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