35 Years of Marine Natural Product Research in Sweden: Cool Molecules and Models from Cold Waters.

Currents efforts in marine biodiscovery have essentially focused on temperate to tropical shallow water organisms. With more than 6000 species of marine plants and animals, the Kosterfjord area has the richest marine biodiversity in Swedish waters, but it remains understudied. The overall objective of our marine pharmacognosy research is to explore and reveal the pharmacological potential of organisms from this poorly explored region. More generally, we wish to understand aspects of structure-activity relationships of chemical interactions in cold-water marine environment (shallow and deep). Our strategy is based on ecologically guided search for compounds through studies of physiology and organism interactions coupled to identification of bioactive molecules guided by especially in vivo assays. The research programme originated in the beginning of the 1980s with a broad screening of Swedish marine organisms using both in vitro and in vivo assays, resulting in isolation and identification of several different bioactive molecules. Two congenerous cyclopeptides, i.e. barettin and 8,9-dihydrobarettin, were isolated from the deep-sea sponge Geodia barretti, and structurally elucidated, guided by their antifouling activity and their affinity to a selection of human serotonin receptors. To optimize the activity a number of analogues of barettin were synthezised and tested for antifouling activity. Within the EU project BlueGenics, two larger homologous peptides, barrettides A and B, were isolated from G. baretti. Also, metabolic fingerprinting combined with sponge systematics was used to further study deep-sea natural product diversity in the genus Geodia. Finally, the chemical property space model 'ChemGPS-NP' has been developed and used in our research group, enabling a more efficient use of obtained compounds and exploration of possible biological activities and targets. Another approach is the broad application of phylogenetic frameworks, which can be used in prediction of where-in which organisms-to search for novel molecules or better sources of known molecules in marine organisms. In a further perspective, the deeper understanding of evolution and development of life on Earth can also provide answers to why marine organisms produce specific molecules.

Metagenomic Analysis of Genes Encoding Nutrient Cycling Pathways in the Microbiota of Deep-Sea and Shallow-Water Sponges.

Sponges host complex symbiotic communities, but to date, the whole picture of the metabolic potential of sponge microbiota remains unclear, particularly the difference between the shallow-water and deep-sea sponge holobionts. In this study, two completely different sponges, shallow-water sponge Theonella swinhoei from the South China Sea and deep-sea sponge Neamphius huxleyi from the Indian Ocean, were selected to compare their whole symbiotic communities and metabolic potential, particularly in element transformation. Phylogenetically diverse bacteria, archaea, fungi, and algae were detected in both shallow-water sponge T. swinhoei and deep-sea sponge N. huxleyi, and different microbial community structures were indicated between these two sponges. Metagenome-based gene abundance analysis indicated that, though the two sponge microbiota have similar core functions, they showed different potential strategies in detailed metabolic processes, e.g., in the transformation and utilization of carbon, nitrogen, phosphorus, and sulfur by corresponding microbial symbionts. This study provides insight into the putative metabolic potentials of the microbiota associated with the shallow-water and deep-sea sponges at the whole community level, extending our knowledge of the sponge microbiota's functions, the association of sponge- microbes, as well as the adaption of sponge microbiota to the marine environment.

Aquaporin in Chondrosia reniformis Nardo, 1847 and Its Possible Role in the Interaction Between Cells and Engulfed Siliceous Particles.

The sponge Chondrosia reniformis selectively engulfs siliceous particles that, when in crystalline form, become quickly dissolved in its ectosome. The molecular mechanism, identity, and physiological significance of the cells involved in this process are not completely understood. In the present study, we applied light and electronic microscopic techniques to show how the quartz particles in C. reniformis are enveloped through collagen fibers and host cells near the surface of these organisms. As various aquaporins from bacteria, animals, and plants bidirectionally conduct metalloids-including silicon ions--through the cell membrane, the presence and potential involvement of aquaporins in quartz dissolution in C. reniformis have been investigated. An aquaporin-like transcript (CrAQP) was isolated according to the transcriptome sequencing results in C. reniformis The full-length CrAQP cDNA is 907 nucleotides long, with a 795-base pair (bp), open reading frame encoding a protein of 265 amino acids, a 29-bp, 5'-non-coding region, and a 83-bp, 3'-untranslated region. The Bayesian phylogenetic inference suggests that CrAqp is closely related to the Aqp8L grade, which is also implicated in H2O2 transport. Quantification of CrAQP mRNA through qPCR indicated that the transcript level was higher in the ectosome than in the choanosome. Immunofluorescence of a mammalian AQP8 in C. reniformis showed positivity in some cells near the quartz particles, a finding that may support the initial hypothesis of the potential involvement of CrAQP in quartz erosion. However, the features of the primary structure of this protein offer a new viewpoint about the functional role of these molecules in this process: that CrAQP may be involved in the permeation of H2O2 released during silica erosion.

Common genetic denominators for Ca++-based skeleton in Metazoa: role of osteoclast-stimulating factor and of carbonic anhydrase in a calcareous sponge.

Calcium-based matrices serve predominantly as inorganic, hard skeletal systems in Metazoa from calcareous sponges [phylum Porifera; class Calcarea] to proto- and deuterostomian multicellular animals. The calcareous sponges form their skeletal elements, the spicules, from amorphous calcium carbonate (ACC). Treatment of spicules from Sycon raphanus with sodium hypochlorite (NaOCl) results in the disintegration of the ACC in those skeletal elements. Until now a distinct protein/enzyme involved in ACC metabolism could not been identified in those animals. We applied the technique of phage display combinatorial libraries to identify oligopeptides that bind to NaOCl-treated spicules: those oligopeptides allowed us to detect proteins that bind to those spicules. Two molecules have been identified, the (putative) enzyme carbonic anhydrase and the (putative) osteoclast-stimulating factor (OSTF), that are involved in the catabolism of ACC. The complete cDNAs were isolated and the recombinant proteins were prepared to raise antibodies. In turn, immunofluorescence staining of tissue slices and qPCR analyses have been performed. The data show that sponges, cultivated under standard condition (10 mM CaCl(2)) show low levels of transcripts/proteins for carbonic anhydrase or OSTF, compared to those animals that had been cultivated under Ca(2+)-depletion condition (1 mM CaCl(2)). Our data identify with the carbonic anhydrase and the OSTF the first two molecules which remain conserved in cells, potentially involved in Ca-based skeletal dissolution, from sponges (sclerocytes) to human (osteoclast).

Molecular cloning of silicatein gene from marine sponge Petrosia ficiformis (Porifera, Demospongiae) and development of primmorphs as a model for biosilicification studies.

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.

Origin of metazoan stem cell system in sponges: first approach to establish the model (Suberites domuncula).

It is established that Porifera (sponges) represent the earliest phylum which branched off from the common ancestor of all multicellular animals, the Urmetazoa. In the present study, the hypothesis is tested if, during this transition, pluripotent stem cells were formed which are provided-similar to the totipotent cells (archaeocytes/germ cells)-with a self-renewal capacity. As a model system, primmorphs from the sponge Suberites domuncula were used. These 3D-cell aggregates were cultivated in medium (RPMI 1640/seawater) either lacking silicate and ferric iron or in medium which was supplemented with these 'morphogenetic' factors. As molecular markers for the potential existence of stem cells in primmorphs, two genes which encode proteins found in stem cells of higher metazoan species, were cloned from S. domuncula. First, the noggin gene, which is present in the Spemann organizer of amphibians and whose translation product acts during the formation of dorsal mesoderm derivatives. The second gene encodes the mesenchymal stem cell-like protein. Both cDNAs were used to study their expression in primmorphs in dependence on the incubation conditions. It was found that noggin expression is strongly upregulated in primmorphs kept in the presence of silicate and ferric iron, while the expression of the mesenchymal stem cell-like protein was downregulated. These data are discussed with respect to the existence of stem cells in sponges.

Retinoid X receptor and retinoic acid response in the marine sponge Suberites domuncula.

To date no nuclear receptors have been identified or cloned from the phylogenetically oldest metazoan phylum, the Porifera (sponges). We show that retinoic acid causes tissue regression in intact individuals of the demosponge Suberites domuncula and in primmorphs, special three-dimensional cell aggregates. Primmorphs were cultivated on a galectin/poly-L-lysine matrix in order to induce canal formation. In the presence of 1 or 50 micromol l(-1) retinoic acid these canals undergo regression, a process that is reversible. We also cloned the cDNA from S. domuncula encoding the retinoid X receptor (RXR), which displays the two motifs of nuclear hormone receptors, the ligand-binding and the DNA-binding domains, and performed phylogenetic analyses of this receptor. RXR expression undergoes strong upregulation in response to treatment with retinoic acid, whereas the expression of the sponge caspase is not increased. The gene encoding the LIM homeodomain protein was found to be strongly upregulated in response to retinoic acid treatment. These data indicate that the RXR and its ligand retinoic acid play a role in the control of morphogenetic events in sponges.

Expression of one sponge Iroquois homeobox gene in primmorphs from Suberites domuncula during canal formation.

Sponges (Porifera) represent the evolutionary oldest multicellular animals. They are provided with the basic molecules involved in cell-cell and cell-matrix interactions. We report here the isolation and characterization of a complementary DNA from the sponge Suberites domuncula coding for the sponge homeobox gene, SUBDOIRX-a. The deduced polypeptide with a predicted Mr of 44,375 possesses the highly conserved Iroquois-homeodomain. We applied in situ hybridization to localize Iroquois in the sponge. The expression of this gene is highest in cells adjacent to the canals of the sponge in the medulla region. To study the expression of Iroquois during development, the in vitro primmorph system from S. domuncula was used. During the formation of these three-dimensional aggregates composed of proliferating cells, the expression of Iroquois depends on ferric iron and water current. An increased expression in response to water current is paralleled with the formation of canal-like pores in the primmorphs. It is suggested that Iroquois expression is involved in the formation of the aquiferous system, the canals in sponges and the canal-like structures in primmorphs.

Genomic structure of the sponge, Halichondria okadai calcyphosine gene.

Calcyphosine is an EF-hand Ca(2+)-binding protein, which was first isolated from the canine thyroid. It is phosphorylated in a cyclic AMP (cAMP)-dependent manner; then it is thought to be implicated in the cross-signaling between the cAMP and calcium-phosphatidylinositol cascades. Here, we isolated the DNA complementary to RNA (cDNA) of an EF-hand Ca(2+)-binding protein from the sponge, Halichondria okadai and determined its genomic structure. The deduced sequence of the sponge Ca(2+)-binding protein showed significant similarity (about 40% identity) with those of mammal calcyphosines, and the intron positions were well conserved between the sponge and human calcyphosine genes. We considered that the isolated cDNA was that of sponge calcyphosine, and that sponge and mammalian calcyphosines evolved from a common ancestor gene. Recent cDNA projects have revealed that a calcyphosine cDNA is also expressed by human, mouse, and the ascidia. These cDNAs have more than 60% identity with sponge calcyphosine and each other, and all are composed of 208 amino acid residues. On the constructed phylogenetic trees, calcyphosines are essentially divided into two groups, types-I and -II calcyphosines. Type-I calcyphosine may be specific to mammals, and type-II is widely distributed among metazoan species. This suggests that type-II calcyphosine is a rather ancient gene with some essential function.

Sponge homologs of vertebrate protein tyrosine kinases and frequent domain shufflings in the early evolution of animals before the parazoan-eumetazoan split.

The protein tyrosine kinases (PTKs) diverged specifically in animal lineages by gene duplications and domain shufflings to form a large protein family comprising diverse subfamilies with distinct domain organizations and functions. On the basis of a phylogenetic tree inferred from a comparison of the shared kinase domains, we previously showed that gene duplications that gave rise to diverse subfamilies predate the divergence of parazoans and eumetazoans. There is, however, still a possibility that, although the kinase domain duplications are ancient events, the domain shufflings that gave rise to different subfamilies with distinct domain organization are more recent event than the kinase domain duplications. To clarify this problem, we have determined the complete sequences of 15 sponge PTKs and have compared the domain organizations of these sponge PTKs and those of eumetazoans. For each of ten sponge PTKs out of 15 analyzed here, a possible eumetazoan (human and Drosophila) ortholog has been identified. The sponge and eumetazoan orthologs are virtually identical in domain organization and belong to the same subfamily in the PTK family tree for each of ten orthologous pairs, except for one subfamily in which a considerable deletions and/or insertions of domains are observed. This result suggests that most, if not all, of the domain shufflings, together with gene duplications, are very old, going back to dates before the parazoan-eumetazoan split, the earliest divergence among extant animal phyla.

Structural organization of lower marine nonvertebrate calmodulin genes.

The troponin C (TnC) superfamily genes generally possess five introns, and the positions where they are inserted are well conserved except for the fourth intron. Based on a structural comparison of TnC genes, we proposed that the common ancestor of TnC or TnC superfamily genes had no intron corresponding to the modern fourth intron, and therefore members of the superfamily have gained the fourth intron independently within each lineage. Here, we cloned calmodulin (CaM, one of the members of the TnC superfamily) cDNAs from two lower marine nonvertebrates, the sea anemone, Metridium senile, belonging to the Cnidaria, and the sponge, Halichondria okadai, belonging to the Porifera, and also determined their genomic organization. Chordate CaM genes generally possess five introns, but neither sea anemone nor sponge CaM has anything corresponding to the fourth intron of chordate CaMs, suggesting that the early metazoan CaM must have had only four introns. The modern fourth intron of chordate CaMs was acquired within the chordate lineage after nonvertebrate/chordate divergence. This notion concurs with our proposal explaining the evolution of the TnC superfamily genes.

Identification of highly conserved genes: SNZ and SNO in the marine sponge Suberites domuncula: their gene structure and promoter activity in mammalian cells(1).

Recently, we reported that cells from the sponge Suberites domuncula respond to ethylene with an increase in intracellular Ca(2+) level [Ca(2+)](i), and with an upregulation of the expression of (at least) two genes, a Ca(2+)/calmodulin-dependent protein kinase and the potential ethylene-responsive gene, termed SDSNZERR (A. Krasko, H.C. Schröder, S. Perovic, R. Steffen, M. Kruse, W. Reichert, I.M. Müller, W.E.G. Müller, J. Biol. Chem. 274 (1999)). Here, we describe for the first time that also mammalian (3T3) cells respond to ethylene, generated by ethephon, with an immediate and transient, strong increase in [Ca(2+)](i). Next, the promoter for the sponge SDSNZERR gene was isolated from S. domuncula. It was found that the SDSNZERR gene is positioned adjacent to the SNZ-related gene (SNZ-proximal open reading frame) (SDSNO) and linked, as in Saccharomyces cerevisiae, in a head-to-head manner. Until now, neither homologues nor orthologues of these two genes have been identified in higher metazoan phyla. The full-length genes share a bidirectional promoter. 3T3 cells were transfected with this promoter; the activity of the SDSNZERR promoter was strong and twice as high as that of the SV40 promoter, while the SDSNO promoter was less active. Surprisingly, the activity of the SDSNZERR promoter could not be modulated by ethylene or salicylic acid while it is strongly upregulated, by 4-fold, under serum-starved conditions. It is concluded that the modulation of the level of [Ca(2+)](i) by ethylene in mammalian cells is not correlated with an upregulation of the ethylene-responsive gene SDSNZERR. The data indicate that in mammalian cells, the activity of the SDSNZERR promoter is associated with the repression of serum-mediated growth arrest.

Origin of insulin receptor-like tyrosine kinases in marine sponges.

One autapomorphic character restricted to all Metazoa including Porifera [sponges] is the existence of transmembrane receptor tyrosine kinases (RTKs). In this study we screened for molecules from one subfamily within the superfamily of the insulin receptors. The subfamily includes the insulin receptors (InsR), the insulin-like growth factor I receptors, and the InsR-related receptors--all found in vertebrates--as well as the InsR-homolog from Drosophila melanogaster. cDNAs encoding putative InsRs were isolated from the hexactinellid sponge Aphrocallistes vastus, the demosponge Suberites domuncula, and the calcareous sponge Sycon raphanus. Phylogenetic analyses of the catalytic domains of the putative RTKs showed that the sponge polypeptides must be grouped with the InsRs. The relationships revealed that all sponge sequences fall into one branch of this group, whereas related sequences from mammals (human, mouse, and rat), insects and molluscs, and polypeptides from one cephalochordate, fall together into a second branch. We have concluded that (i) the InsR-like molecules evolved in sponges prior to the "Cambrian Explosion" and contributed to the rapid appearance of the higher metazoan phyla; (ii) the sponges constitute a monophyletic taxon, and (iii) epidermal growth factor (EGF)-like domains are present in sponges, which allows the insertion of this domain into potential receptor and matrix molecules.

Increased expression of integrin and receptor tyrosine kinase genes during autograft fusion in the sponge Geodia cydonium.

Recently cDNAs coding for cell surface molecules have been isolated from sponges. The molecules for alpha-integrin, galectin, and receptor tyrosine kinase (RTK), obtained from the marine sponge, Geodia cydonium, have been described earlier. In the present study also the cDNA for one putative beta-integrin has been identified from G. cydonium. The deduced aa sequence comprises the characteristic signatures, found in other metazoan beta-integrin molecules; the estimated size is 95,215 Da. To obtain first insights into the molecular events which proceed during autograft fusion, the expressions of these genes were determined on transcriptional and translational level. The cDNAs as well as antibodies raised against the recombinant sponge proteins alpha-integrin, RTK and galectin were used and Northern blot experiments and immunocytochemical analyses have been performed. The results show that transcription of the two subunits of an integrin receptor as well as of the RTK are strongly upregulated after grafting; levels of > 10-fold have been determined in the fusion zone of the grafts after a 10 days incubation. Immunofluorescence studies of sections through the fusion zone support these data. In contrast the transcription of the gene encoding galectin is drastically downregulated after grafting. In a parallel series of experiments the level of the heat-shock protein-70 was determined and it was found that it remained unchanged after grafting. We conclude that integrin subunits and the RTK molecule are involved in self-self recognition of sponge.

Evolutionary relationships of Metazoa within the eukaryotes based on molecular data from Porifera.

Recent molecular data provide strong support for the view that all metazoan phyla, including Porifera, are of monophyletic origin. The relationship of Metazoa, including the Porifera, to Plantae, Fungi and unicellular eukaryotes has only rarely been studied by using cDNAs coding for proteins. Sequence data from rDNA suggested a relationship of Porifera to unicellular eukaryotes (choanoflagellates). However, ultrastructural studies of choanocytes did not support these findings. In the present study, we compared amino acid sequences that are found in a variety of metazoans (including sponges) with those of Plantae, Fungi and unicellular eukaryotes, to obtain an answer to this question. We used the four sequences from 70 kDa heat-shock proteins, the serine-threonine kinase domain found in protein kinases, beta-tubulin and calmodulin. The latter two sequences were deduced from cDNAs, isolated from the sponge Geodia cydonium for the phylogenetic analyses presented. These revealed that the sponge molecules were grouped into the same branch as the Metazoa, which is statistically (significantly) separated from those branches that comprise the sequences from Fungi, Plantae and unicellular eukaryotes. From our molecular data it seems evident that the unicellular eukaryotes existed at an earlier stage of evolution, and the Plantae and especially the Fungi and the Metazoa only appeared later.

Phospholipase C cDNAs from sponge and hydra: antiquity of genes involved in the inositol phospholipid signaling pathway.

To know whether or not the set of genes involved in the inositol phospholipid signaling pathway already existed in the early evolution of animals, we carried out cloning of cDNAs encoding phospholipase Cs (PLCs) from Ephydatia fluviatilis (freshwater sponge) and Hydra magnipapillata strain 105 (hydra). We isolated two PLC cDNAs, PLC-betaS and PLC-gammaS, from sponge and three cDNAs, PLC-betaH1, PLC-betaH2, and PLC-deltaH, from hydra. From the domain organization and the divergence pattern in the PLC family tree, the sponge PLC-betaS and PLC-gammaS and the hydra PLC-deltaH are possibly homologous to the vertebrate PLC-beta, PLC-gamma and PLC-delta subtypes, respectively. A detailed phylogenetic analysis suggests that the hydra PLC-betaH1 and PLC-betaH2 are homologs of the vertebrate PLC-beta1/2/3/Drosophila PLC21 and the vertebrate PLC-beta4/Drosophila norpA, respectively. A phylogenetic analysis of the PLC family and the protein kinase C (PKC) family, together with that of the G protein alpha subunit (Galpha) family, revealed that the origin of the set of genes G(alpha)q, PLC, PKC involved in the inositol phospholipid signaling pathway is very old, going back to dates before the parazoan-eumetazoan split, the earliest branching among extant animal phyla.

Characterization and phylogenetic analysis of a cDNA encoding the Fes/FER related, non-receptor protein-tyrosine kinase in the marine sponge sycon raphanus.

In search of ancient versions of phylogenetically conserved genes/proteins, which are typical for multicellular animals, we have decided to analyse marine sponges (Porifera), the most ancient and most primitive metazoan organisms. We report here the complete nucleotide sequence of Sycon raphanus cDNA coding for a 879 aa long protein, which displays high overall similarity in primary structure and organization of domains with non-receptor tyrosine kinases (TKs) from the Fes/FER family. The encoded protein, which we named Fes/FER_SR, has a highly conserved, 260 aa long tyrosine kinase domain at the C-terminus. Amino-terminal to the catalytic domain is an 85 aa long SH2 domain. The N-terminus is over 500 aa long and displays homology only with N-terminal domains of protein-tyrosine kinases (PTKs) from the Fes/FER family. Mammalian Fes/FER proteins show around 58% overall homology with Fes/FER_SR (identity and similarity) and lower homology was found with Drosophila melanogaster Fps (FER) protein (49%). Homologies in TK, SH2 and N-terminal domains are on average 78%, 65% and 49%, respectively. Fes/FER_SR shows next to best homology with the Abl family of non-receptor PTKs, while Src-related PTKs from the fresh-water sponge Spongilla lacustris are related only distantly to Fes/FER_SR. Phylogenetic analysis shows that the S. raphanus TK is indeed the most ancient known member of the Fes/FER family of non-receptor PTKs. The role of these PTKs in signal transduction in higher animals is still enigmatic; they are present in the nucleus as well as in the cytoplasm and FER is found in all cell types examined. The function of Fes/FER_SR in sponge, the most primitive multicellular animal which lacks specialized organ systems, remains to be elucidated.

Phylogenetic position of the Hexactinellida within the phylum Porifera based on the amino acid sequence of the protein kinase C from Rhabdocalyptus dawsoni.

Recent analyses of genes encoding proteins typical for multicellularity, especially adhesion molecules and receptors, favor the conclusion that all metazoan phyla, including the phylum Porifera (sponges), are of monophyletic origin. However, none of these data includes cDNA encoding a protein from the sponge class Hexactinellida. We have now isolated and characterized the cDNA encoding a protein kinase C, belonging to the C subfamily (cPKC), from the hexactinellid sponge Rhabdocalyptus dawsoni. The two conserved regions, the regulatory part with the pseudosubstrate site, the two zinc fingers, and the C2 domain, as well as the catalytic domain were used for phylogenetic analyses. Sequence alignment and construction of a phylogenetic tree from the catalytic domains revealed that the yeast Saccharomyces cerevisiae and the protozoan Trypanosoma brucei are at the base of the tree, while the hexactinellid R. dawsoni branches off first among the metazoan sequences; the other two classes of the Porifera, the Calcarea (the sequence from Sycon raphanus was used) and the Demospongiae (sequences from Geodia cydonium and Suberites domuncula were used), branch off later. The statistically robust tree also shows that the two cPKC sequences from the higher invertebrates Drosophila melanogaster and Lytechinus pictus are most closely related to the calcareous sponge. This finding was also confirmed by comparing the regulatory part of the kinase gene. We suggest, that (i) within the phylum Porifera, the class Hexactinellida diverged first from a common ancestor to the Calcarea and the Demospongiae, which both appeared later, and (ii) the higher invertebrates are more closely related to the calcareous sponges.

Silicatein alpha: cathepsin L-like protein in sponge biosilica.

Earth's biota produces vast quantities of polymerized silica at ambient temperatures and pressures by mechanisms that are not understood. Silica spicules constitute 75% of the dry weight of the sponge Tethya aurantia, making this organism uniquely tractable for analyses of the proteins intimately associated with the biosilica. Each spicule contains a central protein filament, shown by x-ray diffraction to exhibit a highly regular, repeating structure. The protein filaments can be dissociated to yield three similar subunits, named silicatein alpha, beta, and gamma. The molecular weights and amino acid compositions of the three silicateins are similar, suggesting that they are members of a single protein family. The cDNA sequence of silicatein alpha, the most abundant of these subunits, reveals that this protein is highly similar to members of the cathepsin L and papain family of proteases. The cysteine at the active site in the proteases is replaced by serine in silicatein alpha, although the six cysteines that form disulfide bridges in the proteases are conserved. Silicatein alpha also contains unique tandem arrays of multiple hydroxyls. These structural features may help explain the mechanism of biosilicification and the recently discovered activity of the silicateins in promoting the condensation of silica and organically modified siloxane polymers (silicones) from the corresponding silicon alkoxides. They suggest the possibility of a dynamic role of the silicateins in silicification of the sponge spicule and offer the prospect of a new synthetic route to silica and siloxane polymers at low temperature and pressure and neutral pH.

Early evolution of metazoan serine/threonine and tyrosine kinases: identification of selected kinases in marine sponges.

The phylum Porifera (sponges) was the first to diverge from the common ancestor of the Metazoa. In this study, six cDNAs coding for protein-serine/threonine kinases (PS/TKs) are presented; they have been isolated from libraries obtained from the demosponges Geodia cydonium and Suberites domuncula and from the calcareous sponge Sycon raphanus. Sequence alignments of the catalytic domains revealed that two major families of PS/TK, the "conventional" (Ca(2+)-dependent) protein kinase C (PKC), the cPKC subfamily, as well as the "novel" (Ca(2+)-independent) PKC (nPKC), form two separate clusters. In each cluster, the sequence from S. raphanus diverges first. To approach the question about the origin of protein-tyrosine kinases (PTK), which are found only in Metazoa, we analyzed two additional PS/TKs which have been cloned from S. domuncula: the stress-responsive protein kinase (KRSvSD) and the protein-kinase-C-related kinase (PRKvSD). The construction of the phylogenetic tree, comprising the eight PS/TKs and the PTK cloned previously from G. cydonium, revealed that the PTK derived from the branch including the KRSvSD kinase. These data facilitate the first molecular approach to elucidate the origin of metazoan PTK within the PS/TK superfamily.