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.

Recent studies have shown that the Porifera, with the examples of the demosponges Suberites domuncula and Geodia cydonium, comprise a series of pathways found also in the immune system of Deuterostomia, such as vertebrates, but are absent in Protostomia, with insects or nematodes as examples. One pathway is the (2¢)5¢)oligoadenylate synthetase [(2-5)A synthetase] system. In the present study we show that crude extracts from tissue of S. domuncula collected from the sea display a considerable amount of (2-5)A synthetase activity; 16% of the ATP substrate is converted to the (2-5)A product, while tissue from specimens which were kept for 6 months in an aquarium shows only 1% of conversion. As aquarium animals show a lower bacterial load, those specimens were treated for the experiments with the bacterial endotoxin lipopolysaccharide (LPS); they responded to LPS with a stimulation of the (2-5)A synthetase activity. To monitor if this effect can be obtained also on the in vitro level, primmorphs which comprise proliferating and differentiating cells, were incubated with LPS. Extracts obtained from LPS-treated primmorphs also convert ATP to the (2-5)A products mediated by the synthetase. In parallel to this effect on protein level, LPS causes after an incubation period of 12 h also an increase in the steady-state level of the transcripts encoding the putative (2-5)A synthetase. It is postulated that in sponges the (2-5)A synthetase is involved in antimicrobial defense of the animals.

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.

Species belonging to the lowest metazoan phylum, the sponges (Porifera), exhibit a surprisingly complex and multifaceted Bauplan (body plan). Recently, key molecules have been isolated from sponges which demonstrate that the cells of these animals are provided with characteristic metazoan adhesion and signal transduction molecules, allowing tissue formation. In order to understand which factors control the spatial organization of these cells in the sponge body plan, we screened for a cDNA encoding a soluble modulator of the behaviour of endothelial cells. A cDNA encoding a putative protein, which is highly similar to the human and mouse endothelial monocyte-activating polypeptide (EMAP) II has been isolated from a library of the marine sponge Geodia cydonium. The sponge EMAP-related polypeptide (EMAPR) has been termed EMAPR1_GC. The full-length cDNA clone, GCEMAPR1, has a size of 592 nucleotides (nt) and contains a 447 nt-long potential open reading frame; the molecular weight (MW) of the deduced amino acid sequence, 16,499 Da, is close to that of mature mammalian EMAP II (ca. 18 kDa). The sponge polypeptide is also closely related to a deduced polypeptide from the cosmid clone F58B3 isolated from Caenorhabditis elegans. A phylogenetic analysis revealed that the sponge and the nematode EMAPR molecules form a cluster which is significantly separated from the corresponding mammalian EMAP molecules. The function of the first cloned putative soluble modulator of endothelial cells in sponges remains to be determined.

Sponges (phylum Porifera) have extensively been used as a model system to study cell-cell interaction on molecular level. Recently, we identified and cloned the putative aggregation receptor (AR) of the sponge Geodia cydonium, which interacts in a heterophilic way with the aggregation factor (AF) complex. In the present study, antibodies against this complex have been raised that abolish the adhesion function of the enriched sponge AF, the AF-Fraction 6B. Using this antibody as a tool, a complete 1.7 kb long cDNA, GEOCYAF, could be isolated from a cDNA library that encodes the putative AF. Its deduced aa sequence in the N-terminal section comprises high similarity to amphiphysin/BIN1 sequences found in Protostomia and Deuterostomia. However, the C-terminal portion of the sponge sequence lacks the SH3 domain characteristic for amphiphysin/BIN1. The polypeptide with a calculated size of 47 kDa was expressed in Escherichia coli. The recombinant, soluble 36 kDa putative AF was prepared and found to compete with the AF complex-associated adhesion protein of the AF-Fraction 6B for the binding sites at the cell surface. Furthermore, the recombinant putative AF was recognized by the antibody used to screen the cDNA library by western blotting. In addition, there is evidence that the recombinant putative AF binds to the G. cydonium galectin. It is concluded that the putative G. cydonium AF - a further autapomorphic molecule characteristic for Metazoa - binds to the AR present on the cell surface in association with the homologous galectin.