ARTICLE |
Correspondence to: Kazunori Nakagawa, Dept. of Pathology, Faculty of Medicine, Kyushu Univ. 60, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-82, Japan.
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Summary |
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We have previously isolated rabbit liver microsomal metalloendopeptidase (MEP) as a candidate for the processing enzyme of vitamin K-dependent plasma proteins. A cDNA coding for MEP has revealed that it is structurally related to metalloendopeptidase-24.15, which catalyzes the proteolytic processing of several bioactive peptides. In this study we examined the tissue distribution and subcellular localization of MEP by light and electron microscopic immunohistochemical methods, in addition to Northern blot analysis. Chicken polyclonal antibodies were raised by using synthetic peptides AG1 (Met31-Asn46) and AG3 (Asp537-Gly551) derived from the sequence of MEP. Both anti-AG1 and anti-AG3 antibodies reacted specifically with MEP, as judged by Western blotting and immunohistochemical methods. Both antibodies gave an identical staining distribution, which was localized on the luminal cell surfaces and in the cytoplasm of the following organs: liver, brain, lungs, kidneys, esophagus, stomach, duodenum, pancreas, placenta, epididymis, uterus, ovary, and oviduct. Northern blot analysis revealed that the expression of MEP mRNA is similar to its immunohistochemical distribution except in the heart. These results suggest that MEP may participate more closely in a degradation role in peptide metabolism in various tissues than in a processing role of the proprotein, like metalloendopeptidase-24.15. (J Histochem Cytochem 45:41-47, 1997)
Key Words: Zinc peptidase, Processing protease, Endopeptidase-24.15, Endopeptidase-24.16, Thimet oligoendopeptidase
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Introduction |
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Rabbit liver metalloendopeptidase (MEP) has been isolated as a candidate for the enzyme responsible for precursor processing of proproteins of vitamin K-dependent proteins (
On the other hand, works aimed at the isolation of an angiotensin II receptor have led to purification of a soluble angiotensin binding protein (sABP) from the cytosol fraction of rabbit (-helices analogous to the targeting signal of mitochondrial precursor proteins (
This article describes the immunohistochemical tissue distribution and subcellular localization of MEP in rabbit by use of polyclonal antibodies raised in chicken against synthetic peptides derived from the partial sequences of MEP.
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Materials and Methods |
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Preparation of Polyclonal Antibodies
Polyclonal antibodies against rabbit microsomal MEP were raised in hens (Japanese White Leghorn, l50 days old) using synthetic peptides as immunogens. Ag1, Ag2, and Ag3 of 15-16 amino acid residues (corresponding to the amino acid Met31-Asn46, Asp359-Ser373, Asp537-Gly551, respectively, as shown in Figure 1) were synthesized as multiple antigenic peptides (
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Tissue Preparation and Light Microscopic Immunohistochemistry
Japanese White rabbits (2.5-3.5 kg) were sacrificed and their organs immediately excised, rinsed, cut into small pieces, and immersed in freshly prepared 4% (w/v) paraformaldehyde in 0.1 M sodium phosphate, pH 7.4, at 4C overnight. The fixed tissues were then embedded in paraffin and sectioned at 3-µm thickness with a microtome. The sections were mounted on poly-L-lysine-coated slides and then deparaffinized and blocked with 1.5% dry milk in 10 mM PBS, pH 7.4. The sections were incubated with anti-Ag1 or anti-Ag3 (10 µg/ml) overnight at 4C, followed by incubation for 30 min with biotinylated anti-chicken antibody (Zymed; Burlingame, CA). Endogenous peroxidase activity was blocked by treatment with 0.3% H2O2 in methanol for 30 min at room temperature. The sections were incubated with horseradish peroxidase-labeled streptavidin (Nichirei; Tokyo, Japan) and the peroxidase reaction was developed in PBS containing 0.004% H2O2 and 0.6 mg/ml 3,3'-diaminobenzidine (Merck; Darmstadt, Germany). Each step was followed by three 5-min washes with PBS. For controls, sections were incubated overnight at 4C either with preimmune IgY instead of the primary antibody (anti-Ag1 or anti-Ag3) or with the primary antibody pretreated with a 100-fold excess of the corresponding synthetic peptides. In addition, the sections were counterstained with hematoxylin.
Electron Microscopic Immunohistochemistry
The fixed samples were incubated with 50 mM NH4Cl in PBS for 20 min, dehydrated in a graded series of dimethylformamide, and embedded in Lowicryl K4M (Chemische Werke Lowi; Waldkraiburg, Germany) at 4C. Ultrathin sections were mounted on formvar-coated nickel grids, blocked with 1% bovine serum albumin in PBS, and then incubated for 2 hr with anti-Ag1 or anti-Ag3 (10 µg/ml). The sections were incubated with biotinylated anti-chicken antibody for 1 hr, followed by incubation with 10-nm colloidal gold-labeled streptavidin (Amersham International; Poole, UK). Each step was followed by three 5-min washes with PBS. In addition, the sections were stained with uranyl acetate and lead tartrate, and examined with a JEM 1200EX electron microscope (JEOL; Tokyo, Japan).
Preparation of the cRNA Probe
The EcoRI-EcoRV fragment derived from rabbit pPE13 (
RNA Extraction and Northern Blot Analysis
The RNA was prepared by the acid guanidium thiocyanate-phenol-chloroform extraction method (
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Results |
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Northern Blot Analysis of the MEP mRNA in Rabbit Tissues
Northern blot analysis was performed with polyA+ RNA prepared from various normal rabbit tissues to examine the tissue distribution and to compare the relative amount of mRNA of MEP. MEP was encoded by a single species of mRNA with 4.4 KB and the mRNA was ubiquitously expressed, but at higher levels in the brain, heart, and testis than in the liver (Figure 2), although MEP was isolated from the liver at first.
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Specificity of Antibodies and Tissue Distribution of MEP
Three peptides based on the sequences of MEP, Ag1, Ag2, and Ag3, were synthesized by a multiple antigenic peptide system, as described in Materials and Methods. These peptide sequences had been selected with lower sequence similarities to EP-24.15 to minimize any crossreaction of the antibodies (Figure 1). In particular, it was unlikely that anti-Ag1 IgY recognizes the EP-24.15 because there is an eight-amino-acid deletion compared with the counterpart region of MEP. The immunoblot analysis of MEP purified from the rabbit liver showed that both anti-Ag1 and anti-Ag3 antibodies purified from yolk (IgY) recognized the intact MEP with an apparent molecular mass of 70 kD under reduced conditions (Figure 3). On the other hand, the peptide Ag2 failed to raise an antibody to recognize the peptide and the intact protein, and preimmune IgY did not show any positive reactions (data not shown). The immunochemical specificity of the two antibodies to intact MEP enabled us to examine the tissue-specific localization of MEP as follows. Anti-Ag1 and anti-Ag3 antibodies produced an identical staining pattern and pretreatment of the antibodies with an excess of the corresponding synthetic peptides apparently abrogated the staining of the antigen in tissues.
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Localization of MEP in various tissues by light microscopic immunohistochemistry is summarized in Table 1.
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Brain. The luminal surface of the ependymal cells of the third ventricle was stained positively with anti-Ag1 IgY (Figure 4a). Immunopositive reactions for MEP could also be found in the cytoplasm of the nerve cells of cerebral cortex (Figure 4b). Normal IgY gave no positive staining of the antigen (Figure 4c).
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Lung. Luminal surfaces of bronchiolar and bronchial epithelial cells, including nonciliated and ciliated epithelial cells, showed specific staining (Figure 5a and Figure 5b). In contrast, no immunoreactivity was observed in other alveolar lining cells, alveolar macrophages, or blood vessel cells. Immunoelectron microscopic examination of the bronchiolar epithelium indicated that the reaction products of MEP were predominantly associated with the plasma membrane, including the cilia (Figure 5c).
Kidney. Positive immunoreactions were localized in the luminal surface and in the cytoplasm of the epithelium of the distal convoluted tubules (Figure 6a and Figure 6b), collecting tubules, and papillary ducts (data not shown). The other components of the nephron, including the renal glomerulus and the proximal tubules, were not stained. Blood vessel cells were also negative for the staining (Figure 6a). Immunoelectron microscopic examination of the epithelium showed that the reaction products of MEP were predominantly associated with the plasma membrane of distal convoluted tubules (data not shown).
Stomach. Strong immunoreactions were observed in the parietal cells and the surface epithelial cells of the gastric glands (Figure 7a and Figure 7b). Mucous neck cells were also weakly stained. However, chief cells were negative. Electron microscopic immunochemistry showed subcellular localization at the plasma membrane of the intracellular canalicular system (Figure 7c, arrow).
Duodenum. Positive reactions were localized on the intratubular surface and in the cytoplasm of plasma cells and the cytoplasm of Brunner's glandular cells (Figure 8).
Liver. MEP antigen was located on the surface and within the cytoplasm of the bile duct epithelia in the portal triads (Figure 9) and intralobular interstitium, but not in the parenchymal cells or the sinusoidal lining cells.
Testis and Epididymis. The pseudostratified columnar epithelium of epididymis showed positive reactions on the luminal surface of the plasma membrane, in the cytoplasm, and along the surfaces of the stereocilia (Figure 10). However, the cells in the seminiferous tubule of the testis were negative (data not shown).
Other Tissues. Immunopositive reactions of MEP were also observed in some other tissues, including trophoblasts in the placenta, pseudostratified columnar ciliated epithelium in the Fallopian tube, the epithelial cells of the endometrial glands, all of the islet cells of Langerhans in pancreas, and the stratified squamus epithelium in the esophagus. In addition, plasma cells were also immunohistochemically positive in several organs (Figure 8).
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Discussion |
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The distribution of MEP in brain was predominantly on the luminal surface of the ependymal cells and in the cytoplasm of the nerve cells (Figure 4). A similar membrane-associated localization in rat midbrain has been reported for EP-24.16, a neurotensin-degrading enzyme (neurolysin). Immunohistochemical studies of EP-24.16 in the mesencephalon showed that the enzyme is located in the restricted zones of the plasma membrane and is also present in the cytoplasm of neurons, and predominantly in the cytoplasm of the glia (
MEP was also localized in the bronchiolar epithelium and was predominantly associated with the plasma membrane, including the cilia (Figure 5). Several bioactive peptides, such as substance P and bradykinin in the terminal bronchioles, are believed to regulate airway function, including modulation of bronchomotor tone, bronchial secretion, and bronchial circulation (
Localization of EP-24.15 in lung tissue and the cytoplasm of ciliated epithelial cells of tracheobronchial mucosa has also been reported (
The localization of MEP in the kidney is consistent with the hypothesis that it interacts with urinary peptides filtered through the glomeruli and the proximal renal tubules. Recent studies also indicate that peptidases play an important role in the metabolism of such biologically active peptides, as angiotensin, bradykinin, neurotensin, and parathyroid hormone in the proximal renal tubules (
MEP was localized mainly in the cytoplasm and the plasma membrane of epithelial cells. Although the physiological significance of MEP remains to be determined, the present data suggest that MEP might play a role in the functional inactivation of bioactive peptides on the cell surface in various tissues, together with other oligopeptidases (-neoendorphin, substance P, neurotensin, BAM-2P (bovine adrenal medulla dodecapeptide), degraded by MEP are also cleaved by EP-24.15 (
The deduced amino acid sequence of rat EP-24.16 has been recently reported (
In conclusion, we report here the tissue distribution and subcellular localization of MEP in the rabbit. The parallel co-localization of some bioactive peptides and the shared localization of other peptidases suggest a role of MEP in the functional inactivation of bioactive peptides. This does not preclude the possibility that MEP participates in processing of peptide hormones and in modulation of the tissue differentiation or maturation processes, which remain to be investigated.
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Acknowledgments |
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Supported in part by a Grant-in-Aid for Developmental Scientific Research (B) (No. 04558025) from the Ministry of Education, Science and Culture of Japan, and by a grant from the Kurata Foundation (SK).
We thank Dr T. Harano (Department of Biology, Kyushu University) for valuable advice in preparing the chicken antibodies, H. Fujii and M. Noguchi for tissue preparation, and S. Yugawa for experimental assistance. We also thank Dr B. T. Quinn for comments on the manuscript.
Received for publication August 8, 1996; accepted August 27, 1996.
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