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Involvement of aquaporin channels in water extrusion from biosilica during maturation of sponge siliceous spicules.

Abstract. Aquaporins are a family of small, pore-forming, integral cell membrane proteins. This ancient protein family functions as water channels and is found in all kingdoms (including archaea, eubacteria, fungi, plants, and animals). We discovered that in sponges aquaporin plays a novel role during the maturation of spicules, their skeletal elements. Spicules are synthesized enzymatically via silicatein following a polycondensation reaction. During this process, a 1:1 stoichiometric release of water per one Si-O-Si bond formed is produced. The product of silicatein, biosilica, is a fluffy, soft material that must be hardened in order to function as a solid rod. Using the model of the demosponge species Suberites domuncula Olivi, 1792, which expresses aquaporin, cDNA was cloned and the protein was heterologously expressed. The sponge aquaporin is grouped with the type 8 aquaporins. The function of the sponge aquaporin can be blocked by Mn-sulfate (MnS[O.sub.4]) and mercury chloride (Hg[Cl.sub.2]). Microscopic and functional studies suggest that aquaporin is involved in removal of the reaction water at the site where siliceous spicules are formed. Another molecule that is likely to be involved in biosilica maturation is the mucin/nidogen-like polypeptide. cDNA has also been cloned from S. domuncula. Experimental studies suggest that water extrusion/suctioning from biosilica after enzymatic synthesis during spicule formation involves both aquaporin-mediated water channeling and "polymerization-induced phase separation" facilitated by the mucin/nidogen-like polypeptide.

Introduction

Every organism has its specific intracellular physical and (bio)chemical milieu, which includes intracellular water content (Ho, 2006). Single cells are especially dependent on mechanisms that permit adaptation to external osmotic stress; these cells are directly exposed to the external aqueous environment. In contrast, Metazoa, including sponges, have a built-in organismic system that regulates osmosis. Deviation from optimal conditions results in suboptimal functions, causing cellular stress. These variations are subject to the balancing sensor that readjusts the regulatory circuit. Metazoa have acquired a sophisticated, homeostatically regulated internal environment; for example, a change in the homeostatic balance of the kidney by more than 2%-3% results in pathophysiological symptoms (Guyton and Hall, 1996). It is important to highlight that the components within the cytoplasm are not freely diffusible, but are "embedded" into a gel-like scaffold; alterations of the sol-gel cycles importantly influence the metabolic cycles (Woolf and Hameroff, 2001).

One critical parameter controlling intra-organismic aqueous homeostasis is water movement. Osmosis through the lipid bilayer might be a contributing factor via passive cotransporters with other ions and solutes (Loo et al., 1996). The main route of water exchange is via aquaporin(s) channels, which display an exquisite specificity for water; it is a fast-reacting system that allows water to import and export to the cells, and it also contributes to transcellular water flow (Carlsson et al., 1996). Since the discovery of the first aquaporin by Peter Agre in 1988 (Denker et al., 1988), an additional 13 types of human aquaporins have been identified (Murata et al., 2000). Aquaporins share a common structural architecture: the functional aquaporin unit is a homotetramer (Tornroth-Horsefield et al., 2010), and each monomer is composed of six transmembrane helices connected by alternating intracellular and extracellular loops (Kosinska Eriksson et al., 2013). The specificity of the channel for water is based on direct hydrogen bonding between a single water molecule and the aquaporin family's signature Asn-Pro-Ala motif within the narrowest part of the pore. In addition, interactions with the aromatic/arginine constriction sites are control sites within the pore. Aquaporins are expressed in a wide range of tissues and are often located within a certain region of the cell (Day et al., 2014). In turn, those channels establish a network of water flow through the tissues, triggering cell volume regulation mechanisms. Transport through the aquaporins utilizes a common passive mechanism (Conner et al., 2013).

We recently discovered that in sponges an aquaporin channel exists that is crucial to the formation of spicules. These data are reviewed.

Biosilica: Skeletal Scaffold Material for Spicules

Sponges have two inorganic scaffold sources that form their skeletons: first, biosilica and second, biocalcite. The major inorganic material that forms the skeletal elements in Demospongiae (Fig. 1A and B) and in Hexactinellida (Fig. 1C) is amorphous biosilica. Spicules occur in the cytoplasm (Levi, 1963; reviewed in Uriz et al., 2003) and in the nucleus (as silicate crystals) of some sponge cells; the function of spicules in the nucleus is unknown (Imsiecke et al., 1995). Solid evidence shows that spicule formation is mediated by the enzyme silicatein (reviewed in Wang et al., 2012a; Muller et al., 2013). cDNA as well as the gene encoding this enzyme were first cloned from Tethya aurantium (Cha et al., 1999; Murr and Morse, 2005) and Suberites domuncula (Krasko et al., 2000; Muller et al., 2003). The recombinant silicatein catalyzed the synthesis of amorphous silica using tetraethoxysilane as substrate. In addition to silicate polymerizing activity, silicatein comprises proteolytic (cathepsin-like) activity.

The biosilica matrix of siliceous spicules is formed of amorphous hydrated silica (Si[O.sub.2] * n[H.sub.2]O), similar to opal (Hartman, 1981) or silica gel (Schwab and Shore, 1971; reviewed in Belton et al., 2012). All siliceous spicules have a central canal, the axial canal, in which an axial filament is embedded (Fig. IB). Growth of hard sponge spicules is a rapid process; growth rates of 1-10 [micro]m * [h.sup.-1] were calculated for spicules of the demosponge Ephydatia fluviatilis, with a size of 200-350 [micro]m in length and a diameter of 15 [micro]m (Imsiecke et al., 1995). The properties of sponge spicules as rigid biological materials can be characterized best as structural hybrid composites, possessing unusual combinations of mechanical properties such as strength, stiffness, and toughness (Mayer, 2005). These properties have been studied in detail in the giant basal spicules of Monorhaphis chuni (Muller et al., 2008a; Miserez et al., 2008). Those spicules are composed of up to one thousand 8-10-[micro]m thick lamellae concentrically arranged around the axial cylinder whose diameter is about 100 [micro]m. Hardness was as high as 4 GPa and Young's modulus (E) was 45 GPa. Applying the three-point bending test, it was found that during fracture the cracks did not penetrate straight through the spicule, but rather traversed several lamellae. This finding reflects the exceptional biomechanical properties of biosilica-fabricated spicules: great strength and stiffness combined with unusual toughness. The spicules' biosilica matrix was found to be a hybrid material composed of biosilica as the inorganic component and of protein (Muller et al., 2010).

Until 1999/2000 (Cha et al., 1999; Krasko et al., 2000), it was unknown if inorganic polymers, e.g., biosilica, could be formed enzymatically. The first enzyme to show this reaction was silicatein, which was proven to mediate, with the same enzymatic parameters as other enzymes, the polycondensation reaction of orthosilicate to biosilica (reviewed in: Wang et al., 2012a; Muller et al., 2013). It was discovered that the axial filaments within the axial canals are composed of one protein (or protein family), silicatein(s), which is related to cathepsin(s), a hydrolytic enzyme that was earlier identified in the sponge species Geodia cydonium (Krasko et al., 1997).

A comprehensive description of the biosilica reaction during the use of silicic acid as a starting substrate was published recently (Schroder et al., 2012). The proposed reaction is based on the assumption that all three active amino acid residues--Ser, Asn, and His--within the active center of silicatein are essential for the catalytic mechanism (Fig. 2E). The initial catalytic step consists of a nucleophilic attack ([S.sub.N]2 type) of the partial electronegative oxygen atom of the hydroxyl group of the Ser at the (electropositive) silicon atom of the silicic acid molecule. After proton transfer from His nitrogen (Ser--His hydrogen bond) to one of the silicon OH ligands, the pentavalent intermediate is formed, resulting in release of a water molecule. Another water molecule is released during polycondensation, which is facilitated by proton transfer from the nitrogen imidazole of His, and generates a disilicic acid molecule that remains bound to silicatein by an ester-like linkage (Fig. 2E). The subsequent rotation of the ester bond allows interaction of a second OH ligand of the enzyme-bound silicic acid unit with the nitrogen imidazole of the catalytic center His residue. This process (nucleophilic attack, proton transfer, loss of water, rotation) is then repeated many times, implying that during each Si-O-Si bond formation, one water molecule is released.

Based on these data, it is clear that polycondensation of biosilica is paralleled by a 1:1 stoichiometric release of water per one Si-O-Si bond formed (Wang et al., 2011). This suggestion, that shrinking of the biosilica formed during the enzymatic process also occurs under in vivo conditions, is supported by experimental findings. As described earlier (Eckert et al., 2006), the growth of demosponge spicules proceeds by appositional layering of biosilica shells that are formed within concentric organic cylinders arranged around the axial canal. A typical immature, growing spicule is shown in Fig. 2A. Subsequently, during maturation these concentric biosilica shells fuse to solid rods. During this process, the seven layers of immature spicule mature to solid rods (Fig. 2B); at the same time, the primordial growing spicule rod with a diameter of about 10-12 [micro]m shrinks to a dimension of about 45 [micro]m. During completion of silica spicules, dense biosilica deposition occurs that also results in substantial (50%) shrinkage of the diameter of the rod to 5 [micro]m. This spicule shrinkage has been corroborated by theoretic calculations. The diameter of a silicate monomer is 4.48 A, whereas a silica molecule in a close to completely polycondensed mesh is only 3.56 A (Thompson et al., 1998). As a consequence, the molecular diameter of one silicate/silica molecule decreases by 21%. Assuming that 30 molecules of silicate monomers are arranged in a space-filling manner (Fig. 2C), the same number of silica molecules organized in a completely polycondensed mesh occupy 30% less space (Fig. 2D).

There are strong experimental indications that the aquaporin channel is involved in removal of reaction water during the silicatein-mediated polycondensation reaction, as discussed below (reviewed in Uriz et al., 2003).

Effect of Aquaporin Inhibitors on Morphogenesis of Sponge Primmorphs

The sponge's three-dimensional cell/tissue culture system, termed primmorphs (Muller et al., 1999), has been widely accepted as a useful tool for performing in vitro experiments with sponges under controlled laboratory conditions. In Suberites domuncula, primmorphs are composed of proliferating and differentiating cells and reach a diameter of up to 10 mm (LePennec et al., 2003). In an extensive screening program, we showed that the addition of Mn-sulfate (MnS[O.sub.4]) at concentrations as low as 50 [micro]mol [l.sup.-1], causes a drastic change in morphology of the primmorphs (Muller et al., 2011). Detached single cells from S. domuncula regrouped into small clumps during the first 6 h (100 [micro]m; Fig. 3A), and developed into spheres between 100-200 [micro]m in size during the following 24 h. Over the next 2 d, they continued to grow to sizes ranging between 500-700 [micro]m (Fig. 3G), or after 5 d, to spheres between 1-6 mm in diameter (Fig. 3J); after 7 d, the spheroidal aggregates ranged from 4-6 mm.

If these reaggregation experiments are performed with 50 [micro]mol [l.sup.-1] Mn-sulfate, the morphologies of the aggregates formed differ. The aggregates never formed spherical bodies; their shape was flat. This pattern was seen as early as a 1-d incubation period (Fig. 3B); during this flat time, starlike primmorphs were formed. These aggregates increased in size in culture over the following days (Fig. IE), ultimately forming large flat cell cushions 5 x 15 [mm.sup.2] in size (Fig. 3H and K). Interestingly, the cells in those primmorphs, regardless of whether they were left untreated or incubated with Mn-sulfate, showed similar viability, as can be inferred from the [.sup.3.H]-Thd incorporation studies (Muller et al., 2011).

Further inspection of the primmorphs revealed that the morphology of spicules after cultivation in the presence of Mn-sulfate does not show the typical size and shape. In the absence of Mn-sulfate, two characteristic monaxonal spicules form: first, the oxeas with pointed tips at both ends; and second, more abundantly, tylostyles, which feature a knob at one end and a pointed tip at the other end (Arndt et al., 1935). They range between 50-350 [micro]m in size, with a diameter of between 4-8 [micro]m. Their surfaces are smooth and the knobs are strikingly uniform in size, between 6.5-7.3 [micro]m (Fig. 1A and B). Lengths of spicules formed in Mn-sulfate-enriched medium were identical to those developed in a medium lacking Mn-sulfate, around 150 [micro]m (Fig. 2D). However, their surfaces were not smooth (Fig. 1D-F), but instead showed irregular deposits that were tightly attached to the spicule surfaces. The texture of the deposits was fluffy, rough, and porous; very often spicules fused onto those protruding deposits. Similar structures have been identified in spicules isolated from the tissue of physically impaired S. domuncula specimens (Eckert et al., 2006). Energy dispersive X-ray spectroscopy (EDX) data revealed that the deposits are silica and not organic matter.

The suggestion was that these irregularly formed spicules, especially their porous surface, were caused by insufficient water removal; water was thought to accumulate during the polycondensation reaction. In addition, the flat morphology of the primmorphs, which are formed during Mn-sulfate treatment, is the result of a breakup of the coordinated architecture of the spicular skeletal framework within the primmorphs. It was found that Mn-sulfate inhibits the function of the cell membrane-integrated aquaporin channels. To test this hypothesis, the effect of Mn-sulfate and an introduced inhibitor of aquaporin function, mercury chloride, was studied (Ishibashi et al., 1997; Liu et al., 2006). Mercury chloride is a relatively specific inhibitor of aquaporin type 8. The uptake of intact S. domuncula cells in the absence or presence of the salts has been measured using [.sup.14.C]methylammonium chloride as a tracer. The influx of methylammonium chloride was determined after 30 min. The results showed that both salts reduced the influx of methylammonium chloride in a concentration-dependent manner (Fig. 4). There was a 46% reduction of this influx in the presence of Mn-sulfate (30 [micro]mol [l.sup.-1]) and an 86% reduction with Mn-sulfate (50 [micro]mol [l.sup.-1]), respectively. Similarly strong was inhibition by mercury chloride, with 62% (100 [micro]mol [l.sup.-1]) and 73% (300 [micro]mol [l.sup.-1]) reductions noted, respectively. The sponge aquaporin was impaired in this assay. The antibody raised against the S. domuncula aquaporin was applied. Under the conditions used, a 52% reduction of the influx was measured. We cannot exclude the possibility that Mn-sulfate also impairs overall cell metabolism. Published data on plant aquaporin revealed that manganese ions are relative inhibitors of those channels (Verdoucq et al., 2008). When applying mercury chloride as an inhibitor during the reaggregation of sponge cells to primmorphs, an almost identical change in morphology was seen, compared with Mn-sulfate. Exposure of the culture to 300 [micro]mol [l.sup.-1] mercury chloride again showed the same flat aggregation pattern (Fig. 3C, F, I, L) that was described for Mn-sulfate.

The Sponge Aquaporin

Gene encoding of Suberites domuncula aquaporin was identified by applying the technique of differential display. Using this approach, cDNA was identified (accession number CBY89223) in the presence of Mn-sulfate, the band reflecting the sequence that corresponds to an aquaporin. Complete cDNA was obtained by 5'-race and 3'-race (Muller et al., 2011). The complete sequence is 1123 nucleotides (nts) long and comprises an open reading frame localized between [nt.sub.64] and [nt.sub.1,008] From this sequence, the putative sponge aquaporin protein (AQP_ SUBDO) was deduced (Fig. 5A). To confirm that the complete sequence was obtained. Northern blot analysis was performed; the expected 1.2 kb long transcripts were identified (Muller et al., 2011). The deduced protein (Fig. 5A) is 314 amino acids (aa) long and has a predicted size of 33,298.8 Da, with a theoretical isoelectric point (pI) of 8.46. The sponge aquaporin comprises the Aquaporin Z domain (accession number HAMAP: AQPZ_MF_01146; http://myhits.isb-sib.ch/cgi-bin/motif_scan) within positions a[a.sub.48] and a[a.sub.293] (Fig. 5A); the similarity is high, with an expected E-value of 3.4[e.sup.-07].

A sequence homology/similarity search showed that the sponge aquaporin displayed the greatest similarity to the sea urchin Strongylocentrotus purpuratus protein aquaporin-8 (accession number XP_794577.1) with an E-value of 2[e.sup.-37], reflecting a similarity/identity score of 46%/17%, followed by the human aquaporin-8 (BAD96684.1), with an E-value of 7[e.sup.-27] (similarity/identity: 44%/16%). From this result, we conclude that the Suberites domuncula aquaporin is an aquaporin type 8. The deduced sponge aquaporin comprises the characteristic six transmembrane helices (Kruse et al., 2006). They are located within regions a[a.sub.51] - a[a.sub.74], a[a.sub.87] - a[a.sub.110], a[a.sub.129] - a[a.sub.152], a[a.sub.190] - a[a.sub.209], a[a.sub.22] - a[a.sub.246], and a[a.sub.277] -a[a.sub.296] (Fig. 5A). The key residues, which are thought to reside within the pore region (i.e., [Thr.sup.94], [Ile.sup.236], [Gly.sup.244], Ser/[Cys.sup.245], and [Arg.sup.251]; (Liu et al., 2006)) and control the water exchange, were also identified.

A rooted phylogenetic tree was computed to support the notion that the sponge aquaporin belongs to the type 8 aquaporins (Fig. 5B). For this analysis, the other human aquaporins, aquaporin-1 to aquaporin-7 (AQP1-AQP7) and aquaporin-9 to aquaporin-12 (AQP9-AQP12) were included. After rooting with the distantly related human aquaporin 1, it was evident that the cloned sponge aquaporin forms one branch with the other two type 8 aquaporins.

Localization of the Sponge Aquaporin Channel

As a tool to identify the localization of the sponge aquaporin within the sponge tissue, the recombinant protein was expressed in Escherichia coli. cDNA (complete open reading frame) was expressed in the vector pTrcHis2-TOPO with a C'-terminal 6xHis-tag. After transformation, protein expression was induced with isopropyl 1-thio-D-galactopyranoside for 24 h at 20'C. The bacterial pellet was lysed, unfolded, then refolded and purified by affinity chromatography on Ni-IDA columns (Muller et al., 2011). The resulting recombinant protein was used to raise polyclonal antibodies in rabbits (White New Zealand). The pre-immune serum was taken from the same animal as a control; this sample did not cross-react with the immunogen (Muller et al., 2011).

The antibodies were used to localize the sponge aquaporin within sponge tissue samples. After cutting and fixation, the samples were incubated with the anti-aquaporin antibodies; the immunocomplexes were visualized with the labeled Cy3-conjugated F(ab')2 goat anti-rabbit IgG secondary antibody. The specimens were visualizedby fluorescence microscopy at an excitation wavelength of 546 nm; at the same time, they were stained with 4',6-diamidino-2phenylindole [DAPI] (360 nm excitation wavelength). Immunofluorescence analyses revealed that the highest expression is seen around spicule clusters, which are highlighted and marked in Figure 6A to C. At a higher magnification, it was apparent that the cells surrounding spicules in particular cross-react with the antibodies (Fig. 6D to F). In addition, the immune reaction in tissue from Mn-sulfate treated animals, i.e., the reaction of the antibodies, was much weaker (Muller et al., 2011).

Based on these data, we concluded (Wang et al., 2011, 2013a) that the secondary silica deposits formed on spicules under physiological conditions in the presence of silicate fuse together and subsequently undergo syneresis, which is facilitated by removal of water through aquaporin channels. In growing spicules, biosilica formation and syneresis in the lamellar monolithic structures precede the final step of "biosintering," during which the massive biosilica rods of the spicules are formed.

It was interesting to determine the steady-state expression level of silicatein and aquaporin during maturation of spicules in the presence and absence of Mn-sulfate (Wang et al., 2011). The results of the quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) experiments are summarized in Figure 7. The primmorphs were incubated in the absence/presence of orthosilicate (60 [micro]mol [l.sub.1] sodium silicate) and/or absence/presence of Mn-sulfate (50 [micro]mol [l.sub.1]). In the absence of both silicate and manganese sulfate, expression of the silicatein-[alpha] gene could not be detected at time point 0 and did not change in culture over the next 7 d. After the addition of silicate, the silicatein-[alpha] expression level strongly increased during the incubation period. No drastic gene induction was measured in the presence of 50 [micro]mol [l.sup.-1] Mn-sulfate; the transcripts remained at a high level. If the two components were added together, almost the same increase in gene expression was seen as in the experiments with silicate alone; the silicatein-[alpha] level strongly increased, while the level for aquaporin decreased by about 30%. However, if Mn-sulfate was added to the cultures, an almost complete down-regulation of aquaporin gene expression was observed after 3-7 d. The latter finding indicates that Mn-sulfate not only inhibits influx of the aquaporin channel, it also reduces expression of the underlying gene.

Phase Separation a Major Issue during Hardening of Biosilica

The maturation process of biosilica from the initially formed soft biosilica product to structured, solid spicules has only recently been experimentally approached and understood to some extent.

Biosilica formed by silicatein is not a solid, stiff material like that which is present in mature spicules; it has a solution-like (sol-like) consistency (Muller et al., 2008b). It is important to note that the polycondensation reaction, catalyzed by silicatein, proceeds at a concentration of 0.1 mmol [l.sup.-1] to 1 mmol [l.sup.-1], a concentration range that is too low to allow purely chemically controlled silica polymerization to proceed (Belton et al., 2010). As mentioned above, in vivo spicule synthesis is a rapid process, implying that the processes of (i) silicatein-mediated biosilica synthesis, (ii) syneresis of biosilica, and (iii) hardening/structure formation of spicules must be efficient and fast. Experimental data showing that during maturation of spicules their sizes shrink (Wang et al., 2011) imply that reaction water accumulating during biosilica synthesis is removed from the extracellular space by channeling into cells via aquaporin pores (Fig. 2C and D).

To understand the two-phase separation experimentally, a model system was introduced (Sumper, 2002; Wang et al., 2013b). The lower phase was formed by a Ficoll cushion (8.6% (w/v)) giving the higher density, and by polyethylene glycol (PEG) (10% (w/v)), while silicatein and its substrate, orthosilicate formed from tetraethyl orthosilicate (TEOS) by acid hydrolysis, were added at the upper phase, where the enzymatic polycondensation reaction to biosilica occurred. The separation studied was performed at 20' C. PEG interacts via hydrogen bonds with and between silica nanoparticles, causing gelation of the silica nanoparticles (Park et al., 2007). Furthermore, PEG promotes phase separation resulting in the formation of a silica-rich phase (Bideau et al., 2010). Of note, depending on the nature of the polymers and the size of the particles, PEG causes phase segregation, resulting in an unequal distribution of the two phases within the same particle/droplet (Biswas et al., 2012). Applying this PEG-based, two-phase separation model system, we showed that PEG causes large-scale gelation and phase separation of biosilica particles that were formed enzymatically from orthosilicate at concentrations of around 1 mmol [l.sup.-1] (Wang et al., 2013b). Figure 8 shows how biosilica that was formed enzymatically undergoes solidification from an almost sol (time 0) to a voluminous gel during a 10-h incubation period.

Mucin-Related Protein--a Component Involved in Hardening of Biosilica

Along with phase separation/phase segregation processes such as the PEG model above, during which polymers interact with water through hydrogen bonds, there is a second principle in biology, termed "polymerization-induced phase separation" (Dubinsky et al., 2010a, b). Polymerization of the component can be of the first order if only one reagent participates in the process, or of a higher order if two or more partners are involved. Animal mucins are typical examples of a higher-order polymerization component (Sheehan and Thornton, 2000). Polymerized gelforming mucins are linear and adsorb water during polymerization (Toribara et al., 1991; Sheehan and Thornton, 2000). Moreover, these reactions are driven by "depletion forces," with actin as an example. In a previous study we screened the sponge S. domuncula for mucin-like polypeptide and described such a polymer (Wang et al., 2013b).

Identification of cDNA encoding for the putative mucin-like polypeptide was successful after applying a N-terminal sequence fragment, F-L-Q-E-I-I-L-V-A-I, of a spicule-binding polypeptide. Degenerate forward and reverse primers were constructed to identify those tags in the S. domuncula expressed sequence tag (EST) database. Complete cDNA was obtained and termed, according to characteristic domain region "Nidogen," as a nidogen-like polypeptide (SDNIDOGEN-1; accession number HE971726) (Wang et al., 2013b). The cDNA had one open reading frame (ORF), encoding a 205 aa polypeptide (Fig. 9A). The highest similarity score of the sponge nidogen was found to be with the related mucin sequences, the predicted mucin-4-like polypeptide from Amphimedon queenslandica (XP_003389333.1), and the nidogen and EGF-like domains comprising protein from Sus scrofa (XP_003133861.2). The deduced sequence comprises the partial nidogen domain within parts a[a.sub.108] and a[a.sub.205]. No other domain could be identified; in higher metazoan phyla the nidogen domain exists in mosaic proteins together with other functional domains. The nidogen domain comprises in its helix G2 region binding sites for collagen IV, perlecan, and [Ca.sup.2+]. The two potential [Ca.sup.2+] binding aa moieties are clustered within the sponge nidogen (Fig. 9A). Phylogenetic tree analysis revealed the high sequence similarity of the vertebrate (AF218265J) and the sponge (XP_003389333.1) mucin-4 sequence (Fig. 9B).

Mucin-Related Sponge Nidogen Polypeptide Induces Biosilica Gel Formation

The recombinant protein of the spicule-binding protein/nidogen was prepared in a recombinant way in Escherichia coliy in the same manner as for aquaporin (Wang et al., 2013b), and tested for its potential to induce gel formation. The recombinant protein has the expected size of 23.5 kDa. If a sample (30 [micro]g/ml) was added to 3 mmol [l.sup.-1] prehydrolyzed TEOS, together with silicatein, small silica aggregates were visible only after a 3-h incubation period and increased in size (Fig. 10). In the absence of the nidogen-like protein, no floes were seen.

Conclusion

Young spicules, from 5-day-old primmorphs, contain 16% water, a concentration that continuously decreases to 10% in adult spicules (Wang et al., 2011). These spicules are formed enzymatically via silicatein, whose product, biosilica, undergoes syneresis, during which water is extruded from the scaffold after the polycondensation reaction. During syneresis, the immature spicules shrink, very likely due to the action of aquaporin. Based on microscopic analyses, it can be concluded that during syneresis, which corresponds to the the shrinkage process, the non-structured biosilica product of the silicatein reaction undergoes a structure forming and hardening process (Wang et al., 2012b). As one consequence of the syneresis reaction, spicules, we hypothesize, not only shrink in diameter but also elongate axially, the result of injection of the semi-solid (i.e., not yet hardened) biosilica material into an organic casting mold, and leading to the formation of richly ornamented spicules featuring hooks or thorns (Wang et al., 2012a; see Fig. 11).

In the last few years, we showed that aquaporin-mediated extrusion of the reaction water is the first step during hardening of silicatein. If the channel is blocked, formation of smooth spicules is also blocked. We also showed that in the two-phase separation assays, biosilica condenses in the presence of PEG, allowing acceleration of the sol-to-gel phase.

In the next step, we screened for natural polymers in S. domuncula that alter the condensation and sol-gel state of biosilica. Applying molecular biological techniques (Wang et al., 2011), a gene encoding the spicule-binding protein was identified as a nidogen-like protein. In the presence of this protein, which had been prepared in a recombinant manner, biosilica hardening was accelerated.

The biosilica product of silicatein is amorphous and highly enriched with water that originates from the polycondensation reaction. Over the following stages of spicule formation, the biosilica polymer undergoes an aging process, during which the biosilica material contracts in volume as a result of syneresis. This process is driven both by aquaporin channeling and by mucin/nidogen-like, proteininduced contraction of the biosilica product. This process might be used as a blueprint for the fabrication of biosilicabased solid rods that allow light transmission, as with natural sponge spicules (Cattaneo-Vietti et al., 1996). In summary, studies of the sponge biosilica polymer continue to open new avenues for the application of biosilica both in biomedicine and in nano-optics (reviewed in Schroder et al., 2008).

Acknowledgments

W.E.G.M. is a holder of an ERC Advanced Investigator Grant (No. 268476 BIOSILICA). This work was supported by grants from the European Commission ("Bio-Scaffolds", No. 604036; "CoreShell": No. 286059; "MarBioTec*EU-CN*": No. 268476; and "BlueGenics": No. 311848) and the International Human Frontier Science Program.

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XIAOHONG WANG AND WERNER E. G. MULLER (*)

ERC Advanced Investigator Grant Research Group at Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Duesbergweg 6, D-55128 Mainz, Germany

Received 28 January 20 15; accepted 18 March 2015.

(*) To whom correspondence should be addressed. E-mail: wmueller@uni-mainz.de

Abbreviations: Hg[Cl.sub.2], mercury chloride; MnS[O.sub.4], Mn-sulfate; PEG, polyethylene glycol.
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