The cDNA for the full-length lectin from the marine sponge Geodia cydonium was cloned. Analysis of the deduced aa sequence revealed that this lectin belongs to the group of galectins. The full-length galectin, which was obtained also in a recombinant form, has an M(r) of 20,877; in the processed form it is a 15 kDa polypeptide. The enriched aggregation factor from G.cydonium also was determined to contain, besides minimal amounts of the galectin, a 140 kDa polypeptide which is involved in cell-cell adhesion. Monoclonal antibodies have been raised against this protein; Fab' fragments prepared from them abolished cell-cell reaggregation. Cell reaggregation experiments revealed that the aggregation factor is an essential component in the aggregation process but it requires likewise the presence of the galectin. Antibodies against the galectin blocked the aggregation factor-mediated cell adhesion. A plasma membrane component was identified (a 29 kDa polypeptide) which interacted with the aggregation factor in the presence of galectin; binding could be blocked both by antibodies against the galectin as well as against the aggregation factor. Immunohistochemical analysis revealed that spherulous cells contain the galectin but not the aggregation factor. By laser scanning microscopy, it is shown that both the aggregation factor and the galectin are located at the rim of the cells. From these data we conclude that the G.cydonium aggregation factor binds to the cells via a galectin bridge.

A cDNA encoding a receptor tyrosine kinase (RTK) was previously cloned and expressed from the marine sponge (Porifera) Geodia cydonium. In addition to the two intracellular regions characteristic for RTKs, two immunoglobulin (Ig)-like domains are found in the extracellular part of the sponge RTK. In the present study it is shown that no further Ig-like domain is present in the upstream region of the cDNA as well as of the gene hitherto known from the sponge RTK. Two different full-length cDNAs have been isolated and characterized in the present study, which possess two Ig-like domains, one transmembrane segment, and only a short intracellular part, without a TK domain. The two deduced polypeptides were preliminarily termed sponge adhesion molecules (SAM). The longer form of the SAM, GCSAML, encodes a deduced aa sequence, GCSAML, which comprises in the open reading frame 505 amino acids (aa) and has a calculated Mr of 53911. The short form, GCSAMS, has 313 aa residues and an Mr of 33987. The two Ig-like domains in GCSAML and GCSAMS are highly similar to the corresponding Ig-like domains in the RTKs from G. cydonium; the substitutions on both the aa and nt level are restricted to a few sites. Phylogenetic analyses revealed that the Ig-like domain 1 is similar to the human Ig lambda chain variable region, while the Ig-like domain 2 is related more closely to the human Ig heavy chain variable region. Transplantation experiments (autografting) were performed to demonstrate that the level of expression of the two new genes, GCSAML and GCSAMS, is upregulated during the self/self fusion process. Immunohistochemical analyses using antibodies raised against the two Ig-like domains demonstrate a strong expression in the fusion zone between graft and host. This finding has been supported by northern blotting experiments that revealed that especially GCSAML is strongly upregulated after autografting (up to 12-fold); the expression of GCSAMS reaches a value of 5-fold if compared with the controls. The results presented here demonstrate that the expression of the new molecules described, comprising two Ig-like domains, is upregulated during the process of autograft fusion.

Sponges (Porifera) representing the simplest metazoan phylum so far have been thought to possess no basal lamina tissue structures. One major extracellular matrix protein that is also a constitutive glycoprotein of the basal lamina is fibronectin. It was the aim of the present study to identify the native protein from the marine sponge Geodia cydonium and to isolate the corresponding cDNA. In crude extracts from this sponge protein(s) of M(r) of approximately 230 and approximately 210 kDa could be visualized by Western-blotting using an anti-fibronectin [human] antibody. By PCR cloning from a cDNA library of G. cydonium we isolated a cDNA comprising one element of fibronectin, the type-III (FN3) module. The cDNA (2.3 kb long), encoding a 701 amino acid [aa] long putative "multiadhesive protein" termed MAP_GEOCY, was found to contain (i) a fibronectin-, (ii) a scavenger receptor cysteine-rich [SRCR]-, and (iii) a short consensus repeat [SCR] module. The 89 aa long fibronectin module comprises the characteristic topology and conserved aa found in fibronectin type-III (FN3) elements. The SRCR module (101 aa) features the characteristics of group B SRCR molecules. The predominant proteins belonging to this group are the mammalian WC1-, M130-, CD6- and CD5 antigens that probably are involved in immunological reactions. The SCR module (54 aa) shows the characteristics of type III SCR modules found in complement receptors. Phylogenetic analyses performed with all three building blocks of the "multiadhesive protein" showed that the respective sponge modules form independent, possibly basal, lineages in trees that include the corresponding modules from higher metazoan animals. In summary, these data demonstrate for the first time that the phylogenetically oldest Metazoa, the sponges, contain protein modules seen in higher animals in proteins of the extracellular matrix and in molecules involved in cell-mediated immune reactions in vertebrates.

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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 M r of 24 970, possesses in contrast to all known mammalian death domaincontaining 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 LTB 4 drastically increases from 2.5 pg/mg of protein (controls) to 389 pg LTB 4 /mg during a period of 5 days after transplantation; the level of LTB 4 in autografts does not change. Very likely in response to inflammatory reactions the LTB 4 metabolizing enzyme LTB 4 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 LTB 4 is one metabolite which is involved in the initiation of apoptosis. It is postulated that the potential proapoptotic effect of LTB 4 is prevented in autografts by the expression of the LTB 4 12-hydroxy-dehydrogenase. Cell Death and Differentiation (2000) 7, 461 ± 469.