Journal of Histochemistry and Cytochemistry, Vol. 45, 41-48, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

Tissue Distribution and Subcellular Localization of Rabbit Liver Metalloendopeptidase

Kazunori Nakagawaa, Shun-ichiro Kawabatab, Yutaka Nakashimaa, Sadaaki Iwanagab, and Katsuo Sueishia
a Department of Pathology, Faculty of Medicine, Kyushu University, Fukuoka, Japan
b Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan

Correspondence to: Kazunori Nakagawa, Dept. of Pathology, Faculty of Medicine, Kyushu Univ. 60, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-82, Japan.


  Summary
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Summary
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Materials and Methods
Results
Discussion
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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


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Rabbit liver metalloendopeptidase (MEP) has been isolated as a candidate for the enzyme responsible for precursor processing of proproteins of vitamin K-dependent proteins (Kawabata and Davie 1992 ). MEP cleaves synthetic decapeptides that mimic the processing sites of the vitamin K-dependent proteins. The nucleotide and deduced amino acid sequences of MEP (Kawabata et al. 1993 ) revealed that MEP contains a putative active site of zinc metallopeptidase, -His-Glu-X-X-His-, and that it is structurally related to either rat testis metalloendopeptidase-24.15 (EC 3.4.24.15; EP-24.15) (Pierotti et al. 1990 ) or thimet oligopeptidase (thiol- and metal-dependent oligopeptidase) (Tisljar 1993 ), which cleaves several bioactive peptides in vitro, including bradykinin, angiotensin I, neurotensin, leuteinizing hormone-releasing hormone, and gonadotropin-releasing hormone, and converts enkephalin-containing peptides to enkephalins (Dando et al. 1993 ; Orawski and Simmons 1989 ; Molineaux et al. 1988 ; Orlowski et al. 1983 ).

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 (Bandyopadhyay et al. 1988 ) and porcine liver (Hagiwara et al. 1989 ). The nucleotide sequence of porcine sABP was determined (Sugiura et al. 1992 ) and thereafter detailed sequence comparisons indicated the identity of sABP to MEP (Kato et al. 1994 ; McKie et al. 1993 ). EP-24.15 is reported to be mainly located in the brain and testis but is also ubiquitously present in kidney, heart, lungs, and liver (Kato et al. 1994 ; Choi et al. 1990 ; Pierotti 1990). MEP has no hydrophobic signal sequence for the endoplasmic reticulum, but the NH2-terminal portion of the longest reading frame is rich in positively charged residues, which would most likely fold into separate amphiphilic {alpha}-helices analogous to the targeting signal of mitochondrial precursor proteins (Kawabata et al. 1993 ). Although MEP (or sABP) has been purified from both a membrane and a soluble cytoplasmic fraction of the liver, the tissue distribution and subcellular localization are still unknown.

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.


  Materials and Methods
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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 (Tam 1988 ), using an eight-branched lysine core and an ABI 430A peptide synthesizer and the chemicals and program supplied by the manufacturer (Applied Biosystems Japan; Tokyo, Japan). Each synthetic peptide (250 µg) was emulsified in complete Freund's adjuvant and administered intradermally. A booster with 250 µg in incomplete Freund's adjuvant was given at 2-week intervals for 2 months. The eggs were then collected for 1 week after the last injection. The antibody fractions (IgY) from the immune and preimmune egg yolks were prepared after treatment with dextran sulfate according to a procedure previously described (Jensenius et al. 1981 ).



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Figure 1. Comparison of the sequences of synthetic multiple-antigen peptides and EP-24.15. The position numbers are based on the sequence of the MEP. Identical residues between two proteins are shaded. The peptide sequences selected for synthesis are boxed.

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 (Kawabata et al. 1993 ) was cloned onto the poly linker of Bluescript II SK(+) (Toyobo; Tokyo, Japan) to serve as template. A cRNA probe specific to rabbit MEP was generated by in vitro transcription of anti-sense or sense probes of a truncated PE13 cDNA clone using digoxigenin (DIG)-labeled-UTP (Boehringer Mannheim; Mannheim, Germany) and T3 or T7 RNA polymerase (Gibco; Gaithersburg, MD). This clone contained 700-2050 of the rabbit MEP coding region. After DNAse I digestion of the template DNA, the cRNA probe was degraded by mild alkaline treatment and purified by ethanol precipitation.

RNA Extraction and Northern Blot Analysis
The RNA was prepared by the acid guanidium thiocyanate-phenol-chloroform extraction method (Chomczynski and Sacchi 1987 ). Poly(A)+ RNA was then selected by Oligotex-dT30 (Takara Shuzo; Kyoto, Japan). Poly(A)+ RNA aliquots were electrophoresed in a 1% agarose-formaldehyde gel and transferred to nylon membranes (GeneScreen; Dupont, Boston, MA) by the capillary transfer method. Detection of DIG-labeled ribonucleotides was performed using the immunological luminescent detection protocol proposed by the manufacturers (Boehringer Mannheim).


  Results
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Materials and Methods
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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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Figure 2. Northern blot analysis of MEP mRNA prepared from rabbit tissues. MEP-encoding transcripts were identified by blot hybridization using a PE13 anti-sense cRNA as a probe. The sizes of the standards run in the same gel are indicated in kilobases.

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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Figure 3. Immunoblot analysis of the native MEP purified from a rabbit liver. The molecular weights of standard proteins in the same gel are indicated at left. Native MEP purified from rabbit liver was transferred to a nitrocellulose membrane and immunoblotted with anti-Ag1 (left) and with anti-Ag3 IgY (right). Arrow indicates the 70-kD MEP present in Lanes 1 and 2.

Localization of MEP in various tissues by light microscopic immunohistochemistry is summarized in Table 1.


 
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Table 1. Summary of the MEP immunohistochemical study in rabbit tissues examineda

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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Figure 4. Immunohistochemical demonstration of MEP in a rabbit brain. (a) Ependymal cells lining the third ventricle. Bar = 25 µm. (b) Cerebral cortex. Bar = 10 µm. (c) Ependymal cells lining the third ventricle with normal IgY (negative control). Bar = 25 µm. E, ependymal cell; 3V, third ventricle; As, astrocyte; N, nerve cell; M, microglia. Counterstained with hematoxylin for the nuclei.

Figure 5. Immunohistochemical demonstration of MEP in rabbit lung. (a) Terminal portion of the respiratory tree. Bar = 100 µm. (b) Bronchial epithelium. Bar = 25 µm. Counterstained with hematoxylin for the nuclei. T, terminal bronchiole; V; blood vessel; A, alveoli. (c) Immunoelectron micrograph demonstrating MEP immunolabeling of the bronchial epithelium. Bar = 500 nm.

Figure 6. Immunohistochemical demonstration of MEP in a rabbit kidney. (a) Renal cortex. Bar = 300 µm. (b) Renal cortex at higher magnification. Bar = 25 µm. (c) With normal IgY (negative control). Bar = 300 µm. DC, distal convoluted tubules; G, renal glomerulus; V, blood vessel. Counterstained with hematoxylin for the nuclei.

Figure 7. Immunohistochemical demonstration of MEP in a rabbit stomach. (a) The gastric glands contain a mixed population of chief cells, parietal cells, and mucus-secreting cells. Bar = 75 µm. (b) The base of a gastric gland. Bar = 25 µm. C, chief (peptic) cell; P, parietal cell. Counterstained with hematoxylin for the nuclei. (c) Immunoelectron micrograph demonstrating MEP immunolabeling of parietal cells. Bar = 250 nm. M, mitochondrion; ic, intracellular canalicular system.

Figure 8. Immunohistochemical demonstration of MEP in a rabbit duodenum. Bar = 50 µm. B, Brunner's gland; arrows, plasma cells. Counterstained with hematoxylin for the nuclei.

Figure 9. Immunohistochemical demonstration of MEP in a rabbit liver (portal tract). Bar = 50 µm. V, hepatic portal vein; A, artery; L, lymphatic; B, bile duct. Counterstained with hematoxylin for the nuclei.

Figure 10. Immunohistochemical demonstration of MEP in a rabbit epididymis. Bar = 200 µm. Counterstained with hematoxylin for the nuclei.

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).


  Discussion
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Materials and Methods
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Literature Cited

Kato et al. 1994 reported that the mRNA for porcine sABP was present at high levels in the liver, kidney, and adrenal gland, using RNAse protection assays. The Northern blot analysis of rabbit MEP in our study revealed that MEP was expressed at significant levels in all tissues examined, especially in the brain and the heart. Moreover, immunohistochemical studies clearly indicated the localization of MEP in the liver, brain, lung, kidney, stomach, duodenum, intestine, pancreas, placenta, epididymis, uterus, and in many other tissues (Table 1). But there is a discrepancy between mRNA and protein expression, which remains to be clarified.

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 (Woulfe et al. 1992 ). Recently, MEP was found to degrade neurotensin (Kojima et al. 1995 ), suggesting a functional inactivation of the endogenous neurotensin in this region.

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 (Barnes 1987 ; Fuller et al. 1987 ).

Localization of EP-24.15 in lung tissue and the cytoplasm of ciliated epithelial cells of tracheobronchial mucosa has also been reported (Choi et al. 1990 ). Substance P and bradykinin are good substrates for EP-24.15 (Dahms and Mentlein 1992 ; Orawski and Simmons 1989 ) and for MEP (Kojima et al. 1995 ). Therefore, these findings suggest that MEP and EP-24.15 play an important role in regulation of airway functions by degrading the bioactive peptides in the lung.

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 (Yamaguchi et al. 1991 , Yamaguchi et al. 1992 ). Neurotensin and bradykinin have been shown to be good substrates for MEP (Kojima et al. 1995 ). Considering these points, MEP might therefore be a candidate for a bioactive peptide-degrading enzyme in distal convoluted tubules and collecting 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 (Sunday et al. 1992 ; Shipp et al. 1990 ), including EP-24.15 and EP-24.16 (Chabry et al. 1990 ). Several bioactive peptides, such as bradykinin, {alpha}-neoendorphin, substance P, neurotensin, BAM-2P (bovine adrenal medulla dodecapeptide), degraded by MEP are also cleaved by EP-24.15 (Dando et al. 1993 ; Dahms and Mentlein 1992 ; Orlowski et al. 1983 ) or EP-24.16 (Barelli et al. 1993 ; Dahms and Mentlein 1992 ). The cleavage sites of the peptides are very similar among the three enzymes but are not identical. Therefore, the function of these enzymes may be regulated in part by their different substrate specificities.

Serizawa et al. 1995 have purified and characterized a mitochondrial metallopeptidase, designated oligopeptidase M, from rat liver. The NH2-terminal sequence of mitochondrial oligopeptidase M contains 19 of 20 residues identical to that of MEP and 17 matching the corresponding segment of porcine sABP. Moreover, the enzyme was crossreacted by a monoclonal antibody against rabbit sABP, and this indicates that MEP may therefore be identical to mitochondrial oligopeptidase M. Indeed, MEP has been reported to have an NH2-terminal sequence analogous to the targeting signal of mitochondrial precursor proteins (Kawabata et al. 1993 ). The present data, however, indicate that MEP is localized in the cytoplasmic regions or the luminal surfaces of the epithelial cells of various tissues, but not in mitochondria (Figure 7c). In addition, Tisljar 1993 showed that thimet oligopeptidase activity was localized in the plasma membrane fraction. The complete primary structure of oligopeptidase M, which except for the mitochondria targeting signal resembles the NH2-terminal region, will permit determination of whether or not this enzyme is identical to MEP.

The deduced amino acid sequence of rat EP-24.16 has been recently reported (Dauch et al. 1995 ). The rabbit MEP possesses 90% sequence identity with rat EP-24.16. Therefore, both anti-Ag1 and anti-Ag3 IgYs might detect not only MEP but EP-24.16. However, the substrate specificity of MEP is similar but not entirely identical to that of EP-24.16. From this point of view, it appears probable that MEP corresponds to rabbit EP-24.16, but this remains to be clarified.

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.


  Acknowledgments

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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Summary
Introduction
Materials and Methods
Results
Discussion
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