Molecular characterisation of the smooth endoplasmic reticulum Ca2+-ATPase of Porcellio scaber and its expression in sternal epithelia during the moult cycle
1 Z.E. Elektronenmikroskopie, Universität Ulm, 89096 Ulm,
Germany
2 Department of Physiology and Biophysics, University of Illinois, Chicago,
IL 60612, USA
3 Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672,
USA
* Author for correspondence (e-mail: andreas.ziegler{at}medizin.uni-ulm.de)
Accepted 20 March 2003
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Summary |
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Key words: Porcellio scaber, smooth endoplasmic reticulum, Ca2+-ATPase, SERCA, biomineralisation, Isopoda, Crustacea, epithelial calcium transport
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Introduction |
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Like most crustaceans, P. scaber has a calcified cuticle, which is
moulted regularly to allow for growth of the animal. During premoult the ASE
transports Ca2+, originating from the posterior cuticle to form
large CaCO3 deposits located within the ecdysial gap of the first
four anterior sternites (Messner,
1965). Since isopods moult the posterior half of the body first,
followed by the anterior half, these deposits are degraded by the ASE within
24 h after posterior moult and used for the mineralisation of the new
posterior cuticle (Steel,
1993
). Ultrastructural investigations have shown that during the
formation and degradation of the CaCO3 deposits the ASE is
differentiated for ion transport
(Glötzner and Ziegler,
2000
; Ziegler,
1996
). Electron microprobe analysis of shock-frozen and
freeze-dried cryosections of the ASE revealed that the increase in cytoplasmic
calcium is not uniform across the cell, but occurs by increasing the number of
areas having high calcium concentrations, which are presumably the SER
(Ziegler, 2002
). This
hypothesis was supported by an in situ assay indicating an increase
in SERCA activity during epithelial Ca2+-transport
(Hagedorn and Ziegler, 2002
).
These results are in agreement with previous findings of a correlation between
enamel mineralisation and activity of a SERCA in vertebrate dental enamel
cells (Franklin et al.,
2001
).
Several SERCA isoforms have been identified in the crustaceans Artemia
franciscana (Escalante and Sastre,
1993) and Procambarus clarkii
(Chen et al., 2002
;
Zhang et al., 2000a
), but the
SERCA isoform involved in epithelial Ca2+-transport has not yet
been characterised, nor is it known if the rise in SERCA activity observed in
the ASE is accompanied by an increase in mRNA abundance. In order to increase
our knowledge about SERCA expression in crustacean Ca2+
transporting epithelia, we identified the SERCA cDNA of the ASE of P.
scaber and tested whether the SERCA mRNA level is upregulated during
epithelial Ca2+-transport. Our results show that the SERCA of the
ASE has a very high similarity to the axial muscle isoform of Procambarus
clarkii and Artemia franciscana. In contrast to the finding in
dental enamel epithelium, we observed an increase in SERCA mRNA abundance from
the non-transporting to the calcium-transporting stages.
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Materials and methods |
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Isolation of a partial SERCA cDNA sequence from anterior sternal
epithelium total RNA
Total RNA was extracted from anterior sternal epithelium (ASE) tissue under
RNAse-free conditions (Chomczynski and
Sacchi, 1987) using reagents obtained from Promega Corporation
(Madison, USA). ASE of 20 animals in the late premoult stage were carefully
dissected and pooled in RNAlater (Ambion, Houston, USA). Reverse
transcription of poly(A)+ mRNA was performed with oligo-dT primer
and Superscript II reverse transcriptase (Invitrogen, Karlsruhe, Germany). On
the basis of published SERCA sequences we designed the degenerate forward
SERCA-F1 (5'-TGG GCN AT(A/C/T) AA(C/T) AT(A/C/T) GGN CA(C/T)
TT-3') and reverse SERCA-R1 (5'-ATN GC(G/A) TT(T/C) TT(T/C) TTN
GCC AT-3') primers. Using the polymerase chain reaction (PCR) we
amplified a putative SERCA cDNA fragment. Following separation on agarose gels
(Fig. 1A), this PCR product was
extracted from the gel slices (Qiagen QiaQuick, Valencia, USA) and sequenced
using SERCA-F1 and SERCA-R1 by the dideoxynucleotide method
(Sanger et al., 1997
) at the
Marine DNA Sequencing Center of Mount Desert Island Biological Laboratory. A
BLAST search (Altschul et al.,
1997
) of GenBank confirmed close matches to published SERCA
sequences of other species.
|
Amplification of full-length SERCA cDNA
An amplified, adapter-ligated cDNA pool (`library') was constructed from
ASE total RNA, as described by Matz
(2002). Using the primer
5'-CGC AGT CGG TAC (T)13-3', reverse transcription of 1
µg total RNA from ASE tissue was done with Powerscript reverse
transcriptase (Clontech, Palo Alto, USA). Second-strand synthesis was
performed with DNA polymerase I (0.3 U/µl), RNase H (0.01 U/µl) and
E. coli DNA ligase (0.06 U/µl). An adaptor consisting of the
5'-CGA CGT GGA CTA TCC ATG AAC GCA ACT CTC CGA CCT CTC ACC GAG TAC
G-3' and the complementary 5'-C GTA CTC GGT-3'
oligonucleotides was ligated to the double-stranded cDNA. The double-stranded
cDNA pool was amplified using the Advantage 2 PCR-kit (Clontech) with the
adaptor primer (TRsa: 5'-CGC AGT CGG TAC (T)13-3') and
the distal adaptor primer (DAP: 5'-CGA CGT GGA CTA TCC ATG AAC
GCA-3'). PCR conditions were: 94°C for 40 s, 63°C for 1 min,
72°C for 2.5 min for 13 cycles. A sample was separated in a 1% agarose gel
electrophoresis to confirm the presence of cDNA.
The method described by Matz
(2002) was used to amplify
full-length cDNAs, with two rounds of step-out and nested PCR. Primers were
used at a concentration of 100 nmol l-1. The gene-specific primers
were synthesized on the basis of the previously obtained sequence of the
Porcellio scaber SERCA (GenBank accession no. AF375959). For
amplification, Advantage 2 Polymerase (Clontech) was used with the supplied
amplification buffer and dNTPs were added at a concentration of 0.75 µmol
l-1. As a template, 1 µl of the cDNA library (diluted 1:50) was
used for a 20 µl PCR mixture. Agarose gels of the PCR products are shown in
Fig. 1BE and the main
strategy of the procedure is outlined in
Fig. 2. For the first PCR
round, the antisense gene-specific primer for 5'-RACE was GSP1
(5'-AAT CGT TTC CTT CTG CTT TGT CT-3') and the adaptor-specific
sense primer DAP-st11 (5'-CGA CGT GGA CTA TCC ATG AAC GCA ACT CTC CGA
CCT CTC ACC GA-3'). The sense specific primer for 3'-RACE was GSP3
(5'-CTT GCT GTA GCC GCT ATT CCT-3') and the adaptor-specific
reverse primer DAP-TRsa (5'-CGA CGT GGA CTA TCC ATG AAC GCA CGC AGT CGG
TAC T13). We used 28 cycles under the following conditions:
94°C for 40 s, 65°C for 1 min and 72°C for 2.5 min.
|
For the second round of PCR, the preceding 5'- and 3'-RACE
reactions were diluted 1:50 with H2O and 1 µl of the diluted mix
was used in a total 20 µl mixture. The adaptor-specific primer DAP was
added to both 5'- and 3'-RACE products. The nested, reverse
gene-specific primer GSP2 (5'-GGA AGA GAA CGC ACA ATA GCA T-3')
was used for 5'-RACE and the nested, forward gene-specific primer GSP4
(5'-ATG CTA TTG TGC GTT CTC TTC C-3') for 3'-RACE. PCR
conditions were as described above and amplification was done for 16 cycles. A
sample (15 µl) of each PCR round was electrophoretically separated on a 1%
agarose gel. The bands detected in the 5'- and 3'-RACE reaction
were cut out of the gel, the DNA cleaned using the DNA-extraction kit (Qiagen)
and sequenced by SequiServe
(www.sequiserve.de).
More SERCA-specific primers were designed to sequence the open reading frames
of the fragments. The cDNA sequence was analysed using BioEdit
(Hall, 1999) and compared with
published sequences in GenBank using the BLAST algorithm
(Altschul et al., 1997
).
Phylogenetic analysis
ClustalW (Thompson et al.,
1994), as implemented in the Biological Workbench
(http://workbench.sdsc.edu),
was used to align full-length SERCA sequences. Phylogenetic and molecular
evolutionary analyses were conducted with MEGA version 2.1
(Kumar et al., 2001
) using the
neighbour joining algorithm (Saitou and
Nei, 1987
). Species and their GenBank accession numbers are given
in Fig. 7.
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In situ hybridisation of SERCA mRNA
The probe was a biotin-labeled 23-mer oligonucleotide (GA GCT GGA TCA TTG
AAG TGA CCA) complementary to a fragment of the mRNA encoding the
Porcellio SERCA (Fig.
3, nucleotides 9771000). An oligonucleotide with the
antisense sequence of the Na+/Ca2+-exchanger (AGA GGT
CGT GTC ATG GTT CCG) was used for control experiments
(Ziegler et al., 2002). All
probes were labeled with two biotins and were synthesized by Dr M. Hinz
(University of Ulm). A total of nine sternites from the early premoult, late
premoult and intramoult stages (3 animals at each moulting stage), including
the ASE and, as a control, the ganglial nervous tissue, were fixed for 1 h in
a mixture of 4% paraformaldehyde, 0.25% glutaraldehyde in 0.1 mol
l-1 cacodylate buffer, pH 7.3. The sternal epithelia were immersed
in 2.3 mol l-1 sucrose in 0.1 mol l-1 sodium cacodylate
buffer (SCB) for 90 min, mounted on aluminium rods and frozen in liquid
nitrogen. Semithin (0.7 µm) sections were cut sagittally with an Ultracut S
microtome equipped with a FCS cryochamber (Leica, Wien Austria), using glass
knives and an antistatic device (Diatome, Biel, Switzerland) at temperatures
of -70°C for the specimen holder, knife and cryochamber. Sections were
transferred to lysine-covered glass slides (Polyprep, Sigma), thawed and
washed with Tris-buffered saline (TBS). The hybridisation procedure was done
as described in detail previously (Ziegler
et al., 2002
). The labelled probes were applied at a concentration
of 1 ng µl-1 and the sections hybridized overnight at 50°C.
Detection was done using the TSATM Cyanin 3 System
(NENTM Life Science Products, Boston, USA) employing horseradish
peroxidase (HRP)-conjugated streptavidin and the fluorescent HRP-substrate
Cy3-Tyramide, diluted 1:50 in 1x amplification buffer, for 10 min in the
dark. After washing, the sections were mounted in 20% glycerol in 80% TBS and
examined with a fluorescence microscope (Axiophot, Zeiss, Jena, Germany). All
solutions were prepared with diethylpyrocarbonate (DEPC)-treated water and all
glassware and instruments had been sterilized.
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Results |
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The deduced full-length amino acid sequence showed high identity with
invertebrate SERCAs, e.g. 87% with the SERCA-sequence of Procambarus
clarkii (GenBank accession no. AAB82291), 80% with the SERCA of
Drosophila melanogaster (AAB00735) and 79% with the SERCA of
Artemia franciscana (CAA35980) (not shown). The submitted sequence
also had high identity to many vertebrate SERCAs, e.g. 71% to human SERCA1
(O14983), 71% to SERCA2 from chicken (QO3669) and 67% to human SERCA3
(Q93084). Putative functional residues are shown in
Fig. 3. Hydrophobicity analysis
of the amino acid sequence (Fig.
4) predicts ten transmembrane domains and a high similarity with
other vertebrate and invertebrate SERCAs (for comparison, see
Chen et al., 2002;
Zhang et al., 2000a
). In
situ hybridisation revealed that the amount of SERCA mRNA in the anterior
sternal epithelial cells of the early premoult stage was below the detection
limit (Fig. 5AD). A
large increase in the SERCA mRNA signal occurs from the early premoult to the
late premoult stage. The magnitude of the signal in the intramoult stage was
similar to that in the late premoult stage
(Fig. 5EH). Varying
amounts of unspecific binding occurred in the thick cuticle of early premoult
animals. In the ganglial nervous tissue no change in the SERCA mRNA abundance
was detected between moulting stages (Fig.
6AF). Sections treated with the control probe were
virtually devoid of any signal.
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Discussion |
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Amplification of a full-length SERCA cDNA along with the in situ
hybridisation results indicate that the sternal epithelium of P.
scaber expresses a SERCA. The SERCA of the ASE of P. scaber is
most similar to the axial muscle SERCA isoform of Procambarus clarkii
(Zhang et al., 2000a), which
shares 87% identical amino acids. Both are the same length, 1002 amino acids,
and have identical putative functional domains, e.g. the phosphorylation site
(Lee and East, 2001
), the
63-67 KILLL residues (Fig. 3)
known as the putative retention-signal for the ER/SR
(Magyar and Varadi, 1990
) and
the residues that are thought to be involved in the formation of two
Ca2+-binding sites (Zhang et
al., 2000b
). However, the C and N termini of these proteins are
less highly conserved. For example, within the first 27 amino acids of the
Porcellio SERCA C terminus, 18 amino acids deviate from the
corresponding Procambarus sequence, four of them from hydrophilic to
hydrophobic residues (S4
A, Q10
A,
E20
I, D26
A). In the last 15 amino acids of
the N terminus of the Porcellio sequence, 7 amino acids differ from
the axial muscle SERCA sequence, including two hydrophobic residues becoming
hydrophilic (A997
E, I999
K).
The lack of any indication of other SERCA isoforms in the sternal tissue
does not exclude their existence, particularly in other tissues of P.
scaber. In addition to the axial muscle isoform already mentioned
(Chen et al., 2002), a second
cardiac-muscle tissue-specific SERCA isoform (AAB82290) has been found in
Procambarus clarkii. The Porcellio SERCA is also highly
homologous to the cardiac-muscle isoform, which has a hydrophobic N terminus
containing an additional 18 amino acids, possibly forming an additional 11th
transmembrane spanning region (Chen et al.,
2002
). Two isoforms have also been found in Artemia
franciscana embryonic and adult tissue, where the 6-amino-acid N terminus
in one isoform is substituted by 30 amino acids in the other. Escalante and
Sastre (1995
) showed that
these isoforms arise from regulated expression by alternative tissue-specific
promotors rather than expression from different genes.
Even though the arthropod SERCAs have a high similarity to vertebrate
SERCAs, they generally cannot be classified as any of the vertebrate SERCA
sequences. Vertebrate SERCAs are encoded by three different genes that are
mainly expressed in a tissue-specific manner. The SERCA1 isoforms are mostly
found in fast twitch muscle, SERCA2a in cardiac and slow twitch skeletal
muscle, and the alternative splicing product SERCA2b is distributed
ubiquitously. SERCA3, which occurs in epithelia and endothelia, has been found
in three alternatively spliced products. As far as it is known, the arthropod
SERCAs, including the new Porcellio SERCA sequence, are encoded by
only one gene, giving rise to the hypothesis that one arthropod and the three
vertebrate genes derived from a common ancestral gene
(Escalante and Sastre, 1996).
The phylogenetic tree in Fig.
7, showing the relationship between full-length arthropod and some
vertebrate SERCAs, supports this hypothesis. It is of interest that the
sequences of the Malacostraca Porcellio and Procambarus are
more closely related to insects than to the branchiopod crustacean
Artemia. The Artemia SERCA sequence differs from the
Malacostraca and insect sequence by an insertion of three amino acids,
D83E84A85, and one amino acid at
P381. At present it is unknown whether this is specific to
Artemia franciscana, or is an apomorph character of the Branchiopoda;
however, it is worth noting that there is increasing controversy about the
monophyletic origin of Crustacea, and some hypotheses even propose that the
Branchiopoda branched off the main stem of Crustacea before the insects. For a
more detailed discussion of this issue, refer to Harzsch
(2002
) for example.
Variations of SERCA-mRNA abundance as a function of the crustacean moulting
cycle as described here for the ASE of P. scaber, have already been
demonstrated in Procambarus cardiac and abdominal axial muscle. These
changes in SERCA mRNA could result from either regulated gene expression or
variable mRNA stability. It has been shown that SERCA expression can be
regulated by certain hormones and growth factors (for a review, see
Hussain and Inesi, 1999). Such
a connection between moulting stage and mRNA abundance therefore suggests that
SERCA mRNA expression may be regulated by moulting hormones, for example
20-hydroxyecdysone (also suggested by
Zhang et al., 2000a
).
Interestingly, the expression pattern of SERCA mRNA observed in the ASE of
P. scaber is opposite to that observed in Procambarus muscle
tissues. In the ASE, SERCA mRNA abundance increases from early premoult to
late premoult and intramoult stages, but decreases in Procambarus
from intermoult to pre-and postmoult stages
(Zhang et al., 2000a
). Thus,
if the same moulting hormones regulate SERCA gene transcription, an increase
of hormone concentration would induce opposite effects in the ASE of P.
scaber and in Procambarus muscle. This hypothesis is supported
by tissue-specific moult-related synthesis of actin protein in cheliped and
walking leg muscle of crayfish (El Haj et
al., 1992
). A high 20-OH ecdysone titer in the haemolymph
corresponds with an elevated actin expression in leg muscle and a decreased
expression of actin in cheliped muscle. Possible explanations for these
tissue-specific inverse effects include differential expression or activation
of the ecdysteroid receptor, or, as suggested by Zhang et al.
(2000a
), a regulated
accessibility of the receptor.
The ASE is specialized for ion transport since cuticle secretion is
retarded within the anterior integument during the formation and resorption of
the sternal CaCO3 deposits
(Messner, 1965;
Ziegler, 1997
). Therefore, the
increase in mRNA abundance within the ASE from early premoult to late premoult
and early premoult to intramoult suggests that the SERCA plays a role in
epithelial Ca2+-transport. This is supported by the lack of such
increases in the ganglial nervous tissue, which indicates that the increase of
SERCA mRNA abundance in the ASE is tissue specific, and not due to general
changes of SERCA expression during the moult cycle. Our results are in
accordance with previous analyses of the relative SERCA activity within
permeabilised epithelial cells of the ASE employing the in
situ calcium oxalate technique
(Hagedorn and Ziegler, 2002
).
SERCA activity was below the detection limit during early premoult and
increased to detectable values in mid premoult (2 weeks after the anterior
moult), and further increased significantly by factors of five and four from
mid-premoult to late premoult and from mid-premoult to intramoult,
respectively (Hagedorn and Ziegler,
2002
). The concomitant increase in SERCA mRNA abundance suggests
that the increase in activity of the protein is correlated with an increase in
SERCA mRNA expression. This is in contrast to results obtained in vertebrate
mineralising epithelial cells (Franklin et
al., 2001
). In the rat dental enamel epithelium, abundance of
SERCA 2b isoform and the activity of the nonmitochondrial
Ca2+-ATPase within the microsome fraction is upregulated to high
expression levels during enamel maturation, consistent with a role of SERCA in
epithelial Ca2+-transport. However, upregulation of SERCA was not
evident at the mRNA level, suggesting post-transcriptional regulation
(Franklin et al., 2001
).
The nature of the SERCA's functional involvement in bulk flow of
Ca2+ through mineralising epithelia is still unknown. Simkiss
(1996) suggested that loading
and discharging of membranous compartments, possibly SER cisterns, would lead
to a vectorial translocation of Ca2+. Such a translocation would
result in only transient micromolar gradients of Ca2+, which can be
tolerated by the cells. However, the frequent discharge and reuptake of
Ca2+ions from and to the SER would require a large amount of
energy. Another, more economic mechanism would be the diffusion of
Ca2+ through the lumen of the SER, possibly facilitated by
low-affinity Ca2+-binding proteins. The latter mechanism is
supported by the finding (Hubbard,
1996
) that in rat dental enamel cells the Ca2+ binding
proteins calreticulin and endoplasmin, both localized in the SER, are
upregulated during epithelial Ca2+-transport. However, this does
not exclude a more passive function of the SER e.g. Ca2+ buffering
to avoid high cytosolic Ca2+ concentrations during SER-independent
Ca2+ transit mechanisms, such as vesicular transport
(Nemere, 1992
) or cosecretion
of Ca2+ with matrix proteins (see
Hagedorn and Ziegler, 2002
,
for a more comprehensive discussion).
An essential function of the SER during bulk calcium flow through the
cytoplasm may be in conflict with its function in cellular Ca2+
signalling. Thus, in our previous study
(Hagedorn and Ziegler, 2002)
we suggested that different SERCA-isoforms and possibly SER subcompartments
could avoid such conflicts. However, in the present study we did not find any
evidence for the presence of multiple SERCA isoforms in the ASE, which raises
the possibility that mechanisms connected with the SER Ca2+ release
channels may separate putative functional conflicts.
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Acknowledgments |
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