Poly(silicate)-metabolizing silicatein in siliceous spicules
and silicasomes of demosponges comprises dual
enzymatic activities (silica polymerase and silica esterase)
Werner E. G. Mu
¨
ller
1
, Ute Schloßmacher
1
, Xiaohong Wang
2
, Alexandra Boreiko
1
, David Brandt
1
,
Stephan E. Wolf
3
, Wolfgang Tremel
3
and Heinz C. Schro
¨
der
1
1 Institut fu
¨
r Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universita
¨
t, Mainz, Germany
2 National Research Center for Geoanalysis, Beijing, China
3 Institut fu

Silicatein has been isolated from a number of sili-
ceous sponges, e.g. Tethya aurantium or Suberites
domuncula [9,10]. If the enzyme is isolated from the
skeletal elements of these animals, the spicules, it can
be used in vitro to catalyze polycondensation of a wide
variety of alkoxides, as well as ionic and organometallic
Keywords
poly(silicate); silica esterase; silica
polymerase; silicatein; 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 39 25243
Tel: +49 6131 39 25910
E-mail:
Website: />(Received 22 October 2007, revised 13
November 2007, accepted 26 November 2007)
doi:10.1111/j.1742-4658.2007.06206.x
Siliceous sponges can synthesize poly(silicate) for their spicules enzymati-
cally using silicatein. We found that silicatein exists in silica-filled cell
organelles (silicasomes) that transport the enzyme to the spicules. We show
for the first time that recombinant silicatein acts as a silica polymerase and

the polycondensation of monomeric silicon alkoxides
to form silica structures on surfaces [13].
It was shown that silicatein is the main component of
the axial filament of the spicules [9,10]. Later, this
enzyme was also detected in the extraspicular space,
where it contributes to the appositional growth of these
skeletal elements [14,15]. Silicatein uses either organo-
functional silanes [9] or orthosilicate (W. E. G. Mu
¨
ller,
unpublished results) for the synthesis of poly(silicate).
As seawater has a low content of silicate (about 5 lm),
the sponges have to transport silicate actively into their
cells, via a putative Na
+
⁄ HCO
3
)
[Si(OH)
4
] co-trans-
porter [16]. Intracellularly, silicate is stored in silica-
somes, organelles with a high content of silicate [17].
These results were obtained using a sponge tissue
culture system (termed a primmorph system) [18] that
comprises a special form of 3D cell aggregates com-
posed of proliferating and differentiating cells. Prim-
morphs allow the investigation of spicule formation
under controlled conditions [19]. Based on electron
microscopic studies presented previously [17], it appears

facilitating attack of the hydroxy group on the silicon
atom of the substrate. This reaction can be monitored
spectroscopically on the basis of the release of p-amino-
phenol. The experimental data show that, in addition
to its silica polymerase activity, silicatein also comprises
a silica esterase function, thus supporting the concept
that silicatein is involved in stabilization of the sol state
of biogenic silica. The esterase reaction can be com-
pletely blocked by sodium hexafluorosilicate and by the
cysteine proteinase inhibitor E-64 (l-trans-epoxysucci-
nyl-leucylamido(4-guanidino)butane) [24]. For these
H
2
N
O
Si
O
NH
2
CH
3
CH
3
+
+ H
H
2
N
OH
HO

FEBS Journal 275 (2008) 362–370 ª 2007 The Authors Journal compilation ª 2007 FEBS 363
studies, resulting in elucidation of a new activity of sili-
catein as a silica esterase, we used recombinant silica-
tein-a from the demosponge S. domuncula [10].
Results
Presence of silicatein in the spicules and cell
organelles, the silicasomes
Sections through primmorphs were exposed to anti-
bodies to silicatein, PoAb-aSILIC, and analyzed by the
transmission electron microscopy immunogold labeling
technique. As expected, strong signals were seen in
the axial filament within the sponge spicule (Fig. 2A),
the site hitherto proposed for major occurrence of the
enzyme [14,25]. The images also show, however, dense
accumulation of gold grains in the extraspicular space,
reflecting dense packaging of silicatein molecules there
also. The silicatein molecules are arranged around the
spicules in concentric rings (Fig. 2B). A closer view of
the axial canal in the center of the spicule reveals local-
ization of silicatein in the axial filament as well as
within the silica shell surrounding the spicule
(Fig. 2C). Controls show that pre-immune serum does
A
B
C
D
E
F
G
H

ller et al.
364 FEBS Journal 275 (2008) 362–370 ª 2007 The Authors Journal compilation ª 2007 FEBS
not react with structures within or around the spicules
(Fig. 2D). Likewise, the adsorbed PoAb-aSILIC prepa-
ration, pre-incubated with recombinant silicatein, did
not react either (as shown previously [14]). Strong
reactions of PoAb-aSILIC are also seen in vesicles of
the sclerocytes, the cells surrounding the spicules
(Fig. 2E,F). These intracellular vesicles, termed silica-
somes, are rich in silica [17], and are additionally den-
sely filled with the enzyme. Extracellularly (Fig. 2G),
the silica vesicles fuse with the concentric ring struc-
tures around the spicule (Fig. 2H). These silica vesicles
often remain as intact entities within the rings ⁄ cylin-
ders, reacting positively to anti-silicatein (Fig. 2H).
Catalytic function of silicatein: silica polymerase
(anabolic enzyme)
Synthesis of polymerized polysiloxane derivatives of
silicic acid, was performed using silicatein and di-
methyldimethoxysilane as substrate. After an incubation
period of 1 h, the sample was analyzed by MALDI-
MS. As shown in Fig. 3B, a stepwise 74–75 Da
increase in mass is recorded above an m ⁄ z of 500,
which is due to stepwise polymerization of -Si(Me)
2
-O-
units to the starter silane substrate. Under the incu-
bation conditions used, synthesis of oligomers with
11 -Si(Me)
2

) was determined using this value [27],
and was calculated to be 22.7 lm. In comparison,
the K
m
value for human recombinant cathepsin L
(EC 3.4.22.15), the enzyme closest related to silicatein,
expressed in Escherichia coli, was 1.1 lm, using the
substrate benzyloxycarbonyl-Phe-Arg-4-methylcouma-
rin-7-amide [28]. The turnover value (molecules of con-
verted substrate per enzyme molecule per second) for
silicatein in the silica esterase assay was 5.2. Although
this catabolic de-polymerization reaction may be sub-
stantially different from the cleavage of peptide bonds
by human cathepsin L, the human enzyme shows only
a slightly higher turnover value of 20 using the same
substrate [29].
The specificity of the reaction was determined in two
series of experiments. First, silicatein was replaced in
the assay by the same amount of BSA. Under other-
wise identical conditions, no significant increase in
absorbance was seen at either 300 or 230 nm over
400 500 600 700 800 900 1000
0
0
10
10
20
20
30
30

n =7
n =6
n =8
n =9
n =10
n =11
Fig. 3. MALDI-MS spectrum of the products formed from
dimethyldimethoxysilane in the absence (A) or presence of
4.5 lgÆmL
)1
silicatein (B). The mass distributions differ significantly.
In the presence of silicatein (B), a distinct increase in chain length
can be observed. The distance of 74–75 Da between each individ-
ual peak corresponds to the mass of a single Si(Me)
2
-O unit; oligo-
meric polymerization of 11 units can be resolved. In contrast, no
polymerization products are observed in the absence of silicatein.
W. E. G. Mu
¨
ller et al. Silicatein comprises dual enzymatic activities
FEBS Journal 275 (2008) 362–370 ª 2007 The Authors Journal compilation ª 2007 FEBS 365
2–60 min incubation periods (20 °C). Second, a direct
interaction between the ester-like substrate BAPD
silane (50 lm) and the silicate monomer sodium hexa-
fluorosilicate (1 mm) was studied in the reaction with
silicatein. In previous studies, sodium hexafluorosili-
cate has been proven to induce growth of sponge cells
in culture and to cause differential gene expression
in vivo and in vitro [10,30]. After addition of a 20-fold

the inorganic silicic acid monomers are converted
intracellularly to organosilicate units. The subsequent
process requires intracellular transport of the silicic
acid, or derivatives of it, to the organelles (silicasomes)
in which initial formation of the spicules proceeds. In
the spicule-forming cells, the sclerocytes, the first layers
of the silica shell of the spicules are formed around the
silicatein-based axial filament in specific organelles
[14].
The prerequisite for intracellular initiation of spicule
synthesis is preferential accumulation of silicic acid in
special organelles. Recently, such vesicles with a high
silica content, the silicasomes, have been identified in
sclerocytes [17]. It is expected that, in silicasomes
240 260 280 300 320 340
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
220
Absorption
Wavelength (nm)
0
2

simultaneous release of water. The data summarized
here demonstrate that silicatein does indeed have such
activity; it mediates cleavage of silicate ester bonds in
the BAPD silane substrate. Furthermore, it is shown
that silicatein also exhibits silica-polymerizing activity,
as previously proposed [9]. We have demonstrated that
the polymerizing growth of the silica chains, mediated
by the silica polymerase activity of silicatein, involves
stepwise addition of single silica monomeric units. This
finding implies that silicatein has two different
enzymatic properties, a silica esterase activity and a
polymerizing ⁄ polycondensing activity (silica polymer-
ase). At present, we are working on elucidation of the
molecular switch controlling these dual enzymatic
functions; initial data indicate that low-molecular-
weight compound(s) direct silicatein to either the cata-
bolic or the anabolic reaction. Enzymatic parameters
of the silica esterase activity were determined. The
Michaelis constant (K
m
22.7 lm) and the turnover
value (5.2 molecules of converted substrate per enzyme
molecule per second) for the silica esterase catalytic
reaction of silicatein are similar to those that have
been determined for the related hydrolytic enzyme
cathepsin L [28,29]. Future studies are required to
determine whether silica polymerase and silica esterase
function in principle by the same mechanism. Both
reactions are initiated by a nucleophilic attack by the
hydroxyl group of the Ser residue in the catalytic cen-

Fig. 6. Localization of silicatein in the extraspicular space. (A) The
spicules (sp), formed from poly(silicate) (sia) are surrounded by
sclerocytes (sc) that harbor special organelles, the silicasomes (sis),
that are rich in silicatein (red circles) and silicic acid (blue circles). In
the center of the spicules runs the axial filament (af), which is built
up from silicatein molecules. (B) The silicasomes are released from
the sclerocytes and transported into the extracellular space, from
where these silica vesicles (siv) are translocated to the ring struc-
tures surrounding the growing spicules (sp). The silica vesicles,
harboring silicatein and monomeric silicic acid, fuse with the con-
centric rings (ri) that are present around the spicules. There the sili-
catein molecules become associated with the ring sheet, while the
poly(silicate) (sia) remains in the siliceous lamellae that are formed
within the rings.
W. E. G. Mu
¨
ller et al. Silicatein comprises dual enzymatic activities
FEBS Journal 275 (2008) 362–370 ª 2007 The Authors Journal compilation ª 2007 FEBS 367
render possible the rational application of silicatein in
the fabrication of (new) biomaterials based on layered
silica, of titania and of zirconia [32]. This view is based
on the finding of a dual role for silicatein as an ana-
bolic (silica polymerase) and catabolic enzyme (silica
esterase), allowing the formation of controlled silica
structures. In addition, patterning of poly(silicate) is
modulated by self-assembly of silicatein molecules in
an organized, fractal manner [33,34]; the fractal pat-
tern probably dictates the initial shape of the spicules
[34]. The finding that silicatein catalyzes two reactions,
acting as silica polymerase and silica esterase, provides

sodium hexafluorosilicate from Sigma-Aldrich (Taufkir-
chen, Germany), and p-aminophenol from Riedel de
Hae
¨
n (Seelze, Germany).
Sponges and primmorphs
Specimens of the marine sponge S. domuncula (Porifera,
Demospongiae, Hadromerida) were collected in the North-
ern Adriatic near Rovinj (Croatia), and then kept in aquaria
in Mainz (Germany) at a temperature of 17 °C for more than
5 months. From these animals primmorphs, a 3D cell system
[10,18,19] was prepared. Primmorphs were kept at 17 °Cin
natural seawater (enriched with 60 lm of silicate), supple-
mented with 1% RPMI-1640 medium (GIBCO, Karlsruhe,
Germany). The primmorphs were used for analysis approxi-
mately 20 days later [14].
Scanning electron microscopy
The SEM analysis of spicules was performed using a Zeiss
DSM 962 digital scanning microscope (Zeiss, Aalen, Ger-
many) as described previously [14].
Electron immunogold labeling
Polyclonal antibodies (PoAb-aSILIC) were used that had
been raised against recombinant silicatein-a from S. domun-
cula [14]. Primmorph samples were treated with 0.1% glu-
taraldehyde ⁄ 3% paraformaldehyde in 0.1 m phosphate
buffer (pH 7.4). After 2 h, the material was dehydrated in
ethanol and embedded in LR-White resin (Electron Micros-
copy Sciences, Hatfield, PA, USA). Slices were cut 60 nm
thick and blocked with 5% BSA in NaCl ⁄ P
i

using a molecular
mass of 21 329 Da [10]) in MOPS buffer was covered with
a layer of dimethyldimethoxysilane dissolved in diethyl
ether (10 lmolÆmL
)1
) in the ratio 10 : 1 (v ⁄ v). Samples were
taken after incubating the assays for 1 h at 20 °C, with
shaking. The aqueous layer, containing decomposition
products, silicatein and buffer, was removed, and the
organic phase, which contained only the substrate and the
siloxane polymer, was dried using Na-sulfate to avoid fur-
ther decomposition. Finally, the products were character-
ized by means of MALDI-MS [38,39] performed in a
Finnigan MAT mass spectrometer 8230 (Midland; Canada).
In a control assay, the reaction was performed in the
absence of silicatein.
Esterase activity
The assay is based on the concentration-dependent
increase in the UV absorption at a wavelength of 300 nm
Silicatein comprises dual enzymatic activities W. E. G. Mu
¨
ller et al.
368 FEBS Journal 275 (2008) 362–370 ª 2007 The Authors Journal compilation ª 2007 FEBS
of the degradation product p-aminophenol that results
from hydrolysis of the substrate BAPD silane [40]. The
contribution of the degradation product p-aminophenol to
the UV ⁄ vis spectra was realized by the same phase trans-
fer principle as mentioned above. During continuous
stirring of the assays in Suprasil mixing cuvettes (Hellma
QS-110, Mu

the recipient of a Konrad Adenauer fellowship.
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