Symposium Papers |
Correspondence to: Nobuteru Usuda, Dept. of Anatomy and Cell Biology, Shinshu University School of Medicine, Matsumoto 390, Japan.
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Summary |
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Immunohistochemistry employing a new hapten antibody that detects the SKL sequence and its variants of the PTS1 C-terminus of peroxisomal enzymes was attempted to visualize peroxisomes across species. Rabbits were immunized with the SKL sequence coupled with KLH, between which an arm molecule was interposed. IgG fractions of antisera were affinity-purified against the hapten and employed for immunochemical analyses and immunoelectron microscopy. The specificity of the antibody was examined by immunoblot analyses for various purified enzymes of rat liver peroxisomes and by dot-blot analyses inhibited by SKL peptide and its variants. Various animal and plant tissues were subjected to immunoelectron microscopy with the protein Agold technique. The antibody reacted with various enzymes in the peroxisome with the SKL motif. The affinity of the antibody for tripeptides, which varied depending on their structures, was higher for SKL than for its variants. Hepatic and renal peroxisomes of vertebrates, peroxisomes in the fat body of an insect, and the cotyledon of a plant were visualized by immunoelectron microscopy. Immunohistochemistry employing this SKL antibody may provide specific staining that can detect peroxisomes across different species. (J Histochem Cytochem 47:11191126, 1999)
Key Words: immunohistochemistry, protein Agold technique, peroxisome, targeting signal, species
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Introduction |
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Peroxisomes are cell organelles defined by a single limiting membrane. They are probably present in all eukaryotes, but their ultrastructure differs according to the organism (
Histochemical detection of peroxisomes has been accomplished most commonly by the DAB reaction for catalase (-hydroxyacid oxidase, and urate oxidase (
Recently, the transport system of peroxisomal matrical enzymes, i.e., how they translocate through the peroxisomal membrane after they are synthesized on free ribosomes (
The new antibody was raised against the tripeptide of SKL. The antibody was characterized for its specificity, and immunoelectron microscopic detection of peroxisomes across different species was attempted with it.
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Materials and Methods |
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Tissues
Electron microscopic analysis was conducted on liver tissues of various animals: Wistar rats (Rattus rattus), slider tortoise (Pseudemys scripta collirostris), bullfrog (Rana catesbeiana), and rainbow trout (Salmo gaird nerii), the fat body of a cicada (Oncotympana maculaticollis), and the cotyledon of a soybean (Glycine max), and on rat and bovine (Bos taurus) kidney tissues. Rats were fed a normal or a 1% DOP diet. Peroxisome fractions obtained by sucrose gradient from normal and DOP-treated rat liver tissues were prepared for immunoblot analysis (
Preparation of Antibodies
A peptide, cysteine-aminocaproic acid-serine-lysine-leucine, was synthesized by the solid-phase method, purified by reverse-phase HPLC, and the sequence confirmed by amino acid analysis. SKL is the C-terminus of the deduced amino acid sequence of acyl-CoA oxidase, based on the rat gene (
The antibody for acyl-CoA oxidase was prepared by immunizing rabbits with its protein purified from rat liver tissues fed a 1% DOP diet as previously described (
Preparation of Peroxisomal Enzymes and Peptides
Acyl-CoA oxidase (AOX), enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (PH), 3-ketoacyl-CoA thiolase (PT), urate oxidase (UOX), D-amino acid oxidase (DAOX), and catalase (CAT) were purified from liver or kidney tissues of normal or DOP-treated rats (
Immunoblot Analyses
The purified rat peroxisomal enzymes, AOX, PH, PT, UOX, DAOX, and CAT, firefly luciferase, and peroxisome fractions were subjected to 12% SDS-PAGE electrophoresis and immunoblot analysis was performed as previously described (
Inhibition Assay
Dot-blot analyses were performed by inhibiting the reaction between SKL-KLH antigen and SKL-Ab with the synthetic peptides. The synthetic tripeptides were dissolved in 10 mM potassium phosphate, (pH 7.5)/150 mM NaCl. The KLH-SKL dissolved in 10 mM potassium phosphate (pH 7.5)/150 mM NaCl/1% SDS was applied at a concentration of 50 ng/ml to small (1 cm x 1 cm) squares of nitrocellulose membrane as focused spots in a volume of 1 ml. Each nitrocellulose square was stained by the same procedure as for the immunoblot analysis. The squares were stained with the mixture of SKL-Ab (2.5 mg/ml) and the tripeptides at various concentrations. After the staining, each square was dried and examined by densitometry. The images were digitized with a computer (Apple Computer; Cupertino, CA) equipped with a scanner (Hewlett Packard Japan; Tokyo, Japan). They were analyzed with Ultimage software (Image and Measurement; Tokyo, Japan). The density was corrected for background density measured outside each spot and was expressed relatively as percent inhibition in terms of the control, i.e., the background density was set as 100% inhibition and the density of the reaction products obtained in the absence of any tripeptide was set as 0% inhibition. The percent inhibition was plotted against the log concentration of tripeptides (
Immunoelectron Microscopy
Tissue processing and immunostaining were done basically as reported previously (
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Results |
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Immunoblot Analysis
Figure 1A shows the detection of various amounts of firefly luciferase by SKL-Ab. The signal was slight with an amount of 8 ng and clearly visible with amounts over 16 ng. Figure 1B shows the reactivity of SKL-Ab with various rat peroxisome-specific enzymes under the same conditions as for luciferase. AOX consists of three subunits, Subunit A (MW 72 kD), Subunit B (MW 51 kD), and Subunit C (MW 21 kD). Whereas Subunits A and C were detected by SKL-Ab when 200 ng was applied, Subunit B was not. In contrast, all three subunits of AOX were detected by anti-AOX antibody. PH was detected clearly at an amount of 20 ng. UOX was not detectable with 20 ng but was detected clearly with 400 ng. CAT was not detectable with 20 ng and was detected very weakly with 400 ng. PT and DAOX were not detected when 20 ng or 400 ng was applied. The immunoblots of peroxisome fractions of normal and DOP-treated livers showed more than 15 signals. The most distinct signals corresponded to the molecular weight of UOX and PH for fractions of normal and DOP-treated livers, respectively (Figure 1C).
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Inhibition Assay
Figure 2A shows the results of the inhibition of the reaction between SKL antigen and SKL-Ab by synthetic tripeptides. The density of the reaction products decreased considerably in parallel with the increase in the concentration of SKL. The reaction became negative at the maximal concentration of 1000 µg/ml. The density of the reaction product also decreased along with the increase in the concentration of SRL, but the reaction products were still visible at a concentration of 1000 µg/ml. In contrast, the decrease in the density of the reaction products was not discernible after addition of PGN. Figure 2B shows the relationship between the concentration of tripeptides and percent inhibition obtained from measuring the result of the dot-blot analysis. The percent inhibition was plotted against the log concentration of tripeptides. The slopes of the approximate lines, indicating the effect of the inhibition, were 31, 26, 25, 18, 9.9, and 1.8 for SKL, AKL, SRL, SHL, ANL, and PGN, respectively. The larger the value, the stronger was the inhibition of the reaction by a peptide.
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Immunoelectron Microscopy
Figure 3 Figure 4 Figure 5 illustrate the reactivity of SKL-Ab to various ultrastructures. Electron micrographs of peroxisomes of normal and DOP-treated rat liver tissues stained by the protein Agold technique with SKL-Ab show that gold particles are located over peroxisomes and that other cell structures are negative for the reaction (Figure 3). The core and the matrix of the peroxisomes are reactive in normal hepatocytes (Figure 3A) and in a DOP-treated hepatocyte (Figure 3B). The labeling density increased with the administration of DOP. In proximal tubule epithelial cells of DOP-treated rat kidney tissues stained by the protein Agold technique, gold particles were located solely over peroxisomes, with other ultrastructures, such as the cell membrane, microvilli, absorption vesicles, lysosomes, mitochondria, and nucleus, being consistently negative for the staining (Figure 4A). High-power magnification showed that most gold particles were located over the peripheral area, but not over the central area, in the matrix of peroxisomes (Figure 4B). In the bovine renal peroxisome, the core and the marginal plates were distinctively labeled (Figure 4C). Figure 5 summarizes the reactivity of peroxisomes of various vertebrate hepatocytes, the insect fat body, and the plant cotyledon. Electron micrographs showed spherical cell organelles, i.e. peroxisomes, to be immunoreactive to SKL-Ab in chicken liver (Figure 5A), tortoise liver (Figure 5B), frog liver (Figure 5C), fish liver (Figure 5D), insect fat body (Figure 5E), and soybean cotyledon (Figure 5F). Other structures were immunonegative for SKL-Ab in the liver, the fat body, and the cotyledon as far as was observed.
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Discussion |
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Data are accumulating on the cDNA sequences of peroxisomal matrix enzymes and their deduced amino acid sequences. Most are now known to possess the SKL sequence and its variants (
Immunoblot analyses of these purified enzymes showed that SKL-Ab detects peroxisomal enzymes but with different detection limits. Firefly luciferase, AOX, and PH were detected clearly by SKL-Ab when small amounts of the samples were applied in SDS-PAGE. UOX and CAT were detected when large amounts were applied, but PT and DAOX were not. Judging from the slopes of the approximate lines obtained in the inhibition test by various peptides, SKL is the strongest inhibitor, SRL and AKL are moderate, and SHL and ANL are weaker. PGN has almost no effect on the reaction between SKL peptide and SKL-Ab. Among the three subunits of AOX, Subunits A and C were reactive to the antibody but B was not. Luciferase, Subunits A and C of AOX, and PH have an SKL sequence at the C-termini. UOX has SRL, CAT has ANL, and DAOX has SHL at the C-terminus. The B subunit of AOX and PT do not have SKL or its variants at their C-termini. From these findings in the inhibition test and the immunoblot analysis, the SKL-Ab was shown to react with the SKL motif and its variants, most strongly with SKL itself but more weakly also with the variants SRL, AKL, SHL, and ANL. SKL-Ab could not detect DAOX with SHL but it did detect CAT with ANL, although SKL-Ab has a higher affinity SHL than ANL. This discrepancy may indicate that the structure of enzymes apart from their C-termini may have some effects on the reaction. These immunochemical data show that SKL-Ab specifically reacts with SKL tripeptide and its variants with different affinities. SKL-Ab could also detect signals in peroxisome fractions. These data indicated that SKL-Ab can detect these enzymes with SKL sequence and its variants in rat liver peroxisomes.
Other examples of the detection of various peroxisomal proteins employing differently designed antibodies for PTS1 and biotin-labeled PTS1 receptor have been reported. Antibodies detecting the SKL sequence, designated anti-PTS antibody, have been reported previously against synthetic peptides of CRYHLKPLQSKL and CRYLAGPHGSKL, which contain nine amino acids of the C-termini of AOX and PH, respectively (
SKL-Ab was reactive specifically with peroxisomes among various ultrastructures in rat hepatocytes and renal tubule epithelial cells by electron microscopy. The core and the matrix were reactive in normal and DOP-treated hepatocytes. The main constituent of the core is UOX (-hydroxyacid oxidase (
-hydroxyacid oxidase (
-hydroxyacid oxidase is not known and we do not have purified protein, we cannot say anything about the strong immunoreaction of the marginal plate. Considering the strong immunoreaction with the crystalline structure of the marginal plate, the magnitude of the immunoreactivity of this antibody may depend not only on the sequence but also on whether or not the crystalline structure is formed in peroxisomes.
As examined by immunoblot analysis on purified proteins of rat peroxisomes and peroxisome fractions of rat liver tissues, by the inhibition assay, and by immunoelectron microscopy, this new antibody reacts with various enzymes with PTS1 targeting signal in peroxisomes, and SKL-Ab can be said to be a peroxisome-specific antibody, at least for rat tissues. Many enzymes have been purified and their cDNAs have been cloned from mammalian tissues, especially from rat tissues. However, no example exists for other vertebrates. The reaction of SKL-Ab to peroxisomal enzymes could not be described for birds, reptiles, amphibians, and fish based on the sequence of C-termini of the enzymes. Peroxisomal proteins with PTS1 signal might also be present in other species, because peroxisomal proteins of various species have the PTS1 signal (
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Acknowledgments |
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Supported in part by grants-in-aid for scientific research from the Ministry of Education of Japan.
We gratefully thank Dr T. Hashimoto (Department of Pathology, Northwestern University Medical School) for kind guidance in purifying enzymes and raising antibodies, and Mr Jun Usuda for obtaining tissue materials.
Received for publication March 26, 1999; accepted March 30, 1999.
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