Tài liệu Báo cáo khoa học: Poly(silicate)-metabolizing silicatein in siliceous spicules and silicasomes of demosponges comprises dual enzymatic activities (silica polymerase and silica esterase) doc

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

¨
r Anorganische Chemie, Universita
¨
t, Mainz, Germany
Silicon is the second most common element in the
Earth’s crust [1]; it possesses semi-metallic as well as
metalloid properties. Silicon exists in nature in combi-
nation with oxygen as silicate ions or as silica; silica
has no negative charge, while silicate anions carry a
negative net electrical charge, which is counterbalanced
by cations. Free silica ⁄ silicate is found both in the
crystalline state (such as quartz) and in the amorphous
state (such as opal). Silica ⁄ silicate is widely used in
industry and medicine for the fabrication of poly(sili-
cate), e.g. in amorphous glasses, ceramics, paints,
adhesives, catalysts and photonic materials [2,3]. Fur-
thermore, poly(silicate) is an important new material
in nano(bio)technology [4,5]. This multidisciplinary
research field is concerned with bio- and electronic
engineering at nanometer, molecular and cellular levels
[4]. Currently, production of silica require high temper-
ature conditions and extremes of pH [6]. Hydrated
amorphous silica, e.g. in the form of opal, has superb
properties such as low density and high porosity. In
nature, amorphous silica can be produced by diatoms
by passive deposition onto an organic matrix. Siliceous
sponges (Demospongiae) have the exceptional ability
to synthesize silica enzymatically via silicatein [7,8].
Based on its protein sequence, silicatein is related to
the proteinases of the cathepsin class [9].

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

also as a silica esterase. The enzymatic polymerization ⁄ polycondensation of
silicic acid follows a distinct course. In addition, we show that silicatein
cleaves the ester-like bond in bis(p-aminophenoxy)-dimethylsilane. Enzy-
matic parameters for silica esterase activity are given. The reaction is com-
pletely blocked by sodium hexafluorosilicate and E-64. We consider that
the dual function of silicatein (silica polymerase and silica esterase) will be
useful for the rational synthesis of structured new silica biomaterials.
Abbreviations
BAPD-silane, bis(p-aminophenoxy)-dimethylsilane; EDTA, ethylenediaminetetraacetic acid; E-64,
L-trans-epoxysuccinyl-leucylamido(4-
guanidino)butane; MOPS, [3-(N-morpholino) propanesulfonic acid]; PoAb, polyclonal antibodies; SEM, scanning electron microscopy; TEM,
transmission electron microscopy; TEOS, tetraethoxysilane.
362 FEBS Journal 275 (2008) 362–370 ª 2007 The Authors Journal compilation ª 2007 FEBS
precursors, to the corresponding metal oxides; these
processes occur at standard, ambient temperature and
pressure and neutral pH [11]. Using site-directed muta-
genesis, those amino acids critical to the condensation
of tetraethoxysilane (TEOS) have been determined; the
catalytic triad is histidine (His), asparagine (Asn) and
serine (Ser) [4,12]. In the active center of silicatein, the
hydroxyl group of Ser26 and the imidazole of His165
(catalytic diad) have been shown to play key roles in
the condensation of TEOS [11]. It has been proposed
that these functionalities participate in the formation of
a transitory pentavalent silicon species, stabilized
through a donor bond to the imidazole nitrogen [11].
Using a nitrilotriacetic acid-terminated alkanethiol,
which had been successfully self-assembled onto gold
surfaces, silicatein could be immobilized on matrices; it
was found to retain its enzymatic function, allowing

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

that the silicasomes are intracellular granules that can
release their content by exocytosis to the mesohyl.
The existence of silicatein in silicasomes with high sil-
ica levels implies that silicatein might be involved in the
storage of silica in these organelles, presumably con-
trolling the gel–sol state of silicate. From diatoms, it is
known that silicate is deposited in special organelles,
the silica deposition vesicles, which, in addition to high
levels of silicate, also contain organic components of
unknown function, e.g. mannose [20–22]. It may be
assumed that these molecules prevent polycondensation
of silicate. As silicate – at neutral pH – polycondenses
at concentrations above 1 mm to poly(silicate) [23],
it may be postulated that (organic) molecules, e.g.
silicatein, contribute to stabilization of the sol state of
silica. One mechanism for the gel to sol conversion
could be hydrolysis of the oxygen bridge of the poly-
merized ⁄ polycondensed silicic acid. The linkage bet-
ween silicate or tetrahedral silica units in poly(silicate)
is an ester-like bond. In order to test whether silicatein
– in addition to being a poly(silicate)-forming enzyme
(silica polymerase) – also functions as an silica esterase,
we studied its hydrolytic function on bis(p-aminophen-
oxy)-dimethylsilane (BAPD silane). This compound
comprises two ester-like bonds between silicon and
p-aminophenol and two methyl silane linkages (Fig. 1).
In line with a previous study [9], we propose that
hydrogen bonding between the imidazole nitrogen of
the conserved His and the hydroxyl of the active-site
Ser increases the nucleophilicity of the Ser oxygen,

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

Si
OH
CH
3
CH
3
+
+
–
O
H
N
N
His
Ser
H
Silicatein
Ester-like bond
Silane bond
Fig. 1. Proposed silicatein-a-mediated reaction mechanism for
hydrolysis of bis(p-aminophenoxy)-dimethylsilane which contains
two silicic ester-like (blue) and two silane bonds (red). In the cata-
lytic center of silicatein, the serine (Ser) oxygen makes a nucleo-
philic attack on the silicon, resulting in displacement of
p-aminophenol and formation of a (alkoxyl)-monosilane. This reac-
tion is facilitated by hydrogen bonding between the imidazole nitro-
gen of the conserved histidine (His) and the hydroxyl of the Ser.
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 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

Fig. 2. Localization of silicatein in spicules
and in intra- and extracellular vesicles by
TEM immunogold labeling. (i) Association of
silicatein with spicules. (A) Strong antibody
reactions are seen within the axial canal (ac)
in the axial filament (af), which is sur-
rounded by the spicule (sp); in addition, a sil-
ica vesicle (siv) within one concentric ring
(ri) is present. (B) Strong antigen–antibody
reactions are also seen on the concentric
rings (ri) surrounding a spicule (sp). (C) In
the axial canal (ac), high levels of signals are
seen in and on the axial filament (af), as
well as the inner rim of the silica spicule
(sp). (D) Control: incubation of a section
with pre-immune serum; no reaction is seen
within the axial canal (ac) and around the
spicule (sp). (ii) Intracellular localization of
silicatein in vesicles. (E,F) The cells around
the spicules, the sclerocytes (sc), are filled
with vesicles, which strongly react with
antibodies. These vesicles are termed
silicasomes (sis). (iii) Extracellular localization
of silicatein in vesicles. (G) In the extra-
cellular space (ex-s), the silica vesicles (siv)
can still be seen. (H) These silica vesicles
(siv) frequently remain intact within
rings ⁄ cylinders (ri).
Silicatein comprises dual enzymatic activities W. E. G. Mu
¨

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

-O- units could be detected. If silicatein is
absent from the sample, no signals above an m ⁄ z of
500 Da are seen (Fig. 3A). This result suggests that
silicatein, via its silica polymerase activity, lowers the
activation energy for the polymerization ⁄ condensation
reaction, resulting in successive addition of monomeric
silica units.
Catalytic function of silicatein: silica esterase
activity (catabolic enzyme)
The temperature optimum was found to be in the
range 20–25 °C; the temperature coefficient (Q
10
)
decreases by 2.5-fold above 25 °C and increases by
2.9-fold below 25 °C. Silica esterase activity was rou-
tinely determined at 20 °C using a substrate range
between 20 and 250 lm of BAPD silane. After cleav-
age of one of the silica ester bonds, the concentration
of the released product p-aminophenol was determined
at a wavelength of 300 nm, which is in the trailing
edge of the main absorption bands under the condi-
tions used. Another maximum is recorded at 230 nm
(Fig. 4). The molar absorption coefficient (e at
k = 300 nm) was determined [26] to be 2096.6 LÆmolÆ
cm
)1
, in enzyme reactions with BAPD silane (20, 100
and 200 lm; non-saturating conditions). The Michaelis
constant (K
m

) 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

40
40
50
50
60
60
70
70
A
B
m/z
m/z
400 500 600 700 800 900 1000
Intensity (%) Intensity (%)
503.2 577.2 623.2 697.2 771.4
429.3
461.2
503.2
577.2
623.3
847.0 925.2
475.1
Si
OMe MeO
Me
Me
Si
Me
O
Me

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

molar excess of sodium hexafluorosilicate with respect
to the ester-like substrate BAPD silane, complete sup-
pression of the ester-like activity of the enzyme was
determined in the photometric test used here. Alterna-
tively, the Ser proteinase inhibitor E-64 was added to
the reaction mixture; at a concentration of 10 lm,an
inhibition of the esterase activity > 95% was deter-
mined.
The dissolution process of spicules, the tylostyles,
can also be followed in vivo in tissue of S. domuncula.
Spicules were isolated from tissue [14] and analyzed by
scanning electron microscopic (SEM) analysis. In this
study, the pointed ends of the spicules were compared
(Fig. 5). Intact spicules have a smooth surface
(Fig. 5A), and the tips of the spicules are closed. Dur-
ing the decomposition process in vivo, their diameters
decrease and the lamellar organization becomes overt
(Fig. 5B). In later phases, the surface of the spicules
becomes wrinkled due to exposure of the silica nano-
particles; the axial canal opens and exposes the axial
filament (Fig. 5C).
Discussion
Sponges have to cope with an energetically highly
expensive chain of reactions to form their siliceous
spicules. The first barrier is the uptake of dissolved
silicic acid from the surrounding aqueous environment;
usually only low concentrations of silicic acid, of
approximately 5 lm, exist in seawater [31]. The uptake
of silicic acid is probably mediated by an ATP-con-
suming pump ⁄ transporter [16]. It is unknown whether

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

4
6
8
20 min
Fig. 4. Change of absorption spectra during incubation of silicatein
in the presence of 140 l
M bis(p-aminophenoxy)-dimethylsilane sub-
strate as described in Experimental procedures. At time zero, the
absorbance at k = 300 nm is very low. The absorbance increases
steadily during the subsequent 20 min of incubation. The molar
absorption coefficient (e) at 300 nm is indicated.
ABC
Fig. 5. Stepwise dissolution of tylostyle spicules (sp) in tissue from Suberites domuncula (SEM analysis). The intact spicules (pointed termi-
nus of the spicules) have a smooth surface (A). (B) Progressive decomposition of the spicules is followed by appearance of the lammelar
organization. Finally, the surfaces of the spicules become wrinkled, and silica nanoparticles can be seen on the surface (C); the axial canal
opens and the axial filament becomes visible.
Silicatein comprises dual enzymatic activities W. E. G. Mu
¨
ller et al.
366 FEBS Journal 275 (2008) 362–370 ª 2007 The Authors Journal compilation ª 2007 FEBS
where the silicic acid content is high, self-polymeriza-
tion or self-condensation processes are facilitated.
Polymerization ⁄ condensation of monomeric silicic acid
to poly(silicate) is a random process [23] that results in
the formation of 3D condensed silica polymers or
nuclei. Our experimental results show that silicic acid
co-exists with silicatein in these silicasomes. Based on
these microscopic analyses, we wished to determine
whether silicatein could function as a silica esterase,
allowing hydrolysis of the silicate ester bonds with

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-

ter of the enzyme. In the silica polymerase reaction,
this step may be followed by a condensation reaction,
which could be facilitated by proton transfer to the
His residue in the catalytic center.
The content of the silicasomes is released into the
extracellular space [17], and transported from there to
the spicules. As shown here, the extracellular silica ves-
icles that contain silicatein fuse with the appositionally
growing spicules in diametral direction, and probably
also in the axial direction [2]. These data show that sil-
icatein is transported in silicasomes into the extracellu-
lar space; there, the silica vesicles contribute to the
appositionally growing spicules. Furthermore, our data
allow development of a functional model that contrib-
utes to understanding of the growth of the siliceous
spicules which apparently lack a template or matrix.
The simultaneous release of silicic acid and silicatein
into the concentric rings around the growing spicules,
which gives rise to lamellar formation of the spicules
[30], allows a controlled polymerization ⁄ condensation
process for silicic acid mediated by silicatein along
galectin strings ⁄ nets [25]. The shape of the poly(sili-
cate) product is probably additionally tailored by
collagen sheathing [19]. A schematic outline of the
localization and transport of silicatein in the extraspi-
cular space is given in Fig. 6.
The data summarized here provide, for the first
time, enzyme kinetic data for silicatein, which will
A
B

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

this enzyme with advantageous properties, e.g. for pro-
duction of a flexible shell around organisms after bio-
encapsulation with silica. Recently, this feature has
been utilized to encapsulate bacteria [35]: E. coli were
transformed with the silicatein gene, and, after expres-
sion of silicatein and subsequent incubation with silicic
acid, the bacteria had been encapsulated with bio-sil-
ica, a viscous cover, which did not reduce their growth
properties [35].
Experimental procedures
Materials
Dimethyldimethoxysilane (C
4
H
12
O
2
Si, relative molecular
mass 120.22) and BAPD silane (C
14
H
18
N
2
O
2
Si, relative
molecular mass 274.39) were obtained from ABCR
GmbH (Karlsruhe, Germany), bovine serum albumin
(Cohn fraction V) from Roth (Karlsruhe, Germany),

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

, and then incu-
bated with the primary antibody PoAb-aSILIC (1 : 1000)
for 12 h at 4 °C. After three washes with NaCl ⁄
P
i
⁄ 1% BSA, sections were incubated with a 1 : 1000 dilu-
tion of the secondary antibody (1.4 nm nanogold anti-rab-
bit IgG) for 2 h. Sections were processed as described
previously [14]; enhancement of the immunocomplexes was
performed using silver [36]. The samples were examined by
transmission electron microscopy (TEM) using a Tecnai 12
microscope (FEI Electron Optics, Eindhoven, the Nether-
lands). As controls, pre-immune serum or PoAb-aSILIC,
adsorbed to recombinant silicatein [15], were used.
Silicatein
Recombinant silicatein-a was prepared in E. coli as
described previously [10,37]. The enzyme was stored at a
concentration of 2 mgÆmL
)1
in 20 mm MOPS [3-(N-mor-
pholino) propanesulfonic acid] buffer (pH 7.5, 50 mm
Na-acetate, 1 mm EDTA). This purified recombinant silica-
tein-a preparation was used to raise antibodies (PoAb-
aSILIC) [37]; it has been shown that such antibodies
reacted specifically with the purified silicatein [2,15,25].
MALDI analysis
Silicatein-a (4.5 l gÆ mL
)1
; 210 pmolÆmL
)1

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

¨
llheim, Germany), the reaction was studied at
20 °C within the absorbance range of 220–800 nm using
a Varian Cary 5G UV-Vis-NIR spectrophotometer
(Mulgrave, Australia). Typical reactions (3.5 mL assays)
contained 0.4 lgÆmL
)1
silicatein-a in 20 mm MOPS buffer;
BSA (50 lg) was used in controls. As substrate, concen-
trations of BAPD silane of 20–200 lm (from a 2 mm
stock solution in diethyl ether) were used. Where indi-
cated, sodium hexafluorosilicate (1 mm) was added to the
reaction mixture containing BAPD silane (20 or 200 lm)
and silicatein. In one series of experiments, E-64 (10 lm)
was added to the reaction mixture. In controls, silicatein
was replaced by BSA (4.5 lgÆmL
)1
assay). Kinetic deter-
minations were commenced 30 s after addition of the
components.
Acknowledgements
This work was supported by grants from the European
Commission, the Deutsche Forschungsgemeinschaft,
the Bundesministerium fu
¨
r Bildung und Forschung
Germany (Center of Excellence project BIO-
TECmarin), the National Natural Science Foundation
of China (grant number 50402023) and the Interna-
tional Human Frontier Science Program. S. E. W. is

the recipient of a Konrad Adenauer fellowship.
References
1 Perry CC (2003) Silicification: the process by which
organisms capture and mineralize silica. Rev Mineral
Geochem 54, 291–327.
2Mu
¨
ller WEG, Wang X, Belikov SI, Tremel W,
Schloßmacher U, Natoli A, Brandt D, Boreiko A, Tahir
MN, Mu
¨
ller IM et al. (2007) Formation of siliceous
spicules in demosponges: example Suberites domuncula.
In Handbook of Biomineralization, Vol. 1: Biological
Aspects and Structure Formation (Ba
¨
uerlein E, ed.),
pp. 59–82. Wiley-VCH, Weinheim, Germany.
3 Schro
¨
der HC, Brandt D, Schloßmacher U, Wang X,
Tahir MN, Tremel W, Belikov SI & Mu
¨
ller WEG
(2007) Enzymatic production of biosilica-glass using
enzymes from sponges: basic aspects and application in
nanobiotechnology (material sciences and medicine).
Naturwissenschaften 94, 339–359.
4 Morse DE (1999) Silicon biotechnology: harnessing bio-
logical silica production to make new materials. Trends

Biotechnol 17, 230–232.
5 Wang X & Wang Y (2006) An introduction to the
study on natural characteristics of sponge spicules and
bionic applications. Adv Earth Sci 21, 37–42.
6 Sto
¨
ber W, Fink A & Bohn E (1968) Controlled growth
of monodisperse silica spheres in the micron size range.
J Colloid Interface Sci 26, 62–69.
7 Morse DE (2000) Silicon biotechnology: proteins, genes
and molecular mechanisms controlling biosilica nano-
fabrication offer new routes to polysiloxane synthesis.
In Organosilicon Chemistry IV: from Molecules to Mate-
rials (Auner N & Weis J, eds), pp. 5–16. Wiley-VCH,
New York.
8 Weiner S & Dove PM (2003) An overview of biominer-
alization processes and the problem of the vital effect.
In Biomineralization. Reviews in Mineralogy & Geochem-
istry, Vol. 54 (Dove PM, DeYoreo JJ & Weiner S, eds),
pp. 1–29. Mineralogical Society of America and the
Geochemical Society, Washington, D.C.
9 Cha JN, Shimizu K, Zhou Y, Christianssen SC, Chm-
elka BF, Stucky GD & Morse DE (1999) Silicatein fila-
ments and subunits from a marine sponge direct the
polymerization of silica and silicones in vitro. Proc Natl
Acad Sci USA 96, 361–365.
10 Krasko A, Batel R, Schro
¨
der HC, Mu
¨

ller IM & Mu
¨
ller
WEG (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.
11 Zhou Y, Shimizu K, Cha JN, Stucky GD & Morse DE
(1999) Efficient catalysis of polysiloxane synthesis by
silicatein a requires specific hydroxy and imidazole
functionalities. Angew Chem Int Ed Engl 38, 780–782.
12 Shimizu K, Cha J, Stucky GD & Morse DE (1998)
Silicatein alpha: cathepsin L-like protein in sponge
biosilica. Proc Natl Acad Sci USA 95
, 6234–6238.
13 Tahir MN, The
´
ato P, Mu
¨
ller WEG, Schro
¨
der HC,
Janshoff A, Zhang J, Huth J & Tremel W (2004)
Monitoring the formation of biosilica catalysed by
histidin-tagged silicatein. Chem Commun 24, 2848–2849.
14 Mu
¨
ller WEG, Rothenberger M, Boreiko A, Tremel W,
Reiber A & Schro
¨
der HC (2005) Formation of siliceous

spicules in the marine demosponge Suberites domuncula.
Cell Tissue Res 321, 285–297.
15 Schro
¨
der HC, Boreiko A, Korzhev M, Tahir MN,
Tremel W, Eckert C, Ushijima H, Mu
¨
ller IM & Mu
¨
ller
WEG (2006) Co-expression and functional interaction
of silicatein with galectin: matrix-guided formation of
siliceous spicules in the marine demosponge Suberites
domuncula. J Biol Chem 281, 12001–12009.
16 Schro
¨
der HC, Perovic
´
-Ottstadt S, Rothenberger M,
Wiens M, Schwertner H, Batel R, Korzhev M, Mu
¨
ller
IM & Mu
¨
ller WEG (2004) Silica transport in the
demosponge Suberites domuncula: fluorescence emission
analysis using the PDMPO probe and cloning of a
potential transporter. Biochem J 381, 665–673.
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 369
17 Schro
¨
der HC, Natalio F, Shukoor I, Tremel W,
Schloßmacher U, Wang X & Mu
¨
ller WEG (2007)
Apposition of silica lamellae during growth of spicules
in the demosponge Suberites domuncula: biological ⁄ bio-
chemical studies and chemical ⁄ biomimetical confirma-
tion. J Struct Biol 159, 325–334.
18 Mu
¨
ller WEG, Wiens M, Batel R, Steffen R, Borojevic
R & Custodio RM (1999) Establishment of a primary
cell culture from a sponge: primmorphs from Suberites
domuncula. Mar Ecol Progr Ser 178, 205–219.
19 Eckert C, Schro
¨
der HC, Brandt D, Perovic-Ottstadt S
&Mu
¨
ller WEG (2006) A histochemical and electron
microscopic analysis of the spiculogenesis in the demo-
sponge Suberites domuncula. J Histochem Cytochem 54,
1031–1040.
20 Li C-W, Chu S & Lee M (1989) Characterizing the
silica deposition vesicle of diatoms. Protoplasma 151,
158–163.

21 Vrieling EG, Poort L, Beelen TPM & Gieskes WWC
(1999) Growth and silica content of the diatoms Tha-
lassiosira weissflogii and Navicula salinarum at different
salinities and enrichments with aluminium. Eur J Phycol
34, 307–316.
22 Ba
¨
uerlein E (2003) Biomineralization of unicellular
organisms: an unusual membrane biochemistry for the
production of inorganic nano- and microstructures.
Angew Chem Int Ed 42, 614–641.
23 Iler KK (1979) The Chemistry of Silica. Wiley & Sons,
New York.
24 Barrett AJ, Kembhavi AA, Brown MA, Kirschke H,
Knight CG, Tama M & Hanada K (1982) l-trans-
Epoxysuccinyl-leucylamido(4-guanidino)butane and its
analogues as inhibitors of cysteine proteinases
including cathepsins B, H and L. Biochem J 201,
189–198.
25 Mu
¨
ller WEG, Belikov SI, Tremel W, Perry CC, Gieskes
WWC, Boreiko A & Schro
¨
der HC (2006) Siliceous spic-
ules in marine demosponges (example Suberites domun-
cula). Micron 37, 107–120.
26 Pace CN, Vajdos F, Fee L, Grimsley G & Gray T
(1995) How to measure and predict the molar absorp-
tion coefficient of a protein. Protein Sci 4, 2411–2423.

27 Michaelis L & Menten M (1913) Die Kinetik der
Invertinwirkung. Biochem Z 49
, 333–369.
28 Nomura T, Fujishima A & Fujisawa Y (1996) Charac-
terization and crystallization of recombinant human
cathepsin L. Biochem Biophys Res Commun 228, 792–
796.
29 Mason RW (1986) Species variants of cathepsin L and
their immunological identification. Biochem J 240, 285–
288.
30 Osinga R, Tramper J & Wijffels RH (1999) Cultivation
of marine sponges. Marine Biotechnol 1, 509–532.
31 Maldonado M, Carmona MC, Velasquez Z, Puig A,
Cruzado A, Lopez A & Young CM (2005) Siliceous
sponges as a silicon sink: an overlooked aspect of ben-
thopelagic coupling in the marine silicon cycle. Limnol
Oceanogr 50, 799–809.
32 Tahir MN, The
´
ato P, Mu
¨
ller WEG, Schro
¨
der HC,
Boreiko A, Faiß S, Janshoff A, Huth J & Tremel W
(2005) Formation of layered titania and zirconia cataly-
sed by surface-bound silicatein. Chem Commun 44,
5533–5535.
33 Murr MM & Morse DE (2005) Fractal intermediates in
the self-assembly of silicatein filaments. Proc Natl Acad

Sci USA 102, 11657–11662.
34 Mu
¨
ller WEG, Schloßmacher U, Eckert C, Krasko A,
Boreiko A, Ushijima H, Wolf SE, Tremel W & Schro
¨
-
der HC (2007) Analysis of the axial filament in spicules
of the demosponge Geodia cydonium: different silicatein
composition in microscleres (asters) and megascleres
(oxeas and triaenes). Eur J Cell Biol 86, 473–487.
35 Mu
¨
ller WEG, Engel S, Wang Xi, Wolf SE, Tremel W,
Thakur NL, Krasko A, Divekar M & Schro
¨
der HC
(2007) Bioencapsulation of living bacteria (Escherichia
coli) with poly(silicate) after transformation with silica-
tein-a gene. Biomaterials, in press.
36 Danscher G (1981) Histochemical demonstration of
heavy metals. A revised version of the sulphide silver
method suitable for both light and electronmicroscopy.
Histochemistry 71, 1–16.
37 Mu
¨
ller WEG, Krasko A, Le Pennec G, Steffen R, Am-
mar MSA, Wiens M, Mu
¨
ller IM & Schro

¨
der HC (2003)
Molecular mechanism of spicule formation in the demo-
sponge Suberites domuncula: silicatein – collagen – myo-
trophin. Progr Mol Subcell Biol 33, 195–222.
38 Bahr U, Deppe A, Karas M & Hillekamp F (1988)
Mass spectrometry of synthetic polymers by UV-
matrix-assisted laser desorption ⁄ ionization. Anal Chem
64, 2866–2869.
39 Bierbaum V (2001) Frontiers in mass spectrometry.
Chem Rev 101, 209–606.
40 Chatterjee S, Pramanik S & Bhattacharya SC (2005)
Spectroscopic study of some photographic developing
agents in reverse micelles of AOT in heptane. J Mol
Liquids 116, 131–137.
Silicatein comprises dual enzymatic activities W. E. G. Mu
¨
ller et al.
370 FEBS Journal 275 (2008) 362–370 ª 2007 The Authors Journal compilation ª 2007 FEBS