Regulation of motility of cells from marine sponges by calcium ions

Sponges are known not to contain muscle and nerve cells. Since sponge cells are characterized by high motility we determined the effect of intracellular calcium ion concentration ([Ca2+]i) on their motility. Addition of the Ca2+ ionophore ionomycin to dissociated cells from the marine sponge Suberites domuncula caused in Ca(2+)-containing artificial seawater (ASW) an increase in motility from 0.2 micron/min (absence of the ionophore) to 3.7 microns/min (presence of ionomycin). When the experiments were performed in Ca(2+)-free medium, no effect of ionomycin could be observed. In parallel experiments the changes of [Ca2+]i using the dye Fura-2 were measured. The experiments revealed that ion…

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

Abstract 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 (100 kDa), 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 doma…

Bauplan of Urmetazoa: Basis for Genetic Complexity of Metazoa

Sponges were first grouped to the animal-plants or plant-animals then to the Zoophyta or Mesozoa and finally to the Parazoa. Only after the application of molecular biological techniques was it possible to place the Porifera monophyletically with the other metazoan phyla, justifying a unification of all multicellular animals to only one kingdom, the Metazoa. The first strong support came from the discovery that cell-cell and cell-matrix adhesion molecules that were cloned from sponges and were subsequently expressed share a high DNA sequence and protein function similarity with the corresponding molecules of other metazoans. Besides these evolutionary novelties for Metazoa, sponges also hav…

The complete set of ribosomal proteins from the marine sponge Suberites domuncula

The siliceous marine sponge Suberites domuncula is a member of the most ancient and simplest extant phylum of multicellular animals-Porifera, which have branched off first from the common ancestor of all Metazoa. We have determined primary structures of 79 ribosomal proteins (r-proteins) from S. domuncula: 32 proteins from the small ribosomal subunit and 47 proteins from the large ribosomal subunit. Only L39 and L41 polypeptides (51 and 25 residues long in rat, respectively) are missing. The sponge S. domuncula is, after nematode Caenorhabditis elegans and insect Drosophila melanogaster the third representative of invertebrates with known amino acid sequences of all r-proteins. The comparis…

Bruton tyrosine kinase-like protein, BtkSD, is present in the marine sponge Suberites domuncula.

Sponges, the simplest and most ancient phylum of Metazoa, encode in their genome complex and highly sophisticated proteins that evolved together with multicellularity and are found only in metazoan animals. We report here the finding of a Bruton tyrosine kinase (BTK)-like protein in the marine sponge Suberites domuncula (Demospongiae). The nucleotide sequence of one sponge cDNA predicts a 700-aa-long protein, which contains all of the characteristic domains for the Tec family of protein tyrosine kinases (PTKs). The highest homology (38% identity, 55% overall similarity) was found with human BTK and TEC PTKs. Sponge PTK was therefore named BtkSD. Human BTK is involved in the maturation of B …

Background Ethylene is a widely distributed alkene product which is formed enzymatically (e.g., in plants) or by photochemical reactions (e.g., in the upper oceanic layers from dissolved organic carbon). This gaseous compound was recently found to induce in cells from the marine sponge Suberites domuncula, an increase in intracellular Ca2+ level ([Ca2+]i) and an upregulation of the expression of two genes, the potential ethylene-responsive gene, SDERR, and a Ca2+/calmodulin-dependent protein kinase.

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 w…

Ubiquitin and ubiquitination in cells from the marine sponge Geodia cydonium.

Marine sponges, e.g. Geodia cydonium, have been intensively used to investigate the biochemical and molecular biological basis of cell-cell- and cell-matrix adhesion. It has been shown that a family of galactose-specific lectins, which are present in the extracellular space of G. cydonium, is a main component involved in cell-matrix adhesion in the sponge system. In the present study it is outlined that the purified 16-kDa lectin-1 binds to a 67-kDa membrane-associated protein. This lectin-binding protein undergoes mono- and diubiquitination after incubation of dissociated sponge cells with the homologous aggregation factor (AF), a molecule involved in cell-cell adhesion. The gene coding fo…

Phylogenetic relationship of ubiquitin repeats in the polyubiquitin gene from the marine sponge Geodia cydonium

Ubiquitin is a 76-residue protein which is highly conserved among eukaryotes. Sponge (Porifera) ubiquitin, isolated from Geodia cydonium, is encoded by a gene (termed GCUBI) with six repeats, GCUBI-1 to GCUBI-6. All repeat units encode the same protein (with one exception: GCUBI-4 encodes ubiquitin with a change of Leu to Val at position 71). On the nt level the sequences of the six repeats differ considerably. All changes (except in GCUBI-4) are silent substitutions, which do not affect the protein structure. However, there is one major difference between the repeats: Codons from both codon families (TCN and AGPy) are simultaneously used for the serine at position 65. Using this characteri…

Molecular evolution of the metazoan protein kinase C multigene family

Protein kinases C (PKCs) comprise closely related Ser/Thr kinases, ubiquitously present in animal tissues ; they respond to second messengers, e.g., Ca2+ and/or diacylglycerol, to express their activities. Two PKCs have been sequenced from Geodia cydonium, a member of the lowest multicellular animals, the sponges (Porifera). One sponge G. cydonium PKC, GCPKC1, belongs to the ''novel'' (Ca2+-independent) PKC (nPKC) subfamily while the second one, GCPKC2, has the hall-marks of the ''conventional'' (Ca2+-dependent) PKC (cPKC) subfamily. The alignment of the Ser/Thr catalytic kinase domains, of the predicted aa sequences for these cDNAs with respective segments from previously reported sequence…

Immunoglobulin-like domain is present in the extracellular part of the receptor tyrosine kinase from the marine sponge Geodia cydonium.

We have isolated and characterized two cDNAs from the marine sponge Geodia cydonium coding for a new member of a receptor tyrosine kinase of class II. The deduced amino acid sequence shows two characteristic domains: (i) the tyrosine kinase domain; and (ii) and immunoglobulin-like domain. The latter part shows high homology to the vertebrate C2 type immunoglobulin domain. This result demonstrates that immunoglobulin domains are not recent achievements of higher animals but exist also in those animals which have diverged from other organisms about 800 million years ago.

Sponge proteins are more similar to those of Homo sapiens than to Caenorhabditis elegans

We compared 42 phylogenetically conserved proteins from four marine sponges [Porifera] with almost the complete set of Caenorhabditis elegans proteins and all known proteins from humans. The majority of the sponge proteins are significantly more similar to human than to C. elegans orthologues/homologues. This finding reflects the accelerated evolutionary rate in the C. elegans lineage, since sponges split off first from the common ancestor of all multicellular animals. Furthermore, three sponge/human proteins were not found in C. elegans: (2–5)A synthetase, DNA repair helicase and lens βγ -crystallin. Sponges are the source of the most ancient proteins already present in the common ancestor…

Evolution of osmosensing signal transduction in Metazoa: stress-activated protein kinases p38 and JNK.

Sponges (Porifera) represent the most basal branch of the Metazoa alive today. We show that two central stress-activated protein kinases involved in the osmosensing pathway, p38 mitogen-activated protein kinase (MAPK) and JNK, can complement for the ancestral MAPK Hog1 in the yeast Saccharomyces cerevisiae. S. cerevisiae mutants lacking Hog1 (hog1-Delta 1) have been complemented with the sponge SDJNK and SDp38 genes. Western blotting has revealed that, after transformation, the hog1-Delta 1+ SDJNK(sense) and hog1-Delta 1+ SDp38(sense) clones express the sponge proteins. Functional studies have demonstrated that the complemented clones grow under hyperosmotic conditions (0.6 M NaCl). Further…

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

Abstract 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. Schroder, S. Perovic, R. Steffen, M. Kruse, W. Reichert, I.M. Muller, W.E.G. Muller, 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 …

Protein SRP54 iz morske spužve Geodia cydonium

In the systematic search for phylogenetically conserved proteins in the simplest and most ancient extant metazoan phylum – Porifera, we have identified and analyzed a cDNA encoding the signal recognition particle 54 kD protein (SRP54) from the marine sponge Geodia cydonium (Demospongiae). The signal recognition particle (SRP) is a universally conserved ribonucleoprotein complex of a very ancient origin, comprising SRP RNA and several proteins (six in mammals). The nucleotide sequence of the sponge cDNA predicts a protein of 499 amino acid residues with a calculated Mr of 55175. G. cydonium SRP54 displays unusually high overall similarity (90 %) with human/mammalian SRP54 proteins, higher th…

Emergence and Disappearance of an Immune Molecule, an Antimicrobial Lectin, in Basal Metazoa

Sponges (phylum Porifera) represent the evolutionarily oldest metazoans that comprise already a complex immune system and are related to the crown taxa of the protostomians and the deuterostomians. Here, we demonstrate the existence of a tachylectin-related protein in the demosponge Suberites domuncula, termed Suberites lectin. The MAPK pathway was activated in response to lipopolysaccharide treatment of the three-dimensional cell aggregates, the primmorphs; this process was abolished by the monosaccharide D-GlcNAc. The cDNA encoding the S. domuncula lectin was identified and cloned; it comprises 238 amino acids (26 kDa) in the open reading frame. The deduced protein has one potential trans…

Intracellular signal transduction pathways in sponges.

Abstract Sponges are the lowest multicellular eukaryotic organisms. Due to the relatively low specialization, and concomitantly the high differentiation and dedifferentiation potency of their cells, the sponge cell system has proven to be a useful model to study the mechanism of cell-cell adhesion on molecular levels. Results of detailed biochemical and cell biological studies with the main cell adhesion molecules, the aggregation factor (AF) and the aggregation receptor, led to the formation of the modulation theory of cell adhesion. The events of cell adhesion are contigent on a multiplicity of precisely coordinated intracellular signal transduction pathways. Using the marine sponge Geodi…

S-type lectins occur also in invertebrates: high conservation of the carbohydrate recognition domain in the lectin genes from the marine sponge Geodia cydonium.

The marine sponge Geodia cydonium contains several lectins. The main component, called lectin-1, is composed of three to four identical subunits. The subunits of the lectins were cloned from a cDNA library; two clones were obtained. From the deduced aa sequence of one clone, LECT-1, a mol. wt of 15,313 Da is calculated; this value is in good agreement with mass spectrometric analysis of 15,453 +/- 25 Da. The sequence of another clone, LECT-2, was analysed and the aa sequence was deduced (15,433 Da). The two subunits have a framework sequence of 38 conserved aa which are characteristic for the carbohydrate-binding site of vertebrate S-type lectins. Clustering of lectin sequences of various s…

Src proteins/src genes: from sponges to mammals

The genome of marine sponge Suberites domuncula, a member of the most ancient and most simple metazoan phylum Porifera, encodes at least five genes for Src-type proteins, more than, i.e., Caenorhabditis elegans or Drosophila melanogaster (two in each). Three proteins, SRC1SD, SRC2SD and SRC3SD, were fully characterized. The overall homology (identity+similarity) among the three S. domuncula Srcs (68-71%) is much lower than the sequence conservation between orthologous Src proteins from freshwater sponges (82-85%). It is therefore very likely that several src genes/proteins were already present in the genome of Urmetazoa, the hypothetical metazoan ancestor. We have identified in the S. domun…

Experimental indication in favor of the introns-late theory: the receptor tyrosine kinase gene from the sponge Geodia cydonium.

Abstract We have analyzed the gene that encodes receptor tyrosine kinase (RTK) from the marine sponge Geodia cydonium, which belongs to the most ancient and simple metazoan groups, the Porifera. RTKs are enzymes found only in metazoa. The sponge gene contains two introns in the extracellular part of the protein. However, the rest of the protein (transmembrane and intracellular part), including the tyrosine kinase (TK)-domain, is encoded by a single exon. In contrast, all TK genes, so far known only from higher animals (vertebrates), contain several introns especially in the TK-domain. The TK-domain of G. cydonium shows similarity with numerous members of receptor as well as nonreceptor TKs.…

A (1->3)-beta-D-glucan recognition protein from the sponge Suberites domuncula. Mediated activation of fibrinogen-like protein and epidermal growth factor gene expression

Sponges (phylum Porifera) live in a symbiotic relationship with microorganisms, primarily bacteria. Until now, molecular proof for the capacity of sponges to recognize fungi in the surrounding aqueous milieu has not been available. Here we demonstrate, for the demosponge Suberites domuncula (Porifera, Demospongiae, Hadromerida), a cell surface receptor that recognizes (1--3)-beta-D-glucans, e.g. curdlan or laminarin. This receptor, the (1--3)-beta-D-glucan-binding protein, was identified and its cDNA analysed. The gene coding for the 45 kDa protein was found to be upregulated in tissue after incubation with carbohydrate. Simultaneously with the increased expression of this gene, two further…

Mitochondrial genome of Suberites domuncula: palindromes and inverted repeats are abundant in non-coding regions.

The 26,300-nucleotide sequence of the mitochondrial DNA (mtDNA) molecule of the demosponge Suberites domuncula (Olivi, 1792), the largest in size yet found in Porifera, has been determined. We describe the second hadromerid sponge mitochondrial genome that contains the same set of 41 genes as the hadromerid sponge Tethya actinia, including trnMe(cau), trnI2(cau), trnR2(ucu), and atp9, all of which are transcribed in the same direction. Furthermore, rRNA genes for the small and large ribosomal subunit are very long, rns is indeed the longest among Metazoa (1833 bp). Intergenic regions (IGR) comprise about 25% of S. domuncula mtDNA and include numerous direct and inverted repeats, as well as …

Regulated expression and phosphorylation of the 23-26-kDa ras protein in the sponge Geodia cydonium.

We have cloned, sequenced and examined the sponge Geodia cydonium cDNA encoding a protein homologous to ras proteins. The sponge ras protein has a more conserved N-terminal region and a less conserved C-terminal region, especially in comparison to Dictyostelium discoideum; the similarity to human c-Ha-ras-1 and to Saccharomyces cerevisiae is less pronounced. The sponge ras cDNA comprises five TAG triplets; at the translational level these UAG termination codons are suppressed by a Gln-tRNA. The sponge ras protein was isolated and partially purified (23-26 kDa) and found to undergo phosphorylation at a threonine moiety, when dissociated cells were incubated in the presence of a homologous ag…

Conservation of the positions of metazoan introns from sponges to humans

Abstract Sponges (phylum Porifera) are the phylogenetic oldest Metazoa still extant. They can be considered as reference animals (Urmetazoa) for the understanding of the evolutionary processes resulting in the creation of Metazoa in general and also for the metazoan gene organization in particular. In the marine sponge Suberites domuncula , genes encoding p38 and JNK kinases contain nine and twelve introns, respectively. Eight introns in both genes share the same positions and the identical phases. One p38 intron slipped for six bases and the JNK gene has three more introns. However, the sequences of the introns are not conserved and the introns in JNK gene are generally much longer. Intron…

Molecular markers for germ cell differentiation in the demosponge Suberites domuncula

Sponges (phylum Porifera) are simple metazoans for which no molecular information on gametogenesis and larval development is available. To support the current study, it was confirmed by histology that oocytes and larvae were produced by the demosponge Suberites domuncula. Three genes/expressed products from S. domuncula whose expression correlated with sexual reproduction were identified and characterized (they are used here as marker genes): i) a receptor tyrosine kinase (RTK) with sequence similarity in the tyrosine kinase domain to fibroblast growth factor receptors; ii) the sex-determining protein FEM1 and iii) the sperm associated antigen (SAA) of triploblasts. Antibodies against the e…

Ubiquitins (polyubiquitin and ubiquitin extension protein) in marine sponges: cDNA sequence and phylogenetic analysis

The complete nucleotide sequences of twoSuberites domunculacDNAs and oneSycon raphanuscDNA, all encoding ubiquitin, have been determined. One cDNA fromS. domunculacodes for polyubiquitin with four tandemly repeated monomeric units and the second cDNA encodes ubiquitin fused to a ribosomal protein of 78 amino acids (aa).S. domunculapossesses at least one additional polyubiquitin gene, from which the last two monomers were also sequenced. All analysed genes fromS. domunculaencode identical ubiquitin proteins, with only one aa difference (Ala19) to the human/higher animals ubiquitin (Pro19). Ubiquitin inS. domunculais identical with the ubiquitin found in another Demospongia,Geodia cydonium. T…