ARTICLE |
Correspondence to: Anna Filipek, Nencki Institute of Experimental Biology, 3 Pasteur, 02-093 Warsaw, Poland. E-mail: anfil@nencki.gov.pl
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Summary |
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The expression of a novel calcyclin (S100A6) binding protein (CacyBP) in different rat tissues was determined by Western and Northern blotting. Polyclonal antibodies against recombinant CacyBP purified from E. coli exhibited the highest reaction in the brain and weaker reaction in liver, spleen, and stomach. CacyBP immunoreactivity was also detected in lung and kidney. Densitometric analysis showed that the concentration of CacyBP in the soluble fractions of total brain and cerebellum is approximately 0.17 and 0.34 ng/µg protein, respectively. Northern blotting with a specific cDNA probe confirmed the high level of CacyBP expression in the rat brain and lower levels in other tissues examined. Immunohistochemistry and in situ hybridization of rat brain sections revealed strong expression of CacyBP in neurons of the cerebellum, hippocampus, and cortex. The in situ hybridization detected CacyBP in hippocampus as early as P7 (postnatal day 7) and a peak of expression at P21, and the expression signal was preserved until adulthood. In the entorhinal cortex, the peak of expression was observed at P7, whereas in the cerebellum it was seen at P21. The results presented here show that CacyBP is predominantly a neuronal protein. (J Histochem Cytochem 48:11951202)
Key Words: CacyBP, tissue and cell expression, neuronal localization
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Introduction |
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Calcyclin (S100A6) is an EF-hand calcium binding protein belonging to the S100 family (reviewed in
The new search for Ca2+-dependent targets of calcyclin has led us to the identification of a novel protein (
In this study we analyzed the distribution of this novel calcyclin binding protein, CacyBP, in different rat tissues using specific antibodies developed against the recombinant CacyBP and using a cDNA probe encoding this protein. We also performed in situ hybridization and immunohistochemistry on rat brain sections to identify CacyBP-positive cells. Because the strongest expression of calcyclin binding protein was found in the brain, particularly in neurons, we estimated the amount of CacyBP in this tissue.
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Materials and Methods |
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Antibodies
Polyclonal antibodies were raised in rabbits. Recombinant GSTCacyBP fusion protein was expressed in E. coli as described by
Tissue Extracts
Various rat tissues, as well as parts of the brain such as hippocampus, cerebellum, cortex, and striatum, were homogenized using a Polytron at 6000 rpm in a solution containing 1 mM EDTA, 1 mM dithiothreitol, 10 mg/liter leupeptin, 5 mg/liter aprotinin, 20 mg/liter soybean trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride, and 40 mM Tris, adjusted to pH 7.5 with HCl. The extracts were centrifuged for 1 hr at 4C and 12,000 rpm in an Eppendorf centrifuge. The supernatants were subjected to SDS-PAGE. In all studies that employed animals, the rules established by the Ethical Committee on Animal Research of the Nencki Institute and based on disposition of the President of Polish Republic were strictly followed.
Electrophoresis and Immunoblotting
Gel electrophoresis with 15% (w/v) polyacrylamide containing 0.1% SDS was performed by the method of
Northern Blots
RNA was prepared from various rat tissues using an RNA purification kit obtained from Strategene (La Jolla, CA) and applied on a 1.2% agarose gel in the presence of formaldehyde. Ethidium bromide staining showed similar amounts of RNA in each lane. For detection of CacyBP mRNA, a specific cDNA probe (0.9-kb fragment cDNA between 26 and 931 bp of the original clone) was labeled with [32P]-deoxycytidine 5'-triphosphate ([32P]-dCTP) according to the method of
Measurement of CacyBP Concentration in Rat Brain
The proteins from the supernatant fractions of a total brain and cerebellum obtained after centrifugation at 12,000 x g in an Eppendorf centrifuge were separated by 15% SDS-PAGE and transferred to nitrocellulose filters under the conditions described above. Protein concentration was estimated by Bradford's procedure (BioRad reagent; Hercules, CA) with bovine serum albumin as a standard. The intensity of bands identified by the CacyBP antibody were estimated on films after developing the blots with a chemiluminescent kit (Kirkegaard & Perry Laboratories; Gaithersburg, MD). Different time exposures from two independent blots were used for optical density measurements using a Molecular Dynamics (Sunnyvale, CA) densitometer. The concentration of CacyBP expressed in brain and cerebellum was calculated using different amounts of native CacyBP purified as described by
Immunohistochemistry
Immunohistochemical studies were performed on the rat brain sections. The brain was snap-frozen on dry ice, sliced into 20-µm sections with a cryostat (ReichertJung), melted onto slides covered with poly-L-lysine, and stored at -70C. Then the sections were fixed for 15 min with 4% paraformaldehyde. After washing, the aldehyde groups were reduced by treating the sections for 10 min with 5% NaBH4 in H2O. Sections were washed three times with PBS (pH 7.4), incubated for 20 min with 0.3% H2O2 in PBS, washed twice in PBS, then incubated with 5% bovine serum albumin for 30 min, washed with PBS, and incubated overnight at 4C with serum containing the antibodies against CacyBP diluted 1:2500 in PBS and 3% bovine serum albumin. After washing (three times for 10 min in PBS), the distribution of CacyBP was visualized by incubation with secondary antibodies conjugated with horseradish peroxidase, diluted 1:1000 in PBS and 2% bovine serum albumin. Sections were washed five times for 10 min in PBS and color development was performed with PBS containing 0.5 mg/ml diaminobenzidine, 0.024% cobalt chloride, and 0.02% nickel ammonium sulfate for 5 min. Then H2O2 was added to 0.005% and the incubation was prolonged for another 10 min. The first washing was performed in PBS containing 0.024% cobalt chloride and 0.02% nickel ammonium sulfate for 5 min and the next four washes with PBS only. Control sections were incubated with preimmune serum or with PBS. After the reaction was complete, the sections were dehydrated in ethanol steps and mounted. The sections were analyzed with the use of a computer-assisted image analysis system, MCID 4.
In Situ Hybridization
For in situ hybridization analysis (35S-labeled cDNA (0.9 kb fragment cDNA between 26 and 931 bp of the original clone) as a probe at 37C. The sections were washed for 15 min and then for 1 hr in 50% formamide, 2 x SSC at room temperature. Then the sections were exposed for 2 weeks against ß-Max Hyperfilm (Amersham). The autoradiograph was analyzed with the aid of the PC-based computer program ProVision.
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Results |
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Tissue Distribution of CacyBP in the Rat
The immune serum was generated against GSTCacyBP fusion protein. Therefore, it contained antibodies against both GST and CacyBP. When immune serum was preabsorbed with CacyBP, it stained only recombinant GST (Fig 1A), but when it was preabsorbed with GST, staining only of purified CacyBP was seen (Fig 1B). Neither GST nor CacyBP was stained on the blots incubated with preimmune serum (not shown). The immune serum recognized only one band of molecular weight 30,000 in the brain extract (Fig 1C). As shown in Fig 1D, this band migrates at the same level as the CacyBP purified from Ehrlich ascites tumor cells and recombinant CacyBP obtained after removing of GST. Thus, despite the fact that immune serum represents a mixture of antibodies specific to both CacyBP and GST, it does not stain proteins other than CacyBP in the brain extract. Therefore, it could be used for immunohistochemical localization in this tissue.
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When various rat tissues were tested with the immune serum, the 30,000 immunoreactive protein band was found mainly in the brain, liver, spleen, and stomach (Fig 2A). A moderate level of CacyBP was found in lung and kidney, but in heart and skeletal muscle a band with higher molecular weight (~38,000) was stained. This higher molecular weight protein stained by immune serum may represent a new muscle-specific isoform of CacyBP. However, Northern blotting with the CacyBP cDNA probe (Fig 2B) showed that in all tissues examined a band at the same level (1.8 kb) was observed. The highest mRNA level of CacyBP was found in the brain and a good correlation between the CacyBP protein and its mRNA is observed in this tissue. There was not such a good correlation in the protein and mRNA level in other tissues, such as spleen, liver, stomach, and lung.
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CacyBP Level in Different Parts of Rat Brain
Because CacyBP appears to be expressed predominantly in the brain, we employed Western blotting to analyze its level in different parts of the brain. A nitrocellulose blot containing equal amounts of proteins derived from the soluble fraction of cerebellum, hippocampus, cortex, and striatum was incubated with immune serum containing specific polyclonal antibodies. As shown in Fig 3A, a high level of CacyBP was found in the cerebellum and hippocampus and a low level in the cortex. No positive reaction was seen in the striatum. Similar results were obtained by Northern blotting, confirming the different expression level of CacyBP in various brain regions (Fig 3B).
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Estimation of CacyBP Concentration in Rat Brain and Cerebellum
To estimate the concentration of CacyBP, the proteins from the supernatant fraction derived from total brain and cerebellum were blotted from SDS-PAGE onto nitrocellulose and CacyBP was detected with an immune serum containing specific polyclonal antibodies. Different amounts (3 µg, 15 µg, and 75 µg) of total brain and cerebellum protein were loaded onto the gel. Densitometric analysis of bands from the Western blot containing a known amount of CacyBP were used to obtain a calibration curve from which the amount of CacyBP was calculated (not shown). It was found that the CacyBP concentrations in the soluble fractions of total brain and cerebellum were 0.17 and 0.34 ng/µg protein, respectively.
Cell-specific Expression of CacyBP in Rat Brain
To identify the CacyBP-positive cells, we employed both in situ hybridization and immunohistochemistry. Cryostat sections of adult rat brain, as well as brains at different stages of development, were hybridized with a 35S-labeled cDNA probe. As shown in Fig 4, a high level of expression was observed in the entorhinal cortex (Ent) at P7 (postnatal day 7). At P14 and later until adulthood, no obvious differences in the mRNA level in cortical regions could be seen. The expression was quite uniform throughout the thickness of the cortex (Ctx). No laminar pattern of CacyBP mRNA expression could be revealed. In cerebellum (Cb) at P14, a high level of mRNA was observed in the internal granule cell layer (IGL) and disappeared in the external granule cell layer (EGL). At P21, a marked increase of CacyBP mRNA expression was seen in the granule cells (Gr). This increase was transient; expression declined and reached an adult level as early as P28. In hippocampus, the specific mRNA was present in all fields of Ammon's horn and dentate gyrus (DG). No clear developmental changes of either pattern or intensity of labeling were observed. The immunoreactivity pattern was similar to that obtained with in situ hybridization. Slight differences were observed in the hippocampus, in which the most intense staining was seen in the CA3 region, whereas in situ hybridization revealed the highest level of CacyBP mRNA in the dentate gyrus.
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The high-magnification micrographs demonstrated that, in the dentate gyrus, CacyBP-immunopositive cells were neurons located in the granule cell layer (Gr). Many immunopositive cells were also found in the polymorphic cell layer (Po) (Fig 5A). In the CA1 field of the hippocampus, virtually all pyramidal layer neurons (Py) were immunostained (Fig 5C). Some cells in the molecular layer (M) were also positive (Fig 5C, arrow). Pyramidal neurons (Py) of the CA3 region were very intensely stained (Fig 5D), but cells located outside the pyramidal layer were also clearly positive (Fig 5D, arrow).
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In the cerebellum, the most intense immunostaining was found in Purkinje cells (Pu) (Fig 5B). A lower level of immunoreactivity was observed in granule cells (Gr). In the neocortex, no specific layer was distinguished. Positive cells were both pyramidal and nonpyramidal in shape and were uniformly distributed (Fig 5E). In all experiments, control sections were incubated with preimmune serum or with PBS and no positive reaction was observed (not shown).
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Discussion |
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In this study we investigated the expression pattern of CacyBP, recently cloned and showed to bind to calcyclin in a physiological range of calcium concentration (
Northern analysis on the rat brain was performed using mouse cDNA. Although the sequence homology between mouse and rat CacyBP clone is not known (there is no rat sequence of CacyBP in GenBank), there is a high sequence homology between mouse and a human CacyBP clone (accession number AAC 21458). There are also a few sequences found in the EST database that come from different sources and which are highly homologous to the mouse CacyBP clone. Therefore, it was justified to check the possibility of using the mouse cDNA clone to study the expression in rat tissues. This proved to work, because the mouse clone stained only mRNA of CacyBP in the total RNA from rat tissues. Both Western and Northern blotting analysis revealed that the rat brain contains an especially high level of CacyBP, and in this tissue there was the best correlation between protein and mRNA level. Within this tissue, the cerebellum was found to be particularly abundant in CacyBP protein, the level of which was established by densitometric analysis to be in the range reported for brain-specific proteins such as calretinin (
Using immunohistochemistry and in situ hybridization, we have established that CacyBP is expressed predominantly in neuronal cell bodies, as indicated by high levels noted for Purkinje cells of the cerebellum and for pyramidal and granule cell layers of the hippocampus. In addition, CacyBP-immunopositive cell bodies were also observed scattered throughout the cerebellar cortex, except for Layer I. These data are in agreement with our recent findings showing that CacyBP is highly expressed in the mouse neuroblastoma cell line NB2a but not in glioma C6 cells (not shown).
The pattern of CacyBP expression in the rat brain established by Western and Northern blotting does not correlate well with that of calcyclin (
We have also investigated the developmental pattern of CacyBP mRNA expression in the rat brain. The most striking feature was a peak of expression at P21 in the cerebellum. This is interesting because in the same region of the brain several calcium binding proteins change their pattern of expression postnatally (
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Acknowledgments |
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Supported by grant 6P04 A 05415 from the State Committee on Scientific Research and by statutory funds provided to the Nencki Institute.
We thank Dr P. Groves for critical reading of the manuscript.
Received for publication October 6, 1999; accepted March 8, 2000.
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