The chemokine networks in sponges: potential roles in morphogenesis, immunity and stem cell formation.

Porifera (sponges) are now well accepted as the phylum which branched off first from the common ancestor of all metazoans, the Urmetazoa. The transition to the Metazoa became possible because during this phase, cell-cell as well as cell-matrix adhesion molecules evolved which allowed the formation of a colonial stage of animals. The next prerequisite for the evolution to the Urmetazoa was the establishment of an effective immune system which, flanked by apoptosis, allowed the formation of a first level of individuation. In sponges (with the model Suberites domuncula and Geodia cydonium), the main mediators of the immune responses are the chemokines. Since sponges lack a vascular system and consequently blood cells (in the narrow sense), we have used the term chemokines (in a broad sense) to highlight that the complex network of intercellular mediators initiates besides differentiation processes also cell movement. In the present review, the cDNAs encoding the following chemokines were described and the roles of their deduced proteins during self-self and nonself recognition outlined: the allograft inflammatory factor, the glutathione peroxidase, the endothelial-monocyte-activating polypeptide, the pre-B-cell colony-enhancing factor and the myotrophin as well as an enzyme, the (2-5)A synthetase, which is involved in cytokine response in vertebrates. A further step required to reach the evolutionary step of the integrated stage of the Urmetazoa was the acquisition of a stem cell system. In this review, first markers for stem cells (mesenchymal stem cell-like protein) as well as for chemokines involved in the maintenance of stem cells (noggin and glia maturation factor) are described at the molecular level, and a first functional analysis is approached. Taken together, it is outlined that the chemokine network was essential for the establishment of metazoans, which evolved approximately 600 to 800 million years ago.

Analysis of the sponge [Porifera] gene repertoire: implications for the evolution of the metazoan body plan.

Sponges [phylum Porifera] form the basis of the metazoan kingdom and represent the evolutionary earliest phylum still extant. Hence, as living fossils, they are the taxon closest related to the hypothetical ancestor of all Metazoa, the Urmetazoa. Until recently, it was still unclear whether sponges are provided with a defined body plan. Only after the cloning, expression and functional studies of characteristic metazoan genes, could it be demonstrated that these animals comprise the structural elements which allow the sponge cells to organize themselves according to a body plan. Adhesion molecules involved in cell-cell and cell-matrix interactions have been identified. Among the cell-cell adhesion molecules the aggregation factor (AF) is the prominent particle. It is composed of a core protein that is associated with the adhesion molecules, a 36 kDa as well as a 86 kDa polypeptide. A galectin functions as a linker of the AF to the cell-membrane-associated receptor, the aggregation receptor (AR). The most important extracellular matrix molecules are collagen- and fibronectin-like molecules. These proteins interact with the cell-membrane receptors, the integrins. In addition, a neuronal receptor has been identified, which--together with the identified neuroactive molecules--indicate the existence of a primordial neuronal network already in Porifera. The primmorph system, aggregated cells that retain the capacity to proliferate and differentiate, has been used to demonstrate that a homeobox-containing gene, Iroquois, is expressed during canal formation in primmorphs. The formation of a body plan in sponges is supported by skeletal elements, the spicules, which are composed in Demospongiae as well as in Hexactinellida of amorphous, noncrystalline silica. In Demospongiae the spicule formation is under enzymic control of silicatein. Already at least one morphogen has been identified in sponges, myotrophin, which is likely to be involved in the axis formation. Taken together, these elements support the recent conclusions that sponges are not merely nonorganized cell aggregates, but already complex animals provided with a defined body plan.

Molecular evolution of the metazoan extracellular matrix: cloning and expression of structural proteins from the demosponges Suberites domuncula and Geodia cydonium.

One crucial event during evolution to multicellularity was the development of either direct cell-cell contact or indirect interaction via extracellular matrix (ECM) molecules. The identification of those polypeptides provides conclusive data on the phylogenetic relationship of metazoan phyla and helps us to understand the position of the Metazoa among the other kingdoms. Recently it became evident that the ECM of sponges is amazingly complex; it is composed of fibrous molecules, e.g., collagen, and their corresponding receptors, which are highly similar to those existing in other metazoan phyla. While these data already support the view of monophyly of Metazoa, additional studies are required to understand whether these molecules, which are similar in their primary sequence, also have the same function throughout the metazoan kingdom. In the present study we identified the ligand for one of the autopomorphic characters of Metazoa, the single-transmembrane receptor protein with the receptor tyrosine kinase (RTK) from G. cydonium, as an example: the putative mucus-like protein from G. cydonium. This protein was upregulated during autograft fusion in the homologous system with kinetics similar to those of the RTK. Additionally, a cDNA was isolated from S. domuncula whose deduced polypeptide displays a high sequence similarity to dermatopontin, an ECM molecule found exclusively in Metazoa. Furthermore, it is documented that expression of the fibrous ECM molecule collagen is regulated by the characteristic metazoan morphogens myotrophin and endothelial monocyte-activating polypeptide. These data indicate that the ECM of sponges is not an unstructured ground substance but provides the basis for integrated cell communication.

Contribution of sponge genes to unravel the genome of the hypothetical ancestor of Metazoa (Urmetazoa).

Recently the term Urmetazoa, as the hypothetical metazoan ancestor, was introduced to highlight the finding that all metazoan phyla including the Porifera (sponges) are derived from one common ancestor. Sponges as the evolutionarily oldest, still extant phylum, are provided with a complex network of structural and functional molecules. Analyses of sponge genomes from Demospongiae (Suberites domuncula and Geodia cydonium), Calcarea (Sycon raphanus) and Hexactinellida (Aphrocallistes vastus) have contributed also to the reconstruction of the evolutionary position of Metazoa with respect to Fungi. Furthermore, these analyses have provided evidence that the characteristic evolutionary novelties of Metazoa, such as the extracellular matrix molecules, the cell surface receptors, the nervous signal transduction molecules as well as the immune molecule existing in Porifera, share high sequence and in some aspects also functional similarities to related polypeptides found in other metazoan phyla. During the transition to Metazoa new domains occurred; as one example, the formation of the death domain from the ankyrin is outlined. In parallel, domanial proteins have been formed, such as the receptor tyrosine kinases. The metazoan essentials have been defined by analyzing and comparing the sponge sequences with the related sequences from the metazoans Homo sapiens, Caenorhabditis elegans and Drosophila melanogaster, the fungus Saccharomyces cerevisiae and the plant Arabidopsis thaliana. The data revealed that those sponge molecules grouped to cell adhesion cell recognition proteins are predominantly found in Protostomia and Deuterostomia while they are missing in Fungi and Viridiplantae. Moreover, evidence is presented allowing the conclusion that the sponge molecules are more closely related to the corresponding molecules from H. sapiens than to those of C. elegans or D. melanogaster. Especially surprising was the finding that the Demospongiae are provided with elements of adaptive immunity.

Suppression of allograft rejection in the sponge Suberites domuncula by FK506 and expression of genes encoding FK506-binding proteins in allografts.

Porifera (sponges) are, evolutionarily, the oldest metazoan phylum. Recent molecular data suggest that these animals possess molecules similar to and homologous with those of the innate and adaptive immune systems of higher Metazoa. Applying the biological system of parabiosis and the technique of differential display of mRNA, two cDNAs encoding putative FK506-binding proteins were isolated. FK506 is successfully used in clinics as a drug to prevent allograft rejection and is toxic to Suberites domuncula cells in vitro at doses above 100ng ml(-1). Autograft fusion of transplants from S. domuncula was not affected by FK506. Allograft non-fusion was not affected by FK506 at toxic doses; however, at the non-toxic dose of 20ng ml(-1), the allografts fused with each other. It is shown that at the attachment zone in untreated and (particularly drastic) in FK506-treated allografts, expression of the genes encoding the FK506-binding proteins is upregulated. These data indicate that the drug FK506 suppresses allograft rejection in S. domuncula, most probably via interaction with expression of the gene coding for the FK506-binding proteins.

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.

Potential multidrug resistance gene POHL: an ecologically relevant indicator in marine sponges.

Sponges are sessile filter feeders found in all aquatic habitats from the tropics to the arctic. Against potential environmental hazards, they are provided with efficient defense systems, e.g., protecting chaperones and/or the P-170/multidrug resistance pump system. Here we report on a further multidrug resistance pathway that is related to the pad one homologue (POH1) mechanism recently identified in humans. It is suggested that proteolysis is involved in the inactivation of xenobiotics by the POH1 system. Two cDNAs were cloned, one from the demosponge Geodia cydonium and a second from the hexactinellid sponge Aphrocallistes vastus. The cDNA from G. cydonium, termed GCPOHL, encodes a deduced polypeptide with a size of 34,591 Da and that from A. vastus, AVPOHL, a protein of a calculated M(r) of 34,282. The two sponge cDNAs are highly similar to each other as well as to the known sequences from fungi (Schizosaccharomyces pombe and Saccharomyces cerevisiae) and other Metazoa (from Schistosoma mansoni to humans). Under controlled laboratory conditions, the expression of the potential multidrug resistance gene POHL is, in G. cydonium, strongly upregulated in response to the toxins staurosporin (20 microM) or taxol (50 microM); the first detectable transcripts appear after 1 d and reach a maximum after 3 to 5 d of incubation. The relevance of the expression pattern of the G. cydonium gene POHL for the assessment of pollution in the field was determined at differently polluted sites in the area around Rovinj (Croatia; Mediterranean Sea, Adriatic Sea). The load of the selected sites was assessed by measuring the potency of XAD-7 concentrates of water samples taken from those places to induce the level of benzo[a]pyrene monooxygenase (BaPMO) in fish and to impair the multidrug resistance (MDR)/P-170 extrusion pump in clams. These field experiments revealed that the levels of inducible BaPMO activity in fish and of the MDR potential by the water concentrates are highly correlated with the level of expression of the potential multidrug resistance gene POHL in G. cydonium. This report demonstrates that the detoxification POH pathway, here mediated by the G. cydonium GCPOHL gene, is an additional marker for the assessment of the environmental load in a given marine area.

Isolation and cloning of a C-type lectin from the hexactinellid sponge Aphrocallistes vastus: a putative aggregation factor.

Among the sponges (Porifera), the oldest group of metazoans in phylogenetic terms, the Hexactinellida is considered to have diverged earliest from the two other sponge classes, the Demospongiae and Calcarea. The Hexactinellida are unusual among all Metazoa in possessing mostly syncytial rather than cellular tissues. Here we describe the purification of a cell adhesion molecule with a size of 34 kDa (in its native form; 24 kDa after deglycosylation) from the hexactinellid sponge Aphrocallistes vastus. This adhesion molecule was previously found to agglutinate preserved cells and membranes in a non-species-specific manner (Müller, W. E. G., Zahn, R. K, Conrad, J., Kurelec, B., and Uhlenbruck, G. [1984] Cell adhesion molecules in the haxactinellid Aphrocallistes vastus: species-unspecific aggregationfactor. Differentiation, 26, 30--35). The fact that the aggregation process required Ca(2+) and was inhibited by bird's nest glycoprotein and D-galactose but not by D-mannose or N-acetyl-D-galactosamine suggests that this cell adhesion molecule is a C-type lectin. To test this assumption, two highly similar C-type lectins were cloned from A.vastus. The deduced polypeptides of the two cDNA species isolated classified these molecules as C-type lectins. The calculated M(r) of the 191 aa long sequences were 22,022 and 22,064, respectively. The C-type lectins showed highest similarity to C-type lectins (type-II membrane proteins) from higher metazoan phyla; these molecules are absent in non-Metazoa. The two sponge C-type lectins contain the conserved domains known from other C-type lectins (e.g., disulfide bonds, the amino acids known to be involved in Ca(2+)-binding, as well as the amino acids involved in the specificity of binding to D-galactose) and a hydrophobic N-terminal region. The N-terminal part of the purified C-type lectin was identical with the corresponding region of the deduced polypeptide from the cDNA. It is proposed that the A.vastus lectins might bind to the cell membrane by their hydrophobic segment and might interact with carbohydrate units on the surface of the other cells/syncytia.

Stimulation of protein (collagen) synthesis in sponge cells by a cardiac myotrophin-related molecule from Suberites domuncula.

The body wall of sponges (Porifera), the lowest metazoan phylum, is formed by two epithelial cell layers of exopinacocytes and endopinacocytes, both of which are associated with collagen fibrils. Here we show that a myotrophin-like polypeptide from the sponge Suberites domuncula causes the expression of collagen in cells from the same sponge in vitro. The cDNA of the sponge myotrophin was isolated; the potential open reading frame of 360 nt encodes a 120 aa long protein (Mr of 12,837). The sequence SUBDOMYOL shares high similarity with the known metazoan myotrophin sequences. The expression of SUBDOMYOL is low in single cells but high after formation of primmorph aggregates as well as in intact animals. Recombinant myotrophin was found to stimulate protein synthesis by fivefold, as analyzed by incorporation studies using [3H] lysine. In addition, it is shown that after incubation of single cells with myotrophin, the primmorphs show an unusual elongated, oval-shaped appearance. It is demonstrated that in the presence of recombinant myotrophin, the cells up-regulate the expression of the collagen gene. The cDNA for S. domuncula collagen was isolated; the deduced aa sequence shows that the collagenous internal domain is rather short, with only 24 G-x-y collagen triplets. We conclude that the sponge myotrophin causes in homologous cells the same/similar effect as the cardiac myotrophin in mammalian cells, where it is involved in initiation of cardial ventricular hypertrophy. We assume that an understanding of sponge molecular cell biology will also contribute to a further elucidation of human diseases, here of the cardiovascular system.

Sponge homologue to human and yeast gene encoding the longevity assurance polypeptide: differential expression in telomerase-positive and telomerase-negative cells of Suberites domuncula.

Porifera show a characteristic Bauplan in spite of the fact that (almost) all cells are telomerase-positive and presumably provided with an unlimited potency for cell proliferation. Studies revealed that telomerase-positive cells can be triggered to telomerase-negative cells by dissociating them into single cells. Single cells from the demosponge Suberites domuncula, in contrast to cells present in primmorphs (a special form of cell aggregates), lack the property to proliferate and they undergo apoptosis. One gene, SDLAGL, was identified in primmorphs that showed high sequence similarity to the longevity assurance genes from other Metazoa. In single cells no transcripts of SDLAGL could be identified, while high expression was seen after re-aggregation of single cells and in proliferating cells of primmorphs. We concluded that SDLAGL is involved in the shift of telomerase-positive, proliferating cells to telomerase-negative, non-proliferating cells.

Application of cell culture for the production of bioactive compounds from sponges: synthesis of avarol by primmorphs from Dysidea avara.

Among all metazoan phyla, sponges are known to produce the largest number of bioactive compounds. However, until now, only one compound, arabinofuranosyladenine, has been approved for application in humans. One major obstacle is the limited availability of larger quantities of defined sponge starting material. Recently, we introduced the in vitro culture of primmorphs from Suberites domuncula, which contain proliferating cells. Now we have established the primmorph culture also from the marine sponge Dysidea avara and demonstrate that this special form of sponge cell aggregates produces avarol, a sesquiterpenoid hydroquinone, known to display strong cytostatic activity especially against mammalian cells. If dissociated sponge cells are transferred into Ca(2+)- and Mg(2+)-containing seawater, they form after a period of two to three days round-shaped primmorphs (size of 1 to 3 mm). After longer incubation, the globular primmorphs fuse and form meshes of primmorphs that adhere to the bottom of the incubation chamber. Later, during incubation, freely floating mesh-primmorphs are formed. No bacterial rRNA could be detected in the primmorphs. We were able to prove that the primmorphs produce avarol. Levels (1.4 microg of avarol/100 microg of protein) close to those identified in specimens from the field (1.8 microg/100 microg) are reached. Avarol was extracted from the cells with EtOAc and subsequently purified by HPLC. The identification was performed spectrophotometrically and by thin-layer chromatography. Single cells apparently do not have the potency to produce this secondary metabolite. It is concluded that the primmorph model is a suitable system for the synthesis of bioactive compounds in vitro.

Cloning and expression of the sponge longevity gene SDLAGL.

Porifera show a characteristic Bauplan in spite of the fact that (almost) all cells are telomerase-positive and presumably provided with an unlimited potency for cell proliferation. One gene, SDLAGL, was identified in the marine sponge Suberites domuncula whose deduced polypeptide showed high sequence similarity to the longevity assurance genes from other Metazoa. While in single cells no transcripts of SDLAGL could be identified, high expression was seen after re-aggregation of single cells and in proliferating cells of primmorphs.

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.

Our recent data suggest that during auto- and allograft recognition in sponges (Porifera), cytokines are differentially expressed. Since the mitogen-activated protein kinase (MAPK) signal transduction modulates the synthesis and release of cytokines, we intended to identify one key molecule of this pathway. Therefore, a cDNA from the marine sponge Suberites domuncula encoding the MAPK was isolated and analyzed. Its encoded protein is 366 amino acids long (calculated Mr 42 209), has a TGY dual phosphorylation motif in protein kinase subdomain VIII and displays highest overall similarity to the mammalian p38 stress activated protein kinase (SAPK2), one subfamily of MAPKs. The sponge protein was therefore termed p38_SD. The overall homology (identity and similarity) between p38_SD and human p38alpha (CSBP2) kinase is 82%. One feature of the sponge kinase is the absence of threonine at position 106. In human p38alpha MAPK this residue is involved in the interaction with the specific pyridinyl-imidazole inhibitor; T106 is replaced in p38_SD by methionine. Inhibition studies with the respective inhibitor SB 203580 showed that it had no effect on the phosphorylation of the p38 substrate myelin basic protein. A stress responsive kinase Krs_SD similar to mammalian Ste20 kinases, upstream regulators of p38, had already previously been found in S. domuncula. The S. domuncula p38 MAPK is phosphorylated after treatment of the animal in hypertonic medium. In contrast, exposure of cells to hydrogen peroxide, heat shock and ultraviolet light does not cause any phosphorylation of p38. It is concluded that sponges, the oldest and most simple multicellular animals, utilize the conserved p38 MAPK signaling pathway, known to be involved in stress and immune (inflammatory) responses in higher animals.

Expression of silicatein and collagen genes in the marine sponge Suberites domuncula is controlled by silicate and myotrophin.

The major skeletal elements in the (Porifera) sponges, are spicules formed from inorganic material. The spicules in the Demospongiae class are composed of hydrated, amorphous silica. Recently an enzyme, silicatein, which polymerizes alkoxide substrates to silica was described from the sponge Tethya aurantia. In the present study the cDNA encoding silicatein was isolated from the sponge Suberites domuncula. The deduced polypeptide comprises 331 amino acids and has a calculated size of Mr 36 306. This cDNA was used as a probe to study the potential role of silicate on the expression of the silicatein gene. For these studies, primmorphs, a special form of aggregates composed of proliferating cells, have been used. It was found that after increasing the concentration of soluble silicate in the seawater medium from around 1 microM to approximately 60 microM, this gene is strongly upregulated. Without additional silicate only a very weak expression could be measured. Because silica as well as collagen are required for the formation of spicules, the expression of the gene encoding collagen was measured in parallel. It was also found that the level of transcripts for collagen strongly increases in the presence of 60 microM soluble silicate. In addition, it is demonstrated that the expression of collagen is also upregulated in those primmorphs which were treated with recombinant myotrophin obtained from the same sponge. Myotrophin, however, had no effect on the expression of silicatein. From these data we conclude that silicate influences the expression of the enzyme silicatein and also the expression of collagen, (via the mediator myotrophin).

Increased expression of the potential proapoptotic molecule DD2 and increased synthesis of leukotriene B4 during allograft rejection in a marine sponge.

Sponges (Porifera) are a classical model to study the events during tissue transplantation. Applying the 'insertion technique' autografts from the marine sponge Geodia cydonium fuse within 5 days. In contrast, allografts are rejected and destroyed. Here we show that during allograft rejection the cells in the grafts undergo apoptosis; 5 days after transplantation 46% of the cells show signs of apoptosis. In a previous study it was shown that during this process a tumor necrosis factor-like molecule is induced in allo- and xenografts. Molecules grouped to the superfamily of tumor necrosis factor receptors and a series of associated adapter molecules contain the characteristic death domain. Therefore, we screened for a cDNA encoding such a domain. Here we report on the first invertebrate molecule from Geodia cydonium comprising a death domain. The potential proapoptotic molecule DD2, with a calculated Mr of 24 970, possesses in contrast to all known mammalian death domain-containing proteins two such domains with highest similarity to the death domain present in human Fas/APO-1. The expression of this gene is not detectable in control tissue but strongly upregulated in allografts; only very low expression is seen in autografts. Parallel with the increase of the expression of the potential proapoptotic molecule DD2 in allografts the level of LTB4 drastically increases from 2.5 pg/mg of protein (controls) to 389 pg LTB4/mg during a period of 5 days after transplantation; the level of LTB4 in autografts does not change. Very likely in response to inflammatory reactions the LTB4 metabolizing enzyme LTB4 12-hydroxy-dehydrogenase is expressed both in auto- and allografts. These results demonstrate that sponges are provided with apoptotic pathways, similar to those present in deuterostomes and apparently absent in protostomes, which are composed of molecules comprising a death domain. In addition, it is suggested that in sponges LTB4 is one metabolite which is involved in the initiation of apoptosis. It is postulated that the potential proapoptotic effect of LTB4 is prevented in auto-grafts by the expression of the LTB4 12-hydroxy-dehydrogenase.

Evolution of the innate and adaptive immune systems: relationships between potential immune molecules in the lowest metazoan phylum (Porifera) and those in vertebrates.

Porifera (sponge) form the lowest metazoan phylum and share a common ancestor with other metazoan phyla. In the present study, it is reported that sponges possess molecules that are similar in structure to those molecules involved in the immune system in mammals. Experiments with the marine sponges Geodia cydonium and Suberites domuncula have been performed on tissue (auto- and allografting) as well as on a cellular level. The studies revealed that sponges are provided with elements of the mammalian innate immune system, such as molecules containing scavenger receptor cysteine-rich domains. Furthermore, macrophage-derived cytokine-like molecules have been identified that are up-regulated during the grafting process. In addition, the (2'-5')oligoadenylate synthetase system exists in sponges. "Precursors" of the second type of immune response in mammals, the adaptive immune system, have been traced in sponges. It is shown that the expression of a lymphocyte-derived cytokine from mammals is up-regulated during non-self-recognition in S. domuncula. Finally, in G. cydonium, two classes of receptors that comprise Ig-like domains have been identified: the receptor tyrosine kinases and the non-enzymic sponge adhesion molecules. They contain two polymorphic Ig-like domains that are grouped to the variable set of immunoglobulins. The expression of these molecules is also up-regulated during the grafting process. It is concluded that sponges are already provided with a series of elements used in higher vertebrates for both the innate and the adaptive immune recognition.

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.

Gene structure and function of tyrosine kinases in the marine sponge Geodia cydonium: autapomorphic characters in Metazoa.

Porifera (sponges) represent the most ancient, extant metazoan phylum. They existed already prior to the 'Cambrian Explosion'. Based on the analysis of aa sequences of informative proteins, it is highly likely that all metazoan phyla evolved from only one common ancestor (monophyletic origin). As 'autapomorphic' proteins which are restricted to Metazoa only, integrin receptors, receptors with scavenger receptor cysteine-rich repeats, neuronal-like receptors and protein-tyrosine kinases (PTKs) have been identified in Porifera. From the marine sponge Geodia cydonium, a receptor tyrosine kinase (RTK) has been cloned that comprises the characteristic structural topology known from other metazoan RTKs; an extracellular domain, the transmembrane region, the juxtamembrane region and the TK domain. Only two introns, within the coding region of the RTK gene, could be found, which separate the two highly polymorphic immunoglobulin-like domains, found in the extracellular region of the enzyme. The functional role of this sponge RTK could be demonstrated both in situ (grafting experiments) and in vitro (increase of intracellular Ca2+ level). Upstream of this RTK gene, two further genes coding for tyrosine kinases (TK) have been identified. Both are intron-free. The deduced aa sequence of the first gene shows no transmembrane segment; from the second gene--so far--only half of its catalytic domain is known. A phylogenetic analysis with the TK domains from these sequences and a fourth, from a novel scavenger RTK (all domains comprise the signature for the TK class II receptors), showed that they are distantly related to the insulin and insulin-like receptors. The presented findings support the 'introns-late' hypothesis for such genes that encode 'metazoan' proteins. It is proposed that the TKs evolved from protein-serine/threonine kinases through modularization and subsequent exon shuffling. After formation of the ancestral TKs, the modules lost the framing introns to protect the evolutionary novelty. Since cell culture systems of sponges are now available, it can be expected that soon also those mechanisms that control the developmental programs will be unravelled.

Differential expression of allograft inflammatory factor 1 and of glutathione peroxidase during auto- and allograft response in marine sponges.

Very recently, Porifera (sponges) have been proven to be suitable model systems to study auto- and allograft recognition at the molecular level. Several potential immune molecules have been isolated from the marine sponges Suberites domuncula and Geodia cydonium, among them those which comprise Ig-like domains in their extracellular part. Here we report on the isolation of two cDNAs from S. domuncula that code for molecules involved in mammals in cytokine-mediated graft response; a putative allograft inflammatory factor 1 (AIF-1) and a non-selenium glutathione peroxidase (GPX). Both polypeptides share high similarity with the corresponding mammalian proteins. The expression of the two genes during auto- and allograft recognition in S. domuncula and G. cydonium was determined. It is shown that the expression of the AIF-1-related gene is upregulated only in allografts, while the GPX-related gene is expressed in the fusion zones formed between auto- as well as allografts. Taken together, these findings suggest that besides cell-mediated defense reactions a cytokine-dependent immune response is also elicited during graft recognition in sponges.

Ethylene modulates gene expression in cells of the marine sponge Suberites domuncula and reduces the degree of apoptosis.

Sponges (phylum Porifera) live in an aqueous milieu that contains dissolved organic carbon. This is degraded photochemically by ultraviolet radiation to alkenes, particularly to ethylene. This study demonstrates that sponge cells (here the demosponge Suberites domuncula has been used), which have assembled to primmorphs, react to 5 microM ethylene with a significant up-regulation of intracellular Ca(2+) concentration and with a reduction of starvation-induced apoptosis. In primmorphs from S. domuncula the expression of two genes is up-regulated after exposure to ethylene. The cDNA of the first gene (SDERR) isolated from S. domuncula encodes a potential ethylene-responsive protein, termed ERR_SUBDO; its putative M(r) is 32,704. Data bank search revealed that the sponge polypeptide shares high similarity (82% on amino acid level) with the corresponding plant molecule, the ethylene-inducible protein from Hevea brasiliensis. Until now no other metazoan ethylene-responsive proteins have been identified. The second gene, whose expression is up-regulated in response to ethylene is a Ca(2+)/calmodulin-dependent protein kinase II. Its cDNA, SDCCdPK, encodes a M(r) 54,863 putative kinase that shares 69% similarity with the corresponding enzyme from Drosophila melanogaster. The expression of both genes in primmorphs from S. domuncula is increased by approximately 5-fold after a 3-day incubation period with ethylene. It is concluded that also metazoan cells, with sponge cells as a model, may react to ethylene with an activation of cell metabolism including gene induction.