Bärbel Diehl-Seifert

Innate Immune Defense of the Sponge Suberites domuncula against Bacteria Involves a MyD88-dependent Signaling Pathway INDUCTION OF A PERFORIN-LIKE MOLECULE

Sponges (phylum Porifera) are the phylogenetically oldest metazoa; as filter feeders, they are abundantly exposed to marine microorganisms. Here we present data indicating that the demosponge Suberites domuncula is provided with a recognition system for Gram-negative bacteria. The lipopolysaccharide (LPS)-interacting protein was identified as a receptor on the sponge cell surface, which recognizes the bacterial endotoxin LPS. The cDNA was isolated, and the protein (Mr 49,937) was expressed. During binding to LPS, the protein dimerizes and interacts with MyD88, which was also identified and cloned. The sponge MyD88 (Mr 28,441) is composed of two protein interaction domains, a Toll/interleukin-1 receptor domain (found in MyD88 and in Toll-like receptors) and a death domain (present in MyD88 and interleukin-1 receptor-associated kinase). Northern blot experiments and in situ hybridization studies showed that after LPS treatment, the level of the LPS-interacting protein remains unchanged, whereas MyD88 is strongly up-regulated. A perforin-like molecule (Mr 74,171), the macrophage-expressed protein, was identified as an executing molecule of this pathway. This gene is highly expressed after LPS treatment, especially at the surfaces of the animals. The recombinant protein possesses biological activity and eliminates Gram-negative bacteria; it is inactive against Gram-positive bacteria. These data indicate that S. domuncula is provided with an innate immune system against Gram-negative bacteria; the ligand LPS (a pathogen-associated molecular pattern) is recognized by the pattern recognition receptor (LPS-interacting protein), which interacts with MyD88. A signal transduction is established, which results in an elevated expression of MyD88 as well as of the macrophage-expressed protein as an executing protein.

Emergence and disappearance of an immune molecule, an antimicrobial lectin, in basal metazoa: a tachylectin-related protein in the sponge suberites domuncula

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 transmembrane region, three characteristic Cys residues, and six internal tandem repeats; it shares the highest sequence similarity with lectins from the horseshoe crab Tachypleus trunculus. The steady-state level of expression of the Suberites lectin rises in primmorphs in response to lipopolysaccharide, an effect that was prevented by co-incubation with d-GlcNAc. The natural sponge lectin was purified by affinity chromatography; it has a size of 27 kDa and displays antibacterial activity against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Staphylococcus aureus. The putative protein, deduced from the cloned gene, is identical/similar to the purified natural protein, as demonstrated by immunological cross-reactivity with specific antibodies. We conclude that the S. domuncula lectin acts as an antibacterial molecule involved in immune defense against bacterial invaders.

Transport of mRNA from Nucleus to Cytoplasm

Publisher Summary Transport of mRNP (messenger ribonucleoprotein) from nucleus to cytoplasm plays an important role in gene expression in eukaryotic cells. This chapter focuses on energy-(ATP)-dependent mRNP transport. Nucleocytoplasmic transport of ribosomal RNA can also be induced by ATP, but also occurs by varying [Ca 2+ ]:[Mg 2+ ]. Release of ribosomal RNPs seems to be accompanied by an expansion of the nucleus. Nucleocytoplasmic transport of mRNA seems to be also distinct from the export of tRNA or the exchange of snRNPs and proteins across the nuclear envelope. Nucleocytoplasmic transport of tRNA seems to involve a facilitated diffusion mechanism, showing saturability and sequence specificity; apparently, it does not depend on ATP. In contrast to the transport of mRNPs through the nuclear pore, which appears strictly vectorial, snRNPs can shuttle between nuclear and cytoplasmic compartments. The nuclear uptake of at least some kinds of U-snRNAs in oocytes seems to depend on their association with proteins stockpiled in the cytoplasm. In contrast to the mRNA export, the import of most proteins into the nucleus seems to be energy-independent, although in some cases nucleotides promote this process. The accumulation of karyophilic proteins in the nucleus may be mediated by specific signal sequences recognizing the intranuclear binding sites of these proteins. However, some proteins seem to migrate into the nucleus via a transport mechanism.

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

Abstract 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.

Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro.

Inorganic polymeric phosphate/polyphosphate (polyP) is a natural polymer existing in both pro‐ and eukaryotic systems. In the present study the effect of polyP as well as of polyP supplied in a stoichiometric ratio of 2 m polyP:1 m CaCl2 [polyP (Ca2+ complex)] on the osteoblast‐like SaOS‐2 cells and the osteoclast‐like RAW 264.7 cells was determined. Both polymers are non‐toxic for these cells up to a concentration of 100 µm. In contrast to polyP, polyP (Ca2+ complex) significantly induced hydroxyapatite formation at a concentration > 10 µm, as documented by alizarin red S staining and scanning electron microscopic (SEM) inspection. Furthermore, polyP (Ca2+ complex) triggered in SaOS‐2 cells transcription of BMP2 (bone morphogenetic protein 2), a cytokine involved in maturation of hydroxyapatite‐forming cells. An additional activity of polyP (Ca2+ complex) is described by showing that this polymer impairs osteoclastogenesis. At concentrations > 10 µm polyP (Ca2+ complex) slows down the progression of RAW 264.7 cells to functional osteoclasts, as measured by the expression of TRAP (tartrate‐resistant acid phosphatase). Finally, it is shown that 10–100 µm polyP (Ca2+ complex) inhibited phosphorylation of IκBα by the respective kinase in RAW 264.7 cells. We concluded that polyP (Ca2+ complex) displays a dual effect on bone metabolizing cells. It promotes hydroxyapatite formation in SaOS‐2 cells (osteoblasts) and impairs maturation of the osteoclast‐related RAW 264.7 cells. Copyright

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.

Alginate/silica composite hydrogel as a potential morphogenetically active scaffold for three-dimensional tissue engineering

Pursuing our aim to develop a biomimetic synthetic scaffold suitable for tissue engineering, we embedded bone cells, osteoblast-related SaOS-2 cells and osteoclast-like RAW 264.7 cells, into beads, formed of a Na-alginate-based or a silica-containing Na-alginate-based hydrogel matrix. The beads were incubated either separately (only one cell line in a culture dish) or co-incubated (SaOS-2-containing beads and RAW 264.7 beads). The alginate and alginate/silica hydrogel matrices were found not to impair the viability of the encapsulated cells. In these matrices the SaOS-2 cells retain their capacity to synthesize hydroxyapatite crystallites. The mechanical properties, including surface roughness and hardness, of the hydrogel were determined. If silica is included in the hydrogel matrix, the encapsulated SaOS-2 cells were found to increasingly express the gene encoding for osteoprotegerin in co-cultivation experiments with RAW 264.7 cell beads, suggesting that under the applied conditions the differentiation capacity of the RAW 264.7 cells is impaired. In continuation it was found that under these conditions (SaOS-2 cells cultured together with RAW 264.7 cells) the RAW 264.7 cells show a reduced capacity to express the gene for tartrate-resistant acid phosphatase. It is concluded that the applied bead-encapsulation of bone cells is a useful technique to produce bioactive programmable hydrogels.

Effect of Bioglass on Growth and Biomineralization of SaOS-2 Cells in Hydrogel after 3D Cell Bioprinting

We investigated the effect of bioglass (bioactive glass) on growth and mineralization of bone-related SaOS-2 cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP), administered as polyP•Ca2+-complex, or silica, or as biosilica that had been enzymatically prepared from ortho-silicate by silicatein. These hydrogels, together with SaOS-2 cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, with a size of 55 nm and a molar ratio of SiO2∶CaO∶P2O5 of 55∶40∶5, did not affect the growth of the encapsulated cells. If silica, biosilica, or polyP•Ca2+-complex is co-added to the cell-containing alginate/gelatin hydrogel the growth behavior of the cells is not changed. Addition of 5 mg/ml of bioglass particles to this hydrogel significantly enhanced the potency of the entrapped SaOS-2 cells to mineralize. If compared with the extent of the cells to form mineral deposits in the absence of bioglass, the cells exposed to bioglass together with 100 µmoles/L polyP•Ca2+-complex increased their mineralization activity from 2.1- to 3.9-fold, or with 50 µmoles/L silica from 1.8- to 2.9-fold, or with 50 µmoles/L biosilica from 2.7- to 4.8-fold or with the two components together (100 µmoles/L polyP•Ca2+-complex and 50 µmoles/L biosilica) from 4.1- to 6.8-fold. Element analysis by EDX spectrometry of the mineral nodules formed by SaOS-2 revealed an accumulation of O, P, Ca and C, indicating that the mineral deposits contain, besides Ca-phosphate also Ca-carbonate. The results show that bioglass added to alginate/gelatin hydrogel increases the proliferation and mineralization of bioprinted SaOS-2 cells. We conclude that the development of cell-containing scaffolds consisting of a bioprintable, solid and cell-compatible inner matrix surrounded by a printable hard and flexible outer matrix containing bioglass, provide a suitable strategy for the fabrication of morphogenetically active and biodegradable implants.

Modulation of the initial mineralization process of SaOS-2 cells by carbonic anhydrase activators and polyphosphate.

Ca-phosphate/hydroxyapatite (HA) crystals constitute the mineral matrix of vertebrate bones, while Ca-carbonate is the predominant mineral of many invertebrates, like mollusks. Recent results suggest that CaCO3 is also synthesized during early bone formation. We demonstrate that carbonic anhydrase-driven CaCO3 formation in vitro is activated by organic extracts from the demosponge Suberites domuncula as well as by quinolinic acid, one component isolated from these extracts. Further results revealed that the stimulatory effect of bicarbonate (HCO3−) ions on mineralization of osteoblast-like SaOS-2 cells is strongly enhanced if the cells are exposed to inorganic polyphosphate (polyP), a linear polymer of phosphate linked by energy-rich phosphodiester bonds. The effect of polyP, administered as polyP (Ca2+ salt), on HA formation was found to be amplified by addition of the carbonic anhydrase-activating sponge extract or quinolinic acid. Our results support the assumption that CaCO3 deposits, acting as bio-seeds for Ca-carbonated phosphate formation, are formed as an intermediate during HA mineralization and that the carbonic anhydrase-mediated formation of those deposits is under a positive–negative feedback control by bone alkaline phosphatase-dependent polyP metabolism, offering new targets for therapy of bone diseases/defects.

Interrelation between Extracellular Adhesion Proteins and Extracellular Matrix in Reaggregation of Dissociated Sponge Cells

Publisher Summary This chapter discusses the interrelation between extracellular adhesion proteins and extracellular matrix in the reaggregation of dissociated sponge cells. Sponges (Porifera) are considered as genuine Metazoa and are placed at the base of the animal kingdom. The sponge organism is surrounded by the external epithelium (exopinacoderm), the based epithelial surface (basopinacoderm), and the epithelium of the aquiferous canals. The sponges are used for the isolation and purification of the first soluble aggregation factor (AF). The AF is a key molecule in the cell–cell adhesion apparatus. The AF-mediated cell–cell adhesion system of Geodia cydonium is heterophilic and of the third order. Basically two molecules are involved, the AF and the aggregation receptor (AR). The strength of the interaction between these two molecules is controlled in a tuned manner by enzymatic and nonenzymatic processes. The sponges have invented extracellular elements serving as a guiding matrix for an exact positioning of cells. It comprises the fluidsolid phase (lectin-glucoconjugate system) and the solid phase (collagen).