Claudia Koziol

Sponges (Porifera) model systems to study the shift from immortal to senescent somatic cells: the telomerase activity in somatic cells

Sponges (Porifera) represent the lowest metazoan phylum, characterized by a pronounced plasticity in the determination of cell lineages. In a first approach to elucidate the molecular mechanisms controlling the switch from the cell lineage with a putative indefinite growth capacity to senescent, somatic cells, the activity of the telomerase as an indicator for immortality has been determined. The studies were performed with the marine demosponges Suberites domuncula and Geodia cydonium. It was found that the activity for the telomerase in the tissue of both sponges is high; a quantitative analysis revealed that the extract from S. domuncula contained 10.3 TPG units per 5000 cell equivalents and the one from G. cydonium 8.3 TPG units; hence the activity reached approximately 30-20% of the activity seen in telomerase-positive reference cells. In contrast, dissociated spherulous cells from G. cydonium, after an incubation period of 24 h, contained no detectable telomerase activity. From earlier studies it is known that isolated sponge cells do not proliferate. Based on these findings it is assumed that the separation of the senescent sponge cell lineage from the immortal germ/somatic cell lineage is triggered by the loss of contact with cell adhesion factors. First evidence is included which suggests that the final progress of the senescent, telomerase-negative cells to cell death is caused by apoptosis.

Induction of heat-shock (stress) protein gene expression by selected natural and anthropogenic disturbances in the octocoral Dendronephthya klunzingeri

Previously it was found that the expression of selected heat-shock proteins is upregulated in corals after exposure to elevated temperature. We published that HSPs are suitable markers in sponges to monitor the degree of environmental stress on these animals. In the present study the heat-shock proteins (HSPs) with a molecular weight of 90 kDa have been selected to prove their potential usefulness as biomarkers under controlled laboratory conditions and in the field. The studies have been performed with the octocoral Dendronephthya klunzingeri4.5-fold higher steady-state level of the respective mRNA. Also animals taken from stressed locations in the field showed an increased expression. The amount of HSP90 protein in D. klunzingeri was found to be strongly increased under thermal stress, or exposure to polychlorinated biphenyl (congener 118), but not after treatment with cadmium. Field studies revealed that samples taken from a nonstressed area have a low level of HSP90, but those collected from locations at which the corals are under physical stress (sedimentation through landfilling) show a high expression of HSP90. It is concluded that the chaperone HSP90 might become a suitable biomarker to monitor environmental stress on corals.

Increased Expression of Integrin and Receptor Tyrosine Kinase Genes During Autograft Fusion in the Sponge Geodia cydonium

Recently cDNAs coding for cell surface molecules have been isolated from sponges. The molecules for alpha-integrin, galectin, and receptor tyrosine kinase (RTK), obtained from the marine sponge, Geodia cydonium, have been described earlier. In the present study also the cDNA for one putative beta-integrin has been identified from G. cydonium. The deduced aa sequence comprises the characteristic signatures, found in other metazoan beta-integrin molecules; the estimated size is 95,215 Da. To obtain first insights into the molecular events which proceed during autograft fusion, the expressions of these genes were determined on transcriptional and translational level. The cDNAs as well as antibodies raised against the recombinant sponge proteins alpha-integrin, RTK and galectin were used and Northern blot experiments and immunocytochemical analyses have been performed. The results show that transcription of the two subunits of an integrin receptor as well as of the RTK are strongly upregulated after grafting; levels of > 10-fold have been determined in the fusion zone of the grafts after a 10 days incubation. Immunofluorescence studies of sections through the fusion zone support these data. In contrast the transcription of the gene encoding galectin is drastically downregulated after grafting. In a parallel series of experiments the level of the heat-shock protein-70 was determined and it was found that it remained unchanged after grafting. We conclude that integrin subunits and the RTK molecule are involved in self-self recognition of sponge.

Towards an understanding of the molecular basis of immune responses in sponges: The marine demosponge Geodia cydonium as a model

The phylogenetic position of the phylum Porifera (sponges) is near the base of the kingdom Metazoa. During the last few years, not only rRNA sequences but, more importantly, cDNA/genes that code for proteins have been isolated and characterized from sponges, in particular from the marine demosponge Geodia cydonium. The analysis of the deduced amino acid sequences of these proteins allowed a molecular biological approach to the question of the monophyly of the Metazoa. Molecules of the extracellular matrix/basal lamina, with the integrin receptor, fibronectin, and galectin as prominent examples, and of cell‐surface receptors (tyrosine kinase receptor), elements of sensory systems (crystallin, metabotropic glutamate receptor) as well as homologs/modules of an immune system (immunoglobulin‐like molecules, scavenger receptor cysteine‐rich [SRCR]‐ and short consensus repeats [SCR]‐repeats), classify the Porifera as true Metazoa. As living fossils, provided with simple, primordial molecules allowing cell‐cell and cell‐matrix adhesion as well as processes of signal transduction as known in a more complex manner from higher Metazoa, sponges also show pecularities not known in later phyla. In this paper, the adhesion molecules presumably involved in the sponge immune system are reviewed; these are the basic adhesion molecules (galectin, integrin, fibronectin, and collagen) and especially the highly polymorphic adhesion molecules, the receptor tyrosine kinase as well as the polypeptides comprising scavenger receptor cysteine‐rich (SRCR) and short consensus repeats (SCR) modules. In addition, it is reported that in the model sponge system of G. cydonium, allogeneic rejection involves an upregulation of phenylalanine hydroxylase, an enzyme initiating the pathway to melanin synthesis. Microsc. Res. Tech. 44:218–236, 1999.

Phenylalanine hydroxylase from the sponge Geodia cydonium: implication for allorecognition and evolution of aromatic amino acid HYDROXYLASES

The prophenoloxidase activating system is a defense system, frequently reported both in protostomes and in deuterostomes. The final product of the phenoloxidase activity is melanin which is ubiquitously present throughout the metazoan kingdom. The melanin synthesis pathway starts with the amino acid [aa] phenylalanine which is converted to tyrosine by the phenylalanine hydroxylase [PAH]. We show that after allo-transplantation in the marine sponge Geodia cydonium PAH is upregulated in the grafts. Enzyme determination studies revealed that PAH activity increases by three-fold two d after transplantation and reaches its maximum after 3d (by 3.7-fold). This finding was supported by determining the steady-state level of the mRNA for PAH. Furthermore the cDNA, encoding this enzyme was isolated from G. cydonium. Its deduced aa sequence encodes a protein of 51 kDa. Alignment studies indicate that the sponge PAH shares the consensus pattern as well as one characteristic pterin-binding site with the biopterin-dependent aromatic amino acid hydroxylases. Phylogenetic analysis of sponge PAH shows that all metazoan PAH fall in one group with the sponge PAH as the oldest member. The related classes of enzymes, the tyrosine hydroxylases and the tryptophan hydroxylases are statistically significantly separated from PAH; the tyrosine hydroxylase diverged as the first class from the common ancestor, a process which was calculated to have occurred 500 million years ago. It is concluded that in the sponge model system G. cydonium allogeneic rejection involves an upregulation of PAH, an enzyme initiating the pathway to melanin synthesis.

Evolution of early Metazoa: phylogenetic status of the Hexactinellida within the phylum of Porifera (sponges).

The evolution of the Metazoa from their protozoan ancestors is one of the greatest puzzles of phylogeny (Willmer 1994; Cavalier-Smith et al. 1996). The emergence of multicellular animals has been explained by two major theories: the syncytial theory (Hadzi 1963) - origin from a multinucleated ciliate - and the colonial theory (Haeckel 1868) - origin from a colonial flagellate - both of which assume a di(poly)phyletic origin of the Metazoa. Numerous attempts to resolve whether the Metazoa are of mono- or polyphyletic origin have sought evidence from a wide variety of developmental and morphological data such as body symmetry, type of development (protostome vs. deuterostome), type of body cavities (coelomates vs. pseudocoelomates), cleavage patterns (determinate vs. indeterminate, spiral vs. radial), origin of photoreceptors, evolution of surface cilitation, etc. (see Morris 1993).

Diagnosis of sublethal stress in the marine sponge Geodia cydonium: application of the 70 kDa heat-shock protein and a novel biomarker, the Rab GDP dissociation inhibitor, as probes

Abstract Sponges (Porifera) are among the major phyla inhabiting the marine hard-substrate benthos, both in respect to the number of species and their biomass. Hence reliable biomarkers need to be developed to monitor the environmental load in those animals. Recently, the cDNA for the heat shock protein HSP70 has been isolated from the sponge Geodia cydonium and found to be a reliable indicator for temperature stress. In the present study, we have isolated the Rab GDP-dissociation inhibitor (GDI), which has previously been shown to be a key element in the intracellular traffic system. The 1521 bp long cDNA, encoding sponge GDI, has been isolated and analyzed. The deduced aa sequence ( M r = 50215) displays high homology to vertebrate, invertebrate and also yeast GDIs. Northern blot analysis revealed that the 1.6 kb sponge transcript that hybridized with the sponge GDI is down-regulated under temperature stress; in contrast, the transcription of the sponge HSP70 is strongly enhanced. Due to the high sequence conservation of the GDIs, one can assume that sponge GDI probes can be used as functional biomarkers for temperature stress, especially when applied simultaneously with the highly conserved HSP70 probes.

Immediate early response of the marine sponge Suberites domuncula to heat stress: reduction of trehalose and glutathione concentrations and glutathione S-transferase activity

The marine sponge Suberites domuncula was used to identify early markers for thermal stress. Cubes from sponges have been kept for 30 min at 31°C (10 °C higher than the ambient temperature). After this treatment the sponge cubes were kept again at 21°C. To demonstrate that the animals reacted to the elevated temperature, the expression of heat shock protein (HSP) was determined. Using an antibody raised against HSP70, it was found by Western blotting that the animals specifically express a 45 kDa polypeptide after heat treatment. It was shown that even after 10 min of heat treatment the steady-state concentration of trehalose drops by 40% from a base level of 13 nmol/mg protein. The activity of the trehalose-degrading enzyme, trehalase, remained unchanged. Additional early biomarkers for thermal stress include the enzyme activity of glutathione S-transferase (GST) and the concentration of glutathione (GSH). After 5-min the activity of GST decreased by 40%. Similarly, the concentration of GSH dropped by 50% after 15 to 20 min exposure. The orginal levels of the biomarkers, trehalose, GSH and GST, were reached again after a recovery period of about 180 min. By contrast, the steady-state concentration of polyphosphates did not change during heat treatment. These data show that in S. domuncula the concentrations of trehalose, GSH, and the activity of GST, are biomarkers for immediate early response towards heat stress.

Chapter 14 - Stress Response in Marine Sponges: Genes and Molecules Involved and Their use as Biomarkers

Sponges (Porifera) are sessile filter feeders that are able to accumulate compounds from the surrounding water and thus are highly exposed to environmental stress by pollutants of both anthropogenic and natural origin. However, these animals possess a number of protective mechanisms against environmental stress, such as the expression of heat-shock proteins, the induction of the multi xenobiotic resistance mechanism, and the apoptotic elimination of cells. In the last years, a number of genes involved in the stress response of sponges have been cloned and characterized. In thischapter, the various molecular mechanisms by which cells of the lowest multicellular organisms—the marine sponges—respond to environmental stress and the methods for measurement of stress response in sponges are discussed.

Prolidase in the Marine Sponge Suberites domuncula: Enzyme Activity, Molecular Cloning, and Phylogenetic Relationship

Abstract: The enzyme prolidase hydrolyzes the peptide bond that involves the imino nitrogen of proline or hydroxyproline; hence, it catalyzes the final step in collagen degradation. From mammals it is known that this enzyme plays a major role in the recycling of proline for collagen synthesis and can be considered to be essential for the control of cell growth. The dominant organic exoskeleton in sponges, especially in Demospongiae, is collagen and the collagen-related spongin. Here we demonstrate that crude extracts of the demosponge Suberites domuncula contain prolidase or prolidase-like activity. The complementary DNA encoding the putative prolidase was cloned from a library of the same animal. Two different forms of cDNAs, termed SDPEPD1 and SDPEPD2, were identified, coding for the putative polypeptides PEPD_SD-1 with a molecular mass of 55,805 Da and PEPD_SD-2 with 51,684. Evidence is presented suggesting that the two different transcripts originate from the same gene but are formed by an alternative splicing event. We conclude that demosponges contain the activity as well as the gene for prolidase, a major enzyme involved in collagen metabolism, spicule formation, and cell motility. Phylogenetic analysis revealed that the sponge prolidase branches off first from the common ancestor of metazoan prolidases and later than the yeast prolidase; only distantly related are the bacterial enzymes.