Experimental Indication in Favor of the Introns-Late Theory: The Receptor Tyrosine Kinase Gene from the Sponge Geodia cydonium

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. Phylogenetic analysis of the sponge TK-domain indicates that this enzyme branched off first from the common tree of metazoan TK proteins. Consequently, we assume that introns, found in the TK-domains of genes from higher animals, were inserted into these genes after splitting off the sponge taxa from other metazoan organisms (over 600 million years ago). Our results support the view that ancient genes were not ``in pieces.' Received: 8 August 1996 / Accepted: 4 November 1996     

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.     

Receptor tyrosine kinase, an autapomorphic character of metazoa: identification in marine sponges

In the present review we summarize sequence data obtained from cloning of sponge receptor tyrosine kinases [RTK]. The cDNA sequences were mainly obtained from the marine sponge Geodia cydonium. RTKs (i) with immunoglobulin [Ig]-like domains in the extracellular region, (ii) of the type of insulin-like receptors, as well as (iii) RTKs with one extracellular speract domain, have been identified. The analyses revealed that the RTK genes are constructed in blocks [domains], suggesting a blockwise evolution. The phylogenetic relationships of the sequences obtained revealed that all sponge sequences fall into one branch of the evolutionary tree, while related sequences from higher Metazoa, human, mouse and rat, including also invertebrate sequences, together form a second branch. It is concluded that the RTK molecules have evolved in sponges prior to the "Cambrian Explosion" and have contributed to the rapid appearance of the higher metazoan phyla and that sponges are, as a taxon, also monophyletic. Due to the fact that protein tyrosine kinases in general and RTKs in particular have only been identified in Metazoa, they are, as a group qualified, to be considered as an autapomorphic character of all metazoan phyla.     

Polarity factor 'Frizzled' in the demosponge Suberites domuncula: identification, expression and localization of the receptor in the epithelium/pinacoderm(1)

Until recently, it was assumed that polarity and axis formation have evolved only in metazoan phyla higher than Cnidaria. One key molecule involved in the signal transduction causing tissue polarity is Frizzled, a seven-transmembrane receptor that is activated by the Wnt family of secreted proteins. We report the isolation and characterization of a Frizzled gene from the demosponge Suberites domuncula (Sd-Fz). The deduced polypeptide comprises all characteristic domains known from Frizzled receptors of higher metazoans. In situ hybridization studies show that Sd-Fz is expressed in cells close to the surface of the sponges and in the pinacocytes of some canals. Northern blot analysis demonstrates its upregulation during the formation of three-dimensional sponge cell aggregates in culture. These data provide for the first time experimental evidence that already in the lowest metazoan phylum (Porifera) genes are present which are very likely involved in tissue polarity.     

Receptor protein-tyrosine phosphatases: origin of domains (catalytic domain, Ig-related domain, fibronectin type III module) based on the sequence of the sponge Geodia cydonium

Reversible tyrosine phosphorylation of proteins is one of the major regulatory physiological events in response to cell-cell- and cell-matrix contact in Metazoa. Previously it was documented that the tyrosine phosphorylating enzymes, the tyrosine kinases (TKs), are autapomorphic characters of Metazoa, including sponges. In this paper the tyrosine dephosphorylating enzymes, the protein-tyrosine phosphatases (PTPs), are studied which can be grouped into two subfamilies, the soluble PTPs and the receptor PTPs (RPTPs). PTPs are characterized by one PTPase domain which interestingly comprises sequence similarity to yeast PTPs. In contrast to the PTPs, the RPTPs - which have been found only in Metazoa - are provided with two PTPase domains. To study the evolution of the RPTPs the full-length size RPTP was cloned from the marine demosponge Geodia cydonium, the phylogenetic oldest metazoan taxon. The 3253 bp long sequence has a putative open reading frame coding for a 999 aa long RPTP which is characterized by two fibronectin (type III; FN-III) domains in the extracellular portion, one intracellular immunoglobulin (Ig)-related domain, and two PTPase domains. Phylogenetic analysis revealed that the sponge FN-III domains form the basis of the metazoan FN-III domain with the common metazoan ancestor. The Ig-related, typical metazoan, module is classified to the disulphide lacking Ig members and represents the phylogenetic earliest member of this group. The beta-sheet propensity was calculated and the characteristic amino acids are present in the seven beta-sheets. The analysis of the two PTPase domains of the sponge RPTP demonstrates that the first domain is closely related to the PTPase domains present in the soluble PTPs, while the second PTPase domain is only distantly related to them. By constructing a rooted phylogenetic cladogram it became overt that the duplication of the PTPase domains must have occurred already in yeast. This interesting finding indicates that two conserved PTPase domains originated from a common ancestor in yeast while the evolutionary novelties, the FN-III domains and the Ig-related module, were added during the transition to the Metazoa. Hence, the tyrosine dephosphorylating enzyme, RPTP, is an example for a modular protein which is composed of ancient modules (PTPase domain[s]) and two metazoan novelties, while the tyrosine phosphorylating enzymes, the TKs, evolved only in Metazoa.     

Cloning and expression of new receptors belonging to the immunoglobulin superfamily from the marine sponge Geodia cydonium

 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 M _r of 53911. The short form, GCSAMS, has 313 aa residues and an M _r 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. Received: 17 November 1998 / Revised: 15 March 1999     

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.      

Intracellular pH regulation in ventral horn neurones cultured from embryonic rat spinal cord

Summary

We have isolated and characterized a cDNA from the marine sponge Geodia cydonlum coding for a new member of the tyrosine protein kinase (TK) family. The cDNA encodes a protein of M_r = 68 710, termed GCTK, which is homologous to class II receptor tyrosine kinases (RTKs). GCTK contains conserved amino acids (aa) characteristic of all protein kinases, and the sequences DLATRN and PIRWMATE which are highly specific for TKs. Furthermore, the sequence N-L-Y-x(3)-Y-Y-R Is highly homologous to the sequence D-[LIV]-Y-x(3)-Y-Y-R found only in class II RTKs. The sponge TK, when compared with mammalian class II RTKs, shows maximum 31% homology in the TK domain indicating that this the oldest member of class II RTK started to diverge from the common ancestral protein kinase 650 million years ago. Using GCTK as a probe we identified three mRNA signals ranging from 2μ6 to 0μ6 kb. Kinase activity was localized only in the cell membranes from G. cydonium (M_r = 65 000), and was not detected in the cytosol of this organism. Antibodies raised against a synthetic peptide, corresponding to the aa residues within the catalytic domain of the sponge TK, recognized strongly two proteins of M_r = 65 000; these proteins, present in membrane fractions, also bound to the anti-phosphotyrosine antibody. These data suggest that the TK cloned from the sponge is a membrane-associated 65 kDa protein. Moreover these results demonstrate that RTKs are present from the lowest group of multicellular eukaryotes, sponges, to mammals, and may suggest that RTKs are involved in a signal transduction pathway.     

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. Received: 6 August 1997 / Accepted: 24 October 1997     

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.     

The ancestry and cumulative evolution of immune reactions

The last two decades of study enriched greatly our knowledge of how the immune system originated and the sophisticated immune mechanisms of today's vertebrates and invertebrates developed. Even unicellular organisms possess mechanisms for pathogen destruction and self recognition. The ability to distinguish self from non-self is a prerequisite for recognition of sexual compatibility and ensuring survival. Molecules involved in these processes resemble those found in the phagocytic cells of higher organisms. Recognition of bacteria by scavenger receptors induces phagocytosis or endocytosis. The phagocytic mechanisms characterizing the amoeboid protozoans developed further during the evolution towards innate immunity. The scavenger receptor cysteine-rich domain SRCR is encoded in the genomes from the most primitive sponges to mammals. The immune system of sponges comprises signal transduction molecules which occur in higher metazoans as well. Sponges already possess recognition systems for pathogenic bacteria and fungi, based on membrane receptors (a lipopolysaccharide-interacting protein, a cell surface receptor recognizing β(1 → 3)-d-glucans of fungi). Perforin-like molecules and lysozymes are involved, among others, in defense in sponges. Reactive oxygen and nitrogen species function in the immunity of early metazoan. Genes encoding the family of reactive oxygen-generating NADPH oxidases (Noxes) are found in a variety of protists and plants. The NO synthases of cnidarians, mollusks, and chordates are conserved with respect to the mammalian NOS. The antimicrobial peptides of protozoans, amoebapores, are structural and functional analogs of the natural killer cell peptide, NK-lysin, of vertebrates. An ancestral S-type lectin has been found in sponges. Opsonizing properties of lectins and the ability to agglutinate cells justify their classification as primitive recognition molecules. Invertebrate cytokines are not homologous to those of vertebrate, and their functional convergence was presumably enabled by the general similarity of the lectin-like recognition domain three-dimensional structure. Sponges contain molecules with SCR/CCP domains that show high homology to the mammalian regulators of complement activation (RCA family). A multi-component complement system comprising at least the central molecule of the complement system, C3, Factor B, and MASP developed in the cnidarians and evolved into the multilevel cascade engaged in innate and acquired immunity of vertebrates. The adaptive immune system of mammals is also deeply rooted in the metazoan evolution. Some its precursors have been traced as deep as in sponges, namely, two classes of receptors that comprise Ig-like domains, the receptor tyrosine kinases (RTK), and the non-enzymic sponge adhesion molecules (SAM). The antibody-based immune system defined by the presence of the major histocompatibility complex (MHC), T-cell receptor (TCR), B-cell receptor (BCR) or recombination activating genes (RAGs) is known beginning from jawed fishes. However, genes closely resembling RAG1 and RAG2 have been uncovered in the genome of a see urchin. The ancestry of MHC gene remains unknown. Similarly, no homologue of the protein binding domain (PBD) in MHC molecules has been found in invertebrates. The pathway by which endogenous peptides are degraded for presentation with class I MHC molecules utilizes mechanisms similar to those involved in the normal turnover of intracellular proteins, apparently recruited to work also for the immune system. Several cDNAs coding for lysosomal enzymes, e.g., cathepsin, have been isolated from sponges. All chromosomal duplication events in the MHC region occurred after the origin of the agnathans but before the gnathostomes split from them. The V-domains of the subtype found in the receptors of T and B-cells are known from both agnathans and cephalochordates, although they do not rearrange. The rearrangement mechanism of the lymphocyte V-domains suggests its origin from a common ancestral domain existing before the divergence of the extant gnathostome classes. Activation-induced deaminase (AID) - homologous proteins have been found only in the gnathostomes. It appears thus that the adaptive immunity of vertebrates is a result of stepwise accumulation of small changes in molecules, cells and organs over almost half a billion years.     

Structural Evidence for the Evolution of Pyrogenic Toxin Superantigens

Cells from metazoan organisms are eliminated in a variety of physiological and pathophysiological processes by apoptosis. In this report, we describe the cloning and characterization of molecules from the marine sponges Geodia cydonium and Suberites domuncula, whose domains show a high similarity to those that are found in molecules of the vertebrate Bcl-2 superfamily and of the death receptors. The Bcl-2 proteins contain up to four Bcl-2 homology regions (BH). Two Bcl-2-related molecules have been identified from sponges that are provided with two of those regions, BH1 and BH2, and are termed Bcl-2 homology proteins (BHP). The G. cydonium molecule, BHP1_GC, has a putative size of 28,164, while the related sequence from S. domuncula, BHP1_SD, has a M _r of 24,187. Phylogenetic analyses of the entire two sponge BHPs revealed a high similarity to members of the mammalian Bcl-2 superfamilies and to the Caenorhabditis elegans Ced-9. When the two domains, BH1 and BH2, are analyzed separately, again the highest similarity was found to the members of the Bcl-2 superfamily, but a clearly lower relationship to the C. elegans BH1 and BH2 domains in Ced-9. In unrooted phylogenetic trees the sponge BH1 and BH2 are grouped among the mammalian sequences and are only distantly related to the C. elegans BH domains. The analysis of the gene structure of the G. cydonium BHP showed that the single intron present is located within the BH2 domain at the same position as in C. elegans and rat Bcl-x_L. In addition, a sponge molecule comprising two death domains has been characterized from G. cydonium. The two death domains of the potential proapoptotic molecule GC_DD2, M _r 24,970, share a high similarity with the Fas-FADD/MORT1 domains. A death domain-containing molecule has not been identified in the C. elegans genome. The phylogenetic analysis revealed that the sponge domain originated from an ankyrin building block from which the mammalian Fas-FADD/MORT1 evolved. It is suggested that the apoptotic pathways that involve members of the Bcl-2 superfamily and of the death receptors are already present in the lowest metazoan phylum, the Porifera. Received: 27 July 1999 / Accepted: 28 December 1999     

Conserved intron positions in FGFR genes reflect the modular structure of FGFR and reveal stepwise addition of domains to an already complex ancestral FGFR

We have analyzed the evolution of fibroblast growth factor receptor (FGFR) tyrosine kinase genes throughout a wide range of animal phyla. No evidence for an FGFR gene was found in Porifera, but we tentatively identified an FGFR gene in the placozoan Trichoplax adhaerens. The gene encodes a protein with three immunoglobulin-like domains, a single-pass transmembrane, and a split tyrosine kinase domain. By superimposing intron positions of 20 FGFR genes from Placozoa, Cnidaria, Protostomia, and Deuterostomia over the respective protein domain structure, we identified ten ancestral introns and three conserved intron groups. Our analysis shows (1) that the position of ancestral introns correlates to the modular structure of FGFRs, (2) that the acidic domain very likely evolved in the last common ancestor of triploblasts, (3) that splicing of IgIII was enabled by a triploblast-specific insertion, and (4) that IgI is subject to substantial loss or duplication particularly in quickly evolving genomes. Moreover, intron positions in the catalytic domain of FGFRs map to the borders of protein subdomains highly conserved in other serine/threonine kinases. Nevertheless, these introns were introduced in metazoan receptor tyrosine kinases exclusively. Our data support the view that protein evolution dating back to the Cambrian explosion took place in such a short time window that only subtle changes in the domain structure are detectable in extant representatives of animal phyla. We propose that the first multidomain FGFR originated in the last common ancestor of Placozoa, Cnidaria, and Bilateria. Additional domains were introduced mainly in the ancestor of triploblasts and in the Ecdysozoa.     

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 paraphyly and the origin of Metazoa

In order to allow critical evaluation of the interrelationships between the three sponge classes, and to resolve the question of mono‐ or paraphyly of sponges (Porifera), we used the polymerase chain reaction (PCR) to amplify almost the entire nucleic acid sequence of the 18S rDNA from several hexactinellid, demosponge and calcareous sponge species. The amplification products were cloned, sequenced and then aligned with previously reported sequences from other sponges and nonsponge metazoans and variously distant outgroups, and trees were constructed using both neighbour‐joining and maximum parsimony methods. Our results suggest that sponges are paraphyletic, the Calcarea being more related to monophyletic Eumetazoa than to the siliceous sponges (Demospongiae, Hexactinellida). These results have important implications for our understanding of metazoan origins, because they suggest that the common ancestor of Metazoa was a sponge. They also have consequences for basal metazoan classification, implying that the phylum Porifera should be abandoned. Our results support the upgrading of the calcareous sponge class to the phylum level.     

Polymorphism in the Immunoglobulin-like Domains of the Receptor Tyrosine Kinase from the Sponge Geodia Cydonium

Sponges [Porifera] are the phylogenetically oldest phylum of the Metazoa. They are provided with both cellular and humoral allorecognition systems. The underlying molecules are not yet known. To study allorecognition in sponges we first determined the frequency of graft rejection in a natural population of the marine sponge Geodia cvdonium. We then determined, for the first time at the molecular level, the degree of sequence polymorphism in segments of one molecule which may be related to sponge allorecognition and host defense: the Ig-like domains from the receptor tyrosine kinase [RTK]. Thirty six pairs of auto- and allografts were assayed, either by parabiotic attachment or insertion of grafts. All of the autografts fused, while only two allografts fused and 34 pairs were incompatibile. Rejection among the parabiotic allografts was characterized by the formation of a collagenous barrier, while the allografts that were inserted into the host underwent destruction. At the molecular level we first cloned to completion the 5'-end of sponge RTK, which displays a Pro-Ser-Thr-rich sequence; this is thought to act as a module of cell adhesion proteins. Then we analyzed RT-PCR products of amplification across the two Ig-like domains of RTK (about 500 bp), from two pairs of fusing sponges and one pair of rejecting sponges. High levels of polymorphism were recorded, including 18 nucleotide-substitution positions and a tri-nucleotide deletion, which translate into 13 polymorphic amino acid positions. Two of the six sponges were scored as heterozygotes. Among 9 informative polymorphic sites that were tested for linkage disequilibrium, 11 pairwise comparisons were found to be significant, implying the possibility of distinguishable alleles in this locus. To the best of our knowledge this is the first report of polymorphism in Ig-like domains of a receptor from invertebrates that may be associated with allorecognition. This data attests also that fusion in sponges is not confined to genetically identical individuals.     

Origin of neuronal-like receptors in Metazoa: cloning of a metabotropic glutamate/GABA-like receptor from the marine sponge Geodia cydonium

To date, no conclusive evidence has been presented for the existence of neuronal-like elements in Porifera (sponges). In the present study, isolated cells from the marine sponge Geodia cydonium are shown to react to the excitatory amino acid glutamate with an increase in the concentration of intracellular calcium [Ca2+]i. This effect can also be observed when the compounds L-quisqualic acid (L-QA) or L-(+)-2-amino-4-phosphonobutyric acid (L-AP-4) are used. The effect of L-QA and L-AP-4, both agonists for metabotropic glutamate receptors (mGluRs), can be abolished by the antagonist of group I mGluRs, (RS)-alpha-methyl-4-carboxyphenylglycine. These data suggest that sponge cells contain an mGluR-like protein. A cDNA encoding rat mGluR subtype 1 has been used to identify the complete nucleotide sequence of G. cydonium cDNA coding for a 528-amino-acid-long protein (59 kDa) that displays marked overall similarity to mGluRs and to gamma-aminobutyric acid B receptors. The deduced sponge polypeptide, termed putative mGlu/GABA-like receptor, displays the highest similarity to the two families of metabotropic receptors within the transmembrane segment. The N-terminal part of the sponge sequence shows similarity to mGluR4 and mGluR5. These findings suggest that the earliest evolutionary metazoan phylum, the Porifera, possesses a sophisticated intercellular communication and signaling system, as seen in the neuronal network of higher Metazoa.     

Polymorphism in the Immunoglobulin-like Domains of the Receptor Tyrosine Kinase from the Sponge Geodia Cydonium

Sponges [Porifera] are the phylogenetically oldest phylum of the Metazoa. They are provided with both cellular and humoral allorecognition systems. The underlying molecules are not yet known. To study allorecognition in sponges we first determined the frequency of graft rejection in a natural population of the marine sponge Geodia cvdonium. We then determined, for the first time at the molecular level, the degree of sequence polymorphism in segments of one molecule which may be related to sponge allorecognition and host defense: the Ig-like domains from the receptor tyrosine kinase [RTK]. Thirty six pairs of auto- and allografts were assayed, either by parabiotic attachment or insertion of grafts. All of the autografts fused, while only two allografts fused and 34 pairs were incompatibile. Rejection among the parabiotic allografts was characterized by the formation of a collagenous barrier, while the allografts that were inserted into the host underwent destruction. At the molecular level we first cloned to completion the 5′-end of sponge RTK, which displays a Pro-Ser-Thr-rich sequence; this is thought to act as a module of cell adhesion proteins. Then we analyzed RT-PCR products of amplification across the two Ig-like domains of RTK (about 500 bp), from two pairs of fusing sponges and one pair of rejecting sponges. High levels of polymorphism were recorded, including 18 nucleotide-substitution positions and a tri-nucleotide deletion, which translate into 13 polymorphic amino acid positions. Two of the six sponges were scored as heterozygotes. Among 9 informative polymorphic sites that were tested for linkage disequilibrium, 11 pairwise comparisons were found to be significant, implying the possibility of distinguishable alleles in this locus. To the best of our knowledge this is the first report of polymorphism in Ig-like domains of a receptor from invertebrates that may be associated with allorecognition. This data attests also that fusion in sponges is not confined to genetically identical individuals.