Localization of a Brain Sulfotransferase, SULT4A1, in the Human and Rat Brain : An Immunohistochemical Study
School of Biomedical Sciences, University of Queensland, St Lucia, Queensland, Australia
Correspondence to: Dr. Nancy Liyou, School of Biomedical Sciences, University of Queensland, St Lucia, 4072 Queensland, Australia. E-mail: nancyliyou{at}optushome.com.au
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Summary |
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Key Words: immunohistochemistry sulfotransferase human brain rat brain neuron localization
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Introduction |
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Cytosolic sulfotransferases expressed in the human brain include SULTs 1A1, 1A3, 2A1, and 1E1. However, cloning, enzymological, and immunohistochemical (IHC) studies have provided little information about the distinct localization and level of expression of each isoform in the brain (Rein et al. 1982; Rivett et al. 1982
; Young et al. 1984
; Viani et al. 1990
; Zou et al. 1990
; Duffel et al. 1991
; Homma et al. 1994
; and Beaujean et al. 1999
). Recently, cytosolic sulfotransferase consensus sequences have been used to identify a novel protein from the expressed sequence tag databases (Falany et al. 2000
; Liyou et al. 2000b
). Falany and co-workers (2000)
named this protein BR-STL and reported on its expression in the human and rat brain. No expression was found in other tissues investigated with Northern blotting analysis. This work revealed that the mRNA of this SULT isoform was highly expressed in the cortex of rat and human brain (Falany et al. 2000
). However, the authors were unable to demonstrate sulfotransferase activity.
After cloning the corresponding coding region from human brain cDNA, we expressed the recombinant protein in E. coli. We named this protein, according to the current nomenclature of sulfotransferases, SULT4A1 (Falany 1996). The recombinant protein, containing an N-terminal (His)6 tag, was purified using NiNTA affinity chromatography and used to raise polyclonal antibodies in the goat suitable for IHC studies. This study provides an IHC examination of the distribution and cellular localization of SULT4A1 in the human and rat brain.
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Materials and Methods |
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Bacterial Expression
A single E. coli BL21 (DE3) plysS cell colony, transformed with pET28a(+) containing the SULT4A1 insert, was used to inoculate 10 ml of LB Broth, supplemented with 30 µg/ml kanamycin and 34 µg/ml chloramphenicol, and incubated at 37C overnight. This overnight culture was then used to inoculate 250 ml LB Broth, containing 30 µg/ml kanamycin and 34 µg/ml chloramphenicol, to an OD600 of 0.1 and incubated at 37C with shaking until OD600 reached 0.60.8. Expression of the recombinant protein was induced by the addition of isopropyl-ß-D-thiogalactoside (IPTG) to 1 mM final concentration and the cells were allowed to grow for a further 4 hr at 37C. Cells were collected by centrifugation at 9000 x g for 45 min and the cell pellet stored at -20C until further processing.
Protein Purification
All steps were carried out at 4C in the presence of protease inhibitors (Cmplete EDTA-free protease inhibitor cocktail; Boehringer Mannheim, Mannheim, Germany) unless otherwise stated. Protein analysis was conducted using SDS-PAGE and silver staining as described by Liyou et al. (1999)
. Protein concentrations were estimated either from the absorbance at 280 nm or using the Pierce BCA reagent as described by Liyou et al. (2000a)
.
The frozen cell pellet was resuspended by direct addition of extraction buffer (50 mM Na2PO4, 300 mM NaCl, 5 mM imidazole, pH 7.0) and sonication (three times for 20 sec) on ice. DNase I (2.5 U/µl) was added to the sonicate and incubated for 20 min at 37C, followed by further sonication as above. The soluble fraction was collected via ultracentrifugation at 100,000 x g for 1 hr.
The supernatant obtained above was bound to Nickel Superflow IMAC resin (5 ml; Progen, Brisbane, Australia) with rolling for 2 hr at 4C. After washing with five column volumes of wash buffer (50 mM Na2PO4, 300 mM NaCl, 20 mM imidazole, pH 7.0) in batch form, the resin with bound protein was packed into a column for elution of specifically bound protein using FPLC (Pharmacia; Uppsala, Sweden) with a flow rate of 1.0 ml/min. The resin was washed with a further 10 column volumes of wash buffer. Elution was effected by the application of a 20500 mM linear imidazole gradient over 40 column volumes. The fractions (1.2 ml) were analyzed using A280 measurement and SDS-PAGE as described by Liyou et al. (1999). Based on the absorbance intensity, the recombinant SULT4A1 protein was eluted at around 220 mM imidazole and then concentrated using a Centriprep10 (10-kD cut-off) concentrating cell. SULT4A1 was further purified using anion exchange chromatography (MonoQ; Amersham, Castle Hill, Australia). Concentrated recombinant SULT4A1 was added to an equal volume of wash buffer 1 (50 mM Tris-HCl, pH 8.3) to give a binding NaCl concentration of 150 mM. The sample was applied to the resin at a flow rate of 1.0 ml/min, then washed with 10 column volumes of wash buffer 2 (50 mM Tris, 150 mM NaCl, pH 8.3). Elution of protein was effected using a linear 150300 mM NaCl gradient over 40 column volumes, collecting 1.5 ml fractions. The selected fractions were pooled, concentrated, and stored in 50% glycerol at -80C until further use.
Generation of Polyclonal Antibodies
Purified protein was emulsified with Freund's complete adjuvant for injection into goats. Before immunization, goats were bled to collect control serum (40 ml/animal). Two goats were immunized with approximately 500 µg protein each in multiple sites on the hind legs. Follow-up booster injections with 500 µg protein in incomplete Freund's adjuvant were performed 28 days after the initial injection. The goats were bled 14 days later (40 ml/animal) and serum collected. All animals used in this study were handled according to NHMRC Animal Ethics Committee guidelines. Ethical approval was obtained through the Animal Ethics Committee of the University of Queensland.
Antibodies were purified from the immune serum by affinity chromatography using E. coli recombinant SULT4A1 immobilized on cyanogen bromide-activated Sepharose according to the manufacturer's instructions (Pharmacia). Purified antibodies (in TBS) were concentrated and stored at -20C in 50% glycerol (to a final concentration of 0.46 mg/ml). Corresponding preimmune goat serum was also applied to the affinity resin and the eluate concentrated and stored as above for use as the negative control in the IHC investigation.
Immunoblotting of Purified Recombinant Human SULTs
The specificity of the goat antihuman SULT4A1 antibody was tested using a panel of expressed human sulfotransferases (SULTs 1A1, 1A2, 1A3, 1E1, 2A1, and 1C2). Proteins were separated on gradient SDS-PAGE under reducing conditions and transferred to nitrocellulose as described in Liyou et al. (2000a).
Very slight crossreactivity occurred with SULT1A1 but not with the other SULTs. To correct for this, the purified antibody was applied to an affinity resin of immobilized recombinant SULT1A1 as above. Antibody that did not bind was collected, concentrated, and stored in glycerol. The antibody purified in this manner showed specificity only towards SULT4A1.
Human Tissue Samples
The samples examined in this study were collected according to NHMRC Human Ethics Committee guidelines. Ethical approval was obtained through the Human Ethics Committee of the University of Queensland. Relevant patient information is provided in Table 1.
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Immunohistochemistry
Cases 3 and 4 were examined for this study (Table 1). Immunoperoxidase staining for humSULT4A1 protein was performed on 6-µm sections using the DAKO (Glostrup, Denmark) LSAB+ (labeled streptavidinbiotin) peroxidase system. Sections were deparaffinized in xylene and rehydrated.
Target retrieval was performed using DAKO Target Retrieval Solution (25 min, 97C) and then allowed to cool at room temperature for a further 20 min. Sections were then blocked using 1% H2O2 in buffer A (50 mM Tris-HCl, 0.5 M NaCl, 0.5% Tween-20, pH 7.6), washed in buffer A and incubated overnight (4C) with the goat anti-hum-SULT4A1 IgG (1:200) in buffer A. Sections were washed in buffer A and DAKO biotinylated linking antibody solution was applied (150 µl/section) for 25 min. Sections were then washed in buffer A before application of DAKO enzyme conjugated streptavidin (150 µl/slide, 20 min). Slides were washed in buffer A and Substrate Chromagen (DAKO) added for 5 min. For negative controls, the primary antibody was replaced with the corresponding affinity-purified preimmune serum.
Rat brain sections were obtained from adult male Wistar rats that were deeply anesthetized with sodium pentobarbitol (80 mg/kg IP) and perfused transcardially with 2% sodium nitrite solution (in 0.1 M PBS, pH 7.4) followed by 4% formaldehyde (in 0.1 M PBS, pH 7.4). Brains were quickly removed, postfixed for 2 hr in 4% formaldehyde, then cryoprotected overnight in 10% sucrose (in 0.1 M PBS, pH 7.4, 4C; Buller and Day 2002). Serial forebrain (40-µm) and brainstem (50-µm) sections were collected using a microtome. Two 1-in-5 brainstem series (250-µm interval) and two 1-in-4 forebrain series (160-µm interval) from each rat were immunolabeled, one brain series for the preimmune serum and one brain series for the SULT14A antibody. Sections were stained according to the human procedures described above except that dewaxing and rehydration were omitted. The sections were prepared by rinsing in TBS and blocking in TBS containing 1% H2O2. Labeling then proceeded as described for the human sections. After development the sections were washed in water and placed directly on glass slides to be air-dried overnight, then coverslipped.
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Results |
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Characterization of Polyclonal SULT4A1 Antibodies
Isoform-specific polyclonal antibodies were generated against recombinant humSULT4A1 as described in Materials and Methods. Specificity was determined by probing a range of purified recombinant human cytosolic SULTs via immunoblotting. Figure 1
shows that only recombinant SULT4A1 is recognized by the antibodies that were further purified with an antibody affinity column specific for the proteins of the SULT1A family.
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Localization of SULT4A1 in Human Brain Regions
To identify the specific regions of the brain in which SULT4A1 was expressed, homogenized human brain sections from four individuals were analyzed by immunoblotting. Figure 2
shows the result for one subject, demonstrating that SULT4A1 is expressed strongly, but to various degrees, in all regions examined. The other three subjects studied showed a similar pattern of SULT4A1 expression (results not shown).
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IHC staining of human cerebellum sections reveal localization of SULT4A1 to granular neuronal cell bodies within the folial granular layer of the cerebellum (Figure 3B). Staining was also observed in the Purkinje cells in the lateral lobe of the folia; however, staining was not observed in the vermis. A large number of SULT4A1-positive neurons were also seen in the human pituitary sections (Figure 3F). However, owing to the procedure of brain removal in the rat, the pituitary was not collected for analysis.
SULT4A1-positive neurons were visible in both the human and rat sections in layers of the hippocampus, including Ammon's horn and Sommer's sector. In both the rat and human, many labeled neurons were localized in all divisions of the thalamic nuclei (Figure 3D). In particular, the anterior and subthalamic nuclei were labeled intensely. SULT4A1 was localized to neurons (with and without neuromelanin, which is naturally stained a deep brown) and dendrites in the substantia nigra in human and rat midbrain sections (Figure 3H). Staining was apparent in the pars compacta and pars reticularis. In the hypothalamus, only scattered labeled neurons were found in the paraventricular nucleus. A group of labeled neurons was concentrated dorsal to the paraventricular nucleus in the zona incerta in the rat brain.
SULT4A1-positive neurons were found in the periaqueductal gray and were particularly localized in the dorsal, lateral, and ventrolateral divisions. Analysis of sections of the red nucleus of the midbrain also showed SULT4A1 localized to neurons of both human and rat sections (Figure 4F). The magnocellular neurons of the rat were especially strongly labeled compared to those observed in the human red nucleus.
In both human and rat brainstem sections, SULT4A1 was expressed strongly in neurons of the III cranial nerve (oculomotor nucleus; Figure 4D). Neurons containing SULT4A1 were localized to neuronal cell bodies of the inferior olivary nucleus in the human. In the brainstem, neurons were also strongly labeled in the XII (hypoglossal nucleus, Figure 4H), VII (facial nucleus), and V (trigeminal nucleus) cranial nerves of both human and rat. Many SULT4A1-positive neurons were localized in the lateral reticular nucleus in the brainstem. Labeled cells were also found in the gracile and cuneate nuclei. Very few labeled neurons were observed in the nucleus tractus solitarius. Neurons were labeled in the dorsal motor nucleus of the vagus in both the rat and human. The locus ceruleus was virtually devoid of positive SULT4A1 elements in the human, but in the rat the locus ceruleus was labeled strongly. The parabrachial nucleus of the rat also contained prominent SULT4A1 immunolabeling.
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Discussion |
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The novel sequence identified, cloned, and expressed in the present study establishes criteria for a new SULT family and therefore was named SULT4A1. The generation of isoform-specific polyclonal antibodies towards SULT4A1 in this study represents a powerful tool for examining the role of this enzyme. Immunoblotting analyses showed that SULT4A1 is expressed extensively, and apparently exclusively, in the brain. This is in contrast to other SULT isoforms, which are expressed in brain tissue as well as other tissues (Rein et al. 1982; Young et al. 1984
; Zou et al. 1990
; Duffel et al. 1991
; Homma et al. 1994
; Beaujean et al. 1999
). At present, the physiological substrate of SULT4A1 is unknown. However, the cDNA cloned in this study is 100% identical to that identified by Falany et al. (2000)
and has all the signatory sequences of a sulfotransferase cDNA (Kakuta et al. 1997
; Bidwell et al. 1999
).
The present study collectively demonstrates that SULT4A1 is expressed in the human and rat brain in a restricted manner. Furthermore, the region-specific expression pattern of SULT4A1 in the brain implies a function in the central nervous system, although the nature of this function is not yet clear. In the forebrain we observed the greatest degree of immunolabeling for SULT4A1 in the cortex (motor, cingulate, frontal, somatosensory), globus pallidus, islands of Calleja, septum, thalamus (lateral, medial dorsal, anterior, subthalamic), red nucleus, substantia nigra and pituitary. Therefore, in support of the only other study to have examined brain expression levels, a Northern blotting analysis conducted by Falany and co-workers (2000), we found a high degree of SULT4A1 expression in the cortex of both the human and rat. Interestingly, SULT4A1 expression was observed in neuronal cell bodies and dendrites in all six layers of the cerebral cortex. In the midbrain and brainstem, staining was particularly prominent in the cerebellum, nuclei of the III, V, VII, and XII cranial nerves, red nucleus, and lateral reticular nucleus.
Importantly, we report that many motor nuclei in the brain were found to express SULT4A1. SULT4A1 was localized to granular neuronal cell bodies in the folial granular layer of the cerebellum. The granular cells are the only excitatory neurons in the cerebellum and receive the main input into this region. The neurons of the red nucleus of the midbrain, which express SULT4A1, have cerebral, thalamic cerebellar, and brainstem connections. This pathway is concerned with reflexes involved in motor coordination and maintaining posture. SULT4A1 was also expressed in the III cranial nerve nucleus (oculomotor), which produces certain intrinsic and extrinsic movements of the eyeball. The neuronal cell bodies of the inferior olivary nucleus, which acts on cerebellar circuits to integrate sensory and motor information about movements in real time, also demonstrated the presence of SULT4A1. SULT4A1 was also present in neurons and dendrites in the substantia nigra in human midbrain sections. Many neurons in the substantia nigra send fibers to the basal ganglia, which are involved in the coordination of skeletal muscle movements.
It is notable that there was a high degree of similarity in the localization and level of intensity of SULT4A1 immunolabeling in the human and rat brain. Only relatively minor variations were observed. For example, only a paucity of staining was found in the human locus ceruleus but in the rat the degree of labeling was quite marked. On the other hand, in the human a strong signal was apparent in the amygdala but in the rat very little SULT4A1 immunolabeling was observed. Nevertheless, the high degree of conformity between the rat and the human is favorable if one were to subsequently use the rat as an animal model to investigate human brain SULT4A1 neurochemistry.
The present study greatly extends our knowledge of the distribution and cellular localization of cytosolic sulfotransferases in the brain and provides a foundation for understanding how SULT4A1 might regulate the functions and neurochemistry of different regions of the brain. Our laboratory is now undertaking structural and functional studies to determine the specific role played by SULT4A1.
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Acknowledgments |
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We would like to express our gratitude to Mr Roger Pearson of CSIRO Livestock Industries and to Dr Conrad Sernia and Paul Addison of the School of Biomedical Sciences.
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Footnotes |
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