Expression of two isoforms of the vacuolar-type ATPase subunit B in the zebrafish Danio rerio
Institut für Zoologie und Limnologie, Universität Innsbruck, Austria
* Author for correspondence (e-mail: Bernd.Pelster{at}uibk.ac.at)
Accepted 18 March 2003
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Summary |
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Key words: V-ATPase, subunit B, isoform, hepatocyte, expression, zebrafish, Danio rerio.
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Introduction |
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V-ATPases are multi-subunit enzyme complexes that translocate protons
across membranes using the free energy of ATP hydrolysis. They consist of two
main parts, the so-called head region or the peripheral sector (V1)
for ATP hydrolysis, and the Vo domain, which includes the part of
the protein necessary for proton translocation
(Wieczorek et al., 1999). For
some of the subunits, e.g. a, E and G, tissue- or cell-specific isoforms have
been described, and the different isoforms appear to be involved in different
cellular processes (Murata et al.,
2002
; Sun-Wada et al.,
2002
; Oka et al.,
2001
; Mattsson et al.,
2000
; Toyomura et al.,
2000
).
The head region includes three A and three B subunits, and two isoforms of
the B subunit, which is part of the regulatory/catalytic domain of the enzyme,
have been described in Homo sapiens, Bos taurus and Rattus
rattus (Breton et al.,
2000; Van Hille et al.,
1994
; Puopolo et al.,
1992
). According to tissue-specific expression patterns the two
isoforms are the so-called `kidney isoform' (vatB1) and the `brain isoform'
(vatB2), and the kinetic properties of the mature enzymes appear to be
different (Gluck, 1992
;
Wang and Gluck, 1990
). The
expression of different subunits with different properties may of course be
related to different physiological functions, and one possibility, for
example, could be that one isoform is related to proton transfer through
epithelial membranes, while the second isoform is involved in the
acidification of intracellular vesicles.
In a previous study we cloned and sequenced two isoforms of the B-subunit
of V-ATPase in swimbladder tissue of the European eel Anguilla
anguilla (Niederstätter and
Pelster, 2000), but in trout gill cells only one isoform of the B
subunit has been detected (Perry et al.,
2000
). Comparison of the conserved amino acid residues of the two
isoforms of eel with the mammalian proteins led to the conclusion that the two
eel isoforms may both be related to the mammalian `brain' V-ATPase B subunit
(Cutler and Cramb, 2001
). On
the other hand, comparison of the more variable amino acid residues of the
isoforms clearly revealed that eel vatB1 has a higher homology to the `kidney'
isoform of mammals. If both eel subunits were homologous to the `brain'
subunit of mammals, this would have some interesting implications. Given the
fact that fish are older in evolutionary terms than mammals, the implication
is that the kidney isoform evolved after separation of the fishes, either as a
new isoform, or by modification of one of the two brain isoforms. However,
homology of the single isoform sequenced from trout with the kidney isoform of
mammals has also been shown (Perry et al.,
2000
). Thus two isoforms may also exist in other fish species,
despite the fact that only one isoform has been detected in trout.
Furthermore, immunohistochemical observation of gas gland cells from the eel
swimbladder revelaed that the subunit vatB1 is mainly located in the apical
region, while the vatB2 is also found in basolateral membranes
(Boesch et al., 2003
), which
confirms the idea that these two isoforms may indeed serve partially different
functions. The expression of both isoforms in eel was not limited to the
swimbladder and was detectable by reverse transcriptase polymerase chain
reaction (RT-PCR) in other organs such as gills and kidney (H.
Niederstätter and B. Pelster, unpublished results).
The present study was therefore designed to test the hypothesis that two isoforms of the B-subunit exist in the zebrafish Danio rerio, a member of the large family of cyprinid fish. First, molecular evidence for the expression of two isoforms of the V-ATPase subunit B in the zebrafish was found by cloning and sequencing RNA fragments of both isoforms using degenerate primers by RT-PCR. After determination of the complete coding sequences, zebrafish subunit vatB1 and vatB2 were expressed in a bacterial system. We then investigated the tissue distribution of these two isoforms using RT-PCR, western-blot analysis and immunohistochemistry. The data provide evidence that expression of both isoforms of the vacuolar-type ATPase subunit B is not only restricted to fish species that experience severe changes in water osmolarity, such as the catadromous eel, but may also be a common phenomenon in freshwater fish like the zebrafish.
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Materials and methods |
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RNA isolation
Total RNA was extracted from 50100 mg tissue samples according to
the procedure described by Chomczynski and Sacchi
(1987) using Trizol Reagent
(Life Technologies, Gibco, BRL, Karlsruhe, Germany). After DNAse treatment the
concentration of total RNA was determined using RiboGreen RNA quantification
kit (Molecular Probes, Eugene, OR, USA).
RT-PCR amplification of V-ATPase subunits B1 and B2 fragments
In the first step, degenerate oligonucleotide primers (HATP-1 forward and
reverse) for RT-PCR were used. Approximately 1 µg of total RNA extracted
from zebrafish gill tissue was used in reverse transcription to generate the
first strand cDNA. Then a 1 µl portion of the cDNA was used in a standard
PCR reaction containing 20 nmol l1 of each primer, 10
µmol l1 dNTPs and 1x Advantage-2 Polymerase
(Clontech, Palo Alto, CA, USA) at an annealing temperature of 50°C over 30
cycles. The resulting PCR fragment was purified using a GeneClean kit
according to the manufacturer's instructions (BIO101, Carlsbad, CA, USA) and
ligated into a pCR®4-TOPO cloning vector (Invitrogen, Carlsbad,
CA, USA). The plasmid was transfected and subsequently amplified in E.
coli strain TOPO10 (Invitrogen) according to standard protocols. DNA
sequencing was carried out as described below using M13 forward and reverse
primers (Invitrogen).
RLM-RACE of V-ATPase subunits B1 and B2
Following cloning and DNA sequencing of the first RT-PCR products, gene
specific primers (GSP) for use in 5'-RACE and 3'-RACE (specific
for each isoform) were designed. An RNA ligase-mediated rapid amplification of
cDNA ends (RLM-RACE) Kit (GeneRacerTM, Invitrogen, Carlsbad, CA,
USA) was used to transcribe RNA with AMV reverse transcriptase and
GeneRacerTMOligo dT primers to full-length 5' and
3'-end cDNA. RACE-PCR was performed in a volume of 25 µl consisting
of 1x PCR buffer (Clontech), 0.2 µmol l1 of each
primer, dNTPs and 0.5 µl Advantage 2 Polymerase (Clontech) using a
touch-down protocol in a Gene Amp PCR System 9700 (PE Applied Biosystems,
Norwalk, CT, USA). After gel purification with GeneClean (BIO-101) PCR
fragments were routinely cloned into pCR®4-TOPO vector and
sequenced using M13 forward and reverse primers and sequence-specific primers
where necessary.
DNA sequencing and analysis of the cloned PCR products
For sequencing we used the dye terminator system (PE Applied Biosystems,
Foster City, CA, USA) and an automated sequencer (373A DNA Stretch Sequencer;
PE Applied Biosystems). The templates for sequencing were generated by
purification of plasmid DNA using the alkaline lysis method (QuiaPrep,
Quiagen, Hilden, Germany). DNA sequences were analyzed using ABI Prism
Sequencing Analysis Software (Version 3.0, PE Applied Biosystems) and aligned
with the Sequence Navigator AC software package (Version 1.01, PE Applied
Biosystems) in combination with the GenBank and Swiss-Prot databases for
comparison with other known sequences.
Determination of tissue-distribution by RT-PCR
Total RNA from several tissues (muscle, heart, intestine, liver, spleen,
gills, swimbladder) of the zebrafish was isolated as described above.
First-strand cDNA synthesis was performed with 500 ng total RNA of each
sample, random hexamer primers and MLLV Reverse Transcriptase (Power
ScriptTM, Clontech). Quality of cDNA was controlled by PCR for the
house-keeping gene ß2-microglobulin (for primers, see
Table 1). Using two specific
primer pairs for the two V-ATPase subunit B isoforms, the presence of V-ATPase
and the tissue-specific distribution of these subunits were analyzed.
Sequencing of the resulting PCR products was performed in order to verify the
specificity of the primers. The PCR products obtained were visualized by
agarose gel electrophoresis and ethidium bromide staining.
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Bacterial expression of recombinant zebrafish vatB1 and vatB2
The open-reading frames (ORFs) including the native start codons, but not
the stop codons, were amplified using specific primer pairs for each isoform
of the B subunit of V-ATPase (see Table
1). PCR was performed at a volume of 25 µl consisting of
1x PCR buffer (Clontech); 25 pmol of the corresponding primer, 25
µmol of each dNTP and 0.5 µl Advantage HF-2 mix (Clontech) in a Gene Amp
PCR system 9700 thermocycler (Applied BioSystems). cDNA made from zebrafish
gill tissue served as template. The purified PCR-products were ligated into a
pCR® T7/CT TOPO® vector using T/A cloning
strategy (Invitrogen) and cloned into TOPO 10F' cells (Invitrogen). The
plasmids were analyzed in both directions by sequencing using sequencing
primers T7-forward and V5C-term reverse (Invitrogen). Expression and detection
of both isoforms in BL21(DE3)pLysS (Invitrogen) cells followed the procedures
described by Boesch et al.
(2003). The presence of the
induced protein was verified by western-blot using specific antibodies that
were made against peptides of the two V-ATPase B subunit isoforms of the
European eel. One antibody was directed against an amino acid sequence close
to the 5' end specific for vatB1 (#1035), and a second antibody (#1034)
was specific for a conserved amino acid sequence that is identical for both
isoforms (vatB1 and vatB2). Whereas peptide sequences of zebrafish and eel
were identical for the binding site of antibody #1034, in the binding site of
antibody #1035 at position 3 asparagine was replaced by proline and at
position 5 glutamic acid was replaced by aspartic acid (see
Fig. 1).
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Determination of tissue specific distribution by western-blot
analysis
Tissue samples from zebrafish were excised and immediately soaked in Trizol
(Life Technologies) containing 0.16% of ß-mercaptoethanol (Sigma-Aldrich,
Vienna, Austria) following the instructions of the manufacturer. The resulting
protein pellets were solved in 1x SDS sample buffer containing 1%
proteinase inhibitor cocktail (Sigma-Aldrich), 0.05% PMSF (Sigma-Aldrich) and
1% ß-mercaptoethanol. Proteins were separated under reducing conditions
(125 µmol l1 dithiothreitol) using a 10% Bis-Tris gel and
Mops buffer (Novex, San Diego, USA), and blotted onto PVDF membranes (BioRad,
Hercules, CA, USA) using a constant voltage of 25 V for 1 h. Membranes were
blocked for 1 h with 0.2% I-block (Tropix, USA) and 0.1% Tween 20
(Sigma-Aldrich) in 0.1 mol l1 PBS at room temperature.
Primary antibody incubation was performed overnight at 4°C in blocking
buffer. Use of various antibody concentrations for the western blot revealed
that the optimal signal was obtained at a dilution of 1:250 for antibody
#1034, and of 1:50 for antibody #1035. After additional washing steps the
membranes were probed for 1 h with a horse radish peroxidase (HRP)-conjugated
second antibody (HRP-conjugated anti-rabbit IgG; Sigma-Aldrich). Finally,
proteins were visualized using the enhanced chemiluminescence ECL detection
reagents (Amersham, Buckinghamshire, UK).
Preparation of histological specimens and immunohistochemistry
Tissue preparation and immunohistochemical localization of V-ATPase
subunits B basically followed the procedure described by Boesch et al.
(2003). Briefly, dissected
tissue samples were fixed in 4% buffered paraformaldehyde at 4°C
overnight. The samples were dehydrated in a series of ethanol baths and
finally embedded in paraffin by sequential incubations in methyl benzoate
(1x overnight, 3x 312 h), benzene (2x 30 min),
benzene/paraffin (1x 2 h at 60°C) and paraffin (three changes within
1216 h).
For the immunocytochemical localization paraffin sections (45 µm thickness) from various tissues were cut using an Autocut 2040 (Reichert, Vienna, Austria) and mounted on coated glass-slides (Dimethylsilane, Sigma-Aldrich-Chemie, Vienna, Austria). The sections were dewaxed by series of xylene and ethanol. After antigen-retrieval by proteinase-K digestion and acetylation with 10 min incubation in 0.5% anhydrous acetic acid in 0.1 mol l1 Tris-HCl, pH 8.0, non-specific bindings were blocked with 10% foetal calf serum in Tris-buffered saline (TBS). Incubation with the appropriate (optimal) dilution of the primary antibodies in blocking buffer was undertaken at 4°C overnight. After five washes with TBS slides were incubated with a polyclonal biotinylated anti-rabbit/mouse IgG (Duett-ABC Kit Solution C; Dako, Glostrup, Denmark) for 20 min. Then additional washes were performed and the sections were probed with an anti-biotin alkaline-phosphatase antibody (dilution 1:100; Dako) for 1 h. Finally a purple color reaction was developed at 4°C in a solution of 4-nitro blue tetrasodium chloride (Roche Molecular Biochemicals, Mannheim, Germany) and 5-bromo-4-chloro-3-indolylphosphate-4-toluisin salt (Roche). The sections were washed three times with TBS, mounted in Gel Mount (Lipshaw Immunon, Pittsburgh, PA, USA) and coverslips placed on top. Sections were observed and photographed using bright-field light microscopy (Polyvar, Reichert, Vienna, Austria; Zeiss, Jena, Germany).
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Results |
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Expression of recombinant zebrafish V-ATPase subunits B1 and B2 in E.
coli
The open reading frames of zebrafish vatB1 and vatB2 including native start
codons were cloned and expressed in E. coli BL21(DE3)pLysS
cells. The bacteria were harvested by centrifugation and directly transferred
into sample buffer prior to SDS-gel electrophoresis. Expression was carried
out at 25°C, and addition of 0.5 mmol l1
isopropylthiogalactoside (IPTG) clearly induced the expression of a 60 kDa
protein after 2 h, corresponding to the molecular mass of both recombinant
isoforms, which was increased using 6-Histag by approximately 5 kDa. Western
blot analysis of the recombinant zebrafish vatB1 and vatB2 proteins with
antibodies directed against vatB (#1034) and vatB1 (#1035) showed that the
expressed proteins indeed were vatB1 and vatB2. Recombinant zebrafish vatB1
was recognized by both antibodies, whereas vatB2 was recognized only by
antibody 1034 (see Fig. 2).
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Tissue distribution of V-ATPase subunits B1 and B2 in zebrafish
The tissue distribution of V-ATPase subunits B1 and B2 was assessed at the
RNA and the protein levels. Samples of different tissues from several animals
were dissected and either RNA or the protein isolated. Each experiment was
replicated with at least four different animals. RT-PCR for vatB1 and vatB2
revealed that both isoforms are expressed in all tested tissues
(Fig. 3). The sequence of the
amplified PCR product was verified by sequence analysis.
|
The distribution of vatB1 and vatB2 at the protein level was assessed by western blot analysis. Total protein was isolated, and 30 µg of each protein sample used for each blot. V-ATPase was expressed in all tissues tested, but there were differences in the V-ATPase content of the different tissues. The highest level of expressed protein was detected in gill, liver and heart tissues. Whereas in most tissues the band produced by the two antibodies was similar in size, in the liver the band obtained with antibody #1035 (vatB1) was not as prominent as that obtained with antibody #1034 (vatB1 and vatB2). A high content of both B-subunit isoforms was also found in the intestine, the spleen and the kidney. Very little, however, was detected in swimbladder, although western blot analysis with an increased amount of protein (60 µg) revealed presence of the B-subunit of V-ATPase in this tissue as well. To avoid false positive results due to the expression of V-ATPase in blood cells we also tested samples of zebrafish blood. These samples showed almost no reactivity when probed with the antibodies. We therefore concluded that the discovered B-subunits of V-ATPase indeed did represent protein from the tested tissues (Fig. 4).
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Immunohistochemical localization of the two B subunits in the gills, heart, and kidney cells gave similar results for both antibodies. In the liver, however, the two antibodies yielded very different results. While antibody #1035 gave a very weak signal, antibody #1034 gave a strong signal in virtually all cells (Fig. 5). Controls without primary antibody showed no staining. In the small intestine, localization of the B subunit yielded weak staining in a number of cells, but the antibody #1034 generated a strong signal in large cells located near the lamina propria, which according to their location and size are thought to be neurosecretory cells (enterochomaffin cells, APUD cells; Fig. 6). These cells could not be identified with the antibody #1035, which only binds to isoform vatB1.
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Discussion |
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The B-subunit of the V-ATPase is part of the head region of the protein,
and it is not a transmembrane protein. VatB1, mainly expressed in specialized
proton-translocating intercalated cells (B-cells) in the rat kidney, carries a
C-terminal binding motif (DTAL motif) that enables interaction with the PDZ
domain of the Na+/H+-exchanger regulatory factor
(NHE-RF) (Breton et al., 2000).
The vatB2 isoform, also expressed in various cell types of the kidney, lacks
this binding domain and therefore cannot interact with members of the PDZ
protein family. Thus, interaction between vatB1 and NHE-RF or other PDZ
proteins may contribute to a differential localization of V-ATPases containing
either the B1 or the B2 subunit. NHE-RF has also been shown to interact with
actin filaments of the cytoskeleton
(Murthy et al., 1998
).
Holliday et al. (2000
)
demonstrated that both isoforms of the B subunit of mammalian osteoclasts,
vatB1 and vatB2, include actin binding sites within the first 106 or 112 amino
acid residues of the amino-terminal region. Thus, the B subunit appears to be
involved in an interaction of V-ATPase with the cytoskeleton. Given the high
homology between the eel isoforms and the two mammalian isoforms (see
Niederstätter and Pelster,
2000
) it is quite likely that these binding sites have been
conserved.
The two isoforms apparently differ not only in their kinetic characteristics, but also in the way they interact with other proteins or subunits, which suggests that the mammalian isoforms of the B-subunit included in the V-ATPase may modify the physiological properties of the ATPase. None of the vatB1 isoforms of fish sequenced so far includes the DTAL motif, which is present in the mammalian kidney isoform (see Fig. 1), a possible indication that the physiological function of fish vatB1 might differ from the function of the mammalian kidney isoform.
The expression of enzymes with different properties implies that they may
serve different functions, and this in turn would result in a distinct
localization of the isoforms. In the swimbladder of the European eel we indeed
observed that the vatB1 was located mainly in the apical membrane and in
lamellar bodies, while vatB2 appeared to be mainly located in basolateral
membranes (Boesch et al.,
2003). In the zebrafish we found that mRNA of both isoforms is
present in all tested tissues. RT-PCR, however, is a very sensitive tool and
it cannot be excluded that in some tissues only a few copies of the mRNA were
present, so that the protein in fact is barely expressed.
On the protein level we indeed observed that the expression of the B-subunit is variable between the different tissues analyzed. The gills, for example, which play an important role in osmoregulation, had a relative high expression of V-ATPase.
We also found expression of the B subunit in the liver and the spleen, both organs with a high metabolic activity. In the liver, however, western blot analysis indicated that the vatB1 subunit was not as abundant as vatB2. This was confirmed by immunohistochemistry. Localization of the subunits within the tissue clearly revealed that vatB2 was present in all hepatocytes, while antibodies directed specifically against vatB1 produced only a very weak signal, indicating that this subunit was barely expressed. In the liver, V-ATPase is mainly involved in the acidification of intracellular organelles; there is no epithelial proton transport as, for example, observed in fish gill cells or in tubular kidney cells. The predominance of subunit vatB2 in this tissue therefore suggests that vatB2 might mainly be involved in intracellular proton transport and contribute to an acidification of lysosomes and other cell vesicles.
The small intestine provided a second example of differential localization of the isoforms, since neurosecretory (enterochomaffin cells, APUD cells) could be stained with antibody #1034, but not with the vatB1-specific antibody antibody #1035. Neurosecretory cells are secretory cells, and from our results vatB2 is the predominant isoform in these cells.
As V-ATPases are ubiquitous enzymes it is not surprising that they are also
present in skeletal muscle tissue and the heart. Here the V-ATPase may be
located in the membrane of synaptic vesicles, where it pumps protons from the
cytoplasm into the synaptic vesicle
(Nelson and Harvey, 1999;
Kanner and Schuldiner, 1987
).
Hong (2002
) showed in a
pharmacological study that the release of neurotransmitters from synaptic
vesicles at the nerve-muscle junction is inhibited by bafilomycin A, a
selective inhibitor of the V-ATPase.
In summary, the results of our study demonstrate that the existence of two
isoforms of subunit B of V-ATPase is widespread among fishes, despite the fact
that in rainbow trout only one isoform has been detected. The differential
localization of the two isoforms observed in the swimbladder of the European
eel (Boesch et al., 2003), in
the liver and in small intestine neuroscretory cells of zebrafish suggest that
the two isoforms may serve partly different functions.
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Acknowledgments |
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