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Seleniumaffectsbiosilicaformationinthe demosponge
Suberites domuncula
Effect ongeneexpressionandspicule formation
Werner E. G. Mu
¨
ller
1
, Alexandra Borejko
1
, David Brandt
1
, Ronald Osinga
2
, Hiroshi Ushijima
3
,
Bojan Hamer
4
, Anatoli Krasko
1
, Cao Xupeng
1
, Isabel M. Mu
¨
ller
1
and Heinz C. Schro
¨
der
1
1 Institut fu
¨
r Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universita
¨
t, Mainz, Germany
2 Wageningen University, Fish Culture and Fisheries Group, the Netherlands
3 Department of Developmental Medical Sciences, Institute of International Health, Graduate School of Medicine, University of Tokyo, Japan
4 Center for Marine Research, ‘Ruder Boskovic’ Institute, Rovinj, Croatia
The synthesis of siliceous spicules in sponges (phylum
Porifera) is unique inthe metazoan kingdom. This
form of biomineralization, which results inthe forma-
tion of polymerized amorphous silica, leads to the
production of filigree and highly structured skeletal
elements with morphologies specific to sponge species.
The process of silica ⁄ spiculeformationin sponges can
be designated biologically controlled mineralization [1],
as the reactions are driven by cellular activities that
govern (a) nucleation, (b) growth, (c) morphology, and
(d) location of the spicules within the specimen.
A breakthrough in our understanding of spicule for-
mation in siliceous sponges came from the studies of
Shimizu et al. [2] and Cha et al. [3], who showed that
the formation of the skeletal framework and silica is
enzymatically controlled. The major enzyme that initi-
ates nucleation of spiculeformation was termed silica-
tein [3]. Inthe last few years, several silicatein enzymes
(which catalyze biosilicification) from different demo-
sponges have been identified; according to their protein
sequences they belong to the family of cathepsin L pro-
teolytic enzymes [3,4].
Keywords
selenium; silica; silicatein; spicules; sponges
Correspondence
W. E. G. Mu
¨
ller, Institut fu
¨
r Physiologische
Chemie, Abteilung Angewandte
Molekularbiologie, Universita
¨
t,
Duesbergweg 6, 55099 Mainz, Germany
Fax: +49 6131 3925243
Tel: +49 6131 3925910
E-mail: wmueller@uni-mainz.de
(Received 24 March 2005, revised 18 May
2005, accepted 26 May 2005)
Note
The cDNA sequences for selenoprotein M
(AJ875186) and spicule-associated protein
(AJ872182) have been deposited at
EMBL ⁄ GenBank.
doi:10.1111/j.1742-4658.2005.04795.x
Selenium is a trace element found in freshwater andthe marine environ-
ment. We show that it plays a major role inspiculeformationinthe demo-
sponge Suberites domuncula. If added to primmorphs, an in vitro sponge
cell culture system, it stimulates theformation of siliceous spicules. Using
differential display of transcripts, we demonstrate that, after a 72-h expo-
sure of primmorphs to selenium, two genes are up-regulated; one codes for
selenoprotein M andthe other for a novel spicule-associated protein.
The deduced protein sequence of selenoprotein M (14 kDa) shows charac-
teristic features of metazoan selenoproteins. The spicule-associated protein
(26 kDa) comprises six characteristic repeats of 20 amino acids, composed
of 10 distinct hydrophobic regions ( 9 amino acids in length). Recombin-
ant proteins were prepared, and antibodies were raised against these two
proteins. Both were found to stain the central axial filament, which compri-
ses the silicatein, as well as the surface of the spicules. Inthe presence of
selenium, only the genes for selenoprotein M and spicule-associated protein
are up-regulated, whereas theexpression of the silicatein gene remains
unchanged. Finally we show that, inthe presence of selenium, larger silica
aggregates are formed. We conclude that selenium has a stimulatory effect
on theformation of siliceous spicules in sponges, and it may be involved in
the enzymatic synthesis of biosilica components.
Abbreviations
DMEM, Dulbecco’s modified Eagle’s medium; PoAb, polyclonal antibody.
3838 FEBS Journal 272 (2005) 3838–3852 ª 2005 FEBS
The growth of spicules probably starts intracellularly
[5]. After reaching a crucial size, e.g. in Suberites
domuncula 1 lm in diameter and 8 lm in length, the
spicules are extruded from the cells and their growth
proceeds inthe extracellular space. In S. domuncula
only one type of spicule is formed, megascleres (styles ⁄
oxea), with lengths of up to 450 lm and diameters of
5–7 lm. It is still not known which morphogenetic
processes control the morphology of the spicules. It
has recently been reported that extra-organismic, mor-
phogenetic inorganic elements, e.g. silicate and ferric
iron, and also homeodomain transcription factors, e.g.
Iroquois, are major factors that control the organiza-
tion of the skeletal architecture of spicules [5,6]. The
last step inthe biologically controlled mineralization
process, i.e. the final location of the spicules within the
specimen, involves active transport by specialized cells
[7]. The spicules are finally embedded ⁄ cemented into
an organic matrix which contains collagen [8].
The spicules harbor in their center an organic axial fil-
ament in an 1 lm wide canal. In this study we show
that selenium ⁄ selenite induces genes which lead to the
synthesis of proteins associated with silicatein fibers in
the axial filament. These studies with selenium were trig-
gered by observations that this element is required for
the growth of sponge cells [9]. Serine undergoes chemical
conversion into selenocysteine [10]. In eukaryotes as well
as prokaryotes [11], selenium is biochemically incorpor-
ated into proteins through selenocysteine, a process dur-
ing which a tRNA–EF complex is delivered to a codon
that would normally be read as a stop.
We used differential display to further identify
genes ⁄ proteins inthe siliceous demosponge S. domun-
cula that may be involved in biosilicification in sponges.
The experiments were performed in cultures of sponge
cells, the primmorph system, which represent 3D cell
aggregates; they contain proliferating and differentiating
cells [12,13]. We found that, in response to selenium,
the expression of two genes, selenoprotein M and the
(sponge-specific) spicule-associated protein, is up-regu-
lated. Cell biological data revealed that the increase in
spicule formationin primmorphs caused by selenium
is paralleled by increased expression of these two
genes ⁄ proteins. Finally, we found that, inthe presence
of selenium, larger silica condensation products are
formed inthein vitro assay with recombinant silicatein.
Results
Spicule formationin primmorphs
Light microscopy showed that, after a total incubation
period of 14 days inthe presence of 30 lm silicic acid
and 10 lm Fe(III), many spicules were embedded in
the thin rim region that surrounds the body of the
primmorphs (Fig. 1C,D) and inside the 3D cell aggre-
gates. In contrast, almost no spicules were found in
primmorphs that had been cultured without additional
silicic acid and 10 lm Fe(III) (Fig. 1A,B).
The formation of spicules (monactinal tylostyles) in
primmorphs was also demonstrated by transmission
electron microscopy. Cuts through primmorphs that
had been incubated inthe absence of silicic acid and
Fe(III) did not show any spicules or cells forming
spicules. This is in contrast with primmorphs that
had been incubated for longer than 7 days in the
presence of 30 lm silicic acid and 10 lm Fe(III), under
conditions described in Experimental procedures.
AB
DC
Fig. 1. Light microscopic images of prim-
morphs, grown for 14 days (7 days in RPMI
medium ⁄ seawater and an additional 7 days
in RPMI ⁄ DMEM ⁄ seawater) inthe absence
(A and B) or presence of 30 l
M silicate and
10 l
M ferric citrate (C and D). Spicules can
be seen inthe rim surrounding the body of
the primmorphs (>).
W. E. G. Mu
¨
ller et al. Effect of seleniumonspicule formation
FEBS Journal 272 (2005) 3838–3852 ª 2005 FEBS 3839
Cross-sections showed either cells (sclerocytes) in the
process of forming the axial filaments of the spicules
or regions with extracellularly located spicules. In the
primordial stage, the axial filaments are synthesized
intracellularly in vesicles (Fig. 2A,B); the filaments
vary in size between 0.3 and 1.5 lm in diameter. It
should be highlighted that during formation of the
axial filaments, characterized by rods filled with
highly electron-dense material, these filaments are clo-
sely associated with 15-nm round fibrils (Fig. 2B).
These fibrils may be involved inthe extrusion of
the spicules into the extracellular space. During
maturation, the diameters of the axial filaments
decrease to 0.4 lm (Fig. 2D); in parallel the siliceous
spicules are formed around the filaments (Fig. 2D).
Before the spicules are extruded they grow to lengths
of 8 lm; Fig. 2C shows an intracellular spicule
1.5 lm in length.
Effect of seleniumon glutathione peroxidase
activity
First 14-day-old primmorphs were exposed to 0.1 lm,
1 lm and 10 lm sodium selenite for 0 h (control) or
72 h. Then extracts were prepared and glutathione per-
oxidase activity was determined as described in Experi-
mental procedures. Inthe absence of selenium (time
zero), the enzyme activity was found to be 8.2 ±
1.2 UÆ(mg protein)
)1
. There was no significant change
in activity even when selenium was added to the
primmorphs for 72 h at the indicated concentration
ranges: 7.1 ± 1.1 UÆmg
)1
at 0.1 lm selenium; 7.9 ±
1.0 UÆmg
)1
at 1 lm selenium; 9.1 ± 1.8 UÆ mg
)1
at
10 lm selenium.
Effect of seleniumonspicule formation
in primmorphs
A semiquantitative determination revealed that the
amount of polymerized silica in primmorphs cultured
in the absence of additional silicon was low, amount-
ing to <0.4 mgÆ (g wet weight of primmorphs)
)1
. When
30 lm silicic acid [and 10 lm Fe(III)] was added for
7 days to RPMI medium, the concentration increased
to 13 ± 5 mgÆg
)1
. When it was added for 7 days in
RPMI medium and then 7 days in RPMI ⁄ Dulbecco’s
modified Eagle’s medium (DMEM), the concentration
increased to 15 ± 7 mgÆg
)1
. In comparison, the
amount of silica inthe tissue of adult sponge speci-
mens is 74 ± 18 mgÆ(g wet tissue)
)1
.
If 10 lm sodium selenite was added together with
silicic acid and Fe(III) to the primmorphs, a doubling
of the silica concentration was seen after 7 days
(32 ± 12 mgÆg
)1
), but no further increase was meas-
ured after 14 days (34 ± 12 mgÆg
)1
). If sodium selenite
was added alone, without additional silicic acid and
Fe(III), no significant change inthe amount of silica
was found inthe primmorphs.
AB
DC
Fig. 2. Formation of spicules in primmorphs
(transmission electron microscopic images).
Primmorphs cultured for 14 days were ana-
lyzed. (A, B) Section through a primmorph,
showing theformation of an axial filament
(af), a process that proceeds intracellularly.
At higher magnification (B), the 15-nm round
fibrils (fi) adjacent to the axial filament (af)
become visible. (C) A small spicule that is
still intracellularly located is shown in a
sclerocyte. (D) A more mature spicule sp,
now present extracellularly, is shown which
surrounds the axial filament with its
siliceous material.
Effect of seleniumonspiculeformation W. E. G. Mu
¨
ller et al.
3840 FEBS Journal 272 (2005) 3838–3852 ª 2005 FEBS
Selenium in spicules
To clarify whether selenium is incorporated into spi-
cules (axial filaments andthe surrounding silica) of
S. domuncula, primmorphs were incubated with
75
Se,
as described in Experimental procedures. After incu-
bation for up to 3 days, spicules were isolated,
mechanically disintegrated, andthe radioactivity was
assessed. At time zero, no activity was detected; after
a 1-day incubation, 2500 c.p.m.Æ(5 mg solid mater-
ial)
)1
was measured. The amount increased further
to 5300 c.p.m.Æ5mg
)1
after the extended incubation
period of 3 days. The extract from
75
Se metabolically
labeled spicules was subjected to SDS ⁄ PAGE. As
shown in Fig. 3, the predominant band identified
corresponds to a 14-kDa protein (lanes a and b).
Weaker bands corresponding to sizes of 35 kDa and
23 kDa were also detected if larger amounts of pro-
teins were analyzed (Fig. 3, lane a).
Cloning of the cDNA encoding selenoprotein M
from S. domuncula by differential display
Among the differentially expressed transcripts, one was
found to encode selenoprotein M. The 652-nucleotide
cDNA (accession number AJ875186) contained one
ORF, spanning nucleotides 94–96 to 463–465(stop)
(Fig. 4). One TGA stop codon exists at nucleotides
187–189, which can also function as a codon for seleno-
cysteine [14]. As outlined below, the TGA stop codon
may be suppressed and (very likely) used for the inser-
tion of selenocysteine. The complete protein, with a
calculated molecular mass of 13 918 Da (comprising
the 123-amino-acid ORF) shares the highest sequence
similarity with the 15-kDa selenoprotein M from
humans (accession number NP_536355M) [15]. There-
fore, the sponge molecule was termed selenoprotein M
(SelM_SUBDO) and its cDNA SDSelM. A sequence
comparison revealed that the sponge selenoprotein M
has the highest similarity to human selenoprotein M
(‘expect value’ E of e
)33
); comparatively low is the rela-
tionship to the Drosophila melanogaster putative pro-
tein CG7484-PB (E ¼ 2e
)11
). There are only very
distant – if at all – relationships to the Caenorhabditis
elegans protein with a coiled coil domain (E ¼ 0.13),
the Saccharomyces cerevisiae deduced polypeptide
Yjl049wp (E ¼ 0.56), andthe Arabidopsis thaliana
putative protein At1g08340 (E ¼ 0.23).
Cloning of the cDNA for the spicule-associated
protein
A further differentially expressed transcript was char-
acterized, the cDNA encoding a sponge-specific pro-
tein which was termed spicule-associated protein
(SPIaP_SUBDO). The cDNA, SDSPIaP, is 860-bp long
and comprises one ORF at nucleotides 4–6 to nucleo-
tides 757–759 (stop) (accession number AJ872182).
The 251-amino-acid polypeptide (Fig. 5A) has a calcu-
lated molecular mass of 25 602 Da. This protein dis-
played no striking homology to any protein reported
in the database.
One selection criterion used to study this protein
was the existence of repeats; the spicule-associated
protein has high structural regularity (Fig. 5A). Sec-
ondary-structure analysis revealed a-helical regions
at the C-terminus and N-terminus of the protein, and
the central part of the molecule has extended stret-
ches regularly interspersed with predicted turns and
coil conformations. Very interesting with respect to
the amino-acid sequence is the existence of six highly
similar segments of 20 amino acids (60–79; 80–99;
100–119; 120–139; 140–159; 160–179; Fig. 5A). A
closer analysis of the distribution of polar ⁄ nonpolar
amino acids and calculation of the hydropathicity
within the molecule revealed that the borders of the
spicule-associated protein display highly hydrophobic
(potential transmembrane) regions (Fig. 5B). Further-
more, inthe central part of the deduced protein, the
hydropathicity plot indicates 10 distinct regular
hydrophobic regions of approximately nine amino
acids. The dominant domain consensus sequence (as
in region number 4) reads (Q)TVNVTATPS, with
Ma b
14 -
20 -
30 -
45 -
62 -
90 -
Fig. 3. SDS ⁄ PAGE analysis of
75
Se-labeled protein(s) inthe spi-
cules. Labeled protein(s) were isolated from ground spicules as
described in Experimental procedures. After electrophoresis, the
dried gel was exposed to the film. Positions of the molecular mass
markers are shown onthe left; the arrowhead points to the 14-kDa
protein. Extracts from 10 mg (lane a) and 1 mg (lane b) solid mater-
ial were applied to the gel.
W. E. G. Mu
¨
ller et al. Effect of seleniumonspicule formation
FEBS Journal 272 (2005) 3838–3852 ª 2005 FEBS 3841
the amino acids glutamine, lysine and histidine at
the N-terminus andthe nonpolar amino acid proline
and the hydroxy amino acids serine and threonine at
the C-terminal border.
Recombinant proteins and antibodies
To identify selenoprotein M andthe spicule-associated
protein in sponge extract, the respective recombinant
A
B
13
579
TM TM
1 50 100 200150 250
0
-30
-10
-20
10
20
30
40
50
Fig. 5. Spicule-associated protein SPIaP_
SUBDO. (A) The deduced protein was ana-
lyzed for the predicted secondary structure
as described by Garnier et al. [50]; the
helical conformation (X), the extended
conformation (–), the turn (>) andthe coil
conformation (w) are indicated. The six
highly similar segments of 20 amino acids
are marked in white on black or are
underlined. In addition, the 10 hydrophobic
regions, present inthe six 20-amino-acid
blocks, are indicated and numbered (#).
(B) Hydropathicity plot of the S. domuncula
SPIaP_SUBDO; the calculation was per-
formed by the method of Kyte and Doolittle
[51]. The horizontal axes show the amino
acid numbers along the protein vs. the cor-
responding hydropathicity. The dotted lines
at the )5 value divide hydrophobic regions
(above) from hydrophilic regions (below).
The 10 distinct hydrophobic regions are
consecutively labeled. The two highly
hydrophobic (potential transmembrane; TM)
regions were identified [52]; they range
from amino acids 32–55 and 204–237.
Fig. 4. S. domuncula selenoprotein M. From the S. domuncula nucleotides sequence (SDSelM), selenoprotein M (SelM_SUBDO) is predic-
ted and aligned with the human selenoprotein M precursor (SelM_HUMAN, NP_536355 [2]); the human sequence was shortened between
amino acids 20–35 and 119–124, indicated by square brackets []). Residues conserved (similar or related with respect to their physicochemi-
cal properties) inthe two sequences are shown in white on black. The TGA triplet that encodes selenocysteine (U) is underlined.
Effect of seleniumonspiculeformation W. E. G. Mu
¨
ller et al.
3842 FEBS Journal 272 (2005) 3838–3852 ª 2005 FEBS
proteins were prepared. In parallel, the distribution of
silicatein was analyzed with the tools available [16].
The cDNAs, SDSelM and SDSPIaP, were cloned
into theexpression vector pBAD ⁄ gIII as described in
Experimental procedures. After induction with arabi-
nose, the proteins could be identified inthe bacterial
lysate (Fig. 6A, lane a vs. b). The recombinant protein
was purified (Fig. 6A, lane c) and used to raise anti-
bodies in rabbits. The polyclonal antibody against
SelM (PoAb-SelM) was found to react with the puri-
fied recombinant fusion selenoprotein M (Fig. 6A, lane
d). The size of this fusion protein was 17 kDa, which
is in accordance with the size of the selenoprotein M
fragment of 84 amino acids expressed (10 kDa)
together with the protein stretch covering the myc
epitope and polyhistidine.
Similarly, the antibodies against the spicule-associ-
ated protein (PoAb-SPaP) were tested. Antibodies were
prepared against the 31-kDa recombinant fusion pro-
tein (Fig. 6B, lane a); they reacted inthe western blot
assay with the recombinant protein (Fig. 6B, lane b).
In parallel, the antibodies against the recombinant
silicatein (Fig. 6C, lane a) were subjected to western
blotting and found to react with the 35-kDa protein
(Fig. 6C, lane b). In control assays, it was established
that the adsorbed polyclonal antibodies PoAb-SelM
and PoAb-SPaP did not react with any protein on the
filter (data not shown).
Protein expression of selenoprotein M after
exposure to selenium
In a first series of experiments, 12-day-old primmorphs
(7-day-old primmorphs were incubated for additional
days in RPMI ⁄ DMEM⁄ seawater) were exposed to
10 lm sodium selenite for 24 or 72 h (Fig. 7, lanes a
and b). Primmorphs incubated in seawater ⁄ medium in
the absence of selenium were used as a control.
Extracts were prepared and subjected to western blot
analysis. The blotting results revealed that, in the
absence of selenium, the 14-kDa selenoprotein M is
missing from the extract (not shown). After incubation
for 24 h (lane a) and 72 h (lane b) with sodium selen-
ite, the 14-kDa band, reflecting selenoprotein M, is
clearly present onthe immunoblot.
Gene expression studies
The effect of seleniumontheexpression of the genes
encoding selenoprotein M, spicule-associated protein,
and silicatein was studied (Fig. 8). Inthe absence of
sodium selenite, almost no transcripts were detected
during the 72-h incubation period for selenoprotein M
(the probe SDSelM was used) and spicule-associated
protein (SDSPIaP), whereas a large number of silicatein
(SDSILIC) transcripts could be detected by northern
blotting. However, during the 72-h incubation with
sodium selenite, within the concentration range 10–
SelM SAP SILIC
Mabc d ab a b
14 -
20 -
30 -
45 -
62 -
90 -
17
31
35
-
++
arab
CBA
Fig. 6. Recombinant selenoprotein M, spicule-associated protein
and silicatein. Sponge SDSelM (selenoprotein M), SDSPIaP (spi-
cule-associated protein) and SDSILIC (silicatein) cDNA was
expressed in E. coli. (A) Expression of selenoprotein M (SelM): lane
a, PAGE analysis (10% gels) of bacterial lysate obtained from
E. coli grown inthe absence of arabinose (– arab); lane b, lysate
from bacteria that had been induced with arabinose (+ arab); lane
c, affinity-purified fusion protein; molecular mass 17 kDa; lane d,
western blot analysis of purified fusion protein using PoAb-SelM.
(B) Spicule-associated protein (SAP). The purified fusion protein
(lane a) was used to raise antibodies. The resulting PoAb-SPaP
were found to react with the 31-kDa protein inthe western blot
assay (lane b). (C) The recombinant silicatein (SILIC) was used (lane
a) to prepare antibodies (PoAb-SILIC); they recognized the 35-kDa
recombinant protein (lane b). The size markers (M) are given.
a bM
10
15
25
35
24 72 hrs
Fig. 7. Formation of selenoprotein M in primmorphs after exposure
to selenium. Twelve-day-old primmorphs, which had been incuba-
ted for 7 days in RPMI ⁄ seawater and 5 days in RPMI ⁄ DMEM ⁄ sea-
water were exposed to 10 l
M sodium selenite for 24 h (lane a) or
72 h (lane b). Then the 3D cell aggregates were extracted and the
cleared extract (30 lg per lane) was size separated by SDS ⁄ PAGE
(15% gel). Blotting was performed; the blots were incubated with
PoAb-SelM as described in Experimental procedures. In lane M,
size markers were separated.
W. E. G. Mu
¨
ller et al. Effect of seleniumonspicule formation
FEBS Journal 272 (2005) 3838–3852 ª 2005 FEBS 3843
1000 lm, considerable up-regulation of the expression
of the genes for selenoprotein M and spicule-associated
protein was detected (Fig. 8). Onthe basis of the data
obtained, the steady-state expression was almost identi-
cal within theselenium concentration range chosen. The
expression level of silicatein remained almost unchanged
during the exposure to selenium (Fig. 8).
From these data, we conclude that the steady con-
centration of selenoprotein M and spicule-associated
protein in primmorphs is controlled by selenium at the
transcriptional level, whereas theexpression of silica-
tein is independent of the presence of sodium selenite.
Identification of selenoprotein M, spicule-
associated protein and silicatein in total extract
or in axial filaments by western blotting
To clarify whether selenoprotein M and spicule-associ-
ated protein are associated with the axial filaments of
the spicules from S. domuncula, western blot ⁄ antibody
studies were performed. Extracts from sodium selenite-
treated primmorphs and axial filaments from spicules
were separated by size and subjected to western blot
experiments (Fig. 9). The blots were incubated with
antibodies against selenoprotein M (PoAb-SelM), spi-
cule-associated protein (PoAb-SPaP), or silicatein
(PoAb-SILIC).
It was shown that selenoprotein M (the 14-kDa
band) exists in large amounts inthe soluble extracts
and to a small extent also inthe axial filaments. Like-
wise, the spicule-associated protein was identified in
the total extracts and also in lower amounts in the
axial filament (26-kDa band). Silicatein exists predom-
inantly inthe axial filaments (24-kDa protein) (Fig. 9).
Localization of selenoprotein M and spicule-
associated protein by immunofluorescence
analysis
The proteins were localized in tissue from S. domuncula
using the antibodies PoAb-SILIC, PoAb-SelM and
PoAb-SPaP. Sections were cut through tissue from
which the spicules had not been removed, and incuba-
ted with the antibodies.
The fluorescence images obtained with antibodies
against silicatein show that primarily ⁄ exclusively the
surfaces of the 5–7-lm thick and up to 450-lm long
monactinal tylostyles and to a smaller extent the diac-
tinal oxeas as well as the axial filaments are recognized
by PoAb-SILIC (Fig. 10A); the corresponding Nomar-
sky interference image is shown in parallel (Fig. 10B).
This finding is interesting, as it indicates that silicatein
is not restricted to the axial filament but also exists
around the spicules.
The images obtained with the antibodies raised
against selenoprotein M (Fig. 10C) and spicule-associ-
ated protein (Fig. 10E) surprisingly revealed strong
immunoreaction not only onthe surfaces of the spicules
but also in their canals which harbor the axial filaments.
Parallel Nomarsky interference images (Fig. 10D,F)
show that, in addition to these structures, areas in the
mesohyl of the tissue are decorated by the antibodies.
0 10 100 1000 µM Se
SDSILIC
SDSelM
SDSAP
0.9
0.7
1.4
Fig. 8. Effect of seleniumontheexpression of selenoprotein M,
spicule-associated protein and silicatein. Fourteen-day-old prim-
morphs were exposed to 0–1000 l
M sodium selenite for 72 h. Sub-
sequently, RNA was isolated and equal amounts (5 lg) were
size-separated, transferred, and probed with labeled selenopro-
tein M (SDSelM), spicule-associated protein (SDSPIaP) or silicatein
(SDSILIC) cDNA.
SILIC
45
14
10
20
30
75
37
25
20
26
150
100
75
50
37
25
20
15
24
Ext AF
SelM SAP
M Ext AF
M Ext AF AF
WB WB WBPAGE
Fig. 9. Identification of selenoprotein M and spicule-associated pro-
tein inthe axial filaments by western blotting. Primmorphs (12 days
old) were incubated with 10 l
M sodium selenite for 72 h and then
extracted. The cleared extract was analyzed by SDS ⁄ PAGE (PAGE)
and then by western blotting (WB), using PoAb-SelM, PoAb-SPaP
and PoAb-SILIC. In parallel, axial filaments were prepared and ana-
lyzed inthe same way. The size separated protein pattern of the
extract (Ext) andthe axial filament (AF) is shown for the analysis of
silicatein (SILIC). Western blots ⁄ SDS ⁄ PAGE studies from left to
right show the results for selenoprotein M (SelM), spicule-associ-
ated protein (SAP) and silicatein (SILIC). Signals for selenopro-
tein M (14-kDa band onthe blot) and spicule-associated protein
(26 kDa) are observed inthe axial filament and to a large extent in
the extracts, whereas silicatein (24 kDa) is predominantly found in
the axial filament. Markers were separated in parallel (lane M).
Effect of seleniumonspiculeformation W. E. G. Mu
¨
ller et al.
3844 FEBS Journal 272 (2005) 3838–3852 ª 2005 FEBS
Effect of seleniumon silica formationin vitro
From earlier studies [3,4] it is known that silicatein,
also in its recombinant form, catalyzes silica formation
in vitro; in these studies the colorimetric molybdate
assay was applied.
In the present study, theeffect of seleniumon silica
formation was elucidated using the fluorescence dye
Rhodamine 123 as an indicator. As outlined in Experi-
mental procedures, the reaction was performed under
controlled conditions, using recombinant silicatein. In
the absence of the tetraethoxysilane substrate, only
small aggregates, < 3 lm, were observed under fluor-
escence light (Fig. 11A). If tetraethoxysilane was added
to the assays with recombinant silicatein, the size of
the aggregates increased to 10 lm after an incubation
period of 15 min (Fig. 11B). Longer incubation for 3 h
resulted in further growth of the aggregates to 30–
50 lm (Fig. 11C). If during this 3 h incubation period
1 lm sodium selenite was present inthe reaction
mixture, the sizes of the aggregates reached values of
50–100 lm (Fig. 11D).
Discussion
Selenium is a trace element which is essential for meta-
zoans from humans [11] to sponges [9]. Inthe marine
environment, theselenium concentration varies
between 10 and 100 nm [17]. It is well established that
selenium is found in naturally occurring proteins in
two forms, either – rarely – it is inserted post-trans-
lationally as a dissociable cofactor into molybdenum-
containing enzymes [18], or cotranslationally into
proteins as the amino acid selenocysteine [14].
The experiments described here show that exposure
of primmorphs to selenium results in a significant
increase inspicule formation. After a 7-day exposure
to selenium, a substantial increase inbiosilica content
of the primmorphs was measured. The formation
of new spicules in primmorphs can be monitored in
AB
DC
EF
Fig. 10. Immunofluorescence analysis of sili-
catein, selenoprotein M and spicule-associ-
ated protein in tissue from S. domuncula.
The slices were stained with PoAb-SILIC
(A), PoAb-SelM (C) and PoAb-SPaP (E). In
parallel, the corresponding Nomarsky
interference images (B, D and F) are given;
some spicules are marked inthe paired
images (>). One axial filament (af) in the
center of a spicule is marked in (F).
W. E. G. Mu
¨
ller et al. Effect of seleniumonspicule formation
FEBS Journal 272 (2005) 3838–3852 ª 2005 FEBS 3845
parallel. It is shown that the axial filament, which con-
sists of silicatein [2–4], is formed intracellularly in spe-
cial cells termed sclerocytes [19]. After the initial
intracellular synthesis of small spicules 8 lmin
length and 1.5 lm in diameter, the spicules are prob-
ably extruded into the bulky extracellular space. Then
synthesis is completed extracellularly, again through
the enzymic activity of silicatein, by appositional
growth, as demonstrated here by immunohistochemical
analysis. The antibodies against silicatein not only
reacted with the silicatein of the axial filaments but also
with proteins present onthe surface of the spicules.
Exposure of primmorphs to selenium results in the
expression of two proteins which were shown to be
associated with spicule formation; selenoprotein M
and the spicule-associated protein. Selenoprotein M
has, until now, only been described in metazoa and the
sponge selenoprotein M is the phylogenetically oldest
member. The size of selenoprotein M, deduced from
the cDNA, is 13 918 Da. Western blotting studies per-
formed here show that the native protein has a size of
14 kDa, suggesting that only a small signal peptide
exists at the sponge protein, if at all. A phylogenetic
analysis revealed the closest similarity of the sponge
molecule to the human selenoprotein M, whereas the
proteins of invertebrates (D. melanogaster and C. ele-
gans) are only distantly related.
The finding that the steady-state concentration of
selenoprotein M is regulated at the level of transcrip-
tion was surprising. In vertebrates theexpression of all
selenoproteins is regulated at the level of translation
[20]. In S. domuncula, the increase in selenoprotein M
expression after exposure to 10 lm selenium is large.
The immunochemical analysis shows that a seleno-
cysteine tRNA population exists inthe sponge,
because translation to the full-size protein occurs.
The biological role of selenoprotein M in higher
metazoan phyla is not known in detail. Inthe zebrafish,
selenoprotein M is expressed inthe notochord, the
somites, the spinal cord andthe axial fin fold [21]. These
data interestingly show that theexpression pattern of
the different selenoproteins inthe fish is region-specific.
To obtain an insight into the potential function of
selenoprotein M in S. domuncula, antibodies were
raised. Surprisingly the subsequent immunohistochemi-
cal analysis revealed that selenoprotein M is localized in
the axial filament andonthe surface of the spicules.
Metabolic labeling experiments with
75
Se revealed that
a 14-kDa protein also exists inthe spicules.
It had previously been shown that selenocysteine in
selenoproteins participates in redox reactions, especi-
ally if selenocysteine has a close cysteine partner [11].
Exactly this constellation exists in selenoprotein M;
one cysteine residue is present three amino acids along
from selenocysteine towards the N-terminus. This
intriguing finding may suggest that selenoprotein M
functions as an enzyme.
Electron microscopic images document that forma-
tion of siliceous spicules starts with the synthesis of
90–260-nm silica granules [22]. Granular silica of
AB
DC
Fig. 11. Influence of seleniumonthe size of
silica aggregates, formed in vitro. Recombin-
ant silicatein was incubated inthe standard
reaction assay, inthe absence of the tetra-
ethoxysilane substrate for 3 h (A). If tetra-
ethoxysilane was added to the reaction for
15 min (B) or 3 h (C), the size of the aggre-
gates increased. (D) Larger aggregates of
silica were formed if, during the 3 h
incubation period, 1 l
M sodium selenite was
present inthe reaction mixture. The silica
formed was stained with Rhodamine 123 as
described in Experimental procedures and
inspected by fluorescence microscopy.
Effect of seleniumonspiculeformation W. E. G. Mu
¨
ller et al.
3846 FEBS Journal 272 (2005) 3838–3852 ª 2005 FEBS
exactly this size, 70–300 nm, has recently been synthes-
ized in vitro, using recombinant silicatein from S. do-
muncula [23]. It is still unclear by which process(es)
these silica granules are assembled into silica layers.
Based on experimental data, it has been proposed that
several units of silicatein a form a regular repeating
structure of 5–8 nm [24] or 17 nm [2] periodicity. This
useful information explains the 2D orientation of the
growth of the silica granules ⁄ sheet. For the next phase
in spicule production, forming and shaping has to
occur. Additional factors, e.g. low molecular mass
organic molecules or larger size polypeptides, must be
postulated that guide the packaging of the silica gran-
ules into concentric layers. In elegant studies in diatoms
it was shown that the sophisticated construction of the
diatom shell is the result of high molecular mass pro-
teins, frustulins [25], forming a coat around the dia-
toms, and lower molecular mass proteins present in the
silica shell, the silaffins [26]. The latter 4 to 17-kDa pro-
teins are post-translationally modified at their lysines
and serines. The serine units carry the phosphate
groups, andthe lysines are modified at their a-amino
groups by methylpropylamine units [27].
It must be stressed that the silica formationin diatoms
proceeds nonenzymatically, in contrast with sponges
which form the spicules enzymatically using silicatein.
However, up until now, nobody has been able to iden-
tify the proteins within the silica sheets inthe spicules.
In fact, published data strongly suggest that the thicken-
ing of the spicules is performed enzymatically by apposi-
tion [16]. The immunohistological data presented here
show that silicatein is also present at the surface of the
spicules, supporting this assumption. As the axial fila-
ments of the spicules are not homogeneous and, especi-
ally intracellularly, are associated with membranes and
fibrils, a high-resolution protein analysis of the axial fila-
ments is the only way to further identify (non)enzymatic
proteins involved inspicule synthesis. Here we used
differential display of transcripts to identify the axial
filament-associated protein. The inorganic element
selenium was chosen as inducer, because of its essential
role inthe growth of animals ⁄ sponges, its quasi-enzy-
matic function, and its chemical property of existing in
different valences (II, IV and VI). The transition of the
different co-ordination states allows incorporation into
organic molecules [28].
Using differential display of transcripts, we identified
a second protein that is up-regulated in primmorphs
after incubation with selenium. The deduced protein
shares no significant sequence similarity to any protein
in the database. The characteristic feature of this poly-
peptide is the presence of 10 highly similar repeats,
composed of nine amino acids. Inthe center are
hydrophobic amino acids surrounded by the polar
amino acids glutamine, lysine, serine and threonine.
Taking into account this polar ⁄ hydrophobic ⁄ polar
composition, this protein can be expected to form a
tight association with membranes. This sponge-specific
protein was termed spicule-associated protein because
it exists inthe axial filament and also onthe surface of
the spicules. Theexpression of this protein is not affec-
ted by a change inthe concentration of silicic acid in
the surrounding milieu (not shown). In contrast, as
demonstrated here, theexpression of the silica-poly-
merizing enzyme silicatein is not regulated by selenium
but by silicic acid [4]. Hence, it becomes evident that
morphogenesis of sponges is to a considerable extent
dependent on outside inorganic factors. Both elements,
silicon and selenium, can be considered morphogenetic
factors which control spiculeformationin sponges and
in turn skeleton formationin these animals.
The final interesting finding for the biotechnological
application of silicatein, especially with respect to the
understanding of biosilicaformationin sponges, is
that, inthe presence of selenium, the size of the poly-
merized silica particles formed from recombinant sili-
catein is substantially larger.
Taken together, the data reported show that selen-
ium has a stimulatory effectonformation of siliceous
spicules in sponges. They may also shed new light on
the factors involved inbiosilicaformationin metazoa.
Experiments to elucidate potential catalytic effects of
both free and protein-bound seleniuminthe polymer-
ization process of silica are in progress. Our hypothesis
is that selenium is not only involved in protein ⁄ silica-
tein-controlled silica formationin sponges but func-
tions also as a novel morphogenetic factor during
body plan formationin this oldest metazoan phylum.
Experimental procedures
Chemicals and enzymes
The sources of chemicals and enzymes used were given pre-
viously [4,29]. Natural sterile filtered seawater and sodium
metasilicate were obtained from Sigma-Aldrich (Taufkir-
chen, Germany). Sodium selenite (Na
2
SeO
3
) came from
Sigma (Taufkirchen, Germany). Na
2
[
75
Se]O
3
was from
Amersham Corp. [Little Chalford, Buckinghamshire, UK;
370 MBqÆ(mg Se)
)1
] or Polatom (Otwock Swierk, Poland;
1500 MBqÆmg
)1
).
Sponges
Live specimens of S. domuncula (Porifera, Demospongiae,
Hadromerida) were collected near Rovinj (Croatia) and
W. E. G. Mu
¨
ller et al. Effect of seleniumonspicule formation
FEBS Journal 272 (2005) 3838–3852 ª 2005 FEBS 3847
[...]... 187–189 (amino acid 32 inthe deduced protein) was not included The 252-bp part was cloned into theexpression vector pBAD ⁄ gIIIA (Invitrogen), which contained at the 3¢-terminus the myc epitope andthe polyhistidine region The insert was expressed overnight at 30 °C inthe presence of 0.0002% l-arabinose The fusion protein was extracted and purified with the Histag purification kit (Novagen, Madison, WI,... with the FEBS Journal 272 (2005) 3838–3852 ª 2005 FEBS Effect of seleniumonspiculeformation complete ORF of the SDSPIaP cDNA was used; again the cDNA was inserted into the vector at the same restriction sites, NcoI and HindIII Expressionand purification procedures were the same as for selenoprotein M Raising of antibodies Polyclonal antibodies (PoAbs) were raised against recombinant r-SelM and r-SPaP... Gamulin V (2000) The mitogen-activated protein kinase p38 pathway is conserved in metazoans: cloning and activation of p38 of the SAPK2 subfamily from the sponge Suberitesdomuncula Biol Cell 92, 95–104 43 Grebenjuk VA, Kuusksalu A, Kelve M, Schutze J, Schro¨ ¨ der HC & Muller WEG (2002) Induction of (2¢-5¢) oligo¨ adenylate synthetase inthe marine sponges Suberitesdomunculaand Geodia cydonium by the. .. sponge direct the polymerization of silica and silicones in vitro Proc Natl Acad Sci USA 96, 361–365 4 Krasko A, Batel R, Schroder HC, Muller IM & Muller ¨ ¨ ¨ WEG (2000) Expression of silicatein and collagen genes inthe marine sponge Suberitesdomuncula is controlled by silicate and myotrophin Eur J Biochem 267, 4878–4887 5 Muller WEG (2005) Spatial and temporal expression ¨ patterns in animals In. .. 5¢-AGTGAATGCG-3¢ and one of the dT23N primers inthe assay To avoid DNA-based contamination from the reagents, the assay was incubated (before the addition of the template) with the restriction enzyme Sau3AI for 20 min at 37 °C followed by a final inactivation step at 72 °C for 10 min The PCR parameters used were: 94 °C for 3 min, 45 cycles of 94 °C for 30 s, 40 °C for 2 min, and 72 °C for 30 s, with an additional.. .Effect of seleniumonspiculeformation W E G Muller et al ¨ then kept in aquaria in Mainz (Germany) for more than 2 years before their use grids and analyzed with a Tecnai 12 microscope (FEI Electron Optics, Eindhoven, the Netherlands) Preparation of spicules and axial filaments Silica content of primmorphs Spicules and their axial filaments were prepared as described... Staining of polymerized silica with Rhodamine 123 Enzymatic silica formation was studied using r-silicatein [31] The reactions were performed on a glass slide in a volume of 100 lL The reaction mixture contained 10 lg r-silicatein in NaCl ⁄ Pi (pH 7.2) containing 5 mm Fe(III) and 1 lm ZnCl2 As substrate, 4.5 mm tetraethoxysilane (Sigma) was used The reaction was performed at 22 °C for up to 3 h; the. .. extension step at 72 °C for 10 min After amplification, the labeled fragments were separated on a 6% polyacrylamide sequencing gel using a DNA sequenator (Li-Cor 4000S) The sequencing run was stopped after 3 h, andthe gel was transferred to millimeter paper and vacuum dried The differences inthe banding pattern onthe gel were detected by an infrared scanning device (Odyssey; LiCor, Lincoln, NE, USA) The. .. program from the phylip package [37] The distance matrices were calculated using the Dayhoff PAM matrix model as described [38] The degree of support for internal branches was further assessed by bootstrapping [37] The graphic presentations of the alignments were prepared with genedoc [39] Preparation of recombinant proteins selenoprotein M and spicule- associated protein Expression of selenoprotein M A fragment... ‘shell’ andthe axial filaments The amount of 75Se-labeled material was determined, andthe c.p.m obtained was correlated with 5 mg spicules, before the disintegration In a second series of experiments, the primmorphs (2 g) were incubated for 3 days with 200 nCiÆmL)1 75Se Then spicules were isolated, pulverized and extracted with lysis buffer After centrifugation, the 2000 g supernatant was collected, concentrated . Selenium affects biosilica formation in the demosponge Suberites domuncula Effect on gene expression and spicule formation Werner E. G. Mu ¨ ller 1 , Alexandra Borejko 1 , David Brandt 1 , Ronald. immunoblot. Gene expression studies The effect of selenium on the expression of the genes encoding selenoprotein M, spicule- associated protein, and silicatein was studied (Fig. 8). In the absence. formation in sponges and in turn skeleton formation in these animals. The final interesting finding for the biotechnological application of silicatein, especially with respect to the understanding