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J. Biol. Chem., Vol. 281, Issue 17, 12001-12009, April 28, 2006
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1
From the
Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität Mainz, Duesbergweg 6, D-55099 Mainz, Germany, Institut für Anorganische Chemie und Analytische Chemie, Universität Mainz, Duesbergweg 10-14, D-55099 Mainz, Germany, ¶Museum für Naturkunde, Universität Berlin, Institut für Systematische Zoologie, Invalidenstrasse 43, D-10115 Berlin, Germany, and ||Department of Developmental Medical Sciences, Institute of International Health, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received for publication, November 28, 2005 , and in revised form, February 16, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Major progress in understanding the biochemical mechanism of spicule formation in Demospongiae came from the discovery that silica deposition in T. aurantium is catalyzed by the enzyme silicatein (10, 11); silicatein exists in the axial filament around which the first layer(s) of biosilica are formed. The axial filament had been isolated by hydrogen fluoride (HF),2 which dissolves the biosilica of the spicules. During this procedure O-glycosidic and phosphate ester bonds in proteins are split off. As reported for the silaffins in diatoms, this treatment results in a loss of phosphate groups of the structural molecules and also a modification of their functions (12, 13). Therefore, the (apparently pure) preparation of the axial filaments (composed of silicatein) from T. aurantium studied hitherto (10, 14) may not represent native silicatein. Furthermore, the reported presence of three different but related silicateins (silicatein-
,-
, and -
) in the axial filaments of spicules had not been substantiated on molecular level. Until now, only genes encoding silicateins and could be isolated from T. aurantium (10, 14) or other demosponges, e.g. S. domuncula (5, 15); it may however be possible that the transcript level of the putative silicatein- is too low. Very recently it could be proven that axial filaments of S. domuncula contain, in addition to silicatein, other proteins such as selenoprotein M or the spiculeassociated protein (9). A thorough cytological and electron microscopic study revealed that the axial canal, which harbors the axial filament, is filled with a non-homogenous material (8). It has been reported that the silicateins in S. domuncula are phosphorylated proteins, which are HF-labile. Only in this state are they able to oligomerize (form silicatein dimers and trimers) (8).
Even though the enzymatic activity of silicatein isolated from axial filaments by HF treatment, as well as the enzymatic activity of recombinant silicatein, can be demonstrated to a limited extent, which likely does not reflect the physiological in vivo activity seen in the animal (14, 15). Under optimal conditions, spicules have an astonishing growth rate of 5 µm/h (16), which certainly cannot be attributed to silicatein or recombinant silicatein alone (14). Because silicatein might be technically used to form silica nanoparticles and nanofibers under ambient (low temperature and low pressure) conditions (17-19), a knowledge on the protein composition of the axial filament and of the interactions of these proteins in their native states is also of interest in nanobiotechnology and nanomedicine. Silicatein, when chemically bound to metal surfaces, e.g. gold, retains its biocatalytic activity (18). Even more impressive are studies demonstrating that immobilized silicatein also allows the biosynthesis of titania and zirconia (19).
Here we present a comprehensive study on the role of silicatein from S. domuncula in silica/spicule formation. The question by which molecules silica deposition in sponge spicules is biologically controlled is addressed and partially answered. Evidence is presented that a galectin builds, in concert with collagen, a proteinaceous framework into which silica is deposited. These studies took advantage of (a) the primmorph culture system, a special form of three-dimensional cell aggregates that comprise proliferating and differentiating sponge cells (20), and (b) antibodies against silicatein.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Sponges and Primmorphs—Specimens of the marine sponge S. domuncula (Porifera, Demospongiae, Hadromerida) were collected in the northern Adriatic Sea near Rovinj, Croatia, and then kept in aquaria in Mainz, Germany, at a temperature of 17 °C for more than 5 months.
Primmorphs, the in vitro cell culture system, were prepared as described (15). These three-dimensional cell aggregates were used 5 days after their formation. Primmorphs were kept in natural seawater (containing 4-6 µM silicate) supplemented with 1% RPMI 1640 medium in the absence or presence of 60 µM sodium silicate (as sodium metasilicate) for up to 5 days. Extracts from primmorphs or from sponge tissue were prepared by homogenization in lysis buffer (1x Trisbuffered saline, pH 7.5, 1 mM EDTA, 1% Nonidet-P40, and protease inhibitor mixture (1 tablet/10 ml)) and centrifuged, and the supernatants were subjected to further analysis.
Isolation of the Proteins from Spicules—In earlier studies isolated spicules had been treated with HF to obtain the axial filament (10). In the present study this harsh procedure was replaced by treatment of the spicules with lysis buffer supplemented with 4 M urea. At first the spicules were isolated from tissue, collected (8), and then pulverized thoroughly in a mortar. The lysis-urea buffer was added, and the suspension was stirred for 1 h at 4 °C and subsequently centrifuged at 10,000 x g for 5 min at 4 °C. The supernatant was used for further analysis by SDS-PAGE and Western blotting.
Differential Display and Cloning of Galectin and Collagen cDNAs—The technique of differential display of transcripts was performed as described previously (21). RNA (1 µg) from untreated primmorphs (control, 0 days) and primmorphs treated for 2 days with 60 µM sodium silicate, was reverse-transcribed using the T11CC oligonucleotide as 3' primer. The resulting cDNA was added to the PCR using the arbitrary primer GTGATCGCAG and the T11CC primer in the assay together with [-32P]dATP. Among the dominantly expressed genes, collagen (SDCOLL3) and galectin (SDGALEC2) transcripts as well as transcripts of the silicatein- gene (SDSILCAa) were identified. The 
300-380-nt-long sequences were completed by 5'-RACE with vector primers of the cDNA library from S. domuncula (22). The complete clone for collagen-3 (SDCOLL3) comprises 1302 nts, and that for galectin-2 (SDGA-LEC2) 955 nts.
Northern Blotting—RNA was extracted from liquid nitrogen-pulverized sponge tissue with TRIzol reagent. Total RNA (5 µg) was electrophoresed through a 1% formaldehyde/agarose gel and blotted onto Hybond N+ membrane following the manufacturer's instructions (23). Hybridization was performed with the following probes (complete cDNAs from S. domuncula): silicatein- (SDSILCAa (GenBankTM accession number CAC03737
[GenBank]
.1)), collagen (SDCOLL3), and galectin-2 (SDGALEC2), as well as the housekeeping cDNA for -tubulin (SDTUB (AJ5508069)). Northern blot signals were quantified by the chemiluminescence procedure (24) with CDP-Star as substrate. The screen was scanned with the GS-525 molecular imager (Bio-Rad).
Preparation of Recombinant S. domuncula Galectin-2 and Antibodies—The complete open reading frame of SDGALEC2 was expressed in Escherichia coli using the glutathione S-transferase (GST) fusion system in the pET-41a(+) vector as described (25, 26). The GST fusion protein (65 kDa) was purified by GST-Bind Resign affinity chromatography. The fusion protein was then cleaved with enterokinase to separate the N-terminal GST tag and His tag as well as the S-tag from the recombinant galectin (rGALEC2_SUBDO). This resulting galectin-2 carried at its C terminus a small His tag. Gel electrophoresis of protein extracts was performed in polyacrylamide gels (12% gels) containing 0.1% SDS-PAGE according to Laemmli (27).
Polyclonal antibodies (pAb) were raised against the purified, recombinant galectin-2 (rGALEC2_SUBDO) in female rabbits (New Zealand White) as described (28). 10 µg of recombinant protein per injection was dissolved in phosphate-buffered saline (PBS). After three boosts the serum was collected; the pAb against galectin-2 was termed pAb-aGA-LEC2. The titer of the antibodies was 1:3,000. In the controls, adsorbed pAb-aGALEC2 (100 µl of antibodies were incubated with 20 µg of rGALEC2_SUBDO) was used. Likewise, pAbs against silicatein (pAb-aSILIC) were raised against axial filaments from spicules, containing mainly silicatein, as described earlier (8).
Co-immunoprecipitation and Western Blot Analysis—For co-immunoprecipitation the described protocol (29, 30) was applied. Primmorph samples (200 mg each) were exposed in seawater to 60 µM sodium silicate for 0-5 days. Then they were shock-frozen in liquid nitrogen, subjected to twice the volume of lysis buffer (1x Tris-buffered saline, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.1% Nonidet-P40, 10 mM NaF, protease inhibitor mixture (1 tablet/10 ml), and 1 mM sodium orthovanadate), homogenized, incubated for 60 min at 37 °C, and then brought to 4 °C. Finally the samples were subjected to co-immunoprecipitation or Western blot analyses. For each immunoprecipitation a 100-µl aliquot of the lysate was incubated with 2 µl of undiluted serum (preimmune or immune pAb (pAb-aGALEC2 or pAb-aSILIC)) for 1 h (4 °C). Protein A/G-agarose beads (10 µl) were added and after 3 h were washed with lysis buffer and resuspended in SDS-PAGE sample buffer. SDS-PAGE-fractionated proteins were analyzed on Western blots. The membranes were probed with the pAb (pAb-aGALEC2 or pAb-aSILIC) and detected by anti-rabbit IgG (peroxidase-coupled) using the BM chemiluminescence blotting substrate kit. In control experiments 100 µl of the respective antibodies pAb-aGALEC2 and pAb-aSILIC were adsorbed with 20 µg of the recombinant proteins (rGALEC2_SUBDO or rSILIC_SUBDO) during an incubation period of 30 min (4 °C) prior to their use.
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Silicatein Enzyme Assay—For these studies recombinant silicatein was used; preparation (17, 31) and reaction conditions (14, 31) were as described previously. In a total volume of 500 µl, 100 µg of recombinant silicatein was added to the 25 mM Tris-HCl buffer, pH 7.2 (10 mM DL-dithiothreitol, 100 mM NaCl, 1 µM ZnCl2,5 µM Fe3+ citrate, 5 mM CaCl2,5 mM MgCl2). 1 mM tetraethoxysilane was used as substrate. Reaction proceeded at 22 °C for 0-120 min and was terminated by centrifugation (20,000 x g; 4 °C, 15 min). The sediment was hydrolyzed by NaOH, and the released silicic acid was determined as described (14). Where specified, the standard assays were performed in the absence of silicatein, or they contained 100 µg of galectin-2 (rGALEC2_SUBDO) instead of silicatein or 100 µg of galectin-2 together with 100 µg of silicatein.
Aggregate Formation of Recombinant Galectin-2—To visualize aggregate formation of rGALEC2_SUBDO in vitro recombinant protein (0.1 mg/ml) was dissolved in PBS supplemented with 5 mM CaCl2. After standing on ice for 2-30 min, samples were dropped onto a carboncoated copper grid. After drying, the film was stained with platinum and examined by electron microscope (32).
Electron Microscopy—For transmission microscopic analysis, sponge primmorphs or tissue samples were cut into pieces (1 mm3), incubated in phosphate buffer, and then washed in phosphate-buffered saline, pH 7.4, at room temperature as described (5, 8). After the samples were treated with 1.25% NaHCO3,2%OsO4, and 1% NaCl, they were dehydrated with ethanol. The dried samples were fixed in propylene oxide/araldite, covered with pure araldite, and hardened at 60 °C for 2 days prior to cutting to 60-nm ultrathin slices (Ultracut S; Leica, Wetzlar, Germany). The samples were transferred onto coated copper grids and analyzed with a Tecnai 12 microscope (FEI Electron Optics, Eindhoven, Netherlands).
Electron immunogold labeling was performed with primmorph/tissue samples treated in glutaraldehyde/paraformaldehyde buffered in phosphate buffer, pH 7.4 (5, 8). The material was dehydrated in ethanol and embedded in LR-White resin. 60-nm thick slices were cut and blocked with bovine serum albumin in PBS and then incubated with the primary antibody pAb-aSILIC (1:1,000) for 12 h at 4 °C. In controls, preimmune serum were used. After three washes with PBS, 1% BSA, sections were incubated with a 1:100 dilution of the secondary antibody (1.4-nm nanogold anti-rabbit IgG; diluted 1:200) for 2 h. Sections were rinsed in PBS, treated with glutaraldehyde/PBS, washed, and dried. Subsequently, enhancing of the immunocomplexes was performed with silver as described (33). The samples were examined as described above with the Tecnai 12 microscope.
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Immunohistology—Tissue samples were embedded in paraformaldehyde. After dehydration the samples were embedded in Technovit 8100, and 8-µm-thick sections were cut (8). The sponge tissue was not treated with HF/NaF solution to ensure that siliceous spicules were not removed. The sections were incubated with the anti-galectin-2 antibody (pAb-aGALEC2); rhodamine-conjugated goat anti-rabbit immunoglobulin was used as a secondary antibody. The preimmune rabbit serum was used as a control. Further details were given recently (9). The immunofluorescence analysis was done with an Olympus AHBT3 light microscope together with an AH3-RFC reflected light fluorescence attachment at an emission wavelength of 546 nm (filter G).
Further Analytical Methods—For the quantification of protein, the Bradford method (34) (Roti-Quant solution-Roth) was used. The N-terminal part of the mature galectin-2 protein was determined as described earlier (35). After size separation by SDS-gel electrophoresis the N terminus of galectin-2 was determined by direct protein sequencing.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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gene (SDSILCAa; CAC03737
[GenBank]
.1), two major genes were identified that are expressed in primmorphs in response to sodium silicate treatment. Analysis revealed that one of these transcript codes was for a galectin and the other one for a collagen protein; both cDNAs were completed. Galectin—The 955-nt-long cDNA for galectin (SDGALEC2) comprises one open reading frame between nt70/72 and nt949-951[stop]. The deduced protein (GALEC2a_SUBDO) of 293 aa (Fig. 1) has a predicted size of 33,091 Da. Galectin-2 shows two galactoside-binding lectin domains (Motif Scan), which span aa1 to aa117 (domain 1) and aa118 to aa293 (domain 2). A highly hydrophobic region in galectin-2, predicted according the described algorithm (36), is found between aa275 and aa293. The mature protein, isolated after separation on an SDS-gel, begins at aa10 of the predicted protein. Highest sequence similarity was found in the data base with S. domuncula galectin-1 with an "expect value" (E) (26) of 2e-24 and then with nematode galectin (Hemonchus contortus, AAD11972 [GenBank] .1; E = 9e-18).
Collagen—The cDNA of the fibrillar collagen gene (SDCOLL3) is 1302 nts long. The open reading frame, from nt5-7 to nt1076-1078[stop] encodes the deduced collagen protein, termed COLL3_SUBDO (Fig. 2A), with a predicted size of 357 amino acids and a molecular mass of 36,665 Da. Three segments can be distinguished: (i) the N-terminal non-collagen region (aa1 to aa26); (ii) the region comprising the characteristic G-X-Y collagen triple helix repeats (in COLL3_SUBDO 37 triples; aa27-136); and finally (iii) the non-collagen C-terminal part (aa137-357). This arrangement has been described also for the other collagen proteins from S. domuncula (15, 37). Data bank searches revealed that COLL3_SUBDO shares highest sequence similarity to the fibrillar collagen from Haliotis discus (BAA75668 [GenBank] (38)) with E = 8e-39 and from Xenopus laevis (AAH49829 [GenBank] (39)) with E = 2e-34.
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, Galectin, and Collagen (SDSILCAa, 1.4 kb transcript size), galectin-2 (SDGALEC2, 1.2 kb), and collagen-3 (SDCOLL3, 1.1 kb) were up-regulated, primmorphs were incubated with sodium silicate for at least 2 days. The steady-state expressions of the genes were determined semiquantitatively and showed only very low levels of these transcripts at day 0. A strong increase could be measured for the transcripts after 2 or 5 days (Fig. 3). Control experiments with the housekeeping gene -tubulin (transcript size of 1.4 kb) showed that the same amounts of RNA were loaded onto the gels (Fig. 3).
Recombinant Galectin, Expression, and Aggregate Formation
Galectin-2 was expressed as a fusion protein with a GST tag, His tag, S-tag, and again a His tag. This protein was analyzed by SDS-PAGE and found to display the expected size of 69 kDa (37 kDa for galectin-2 and 32 kDa for the tags; Fig. 4A, lane a). Antibodies (pAb-aGALEC2) raised against recombinant galectin-2 recognized this protein on a Western blot (Fig. 4A, lane b). The fusion protein was subsequently cleaved with enterokinase to remove the N-terminal Tag region, and the final recombinant galectin (rGALEC2_SUBDO) with its C-terminal His tag had the expected size of 34 kDa (data not shown). The antibodies were also used to identify the native protein in the crude sponge extract (Fig. 4B, lane a). In this preparation pAb-aGALEC2 recognized the 35-kDa native galectin-2 (Fig. 4B, lane b). To demonstrate the specificity of pAb-aGA-LEC2, the antibodies were adsorbed with rGALEC2_SUBDO prior to the application in the Western blot. The adsorbed antibodies did not show any visible immunocomplexes (Fig. 4B, lane c).
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Interaction of Silicatein- with Galectin-2 Proteins
The experiments were performed with primmorphs. They remained for 0-5 days in seawater supplemented with 60 µM sodium silicate. Then lysates were prepared, subjected to PAGE (Fig. 6), and analyzed for the presence of a complex between galectin-2 and silicatein, applying the co-immunoprecipitation technique. pAb-aGALEC2 and pAb-aSILIC were used as tools to identify the two proteins, as described under "Experimental Procedures."
In one series of experiments, the primmorph lysate was reacted with pAb-aGALEC2 and in a second series with pAb-aSILIC. After immunoprecipitation, the proteins were size-separated and probed for Western blots with antibody combinations as follows. The proteins precipitated with pAb-aGALEC2 were reacted with pAb-aSILIC (Fig. 6a), or the pAb-aSILIC precipitated material was probed with pAb-aGALEC2 (Fig. 6b). It can be seen that pAb-aGALEC2 also precipitated, in addition to galectin-2 (Fig. 4), the 24-kDa silicatein protein (Fig. 6a). Silicatein was identified by pAb-aSILIC. The immunoreaction was strong in lysates prepared from primmorphs that had been incubated with silicate for 2 or 5 days; at time zero almost no immunocomplexes could be seen. In a control experiment the specificity of the reaction was demonstrated by applying adsorbed pAb-aSILIC; this preparation did not react with any lysate protein (not shown). In the reverse series, lysates were treated with pAb-aSILIC (Fig. 6b). After precipitation and size separation the blots were incubated with pAb-aGALEC2, and the immunocomplexes were visualized with labeled anti-rabbit IgG. At time zero, only a faint band, corresponding to the 35-kDa galectin-2 was detected. After incubation for 2 or 5 days the amount of 35-kDa galectin-2 strongly increased. Again the specificity of the pAb-aGALEC2 antibodies was verified after adsorption; no strong reactions on the blot could be seen (not shown). Further controls were performed with preimmune serum, which revealed no signals on the Western blots (not shown). From these data we conclude that galectin-2 interacts with silicatein in lysates from primmorphs that have been treated for at least 2 days with silicate.
In a further series of experiments the site of interaction between galectin-2 and silicatein was investigated. As outlined above, galectin-2 carries at the C terminus a highly hydrophobic stretch (aa275-293), which can be considered as one segment involved in such an interaction. To test this assumption competition experiments were performed with the synthetic oligopeptide. The peptide was added prior to the incubation step at 37 °C for 60 min. These samples were processed with the two antibody combinations: immunoprecipitation with pAb-aGALEC2 and subsequent Western blotting with pAb-aSILIC (Fig. 6a); or immunoprecipitation with pAb-aSILIC followed by Western blotting with pAb-aGALEC2 (Fig. 6b). The signals visible in those Western blots were considerably lower than those seen with extracts not supplemented with the oligopeptide, e.g. the signal (extracts from primmorph after 5 days in silicate) of the co-precipitation experiment pAb-aGALEC2/pAb-aSILIC was reduced to less than 30%, close to that seen in extracts from primmorphs not treated with silicate. These findings support the presumption that galectin-2 binds to silicatein, very likely through its C terminus.
Effect of Galectin-2 on the Silicatein Reaction
In earlier studies it was shown that silicatein mediates the formation of silica in solution (14, 15). In the present study we determined the effect of galectin-2 on the extent of silica formation in assays containing 5 mM CaCl2 in addition to silicatein and its substrate, tetraethoxysilane (1 mM). As summarized in Fig. 7A, in the absence of silicatein, the detectable amount of silica was low (19 ± 4 ng/assay during the 120-min incubation period). The addition of silicatein (100 µg/assay) resulted in silica production; after 120 min 47 ± 12 ng/assay was formed. The addition of galectin-2 (100 µg) alone also resulted in a significant but smaller increase in the amount of silica (23 ± 5 ng/assay). However, if silicatein (100 µg) was added together with galectin-2 (100 µg), a strong increase in silica formation (115 ± 20 ng/assay) was measured.
In a parallel series, the synthetic oligopeptide was added to the silicatein-containing assay in the presence and absence of galectin. Again it is seen that in the presence of silicatein (42 ± 9 ng/assay; 120-min incubation period) or galectin (16 ± 5 ng) a significant amount of silica was formed (Fig. 7B). However, if galectin-2 was added together with the oligopeptide and silicatein (53 ± 16 ng), no significant stimulatory effect of galectin was measured.
S. domuncula Collagen
Collagen fibrils were isolated from fresh tissue and analyzed electron microscopically (Fig. 2B). After isolation and subsequent staining/shadowing, the banding pattern of the collagen fibrils became visible; the diameter of the fibrils is 15 nm, and the simplified arrangement of bandings has a periodicity of about 20 nm.
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Immunofluorescence Analysis; Identification of Galectin-2 in Spicules
Cryosections were prepared from sponge tissue and reacted with pAb-aGALEC2, and the immunocomplexes were visualized with Cy3-labeled IgG. As shown in Fig. 9A, a bright staining on the sections is seen on the surface of the spicules and also in their axial canal/axial filaments; this localization becomes more obvious at a higher magnification (Fig. 9C). The corresponding Nomarsky interference image shows that spicules are embedded in the bulky mesohyl (Fig. 9D). A control series shows that the preimmune serum did not react with structures on the slides (Fig. 9B).
Embedding of Spicules in the Mesohyl of the Sponge
The initial synthesis of the spicules occurs intracellularly in the sclerocytes (8). After reaching a critical length of 5-10 µm, the spicules are extruded into the bulky extracellular matrix within the mesohyl. Transmission microscopic analysis shows that in this space the spicules are surrounded by cells that never come closer to the spicules than 10 nm. Some of these 10-nm spaces contain string- and net-like structures. In the mature state the axial filament (Fig. 10A), with a diameter of 300-600 nm, is compact. In growing spicules the axial canal is filled with the axial filament, which is surrounded by membranous structures and a matrix consisting of bundles and string- and net-like structures of variable size between 0.5 and 4 nm (Fig. 10B). The extraspicular space likewise contains those small string- and net-like structures as well as solid rods. These rods are rarely curved and have diameters between 10 and 14 nm (Fig. 10C). At higher magnification they show a banding pattern, indicating that they are collagen fibers (Fig. 10D). In contrast to the rods, the string- and net-like structures are often, especially in the axial canal, closely associated with the silica shell of the spicules (Fig. 10E); collagen fibers are never found tightly associated with the silica shell of the spicules. However, frequently the collagen fibers existing in the extraspicular space are intimately associated with the 0.5-4-nm-thick string-/net-like structures (Fig. 10F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The second molecule expressed in S. domuncula after exposure to silicate is collagen. Until now, only genes encoding non-fibrillar collagens had been described in sponges, e.g. as for Ephydatia muelleri (40) or S. domuncula (37). The newly discovered S. domuncula collagen displays the typical structure for fibrillar collagens: the N-peptide, the triple helix segment, and the C-peptide. Fibril-forming collagens are characterized by the ability to assemble into highly oriented supramolecular aggregates with a characteristic suprastructure, the quarter-staggered fibril array (see Ref. 44). Electron microscopic analysis of the S. domuncula collagen molecules revealed a pronounced banding pattern. Those collagens in sponges had been identified earlier from electron microscopic analyses (45). It had been proposed that fibrillar collagens control spicule formation (46). In addition, collagen had been implicated in the directional movement of cells and transport of spicules (46, 47). The collagen fibers have now been demonstrated in S. domuncula electron microscopically, and their role in the morphogenesis of the spicules has become likely, as outlined below.
As a further class of structural proteins occurring in Demospongiae, spongins have been described (reviewed in Ref. 48). This type of proteins was first reported by Croockewit (49); it is rich in glycine (50) but is distinguishable from collagen by the absence of the banding pattern (48). However, until now no genes coding for such (glyco)proteins have been discovered. This is surprising, because at present close to 20,000 ESTs (expressed sequence tags) have already been annotated from S. domuncula to our sponge data base; a domain search in this base gave no indication of the existence of an additional structural/fibrillar protein other than collagen.
The functional interaction between galectin-2 and silicatein has been demonstrated by co-immunoprecipitation studies using antibodies raised against these proteins. These experiments revealed that silicatein (24 kDa) co-precipitates with galectin-2 (35 kDa) in extracts obtained from primmorphs, which had been exposed to 60 µM silicate for more than 2 days. This interaction was abolished in the presence of a peptide corresponding to the hydrophobic C terminus of galectin-2. The assumption that this interaction could alter the biocatalytic activity of silicatein was strongly supported by enzymatic studies. In the presence of galectin-2 the amount of biosilica formed increased by more than 2-fold as judged by analytical determination, suggesting at least an additive effect. This substantial stimulatory effect of galectin-2 on silicateinmediated biosilica formation could be suppressed by the presence of the synthetic peptide.
For a further proof that in the native state galectin-2 and silicatein co-localize, a new extraction procedure was introduced. SDS-PAGE analysis of the axial filaments obtained by this method revealed that they comprise, in addition to the 23-25-kDa silicateins, further distinct proteins with sizes of 35 and >250 kDa. The 35-kDa protein was identified as galectin-2 in Western blots; the >250-kDa polypeptide remains unidentified. The calculated size of the processed form of galectin-2, without the short N terminus, is 32 kDa. This difference to the 35-kDa form is very likely due to the glycosylation of galectin-2; the evidence that galectin-2 is a glycoprotein came from staining studies of polyacrylamide gels with periodic acid-Schiff base reagent (not shown). The existence of galectin in the axial canal and also on the surface of the spicules has been additionally and unequivocally demonstrated here by immunofluorescence. The fact that in S. domuncula the axial filament does not contain only one type of protein (silicatein), but also further proteins, had been demonstrated previously (8, 9).
The finding that the filament present in the axial canal is not homogenous (does not consist only of silicatein) was supported by electron microscopic analysis. Sections through primmorphs revealed that the axial canal contains not only electron-dense material, very likely silicatein, but also membraneous structures, 0.5-4-nm-thick string-like structures and less electron-dense material. The thin strings exist in close association with the silicate shell of the spicules, both inside and outside of the spicules. They are markedly distinguished from the 10- and 14-nm large solid rods, which are striated. Although the rods represent collagen, the thin string- and net-like structures very likely represent self-associated galectin. This assumption was supported experimentally by immunogold electron microscopy investigations. The images show that the silicatein molecules are closely linked to the string- and net-like structures. Only rarely is labeled silicatein, bound to the string-/net-like structures, associated with collagen fibers.
A further protein in the axial filaments is galectin, which undergoes self-association in the presence of Ca2+. In vitro assembly experiments followed by electron microscopic analysis showed that the self-associated proteins are positioned along an axis and not irregularly distributed. Also the next step, which is the formation of the galectin-2-containing string- and net-like structures, could be documented electron microscopically. Immunogold electron microscopic images led to the assumption that collagen fibers mediate the functional orientation of silicatein-galectin-2 complexes by arranging them concentrically around the longer axis of a growing spicule. Then, silicatein mediates silica deposition onto the surface of the existing silica layer. Because the surface of a new siliceous spicule is also covered with silicatein, the appositional growth/thickening of a spicule proceeds from two directions (centrifugal and centripetal) (8, 9); a scheme that outlines the probable growth of the spicules is given in Fig. 12. The lamellar composition of siliceous spicules has not only been demonstrated for Hexactinellida (51) but also for Demospongiae (5).
These data will contribute to the potential application of silicatein in bio(nano)technology. In view of the finding that galectin is used by silicatein as a matrix for an extensive synthesis and deposition of biosilica, embedding of the enzyme in suitable organic polymers/aggregates that support the silicatein-mediated silica formation at ambient temperature will be the prime application for the enzyme.
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FOOTNOTES |
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* This work was supported by grants from the European Commission (SILIBIOTEC), theDeutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung Germany, and the International Human Frontier Science Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
1 To whom correspondence should be addressed: Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, 55099 Mainz, Germany. Tel.: 49-6131-39-25910; Fax: 49-6131-39-25243. E-mail: wmueller{at}uni-mainz.de.
2 The abbreviations used are: HF, hydrogen fluoride; CDP-Star, cytidine 5'-diphosphate(disodium 2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)-tricyclo[3.3.1.13,7]-decan}-4-yl)phenyl phosphate); RACE, rapid amplification of cDNA ends; pAb, polyclonal antibody; pAb-aSILIC, pAb against silicatein; pAb-aGALEC2, pAb against GALEC2; nt, nucleotide; aa, amino acid; GST, glutathione S-transferase; PBS, phosphate-buffered saline.
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