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1.
The enzymatic-silicatein mediated formation of the skeletal elements, the spicules of siliceous sponges starts intracellularly and is completed extracellularly. With Suberites domuncula we show that the axial growth of the spicules proceeds in three phases: (I) formation of an axial canal; (II) evagination of a cell process into the axial canal, and (III) assembly of the axial filament composed of silicatein. During these phases the core part of the spicule is synthesized. Silicatein and its substrate silicate are stored in silicasomes, found both inside and outside of the cellular extension within the axial canal, as well as all around the spicule. The membranes of the silicasomes are interspersed by pores of ≈ 2 nm that are likely associated with aquaporin channels which are implicated in the hardening of the initial bio-silica products formed by silicatein. We can summarize the sequence of events that govern spicule formation as follows: differential GENETIC READOUT (of silicatein) → FRACTAL ASSOCIATION of the silicateins → EVAGINATION of cells by hydro-mechanical forces into the axial canal → and finally PROCESSIVE BIO-SILICA POLYCONDENSATION around the axial canal. We termed this process, occurring sequentially or in parallel, BIO-INORGANIC SELF-ORGANIZATION.  相似文献   

2.
The skeleton of the siliceous sponges (Porifera: Hexactinellida and Demospongiae) is supported by spicules composed of bio-silica. In the axial canals of megascleres, harboring the axial filaments, three isoforms of the enzyme silicatein (-alpha, -beta and -gamma) have been identified until now, using the demosponges Tethya aurantium and Suberites domuncula. Here we describe the composition of the proteinaceous components of the axial filament from small spicules, the microscleres, in the demosponge Geodia cydonium that possesses megascleres and microscleres. The morphology of the different spicule types is described. Also in G. cydonium the synthesis of the spicules starts intracellularly and they are subsequently extruded to the extracellular space. In contrast to the composition of the silicateins in the megascleres (isoforms: -alpha, -beta and -gamma), the axial filaments of the microscleres contain only one form of silicatein, termed silicatein-alpha/beta, with a size of 25kDa. Silicatein-alpha/beta undergoes three phosphorylation steps. The gene encoding silicatein-alpha/beta was identified and found to comprise the same characteristic sites, described previously for silicateins-alpha or -beta. It is hypothesized, that the different composition of the axial filaments, with respect to silicateins, contributes to the morphology of the different types of spicules.  相似文献   

3.
Silicateins are enzymes, which are restricted to sponges (phylum Porifera), that mediate the catalytic formation of biosilica from monomeric silicon compounds. The silicatein protein is compartmented in the sponges in the axial filaments which reside in the axial canals of the siliceous spicules. In the present study silicatein has been isolated from the freshwater sponge Lubomirskia baicalensis where it occurs in isoforms with sizes of 23 kDa, 24 kDa and 26 kDa. Since the larger protein is glycosylated we posit that it is a processed form of one of the smaller size forms. The silicatein isoforms are post-translationally modified by phosphorylation; at least four isoforms exist with pI's of 5.4, of 5.2, of 4.9 and of 4.7. Surprisingly silicatein not only mediates polymerization of silicate, but also displays proteolytic activity which is specific for cathepsin L enzymes, thus underscoring the high relationship of the silicateins to cathepsin L. The cDNAs from L. baicalensis for silicatein and cathepsin L, as well as the respective genes, were cloned. It was found that the five introns present in the sponge genes are highly conserved up to human cathepsin L. This analysis has been completed by sequencing of two silicatein genes (both for silicatein-alpha and -beta) and of cathepsin L from another demosponge, Suberites domuncula. A comprehensive phylogenetic analysis with these new sequences shed new light upon the evolution of cathepsin L and silicatein families which occurred at the base of the metazoan phyla. It is concluded, that in parallel with the emergence of these enzymes at first the number of introns increased, especially in the coding region of the mature enzyme. Later in evolution the number of introns decreased again. We postulate that modification of the catalytic triad, especially of its first amino acid, is a suitable target for a chemical modulation of enzyme function of the silicateins/cathepsin L.  相似文献   

4.
The siliceous spicules of sponges (Porifera) are synthesized by the enzyme silicatein. This protein and its gene have been identified so far in the Demospongiae, e.g., Tethya aurantium and Suberites domuncula. In the Hexactinellida, the second class of siliceous sponges, the mechanism of synthesis of the largest bio-silica structures on Earth remains obscure. Here, we describe the morphology of the spicules (diactines and stauractines) of the hexactinellid Crateromorpha meyeri. These spicules are composed of silica lamellae concentrically arranged around a central axial canal and contain proteinaceous sheaths (within the siliceous mantel) and proteinaceous axial filaments (within the axial canal). The major protein in the spicules is a 24-kDa protein that strongly reacts with anti-silicatein antibodies in Western blots. Its cDNA has been successfully cloned; the deduced hexactinellid silicatein comprises, in addition to the characteristic catalytic triad amino acids Ser-His-Asn and the "conventional" serine cluster, a "hexactinellid C. meyeri-specific" Ser cluster. We show that anti-silicatein antibodies react specifically with the proteinaceous matrix of the C. meyeri spicules. The characterization of silicatein at the genetic level should contribute to an understanding of the molecular/biochemical mechanism of spiculogenesis in Hexactinellida. These data also indicate that silicatein is an autapomorphic molecule common to both classes of siliceous sponges.  相似文献   

5.
The major structural and enzymatically active protein in spicules from siliceous sponges, e.g., for Suberites domuncula studied here, is silicatein. Silicatein has been established to be the key enzyme that catalyzes the formation of biosilica, a polymer that represents the inorganic scaffold for the spicule. In the present study, it is shown, by application of high-resolution transmission and scanning transmission electron microscopy that, during the initial phase of spicule synthesis, nanofibrils with a diameter of around 10 nm are formed that comprise bundles of between 10 and 20 nanofibrils. In intracellular vacuoles, silicasomes, the nanofibrils form polar structures with a pointed tip and a blunt end. In a time-dependent manner, these nanofibrillar bundles become embedded into a Si-rich matrix, indicative for the formation of biosilica via silicatein molecules that form the nanofibrils. These biosilicified nanofibrillar bundles become extruded from the intracellular space, where they are located in the silicasomes, to the extracellular environment by an evagination process, during which a cellular protrusion forms the axial canal in the growing spicule. The nanofibrillar bundles condense and progressively form the axial filament that becomes localized in the extracellular space. It is concluded that the silicatein-composing nanofibrils act not only as enzymatic silica bio-condensing platforms but also as a structure-giving guidance for the growing spicule.  相似文献   

6.
The siliceous skeleton of demosponges is constructed of spicules. We have studied the formation of spicules in primmorphs from Suberites domuncula. Scanning electron microscopy and transmission electron-microscopical (TEM) analyses have revealed, in the center of the spicules, an axial canal that is 0.3–1.6 m wide and filled with an axial filament. This filament is composed of the enzyme silicatein, which synthesizes the spicules. TEM analysis has shown that spicule formation starts intracellularly and ends extracellularly in the mesohyl. At the initial stage, the axial canal is composed only of silicatein, whereas membranous structures and fibrils (10–15 nm in width) can later also be identified, suggesting that intracellular components protrude into the axial canal. Antibodies against silicatein have been applied for Western blotting; intracellularly, silicatein is processed to the mature form (24 kDa), whereas the pro-enzyme with the propeptide (33 kDa) is detected extracellularly. Silicatein undergoes phosphorylation at five sites. Immunohistological analysis has shown that silicatein exists in the axial canal (axial filament) and on the surface of the spicules, suggesting that they grow by apposition. Finally, we have demonstrated that the enzymic reaction of silicatein is inhibited by anti-silicatein antibodies. These data provide, for the first time, a comprehensive outline of spicule formation.This work was supported by grants from the European Commission (SILIBIOTEC), the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung Germany (project: Center of Excellence BIOTECmarin) and the International Human Frontier Science Program.  相似文献   

7.
Siliceous spicules of the freshwater Baikal sponge Lubomirskia baicalensis contain several proteins including silicateins. Existences of four different genes of silicatein alpha (alpha1, alpha2, alpha3, alpha4) which are related to silicatein alpha from the sea sponges were found when cDNA library analysis was made. The intron-exon structure of the full-size silicatein alpha1 gene was determined. This gene has total length of 1988 bp and includes 6 introns (1007 bp) and 7 exons (981 bp). With use of mass-spectrometric analysis of the spicule proteins tryptic digest, two silicateins alpha were authentically found.  相似文献   

8.
The skeleton of demosponges is built of spicules consisting of biosilica. Using the primmorph system from Suberites domuncula, we demonstrate that silicatein, the biosilica-synthesizing enzyme, and silicase, the catabolic enzyme, are colocalized at the surface of growing spicules as well as in the axial filament located in the axial canal. It is assumed that these two enzymes are responsible for the deposition of biosilica. In search of additional potential structural molecules that might guide the mineralization process during spiculogenesis to species-specific spicules, electron microscopic studies with antibodies against galectin and silicatein were performed. These studies showed that silicatein forms a complex with galectin; the strings/bundles of this complex are intimately associated with the surface of the spicules and arranged concentrically around them. Collagen fibers are near the silactein/galectin complexes. The strings/bundles formed from silicatein/galectin display a lower degree of orientation than the collagen fibers arranged in a highly ordered pattern around the spicules. These data indicate that species-specific formation of spicules involves a network of (diffusible) regulatory factor(s) controlling enzymatic silica deposition; this mineralization process proceeds on a galectin/collagen organic matrix.  相似文献   

9.
In some sponges peculiar proteins called silicateins catalyze silica polymerization in ordered structures, and their study is of high interest for possible biotechnological applications in the nanostructure industry. In this work we describe the isolation and the molecular characterization of silicatein from spicules of Petrosia ficiformis, a common Mediterranean sponge, and the development of a cellular model (primmorphs) suitable for in vitro studies of silicatein gene regulation. The spicule of P. ficiformis contains an axial filament composed of 2 insoluble proteins, of 30 and 23 kDa. The 23-kDa protein was characterized, and the full-length cDNA was cloned. The putative amino acid sequence has high homology with previously described silicateins from other sponge species and also is very similar to cathepsins, a cystein protease family. Finally, P. ficiformis primmorphs express the silicatein gene, suggesting that they should be a good model for biosilicification studies.  相似文献   

10.
The skeletal elements (spicules) of the demosponge Lubomirskia baicalensis were analyzed; they are composed of amorphous, non-crystalline silica, and contain in a central axial canal the axial filament which consists of the enzyme silicatein. The axial filament, that orients the spicule in its longitudinal axis exists also in the center of the spines which decorate the spicule. During growth of the sponge, new serially arranged modules which are formed from longitudinally arranged spicule bundles are added at the tip of the branches. X-ray analysis revealed that these serial modules are separated from each other by septate zones (annuli). We describe that the longitudinal bundles of spicules of a new module originate from the apex of the earlier module from where they protrude. A cross section through the oscular/apical-basal axis shows that the bundle rays are organized in a concentric and radiate pattern. High resolution magnetic resonance microimaging studies showed that the silica spheres of the spicules in the cone region contain high amounts of 'mobile' water. We conclude that the radiate accretive growth pattern of sponges is initiated in the apical region (cones) by newly growing spicules which are characterized by high amounts of 'mobile' water; subsequently spicule bundles are formed laterally around the cones.  相似文献   

11.
Botting, J.P., Muir, L.A., Xiao, S., Li, X. & Lin, J.‐P. 2012: Evidence for spicule homology in calcareous and siliceous sponges: biminerallic spicules in Lenica sp. from the Early Cambrian of South China. Lethaia, Vol. 45, pp. 463–475. The relationships of the extant sponge classes, and the nature of the last common ancestor of all sponges, are currently unclear. Early sponges preserved in the fossil record differ greatly from extant taxa, and therefore information from the fossil record is critical for testing hypotheses of sponge phylogenetic relationships that are based on modern taxa. New specimens of the enigmatic sponge Lenica sp., from the Early Cambrian Hetang Biota of South China, exhibit an unusual spicule structure. Each spicule consists of a siliceous core with an axial canal, an organic outer layer and a middle layer interpreted to have been originally calcium carbonate. This finding confirms previous work suggesting the existence of biminerallic spicules in early sponges. Combined with data from other early sponges, the new findings imply that the two fundamental spicule structures of modern sponges were derived from a compound, biminerallic precursor. Spicules are therefore homologous structures in Calcarea and Silicea, and if sponges are paraphyletic with respect to Eumetazoa, then spicules may also have been a primitive feature of Metazoa. □Calcarea, Early Cambrian, Hetang Biota, phylogeny, Silicea, taphonomy.  相似文献   

12.
Silicateins are unique enzymes of sponges (phylum Porifera) that template and catalyze the polymerization of nanoscale silicate to siliceous skeletal elements. These multifunctional spicules are often elaborately shaped, with complex symmetries. They carry an axial proteinaceous filament, consisting of silicatein and the scaffold protein silintaphin-1, which guides silica deposition and subsequent spicular morphogenesis. In vivo, the synthesis of the axial filament very likely proceeds in three steps: (a) assembly of silicatein monomers to form one pentamer; (b) assembly of pentamers to form fractal-like structures; and finally (c) assembly of fractal-like structures to form filaments. The present study was aimed at exploring the effect of self-assembled complexes of silicatein and silintaphin-1 on biosilica synthesis in vitro. Hence, in a comparative approach, recombinant silicatein and recombinant silintaphin-1 were used at different stoichiometric ratios to form axial filaments and to synthesize biosilica. Whereas recombinant silicatein-α reaggregates to randomly organized structures, coincubation of silicatein-α and silintaphin-1 (molecular ratio 4 : 1) resulted in synthetic filaments via fractal-like patterned self-assemblies, as observed by electron microscopy. Concurrently, owing to the concerted action of both proteins, the enzymatic activity of silicatein-α strongly increased by 5.3-fold (with the substrate tetraethyl orthosilicate), leading to significantly enhanced synthesis of biosilica. These results indicate that silicatein-α-mediated biosilicification depends on the concomitant presence of silicatein-α and silintaphin-1. Accordingly, silintaphin-1 might not only enhance the enzymatic activity of silicatein-α, but also accelerate the nonenzymatic polycondensation of the silica product before releasing the fully synthesized biosiliceous polymer.  相似文献   

13.
The formation of spicules is a complicated morphogenetic process in sponges (phylum Porifera). The primmorph system was used to demonstrate that in the demosponge Suberites domuncula the synthesis of the siliceous spicules starts intracellularly and is dependent on the concentration of silicic acid. To understand spicule formation, a cluster of genes was isolated. In the center of this cluster is the silicatein gene, which codes for the enzyme that synthesizes spicules. This gene is flanked by an ankyrin repeat gene at one side and by a tumor necrosis factor receptor-associated factor and a protein kinase gene at the other side. All genes are strongly expressed in primmorphs and intact animals after exposure to silicic acid, and this expression is restricted to those areas where the spicule formation starts or where spicules are maintained in the animals. Our observations suggest that in S. domuncula a coordinated expression of physically linked genes is essential for the synthesis of the major skeletal elements.  相似文献   

14.
The skeleton of siliceous sponges consists of amorphous biogenous silica (biosilica). Biosilica formation is driven enzymatically by means of silicatein(s). During this unique process of enzymatic polycondensation, skeletal elements (spicules) that enfold a central proteinaceous structure (axial filament), mainly comprising silicatein, are formed. However, only the concerted action of silicatein and other proteins can explain the genetically controlled diversity of spicular morphotypes, from simple rods with pointed ends to intricate structures with up to six rays. With the scaffold protein silintaphin-1, a first silicatein interactor that facilitates the formation of the axial filament and, consequently, of the growing spicule was discovered. In this study, a new interactor has been identified by both a conventional yeast two-hybrid library screening and a newly established pull-down assay. For the latter approach, silicatein-α has been bioengineered to carry a Glu tag, which confers binding affinity to hydroxyapatite. After immobilization on a solid-phase matrix (hydroxyapatite), the Glu-tagged silicatein was used as bait for the identification of interactors. Both approaches revealed a 15 kDa polypeptide, and its identity was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Colocalization of silintaphin-2 and silicatein-α within the axial filament and on the spicule surface was shown by immunohistological analyses. Subsequent autoradiography demonstrated the Ca(2+) binding affinity of this silicatein interactor. These findings indicate that both proteins operate in concert during spiculogenesis. Besides binding of calcium, silintaphin-2 shares several structural features with certain acidic, secreted extracellular matrix proteins that facilitate tissue mineralization in Metazoa. Hence, silintaphin-2 might mediate signal transduction during spiculogenesis or may play a more direct role during biosilica formation, in concert with silicatein.  相似文献   

15.
Silicateins are the key enzymes involved in the enzymatic polycondensation of the inorganic scaffold of the skeletal elements of the siliceous sponges, the spicules. The gene encoding pro-silicatein is inserted into the pCold TF vector, comprising the gene for the bacterial trigger factor. This hybrid gene is expressed in Escherichia coli and the synthesized fusion protein is purified. The fusion protein is split into the single proteins with thrombin by cleavage of the linker sequence present between the two proteins. At 23 °C, the 87 kDa trigger factor-pro-silicatein fusion protein is cleaved to the 51 kDa trigger factor and the 35 kDa pro-silicatein. The cleavage process proceeds and results in the release of the 23 kDa mature silicatein, a process which very likely proceeds by autocatalysis. Almost in parallel with its formation, the mature enzyme precipitates as pure 23 kDa protein. When the precipitate is dissolved in an urea buffer, the solubilized protein displays its full enzymatic activity which is enhanced multi-fold in the presence of the silicatein interactor silintaphin-1 or of poly(ethylene glycol) (PEG). The biosilica product formed increases its compactness if silicatein is supplemented with silintaphin-1 or PEG. The elastic modulus of the silicatein-mediated biosilica product increases in parallel with the addition of silintaphin-1 and/or PEG from 17 MPa (silicatein) via 61 MPa (silicatein:silintaphin-1) to 101 MPa (silicatein:silintaphin-1 and PEG). These data show that the maturation process from the pro-silicatein state to the mature form is the crucial step during which silicatein acquires its structure-guiding and structure-forming properties.  相似文献   

16.
Recently it has been discovered that the formation of the siliceous spicules of Demospongiae proceeds enzymatically (via silicatein) and occurs matrix guided (on galectin strings). In addition, it could be demonstrated that silicatein, if immobilized onto inorganic surfaces, provides the template for the synthesis of biosilica. In order to understand the formation of spicules in the intact organism, detailed studies with primmorphs from Suberites domuncula have been performed. The demosponge spicules are formed from several silica lamellae which are concentrically arranged around the axial canal, harboring the axial filament composed of silicatein. Now we show that the appositional growth of the spicules in radial and longitudinal direction proceeds in the extracellular space along hollow cylinders; their surfaces are formed by silicatein. The extracellularly located spicules are surrounded by sclerocytes which are filled with both electron-dense and electron-poor vesicles; energy dispersive X-ray analysis/scanning electron microscopical studies revealed that the electron-dense vesicles are filled of silicon/silica and therefore termed silicasomes. The release of the content of the silicasomes into the hollow cylinder suggests that the newly formed silica lamella originate there; in addition the data are compatible with the view that the silicatein molecules, attached at the centripetal and centrifugal surfaces, mediate biosilica formation. In a chemical/biomimetical approach silicatein is linked onto the organic material-free spicules after their functionalization with aminopropyltriethoxysilane [amino groups]-poly(acetoxime methacrylate) [reactive ester polymer]-N(epsilon)-benzyloxycarbonyl L-lysine tert-butyl ester-Ni(II); finally His-tagged silicatein is immobilized. The matrix-bound enzyme synthesized a new biosilica lamella. These bioinspired findings are considered as the basis for a technical use/application/utilization of hollow cylinders formed by matrix-guided silicatein molecules for the biocatalytic synthesis of nanostructured tubes.  相似文献   

17.
Silicatein genes are known to be involved in siliceous spicule formation in marine sponges. Proteins encoded by these genes, silicateins, were recently proposed for nanobiotechnological applications. We studied silicatein genes of marine sponges Latrunculia oparinae collected in the west Pacific region, shelf of Kuril Islands. Five silicatein genes, LoSilA1, LoSilA1a, LoSilA2, and LoSilA3 (silicatein-α group), LoSilB (silicatein-β group), and one cathepsin gene, LoCath, were isolated from the sponge L. oparinae for the first time. The deduced amino acid sequence of L. oparinae silicateins showed high-sequence identity with silicateins described previously. LoCath contains the catalytic triad of amino acid residues Cys-His-Asn characteristic for cathepsins as well as motifs typical for silicateins. A phylogenetic analysis places LoCath between sponge silicateins-β and L-cathepsins suggesting that the LoCath gene represents an intermediate form between silicatein and cathepsin genes. Additionally, we identified, for the first time, silicatein genes (AcSilA and AcSilB) in nonspicule-forming marine sponge, Acаnthodendrilla sp. The results suggest that silicateins could participate also in the function(s) unrelated to spiculogenesis.  相似文献   

18.
19.
The two sponge classes, Hexactinellida and Demospongiae, comprise a skeleton that is composed of siliceous skeletal elements (spicules). Spicule growth proceeds by appositional layering of lamellae that consist of silica nanoparticles, which are synthesized via the sponge-specific enzyme silicatein. While in demosponges during maturation the lamellae consolidate to a solid rod, the lamellar organization of hexactinellid spicules largely persists. However, the innermost lamellae, near the spicule core, can also fuse to a solid axial cylinder. Similar to the fusion of siliceous nanoparticles and lamella, in several hexactinellid species individual spicules unify during sintering-like processes. Here, we study the different stages of a process that we termed bio-sintering, within the giant basal spicule (GBS) of Monorhaphis chuni. During this study, a major GBS protein component (27 kDa) was isolated and analyzed by MALDI-TOF-MS. The sequences were used to isolate and clone the encoding cDNA via degenerate primer PCR. Bioinformatic analyses revealed a significant sequence homology to silicatein. In addition, the native GBS protein was able to mediate bio-silica synthesis in vitro. We conclude that the syntheses of bio-silica in M. chuni, and the subsequent fusion of nanoparticles to lamellae, and finally to spicules, are enzymatically-driven by a silicatein-like protein. In addition, evidence is now presented that in hexactinellids those fusions involve sintering-like processes.  相似文献   

20.
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