Peptides
The human giGPx-derived peptide (SAISLDGEKVDFNTFRGRAC) (
Immunization of Mice
The giGPx-derived peptide described above was conjugated with maleimide-activated keyhole limpet hemocyanine (Pierce; Rockford, IL). BALB/C mice were injected IP with 50 µg of the immunogen emulsified with Freund's complete adjuvant. Mice were subsequently injected IP on a further four occasions at 2-week intervals with 50 µg immunogen emulsified with Freund's incomplete adjuvant. After 2 more weeks, mice were injected IV with 50 µg of immunogen in PBS. Splenic lymphocytes were isolated from the mice 3 days after the final booster and fused with a SP2/0-Ag14 mouse myeloma cell line as previously described (
Enzyme-linked Immunosorbent Assay
ELISA was based on a modification of a previously described protocol (
Western Blotting Assay
Cell extracts were obtained from 1 x 108 cultured cells in 1 ml of low-salt lysis buffer [10 mM Tris-HCl (pH 8.0), 0.14 M NaCl, 3 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethyl sulfonyl fluoride, and 0.5% (v/v) NP-40] on ice for 30 min. For preparation of human tissue extracts, tissue samples were homogenized and treated with low-salt lysis buffer on ice for 30 min. After centrifugation at 10,000 x g for 10 min at 4C to remove cell debris, lysates were fractionated by SDS-PAGE on a 12.5% (w/v) polyacrylamide gel (
Immunohistochemical Staining
Cultured cells were fixed with 10% (v/v) phosphate-buffered formalin (pH 7.2), and resuspended in warmed 2% (w/v) agar solution after centrifugation at 1000 rpm for 5 min. Cells were centrifuged at 3000 rpm for 5 min and cooled for 30 min at 4C. The cell pellets within solid agar were again fixed with phosphate-buffered formalin and embedded in paraffin. A variety of human tissues to be analyzed for giGPx localization were selected from surgically removed or autopsy materials (patient age 4575 years), fixed with phosphate-buffered formalin, and embedded in paraffin. Immunohistochemical staining was performed using the standard labeled streptavidinbiotinperoxidase complex method (LSAB kit; Dako Japan, Kyoto, Japan). Briefly, after routine deparaffinization, 4-µm-thick sections were pretreated with 0.3% (v/v) H2O2 in methanol for 30 min and incubated with anti-human giGPx MAb overnight in a moist chamber at 4C. For negative controls, normal mouse serum (diluted 1:100) was used instead of primary antibody. Counterstaining of nuclei was achieved with 0.3% (w/v) methyl green solution.
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Results |
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Generation of Hybridomas and Specificity of MAbs
Immune splenocytes were fused with SP2/0-Ag14 murine myeloma cells using polyethylene glycol 2000, and hybridoma cells were generated in hypoxanthine/aminopterin/thymidine culture medium. Hybridoma supernatants were screened for anti-giGPx activity by ELISA, using a giGPx-derived synthetic peptide as antigen. Four anti-giGPx-positive hybridomas secreted antibodies which were specific for a 24-kD protein identified by Western blotting using KATO-III cell lysate as antigen. The apparent molecular weight of this protein corresponded to that predicted from the amino acid sequence of human giGPx. The hybridomas were subcloned twice by the limiting dilution method and all four hybridoma clones were found to secrete IgG1 antibodies.
The four MAbs, denoted FEG-1, -2, -3, and -4, were tested by Western blotting for reactivity with KATO-III and TAB089 cell lysates. All MAbs recognized a 24-kD protein in KATO-III cell lysate but not in TAB089 cell lysate (Fig 1A). Whereas FEG-1 and -4 MAbs reacted strongly with KATO-III cell lysate, FEG-2 and -3 MAbs showed weak reactivity. No reactivity was observed with anti-HIV transmembrane protein MAb (
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As a control for antibody specificity, antibody was pretreated with the giGPx-derived synthetic peptide for 1 hr at 37C and the effect on reactivity with KATO-III lysate observed by Western blotting. A typical pattern using FEG-1 MAb is shown in Fig 1B. Reactivity with the 24-kD protein in KATO-III cell lysates was lost with increasing peptide concentration up to 10 µg/ml, strongly suggesting that the 24-kD protein in KATO-III cells has the same antigenicity as the peptide.
The crossreactivity of mAbs was investigated by Western blotting using a commercially available human cGPx protein (Sigma Chemical; St Louis, MO), an anti-phGPx MAb. The anti-phGPx MAb specifically recognized the 22-kD phGPx protein in lysates of KATO-III and TAB089 cells but did not recognize the 24-kD protein that was detected with the FEG series of MAbs (Fig 2A). Moreover, the FEG series of MAbs did not crossreact with large amounts of the commercial cGPx protein which although also a 24-kD protein, migrates slightly faster than putative human giGPx, as visualized by Amido Black 10B staining. For example, the reactivity of FEG-1 MAb with the commercial cGPx protein is shown in Fig 2A.
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Next, the crossreactivity with eGPx was investigated by using a commercially available anti-eGPx mAb (Seikagaku Kogyo; Tokyo, Japan). Although the anti-eGPx MAb recognized a 23-kD protein in human plasma, it did not detect the 24-kD protein in KATO-III cell lysate (Fig 2B). Several bands at 26120 kD were faintly detected in human plasma with the FEG series of MAbs and the anti-eGPx MAb, but similar bands were also detected in the absence of primary antibody (data not shown). These results strongly suggest that the four FEG MAbs specifically recognize human giGPx without crossreactivity to human phGPx, eGPx, or cGPx antigens. We therefore define FEG MAbs -1, -2, -3, and -4 as anti-human giGPx MAbs. FEG-1 mAb (reacting most strongly with giGPx antigen; see Fig 1A) was used for all subsequent experiments.
Reactivity with Human Tissues as Assayed by Western Blotting
The reactivity of FEG-1 MAb with giGPx antigen in normal human tissues was investigated by Western blotting using 29 human tissue extracts as antigen (Table 1). Although giGPx antigen was readily detected in stomach, duodenum, large intestine, liver, and gallbladder, it was not detected in other tissues such as salivary gland and esophagus. The reactivity of FEG-1 MAb with liver and gallbladder tissue was stronger than that with stomach, duodenum, and large intestine. Perhaps surprisingly, giGPx antigen was not detected in a tissue extract from pancreas. It is conceivable, however, that giGPx was degraded by pancreatic enzyme activity during preparation of the tissue extract, a hypothesis supported by the detection of little ß-actin in extracts of fresh pancreas in a control Western blotting assay using an anti-ß-actin antibody. The data strongly indicate that human giGPx is expressed specifically in the tissues of the gastrointestinal tract.
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Immunohistochemical Localization of giGPx in Human Tissues
Immunohistochemical staining using FEG-1 MAb showed giGPx localization mainly to the neck area of the gastric pits and the basal portion of the crypts in duodenum, ileum, and large intestine (Table 1; Fig 3). giGPx was found in both cytoplasm and nucleus of epithelial cells, the latter showing rather stronger positivity than the former. The intensity was relatively weaker in the large intestine compared to the small intestine, particularly in the ileal mucosa. giGPx was observed in the epithelium of bile duct, pancreatic duct, and salivary gland tissue. Furthermore, positive staining was also seen in the lower portion of the mucosal folds, particularly at the RokitanskyAschoff sinus of the gallbladder. Liver cells showed a relatively weak reaction in the centrilobular area. FEG-2, -3, and -4, were also used for immunohistochemical studies on human tissues. Unlike FEG-1 MAb, some artifacts were detectable using the other FEG MAbs (data not shown). However, the pattern of reactivity with giGPx closely paralleled that observed for FEG-1 MAb. It may be that the specificity of the FEG-2, -3, and -4 MAbs is lower than that of FEG-1 MAb in formalin-fixed samples.
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Reactivity with Other Gastric Carcinoma Cell Lines and Localization of giGPx in Cells
The reactivity of FEG-1 MAb with three gastric carcinoma cell lines, Okajima, OCUM1, and HuG-1 was investigated by Western blotting (Fig 4). giGPx was detected as a 24-kD protein in lysates of all three cell lines, as well as in KATO-III. This suggests that similar forms of giGPx are stably expressed in normal gastrointestinal tissue and in gastrointestinal carcinomas. To confirm the localization of giGPx in cells, each of the three gastric carcinoma cell lines was stained immunohistochemically using FEG-1 MAb. Both the nuclei and cytoplasm of Okajima cells were strongly stained (Fig 5A). Similar results were obtained with OCUM1, HuG-1, and KATO-III cells (data not shown). Twenty to 60% of total cells were immunohistochemically positive, depending on the cell line. When normal mouse serum (diluted 1:100) was used instead of FEG-1 MAb as a negative control, giGPx-positive cells were not detected (data not shown). Moreover, no expression of giGPx was observed in TAB089 cells, a B-lymphoblastoid cell line (data not shown).
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The reactivity of FEG-1 preincubated with its peptide (10 µg/ml) for 1 hr at 37C was tested on Okajima cells (Fig 5B). In confirmation of the effect seen on Western blotting (Fig 1B), pretreatment of FEG-1 with the peptide used to raise it completely negated any interaction. Taken together, these data strongly suggest that FEG-1 MAb is indeed specific for human giGPx.
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Discussion |
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We established four hybridoma clones producing MAbs against human giGPx antigen. These mAbs did not crossreact with commercial human cGPx, human phGPx, or human eGPx, suggesting that p24 detected in KATO-III cell lysate using the MAbs described herein is indeed likely to be giGPx. On the basis of the evidence presented here, we therefore propose that the four MAbs described specifically interact with human giGPx.
The amino acid sequence of human giGPx shares 95% homology with its mouse counterpart (
The human giGPx mRNA has been isolated from human liver, large intestine, and human hepatoma HepG2 cDNA libraries (
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Acknowledgments |
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Supported in part by a grant from Kitasato University Graduate School of Medical Sciences (No. 9901).
We wish to thank Ms K. Hana and Ms Y. Numata for technical support and Dr M. Rau for critical reading of the manuscript.
Received for publication December 28, 2000; accepted January 11, 2000.
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