Arginine kinase in the demosponge Suberites domuncula: regulation of its expression and catalytic activity by silicic acid
-Ottstadt1
1 Institut für Physiologische Chemie, Abteilung Angewandte
Molekularbiologie, Universität, Duesbergweg 6, D-55099 Mainz,
Germany,
2 Center for Marine Research, `Ruder Boskovic' Institute, HR-52210 Rovinj,
Croatia
3 CNR-Direzione Progetto Finalizzato Biotecnologie, Via Leon Battista
Alberti 4, I-16132 Genova, Italy
* Author for correspondence (e-mail: wmueller{at}uni-mainz.de)
Accepted 1 December 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: sponge, arginine kinase, siliceous spicule, Suberites domuncula, primmorph, energy metabolism
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Silica contributes over 60% of the dry demosponge biomass
(Desqueyroux-Faundez, 1990),
sometimes even reaching 90% (Barthel,
1995
). Since the average concentration of silica in seawater is
low, with values of less than 3 µmol l-1
(Maldonado et al., 1999
) close
to the surface, it can be extrapolated that most of the energy generated by
sponges is required to build the siliceous skeleton. This assumption is
supported by a recent study, which demonstrated that sponges are provided with
a (potential) silicic acid transporter that depends on the establishment of
energy-requiring ion gradients; this (potential) transporter comprises a
4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) binding site
(Schröder et al., 2004a
).
DIDS prevents uptake of silicic acid in sponge cells, indicating that the
(potential) silicic acid transporter is involved in the formation of silica in
the spicules. The silica of the spicules is synthesized enzymatically with the
help of silicatein (Cha et al.,
1999
; Krasko et al.,
2000
). The substrate required for silicatein to form silica is
actively taken up by the tissue and finally by the cells, sclerocytes, which
form the spicules (Schröder et al.,
2004a
).
Since the studies of Roche et al.
(Roche and Robin, 1954;
Roche et al., 1957
) it has
been well established that sponges contain the creatine phosphate/creatine
kinase system. These findings were confirmed by molecular biological studies
on the demosponge Tethya aurantia
(Ellington, 2000
;
Sona et al., 2004
). Based on
their data it was hypothesized that the creatine kinase system evolved early
in metazoan evolution in organisms composed of highly polarized cells
(Ellington, 2001
). In line
with these studies, we extend the available biochemical/molecular biological
data for sponges to formulate the pathway involved in the formation of ATP in
the cytoplasm.
In eukaryotic cells the major portion of ATP is generated in the
mitochondria via oxidative phosphorylation; the high energy
equivalents have then to be transported to the cytoplasm. The enzymes that
catalyze the reversible transfer of high-energy phosphoryl groups of ATP to
naturally occurring guanidine compounds, e.g. creatine, glycocyamine,
taurocyamine, lombricine and arginine, are the phosphagen kinases (see
Suzuki et al., 1997). In
vertebrates, phosphocreatine is the only phosphagen, and the corresponding
phosphagen kinase is creatine kinase, while in invertebrates more kinases
corresponding to the respective phosphagen have been described (see
Muhlebach et al., 1994
). On
the grounds of the high sequence similarity among these kinases it has been
proposed that members of the phosphagen kinases evolved from one common
ancestor (Suzuki and Furukohri,
1993
); however, to date the evolutionary processes are not fully
understood (Ellington, 2001
).
Phosphagen kinases are missing in fungi and plants.
In the present study we show that the gene encoding arginine kinase is
differentially highly expressed during exposure of sponges to silicic acid. We
identified the cDNA from the demosponge Suberites domuncula and
determined its expression level. In parallel, the enzymic activity of arginine
kinase was monitored to establish the importance of this enzyme during
siliceous spicule formation. S. domuncula is suitable for such
experimental studies since it can be maintained in aquaria for over 3 years
(Le Pennec et al., 2003) and
proliferating cells from this sponge can be cultivated in 3D-cell cultures,
termed primmorphs (Müller et al.,
1999
); furthermore an Expressed Sequence Tags (EST) library
containing over 15 000 cDNAs is available
(http://spongebase.genoserv.de/).
Searching this database revealed no creatine kinase or other phosphagen
kinases besides the arginine kinase.
The results reported here suggest that silicic acid causes an induction of
the expression of the arginine kinase gene and an increase of enzyme
activity in primmorphs after incubation. This effect could be abolished by
coincubation with DIDS. Recently it was found in sponge cells that DIDS blocks
the uptake of silicic acid (Schröder
et al., 2004a).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sponges
Live specimens of Suberites domuncula Olivi (Porifera,
Demospongiae, Hadromerida) were collected near Rovinj (Croatia) and then kept
in aquaria in Mainz (Germany) for more than 2 years prior to their use.
Formation of primmorphs and incubation conditions
The procedure for the formation of primmorphs (3D-cell cultures) from
single cells was as described previously
(Custodio et al., 1998;
Müller et al., 1999
).
Starting from single cells, primmorphs of 3-7 mm are formed after 5 days.
7-day-old primmorphs were used for the experiments, cultivated in natural
seawater supplemented with 0.2% of RPMI1640 medium. These 3D-cell cultures
were transferred into RPMI1640 medium. Incubation was performed in the
presence of the 2 µmol l-1 silicic acid (ambient concentration,
present in the natural seawater) or in seawater adjusted to a silicic acid
concentration of 60 µmol l-1
(Krasko et al., 2002
) and
incubated for up to 5 days. Then the samples were used for the determination
of arginine kinase activity and for in situ hybridization analysis.
Where indicated, DIDS was added at a final concentration of 100 µmol
l-1; the incubation studies were performed in darkness. In
parallel, samples were taken for extraction of RNA from
liquid-nitrogen-pulverized sponge tissue using TRIzol Reagent (GibcoBRL, Grand
Island, NY, USA), as recommended by the manufacturer.
Isolation of the cDNA for the arginine kinase
These studies were performed with primmorphs incubated either for 5 days in
the absence of additional silicic acid in the RPMI1640/seawater medium, or
cultivated in RPMI1640/seawater supplemented with 60 µmol l-1
silicic acid.
The technique of differential display for identification of the arginine
kinase cDNA was as described (Müller et al.,
2002,
2003a
). RNA (1 µg) from
controls and from specimens treated with silicic acid was reverse-transcribed
using the T11CC oligonucleotide as 3' primer. The resulting
cDNA was added to the polymerase chain reaction (PCR) using the arbitrary
primer GTGATCGCAG and the T11CC primer in the assay, together with
[
32P]-dATP. After amplification the radioactive fragments
were separated on a 6% polyacrylamide sequencing gel and autoradiographed. The
major DNA bands were collected and subsequently reamplified in 50 µl
reactions. Finally, the products were subcloned in pGEM-T vector
(Promega, Madison, WI, USA) and sequenced. Among the over 50 sequences
obtained, five partial cDNAs were isolated whose deduced polypeptide sequence
showed similarity to that of arginine kinase. The complete cDNA was obtained
by primer walking (Ausubel et al.,
1995
; Wiens et al.,
1998
). The arginine kinase cDNA was 1355 nucleotides (nt)
long and was termed SDAK.
Sequence analysis
Sequences were analyzed using computer programs BLAST 2003
(http://www.ncbi.nlm.nih.gov/blast/blast.cgi)
and FASTA 2003
(http://www.ncbi.nlm.nih.gov/BLAST/fasta.html).
Multiple alignments were performed with CLUSTAL W Ver. 1.6
(Thompson et al., 1994).
Phylogenetic trees were constructed on the basis of amino acid (aa) sequence
alignments by neighbour-joining, as implemented in the `Neighbor' program from
the PHYLIP package (Felsenstein,
1993
). The distance matrices were calculated using the Dayhoff PAM
matrix model as described (Dayhoff et al.,
1978
). The degree of support for internal branches was further
assessed by bootstrapping (Felsenstein,
1993
). The graphic presentations of the alignments were prepared
with GeneDoc (Nicholas and Nicholas,
1997
).
RNA preparation and northern blot analysis
RNA was extracted from liquid-nitrogen-pulverized tissue using TRIzol
Reagent (GibcoBRL) as described (Grebenjuk
et al., 2002). Then 5 µg of total RNA was electrophoresed and
blotted onto Hybond-N+ nylon membrane (Amersham; Little Chalfont,
Bucks, UK). Hybridization was performed using a 350 nt segment of the
SDAK cDNA; a 400 nt segment of the house-keeping gene ß-tubulin
of S. domuncula, SDTUB (EMBL/GenBank accession number AJ550806), was
used as an internal standard. The probes were labeled using the
PCR-digoxigenin (DIG)-Probe-Synthesis Kit (Roche, Mannheim, Germany). After
washing, DIG-labeled nucleic acid was detected with anti-DIG Fab fragments and
visualized by chemiluminescence technique using CDP (Roche). For
semiquantitative analysis of the expression levels, the bands on the film were
scanned using the GS-525 Molecular Imager (Bio-Rad, Hercules, CA, USA).
In situ localization studies
The method used was based on a described procedure
(Polak and McGee, 1998;
Perovi
et al., 2003
).
Frozen sections (8 µm) were prepared, fixed, treated with Proteinase K and
subsequently fixed again with paraformaldehyde. To remove the sponge color the
sections were washed with increasing concentrations of ethanol and decreasing
concentrations of acetone. After rehydration the sections were hybridized with
the labeled probe, a 250 nt long SDAK cDNA portion. After blocking,
the sections were incubated with an anti-digoxigenin antibody conjugated with
alkaline phosphatase. The dye reagent NBT/X-Phosphate was used for
visualization of the signals. Antisense and sense ssDNA DIG-labeled probes
were synthesized by PCR using the PCR-DIG-Probe-Synthesis Kit (Roche). Sense
probes were used in parallel as negative controls in the experiments.
Determination of arginine kinase activity
Primmorphs were exposed to silicic acid in the absence or presence of DIDS
as indicated. Samples were homogenized in lysis-buffer [1x TBS
(Tris-buffered saline), pH 7.5, 1 mmol l-1 EDTA and protease
inhibitor cocktail (1 tablet/10 ml; Roche)], centrifuged and the supernatants
subjected to enzyme activity determination.
Arginine kinase activity was determined according to a modified procedure
(Nealon and Herderson, 1976).
The reaction mixture (final volume 1 ml) contained 10 mmol l-1
Tris-maleate buffer (pH 7.0) supplemented with 0.4 mmol l-1
phosphoarginine, 10 mmol l-1 glucose, 5 mmol l-1
MgSO4, 1 mmol l-1 NADP+, 5 units
ml-1 hexokinase, 1 unit ml-1 glucose-6-phosphate
dehydrogenase and 10 µl of enzyme preparation. The appearance of NADPH was
monitored in a spectrophotometer at 340 nm (25°C) and standardized using
known amounts of ATP. Arginine kinase activity was expressed on the basis of
protein content and given as nmol ATP min-1 µg-1
protein. Five parallel experiments were performed.
Microscopical inspections
Transmission electron-microscopic (TEM) observations were performed using a
Zeiss (Aalen, Germany) EM 9A electron microscope. Primmorphs were pre-fixed in
2% glutaraldehyde in 0.05 mol l-1 sodium cacodylate buffer (pH 7.4)
with 0.2 mol l-1 sucrose. After 3 days the specimens were
post-fixed in 1% osmium tetroxide in 0.05 mol l-1 cacodylate buffer
(pH 7.4) at 4°C for 2 h. After dehydration the specimens were embedded in
araldite; thin sections (700 Å) were double stained in uranyl acetate
and lead citrate (Müller et al.,
1986).
Analytical techniques
Protein concentration was measured according to Lowry et al.
(1951) using bovine serum
albumin as standard. The concentration of silicate in the natural seawater
used for the experiments was determined, applying the molybdate (Silicon Test)
method (Simpson et al.,
1985
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The highest similarity of the S. domuncula AK_SUBDO was found with
arginine kinase cDNA from the cnidarian (Anthozoa) Anthopleura
japonica (Suzuki et al.,
1997), with an `Expect value [E]'
(Coligan et al., 2000
) of
6e-65 and an overall score of similar and identical aa residues of
51% and 35%, respectively. Therefore, the sponge sequence was termed arginine
kinase. The sponge polypeptide displays the two characteristic domains of the
ATP:guanido phosphotransferases (ISREC server;
http://hits.isb-sib.ch/cgi-bin/PFSCAN_parser),
the N-terminal (between aa29 to aa109; Pfam PF02807
[http://www.sanger.ac.uk/cgi-bin/Pfam/getacc?])
and C-terminal catalytic domains (aa120 to aa380; Pfam
PF00217). These domains are implicated in the reversible transfer of high
energy phosphate from ATP to the various phosphogens. The aa residues
coordinating transfer of Mg2+ to ATP
(Suzuki et al., 1997
) are also
present (Fig. 1A).
Phylogenetic analysis of S. domuncula arginine kinase
Arginine kinases, like all other members of the guanidino kinases, are
restricted primarily to Metazoa; so no rooting could be performed when
constructing a phylogenetic tree. The phylogenetic tree comprised the `usual'
mono-domain 40-kDa guanidino kinases (arginine kinases) and the `unusual'
two-domain 80 kDa guanidino kinases (arginine kinases). The two-domain kinases
identified in the Cnidarian Anthopleura japonica
(Suzuki et al., 1997) and the
mollusks (Bivalvia) Solen strictus
(Suzuki et al., 2002
),
Corbicula japonica (Suzuki et
al., 2002
) and Ensis directus
(Compaan and Ellington, 2003
),
were dissected into two parts and both parts were aligned in parallel with the
mono-domain guanidino kinases from mollusks, oyster Crassostrea gigas
and Solen strictus, crustaceans Homarus gammarus
(KARG_HOMGA, 538542), Eriocheir sinensis and Artemia
franciscana, and insects, Drosophila melanogaster and
Schistocerca americana. In addition, glycocyamine kinase from the
annelid Nereis virens, human creatine kinase
(Mariman et al., 1987
) and the
holothurian enzyme from Stichopus japonicus were included in the
alignment. The tree shows very strikingly that the duplication of the arginine
kinases occurred in some mollusk species (e.g. S. strictus), but not
in others (e.g. C. gigas; Fig.
1B). It had been proposed recently
(Takeuchi et al., 2004
) that
gene duplication and their subsequent fusion in mollusks occurred separately
from events that took place in hydrozoa (A. japonica).
Induction of arginine kinase gene expression in response to silicic acid
In order to clarify whether the S. domuncula arginine kinase gene
is upregulated after incubation with silicic acid, primmorphs were cultured in
the presence or absence of 60 µmol l-1 additional silicic acid.
Northern blot analyses were performed with the labeled cDNA probe for arginine
kinase (SDAK) and the house-keeping gene ß-tubulin
(SDTUB). These studies revealed that after addition of silicic acid
to the primmorphs, within 1 day a significant (threefold) increase of the
steady-state level of arginine kinase transcripts could already be
measured, a value which further increased after additional 2-4 days
(Fig. 2, left). In contrast, if
the primmorphs remained in culture without additional silicic acid, no
significant change of the expression level could be seen
(Fig. 2, right). Control blots,
performed with the house-keeping gene ß-tubulin, confirmed that the same
amount of RNA was loaded onto the gels. In parallel, expression studies were
performed in the presence of 100 µmol l-1 DIDS for 0-5 days, and
in the presence of this inhibitor the silicic acid-mediated upregulation of
arginine kinase expression was not seen (data not shown).
|
In situ hybridization analyses
As further proof that in primmorphs gene expression of arginine kinase is
positively affected by silicic acid, in situ hybridization studies
were performed. Primmorphs were incubated for 5 days in the absence
(Fig. 3A,B) or presence of 60
µmol l-1 of silicic acid
(Fig. 3C,D). Then cryosections
were hybridization with the labeled probe for arginine kinase (SDAK).
Sections from primmorphs that remained for 5 days in the absence of silicic
acid did not show high signals using antisense SDAK ssDNA probes
(Fig. 3A,B). However, if the
primmorphs had been kept in the presence of silicic acid, high expression of
this gene was seen, especially in the center of these 3D-cell cultures
(Fig. 3C,D). In a further
series, the primmorphs were cultivated in the presence of silicic acid
together with the inhibitor DIDS (100 µmol l-1;
Fig. 3E,F). In sections from
these primmorphs the expression level was almost identical to that seen in
cultures without additional silicic acid. No reaction was observed when the
cells were treated with sense probes (not shown).
|
Arginine kinase activity in sponge primmorphs after silicic acid incubation
Primmorphs were obtained as described under `Materials and methods'. After
formation of the 3D-cell cultures, cultivation in seawater was continued in
the absence of additional silicic acid for 5 days. Under these conditions,
arginine kinase activity was approximately 2.5 nmol ATP generated
min-1 10 µg-1 protein. This value did not change
significantly when primmorphs were incubated with 100 µmol l-1
DIDS (Fig. 4). However, if the
cultures were incubated with an additional 60 µmol l-1 of
silicic acid the enzyme activity had already increased after 1 day to 6 nmol
ATP min-1 10 µg-1 protein, a value which rose to 27
nmol ATP min-1 10 µg-1 protein on further incubation.
Coincubation with DIDS reduced the enzyme activity by more than 70%
(Fig. 4). These data show that
the arginine kinase activity increased strongly in primmorphs in response to
silicic acid.
|
Formation of spicules in primmorphs from S. domuncula
We have previously demonstrated that primmorphs start to form spicules
after incubation with 60 µmol l-1 silicic acid
(Krasko et al., 2000). Here we
cut cross sections through primmorphs that had been cultivated for 5 days in
the absence or presence of 60 µmol l-1 silicic acid. TEM
analysis of primmorphs grown in the absence of silicic acid did not show any
cells that synthesized spicules. However, in primmorphs kept in the presence
of silicic acid, sclerocytes that contained newly growing spicules were
frequently observed (Fig. 5).
In Fig. 5A two sclerocytes are
seen, each surrounding a spicule. At higher magnification it can be observed
that the blunt end of one spicule is tightly associated with two fibrils,
which are very likely collagen fibers (Fig.
5B,D); however such fibers were not always present at the blunt
end (Fig. 5C). Those fibers
surrounded the spicules at all phases (Fig.
5B-D). Note that the sharp, likewise growing, tip of the spicule
showed an inhomogeneous structure, suggesting that the material in this
regions is not densely packed (Fig.
5D).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The technique of differential display of mRNA was used to identify which
energy transferring kinase is involved in one major energy-consuming reaction
in sponges, the formation of the skeleton. Primmorphs were exposed to silicic
acid and their mRNA expression patterns were compared with those in 3D-cell
aggregates, which grew in the low-silicic acid medium. Silicic acid is the
inorganic component of the spicules in Demospongiae. As outlined in the
Introduction, the spicules are the major skeletal elements of sponges; their
framework is connected by filamentous organic fibrils
(Garrone, 1978). Furthermore,
it should be mentioned that the growth of spicules is a fast process
(Elvin, 1972
). The synthesis
and formation of this scaffold require considerable energy reserves.
Analytical studies revealed that arginine is one dominant amino acid in
sponges, reaching intracellular concentrations of 2 mmol l-1 (Marco
Giovine; unpublished observations). The differential display analyses revealed
that 10% of the transcript fragments contribute to the arginine kinase cDNA.
The full-length cDNA was identified. It comprises, as with all other metazoan
phosphagen kinases, the two domains characteristic for these kinases, the
N-terminal domain and the C-terminal catalytic domain. Also the
Mg2+/ATP coordinating residues within the C-terminal domain are
conserved.
To study the phylogenetic aspect of the sponge arginine kinase was equally
interesting. In yeast (Saccharomyces cerevisiae) or plants (e.g.
Arabidopsis thaliana) no arginine kinases, or related enzymes, exist.
Hence the sponge molecule is evolutionarily one of the oldest phosphagen
kinases. This fact is also reflected by the radial phylogenetic tree, which
shows that the human/vertebrate creatine kinases, and the glycocyamine kinase
(Nereis virens) branched off from the sponge common ancestor molecule
(Fig. 1B). Arginine kinases
emerged in the other direction, comprising the `usual' mono-domain 40-kDa
guanidino kinases (arginine kinases) and the `unusual' two-domain 80 kDa
guanidino kinases (arginine kinases). Interestingly, the next closest arginine
kinase family member, the cnidarian A. japonica
(Suzuki et al., 2002;
Takeuchi et al., 2004
) has
already undergone an independent gene duplication in another taxon, the
mollusks (see Fig. 1). While
the guanidino kinase from the mollusk C. gigas belongs to the
mono-domain kinases, other molluskan kinases, from S. strictus, C.
japonica and E. directus, are two-domain 80 kDa guanidino
kinases. It is amazing that within the Bivalvia, and even in the same subclass
Lammellibranchia, this duplication proceeded from mono-domain, C.
gigas (superorder Filibranchia), to two-domain kinases, S.
strictus (Eulamellibranchia), C. japonica (Eulamellibranchia)
and E. directus (Eulamellibranchia). It was suspected that this gene
duplication parallels the higher enzyme activity
(Takeuchi et al., 2004
).
Crystal structure classification (Cheek et
al., 2002
) supports the grouping of the kinases deduced from
alignment studies with protein sequence data. The enzyme activity of arginine
kinase has been detected in the flagellate protozoa Trypanosoma cruzi
(Alonso et al., 2001
) and
Paramecium caudatum (Noguchi et
al., 2001
), indicating that arginine kinases evolved before the
emergence of multicellular animals. Recent phylogenetic analyses also
indicates that the ciliate arginine kinases show a high phylogenetic
relationship to those of sea anemones
(Ellington and Suzuki,
2005
).
The primmorph system was established in order to obtain molecular
biological insights into the proliferation
(Le Pennec et al., 2003),
differentiation (Müller et al.,
2004b
) and immunological
(Müller et al., 2002
)
capacities of primordial sponge cells. Here, this system was used to
demonstrate that the expression level of the gene encoding the arginine kinase
strongly depends on the presence of silicic acid in the medium. The gene
induction is fast; after 1 day a strong upregulation is already seen. This
fast response is characteristic for the primmorph system. Previously it was
shown that the genes for silicatein, the enzyme that causes the formation of
silica in the spicules, and for collagen, the organic cover around the
skeletal elements, also already show strong induction 1 day after silicic acid
incubation (Krasko et al.,
2000
). One piece of evidence that the increased expression of the
arginine kinase gene is indeed dependent on the exogenous silicic acid, came
from inhibition studies using DIDS, an established inhibitor of band
3-mediated anion exchanger through covalent binding to lysine residue(s)
(Kopito, 1990
). The inhibition
is not restricted to the transport of the
exchanger in vitro but also affects transport systems in
vivo, e.g. in rats (Horie et al.,
1993
; Kubota et al.,
2003
). Note that DIDS has been shown to prevent uptake of silicic
acid via the silica transport in this species
(Schröder et al.,
2004a
).
One major differentiation pathway is the transition from the totipotent
stem cells, the archaeocytes, into the spicule-forming sclerocytes
(Müller et al., 2003b).
Archaeocytes are the major cell fraction in primmorphs
(Müller et al., 1999
) and
the inducer of this differentiation direction is silicic acid; in the
archaeocyte cell lineage noggin and MSCP expression are crucial for the fate
of the sclerocyte differentiation
(Schröder et al., 2004b
).
In the present study it is seen that the expression of the arginine
kinase gene in primmorphs, incubated in the presence of silicic acid,
occurs primarily in the center of these 3D-cell aggregates. This localized
expression is in accordance with previous findings that differentiation of
cells, e.g. for the formation of aqueous canals, is first detected in the
center of the primmorphs (Müller et
al., 2004a
). It interesting to observe this specific localization
in the choanocyte-forming region of primmorphs. In fact, these peculiar cells
need high amounts of energy for cilia movement and phosphoarginine should be
primarily involved as energy reservoir. It is intriguing to notice that in
ciliates (Protozoa) arginine kinase activity also plays a fundamental role in
cilia movement (Noguchi et al.,
2001
). The specificity of the silicic acid-mediated induction of
the arginine kinase gene is, again, shown by inhibition studies with
DIDS; this stilbene analogue almost completely prevents this expression.
The increased expression of arginine kinase also correlates with a
strong enhancement of arginine kinase enzyme activity. In primmorphs a strong
increase of activity was measured after 1 day of exposure to silicic acid,
again a process that could be blocked by DIDS. Since this inhibitor blocks
both spicule formation and expression of arginine kinase gene, it can
be assumed that under these conditions a steep electrochemical gradient for
silicic acid transport results. An association of phosphagen kinases with
membrane-associated ATPase is well documented (reviewed in
Ellington, 2001). Final
support for the differentiation-mediating capacity of silicic acid, which
allows formulation of a chain of reactions from increased expression of the
arginine kinase gene to enhanced enzyme activity and finally to the
onset of spicule formation in primmorphs, was documented by transmission
electron-microscopy. This analysis revealed that spicules are formed in those
primmorphs that were exposed to silicic acid.
Taken together, the results of the present work show that arginine kinase
is a crucial enzyme in the high energy-consuming reaction of spicule formation
in primmorphs. In addition, the data demonstrate that the evolutionary oldest
metazoan phylum, the Porifera, comprise the ancestral phosphagen kinase, an
arginine kinase, for all other kinases of higher taxa. Studies are in progress
to elucidate the role of arginine kinase in adult specimens; there the highest
energy consumption should be localized in the choanocytes, which are the
`motor' cells that drive the water through the aqueous canal system. A similar
role for creatine kinase in choanocyte function has recently been suggested
for another sponge (Sona et al.,
2004).
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alonso, G. D., Pereira, C. A., Remedi, M. S., Paveto, M. C., Cochella, L., Ivaldi, M. S., Gerez de Burgos, N. M., Torres, H. N. and Flawiá, M. M. (2001). Arginine kinase of the flagellate protozoa Trypanosoma cruzi: regulation of its expression and catalytic activity. FEBS Lett. 489, 22-25.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A., Seidmann, J. G. and Struhl, K. (1995). Current Protocols in Molecular Biology. New York, John Wiley and Sons.
Barthel, D. (1995). Tissue composition of sponges from the Weddell Sea, Antarctica: not much meat on the bones. Mar. Ecol. Prog. Ser. 123,149 -153.
Cha, J. N., Shimizu, K., Zhou, Y., Christianssen, S. C.,
Chmelka, B. F., Stucky, G. D. and Morse, D. E. (1999).
Silicatein filaments and subunits from a marine sponge direct the
polymerization of silica and silicones in vitro. Proc.
Natl. Acad. Sci. USA 96,361
-365.
Cheek, S., Zhang, H. and Grishin, N. V. (2002). Sequence and crystal structure classification of kinases. J. Mol. Biol. 320,855 -881.[CrossRef][Medline]
Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W. and Wingfield, P. T. (2000). Current Protocols in Protein Science, pp. 2.0.1-2.8.17. Chichester. John Wiley & Sons.
Compaan, D. M. and Ellington, W. R. (2003).
Functional consequences of a gene duplication and fusion event in an arginine
kinase. J. Exp. Biol.
206,1545
-1556.
Custodio, M. R., Prokic, I., Steffen, R., Koziol, C., Borojevic, R., Brümmer, F., Nickel, M. and Müller, W. E. G. (1998). Primmorphs generated from dissociated cells of the sponge Suberites domuncula: A model system for studies of cell proliferation and cell death. Mech. Ageing Dev. 105, 45-59.[CrossRef][Medline]
Dayhoff, M. O., Schwartz, R. M. and Orcutt, B. C. (1978). A model of evolutionary change in protein. In Atlas of protein sequence and structure (ed. M. O. Dayhoff), pp. 345-352. Washington, DC: National Biomedical Research Foundation.
Desqueyroux-Faundez, R. (1990). Silica content of the New Caledonian fauna of Haplosclerida and Petrosiida and its possible taxonomic significance. In New Perspectives in Sponge Biology (ed. K. Rützler), pp.279 -283. Washington DC: Smithonian Institute.
Ellington, W. R. (2000). A dimeric creatine kinase from a sponge. Comp. Biochem. Physiol. 126B, 1-7.
Ellington, W. R. (2001). Evolution and physiological roles of phosphagen systems. Ann. Rev. Physiol. 63,289 -325.[CrossRef][Medline]
Ellington, W. R. and Suzuki, T. (2005). Evolution and divergence of creatine kinases. In Molecular Anatomy and Physiology of Proteins - Creatine Kinase (ed. C. Vial). New York: NovaScience (in press).
Elvin, D. W. (1972). Growth rates of the siliceous spicules of the freshwater sponge Ephydatia fluviatilis (Lieberkühn). Trans. Amer. Microsc. Soc. 90,219 -224.
Felsenstein, J. (1993). PHYLIP, ver. 3.5. Seattle: University of Washington.
Garrone, R. (1978). Phylogenesis of Connective Tissue. Morphological Aspects and Biosynthesis of Sponge Intercellular Matrix. Basel: S. Karger.
Gatti, S., Brey, T., Müller, W. E. G., Heilmayer, O. and Holst, G. (2002). Oxygen microoptodes: a new tool for oxygen measurements in aquatic animal ecology. Marine Biol. 140,1075 -1085.[CrossRef]
Grebenjuk, V. A., Kuusksalu, A., Kelve, M., Schütze, J.,
Schröder, H. C. and Müller, W. E. G. (2002).
Induction of (2'-5')oligoadenylate synthetase in the marine
sponges Suberites domuncula and Geodia cydonium by the
bacterial endotoxin lipopolysaccharide. Eur. J.
Biochem. 269,1382
-1392.
Hartman, W. D. (1958). Natural history of the marine sponges of southern New England. Bull. Peabody Mus. Nat. Hist. (Yale University) 12,1 -155.
Horie, S., Yano, S. and Watanabe, K. (1993). Effects of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, an inhibitor of Cl(-)-HCO3- exchanger, on stress-induced gastric lesions in rats. Res. Commun. Chem. Pathol. Pharmacol. 79,117 -120.[Medline]
Kopito, R. R. (1990). Molecular biology of the anion exchanger gene family. Int. Rev. Cytol. 123,177 -199.[Medline]
Krasko, A., Batel, R., Schröder, H. C., Müller, I. M.
and Müller, W. E. G. (2000). Expression of silicatein
and collagen genes in the marine sponge Suberites domuncula is
controlled by silicate and myotrophin. Eur. J.
Biochem. 267,4878
-4887.
Krasko, A., Schröder, H. C., Batel, R., Grebenjuk, V. A., Steffen, R., Müller, I. M. and Müller, W. E. G. (2002). Iron induces proliferation and morphogenesis in primmorphs from the marine sponge Suberites domuncula. DNA Cell Biol. 21,67 -80.[CrossRef][Medline]
Kruse, M., Müller, I. M. and Müller, W. E. G. (1997). Early evolution of metazoan serine/threonine- and tyrosine kinases: identification of selected kinases in marine sponges. Mol. Biol. Evol. 14,1326 -1334.[Abstract]
Kubota, K., Ishibashi, T., Matsubara, T., Hori, T., Ozaki, K., Yamazoe, M., Yoshida, J., Nishio, M. and Aizawa, Y. (2003.) Effects of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on chlorpromazine on NO3- transport via anion exchanger in erythrocytes: inertness of DIDS in whole blood. J. Phamacol. Sci. 93,505 -508.[CrossRef]
Le Pennec, G., Perovi, S., Ammar, M. S. A., Grebenjuk,
V. A., Steffen, R., Brümmer, F. and Müller, W. E. G.
(2003). Cultivation of primmorphs from the marine sponge
Suberites domuncula: morphogenetic potential of silicon and iron, a
review. Marine Biotechnol.
100,93
-108.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R.
J. (1951). Protein measurement with the folin phenol reagent.
J. Biol. Chem. 193,265
-275.
Maldonado, M., Carmona, M. C., Uriz, M. J. and Cruzado, A. (1999). Decline in Mesozoic reef-building sponges explained by silicon limitation. Nature 401,785 -788.[CrossRef]
Mariman, E. C., Broers, C. A., Claesen, C. A., Tesser, G. I. and Wieringa, B. (1987). Structure and expression of the human creatine kinase B gene. Genomics 1, 126-137.[CrossRef][Medline]
Muhlebach, S. M., Gross, M., Wirz, T., Wallimann, T., Perriard, J. C. and Wyss, M. (1994). Sequence homology structure predictions of the creatine kinase isoenzyme. Mol. Cell. Biochem. 133/134,245 -262.
Müller, W. E. G., Diehl-Seifert, B., Sobel, C., Bechtold, A., Kljajic, Z. and Dorn, A. (1986). Sponge secondary metabolites: biochemical and ultrastructural localization of the antimitotic agent avarol in Dysidea avara. J. Histochem. Cytochem. 34,1687 -1690.[Abstract]
Müller, W. E. G., Wiens, M., Batel, R., Steffen, R., Borojevic, R. and Custodio, M. R. (1999). Establishment of a primary cell culture from a sponge: primmorphs from Suberites domuncula. Marine Ecol. Progr. Ser. 178,205 -219.
Müller, W. E. G., Krasko, A., Skorokhod, A., Bünz, C., Grebenjuk, V. A., Steffen, R., Batel, R., Müller, I. M. and Schröder, H. C. (2002). Histocompatibility reaction in the sponge Suberites domuncula on tissue and cellular level: central role of the allograft inflammatory factor 1. Immunogenetics 54,48 -58.[CrossRef][Medline]
Müller, W. E. G., Klemt, M., Thakur, N. L., Schröder, H. C., Aiello, A., D'Esposito, M., Menna, M. and Fattorusso, E. (2003a). Molecular/chemical ecology in sponges: evidence for an adaptive antibacterial response in Suberites domuncula. Marine Biol. 144,19 -29.[CrossRef]
Müller, W. E. G., Korzhev, M., Le Pennec, G., Müller, I. M. and Schröder, H. C. (2003b). Origin of metazoan stem cell system in sponges: first approach to establish the model (Suberites domuncula). Biomolec. Eng. 20,369 -379.[CrossRef]
Müller, W. E. G., Thakur, N. L., Ushijima, H., Thakur, A.
N., Krasko, A., Le Pennec, G., Indap, M. M., Perovi-Ottstadt, S.,
Schröder, H. C., Lang, G. and Bringmann, G. (2004a).
Matrix-mediated canal formation in primmorphs from the sponge Suberites
domuncula involves the expression of a CD36 receptor-ligand system.
J. Cell Sci. 117,2579
-2590.
Müller, W. E. G., Wiens, M., Adell, T., Gamulin, V., Schröder, H. C. and Müller, I. M. (2004b). Bauplan of Urmetazoa: basis for genetic complexity of Metazoa. Int. Rev. Cytol. 235,53 -92.[Medline]
Nealon, D. A. and Herderson, A. R. (1976). The apparent Arrhenius relationships of the human creatine kinase isoenzymes using Oliver-Rosalki assay. Clin. Chim. Acta 66,131 -136.[CrossRef][Medline]
Nicholas, K. B. and Nicholas. H. B., Jr (1997). GeneDoc: A tool for editing and annotating multiple sequence alignments. Version 1.1.004. Distributed by the author; cris.com/~ketchup/genedoc.shtml; INTERNET.
Noguchi, M., Sawada, T. and Akazawa, T. (2001).
ATP-regenerating system in the cilia of Paramecium caudatum.
J. Exp. Biol. 204,1063
-1071.
Osinga, R., Tramper, J. and Wijffels, R. H. (1999). Cultivation of marine sponges. Marine Biotechnol. 1,509 -532
Osinga, R., Kleijn, R., Groenendijk, E., Niesink, P., Tramper, J. and Wijffels, R. H. (2001). Development of in vivo sponge culture: particle feeding by the tropical sponge Pseudosuberites aff. andrewsi. Marine Biotechnol. 3,544 -554.[CrossRef]
Osinga, R., Belarbi, E. H., Grima, E. M., Tramper, J. and Wijffels, R. H. (2003). Progress towards a controlled culture of the marine sponge Pseudosuberites andrewsi in a bioreactor. J. Biotechnol. 100,141 -144.[CrossRef][Medline]
Perovi, S., Schröder, H. C., Sudek, S., Grebenjuk,
V. A., Batel, R.,
tifani
, M., Müller, I. M. and
Müller, W. E. G. (2003). Expression of one sponge
Iroquois homeobox gene in primmorphs from Suberites
domuncula during canal formation. Evol. Dev.
5, 240-250.[CrossRef][Medline]
Polak, J. M. and McGee, J. D. (1998). In Situ Hybridization. Oxford, Oxford University Press.
Roche, J. and Robin, Y. (1954). Sur les phosphagens des éponges. CR Soc. Biol. 48,1451 -1543.
Roche, J., van-Thoai, N. and Robin, Y. (1957). Sur la presence de creatine chez les invertebres et sa signification biologique. Biochim. Biophys. Acta 24,514 -519.[CrossRef][Medline]
Schröder, H. C., Perovi-Ottstadt, S., Rothenberger,
M., Wiens, M., Schwertner, H., Batel, R., Korzhev, M., Müller, I. M. and
Müller, W. E. G. (2004a). Silica transport in the
demosponge Suberites domuncula: fluorescence emission analysis using
the PDMPO probe and cloning of a potential transporter. Biochem.
J. 381,1
-9.[CrossRef][Medline]
Schröder, H. C., Perovi-Ottstadt, S., Wiens, M.,
Batel, R., Müller, I. M. and Müller, W. E. G.
(2004b). Differentiation capacity of the epithelial cells in the
sponge Suberites domuncula. Cell Tissue Res.
316,271
-280.[CrossRef][Medline]
Simpson, T. L., Gil, M., Connes, R., Diaz, J. P. and Paris, J. (1985). Effects of germanium (Ge) on the silica spicules of the marine sponge Suberites domuncula: transformation of the spicule type. J. Morphol. 183,117 -128.
Sona, S., Suzuki, T. and Ellington, W. R. (2004). Cloning and expression of mitochondrial and protoflagellar creatine kinases from a marine sponge: implications for the origin of intracellular energy transport systems. Biochem. Biophys. Res. Comm. 317,1207 -1214.[CrossRef][Medline]
Suzuki, T. and Furukohri, T. (1993). Evolution of phosphagen kinase: primary structure of glycocyamine kinase and arginine kinase from invertebrates. J. Mol. Biol. 237,353 -357.
Suzuki, T., Kawasaki, Y. and Furukohri, T. (1997). Evolution of phosphagen kinase. Isolation, characterization and cDNA-derived amino acid sequence of two-domain arginine kinase from the sea anemone Anthopleura japonicus. Biochem. J. 328,301 -306.[Medline]
Suzuki, T., Sugimura, N., Taniguchi, T., Unemi, Y., Murata, T., Hayashida, M., Yokouchi, K., Uda, K. and Furukohri, T. (2002). Two-domain arginine kinases from the clams Solen strictus and Corbicula japonica. Exceptional amino acid replacement of the functionally important D62 by G. Int. J. Biochem. Cell Biol. 34,1221 -1229.[CrossRef][Medline]
Takeuchi, M., Mizuta, C., Uda, K., Fujimoto, N., Okamoto, M. and Suzuki, T. (2004). Unique evolution of Bivalvia arginine kinases. Cell Molec. Life Sci. 61,110 -117.[CrossRef][Medline]
Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22,4673 -4680.[Abstract]
Tombes, R. M. and Shapiro, B. M. (1989). Energy transport and cell polarity: relationship of phosphagen kinase activity to sperm function. J. Exp. Zool. 251, 89-90.
Wiens, M., Koziol, C., Hassanein, H. M. A., Batel, R. and Müller, W. E. G. (1998). Expression of the chaperones 14-3-3 and HSP70 induced by PCB 118 (2,3',4,4',5-pentachlorobiphenyl) in the marine sponge Geodia cydonium. Mar. Ecol. Prog. Ser. 165,247 -257.
Related articles in JEB: