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
Trang 1and silicasomes of demosponges comprises dual
enzymatic activities (silica polymerase and silica esterase) Werner E G Mu¨ller1, Ute Schloßmacher1, Xiaohong Wang2, Alexandra Boreiko1, David Brandt1, Stephan E Wolf3, Wolfgang Tremel3and Heinz C Schro¨der1
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: wmueller@uni-mainz.de
Website: http://www.biotecmarin.de/
(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.
Trang 2precursors, 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+⁄ HCO3)[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 silsil-icatein 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
H2N
O
Si O
NH2
CH3
CH 3
+
+ H
H2N
OH HO
CH3
CH3
+
+ –
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.
Trang 3studies, 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
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).
Trang 4not 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 20–25C; the temperature coefficient (Q10)
decreases by 2.5-fold above 25C and increases by
2.9-fold below 25C Silica esterase activity was
rou-tinely determined at 20C 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 (Km) was determined using this value [27],
and was calculated to be 22.7 lm In comparison, the Kmvalue 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 (%) 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)1silicatein (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.
Trang 52–60 min incubation periods (20C) 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
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.
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.
Trang 6where the silicic acid content is high,
self-polymeriza-tion or self-condensaself-polymeriza-tion 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 anacata-bolic reaction Enzymatic parameters
of the silica esterase activity were determined The
Michaelis constant (Km22.7 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.
Trang 7render 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 cataana-bolic 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 (C4H12O2Si, relative molecular
mass 120.22) and BAPD silane (C14H18N2O2Si, 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 17C for more than
5 months From these animals primmorphs, a 3D cell system
[10,18,19] was prepared Primmorphs were kept at 17C in
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⁄ Pi, and then incu-bated with the primary antibody PoAb-aSILIC (1 : 1000) for 12 h at 4C After three washes with NaCl⁄
Pi⁄ 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 lgÆmL)1; 210 pmolÆmL)1using 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 20C, 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
Trang 8of 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
20C 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
2 Mu¨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
Trang 917 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 bio-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 weissflogiiand 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