Sponge Geodia Cydonium Research Articles

Identification of a Cell Surface-Associated Protein Involved in Mouse Neural Cell Aggregation by Means of Antibodies Against the Sponge Aggregation Factor

Polyclonal antibodies were raised against the purified aggregation factor (AF) from the sponge Geodia cydonium to elucidate possible immunological relationships between adhesion molecules of lower multicellular eukaryotic systems (sponges) and those of vertebrates. This anti-AF recognized a series of polypeptides associated with the AF, among them also a polypeptide with a Mr of 47,000 (p47). The formation of the antibody-p47 immunocomplexes could be prevented by adsorbing the anti-AF with a brain extract from DBA/2J mice. Moreover, this brain polypeptide inhibited the AF-mediated aggregation of sponge cells. Interestingly, the anti-AF recognized a p37 molecule in the brains of 2- to 3-day-old mice; no reaction could be traced using brain extracts from animals older than 2 months. The anti-AF failed to interact with polypeptides from mouse liver or spleen. By indirect immunofluorescence staining the p37 was found to be localized on the plasma membranes of brain cells. Moreover, Fab' fragments of the anti-AF inhibited aggregation of mouse brain cells. These data indicate that the sponge anti-AF recognizes a p37 molecule in mouse brain cells which is either directly or indirectly involved in brain cell aggregation.

Induction of ras gene expression by homologous aggregation factor in cells from the sponge Geodia cydonium.

Northern blot data as well as immunocytochemical and immunoblotting experiments indicate that the sponge Geodia cydonium contains the ras gene (or ras-related gene). The ras gene transcript has a size of 1.5 kilobases and the ras gene product has an Mr of 23,000-26,000. While dissociated G. cydonium cells lack ras gene product, ras gene expression can be induced by incubating the cells with the cell-binding fragment of the homologous aggregation factor. Maximal expression was observed 10-15 h after addition of the fragment. The sponge ras protein was partially purified by immunoprecipitation and bound GTP with a dissociation constant of 1.8 x 10(-6) M. It was found to be associated with the cell membrane. Autoradiographic studies revealed that the ras protein binds to the plasma membrane-associated anti-aggregation receptor and thereby mediates the mitogenic response, caused by the homologous lectin. It is hypothesized that sponges are provided in addition to the aggregation factor-caused and protein kinase C-mediated pathway with a second mechanism of transduction of mitotic signals, the lectin-caused and ras-mediated signaling.

Control of the aggregation factor-aggregation receptor interaction in sponges by protein kinase C

By means of immunobiochemical and immunocytological techniques it was found that the aggregation factor (AF) from the sponge Geodia cydonium is stored in vesicles of spherulous cells. During the reaggregation process of dissociated cells, the AF which is present extracellularly was determined to be bound to the cell-surface-associated aggregation receptor (AR) only during the initial phase (0–5 h after addition of the AF to the single cell suspension). At later stages (20 h), the AF colocalized with extracellular structures, e.g., collagen and glycoconjugates. Immobilized to nitrocellulose, the AR, a molecule with M r of 43.5 kDa, displayed its binding affinity to the AF only if it was isolated from early aggregates (5 h). The transition of the AF-susceptible to the AF-deficient state of the plasma membrane was mimicked in vitro by incubation of plasma membranes from early aggregates with purified protein kinase C. This conversion to the AF-deficient state could be prevented by the protein kinase C inhibitor staurosporine. Together with earlier findings, which revealed that the AR is phosphorylated by protein kinase C, we propose that in the sponge system this enzyme controls intercellular processes involved in morphogenesis.

Sponge aggregation factor: identification of the specific collagen-binding site by means of a monoclonal antibody.

The aggregation factor (AF) from the sponge Geodia cydonium is known to be a complex proteinaceous particle, composed of a series of different (glyco)proteins (Mr lower than 150,000) around a 90S sunburst-like core structure. One of the low-Mr proteins is the 47-KD cell binding fragment. We describe a new monoclonal antibody (mAb), III1E6, raised against purified AF particles, which recognizes in tissue slices structures present both on the plasma membrane and in a network-like manner in the extracellular space. By applying immunoelectron microscopical, immunoblotting, and immunoaffinity chromatographical techniques, the mAb III1E6 was shown to recognize the core structure of the AF particle. Cell adhesion studies revealed that the mAb does not inhibit AF mediated cell-cell adhesion but abolishes AF-caused attachment of cells to collagen. Electron microscopic data show that III1E6 prevents association of AF particles with collagen fibrils. By applying the techniques of immunoblotting and of protein-protein recognition on the solid phase in vitro, we could formulate the following series of events: the AF particle recognizes, with its 47-KD cell binding fragment, the aggregation receptor protein in the plasma membrane and with its core structure the collagen fibrils. These fibrils interact optionally, either via the same route or via the collagen assembly factor, with an adjacent cell surface. These findings demonstrate that the AF particle is not only the key molecule for cell-cell adhesion but also a component of cell-matrix interactions.

Specific phosphorylation of proteins in pore complex-laminae from the sponge Geodia cydonium by the homologous aggregation factor and phorbol ester. Role of protein kinase C in the phosphorylation of DNA topoisomerase II.

We have recently shown that the aggregation factor (AF) from the sponge Geodia cydonium stimulates DNA synthesis in quiescent, dissociated cells from the same organism; this event was correlated with the release of the two second messengers: inositol trisphosphate and diacylglycerol. Here we describe that after binding of the AF to the plasma membrane-bound aggregation receptor, a rapid and drastic increase in the incorporation of 32Pi into a series of proteins in the pore complex-lamina fraction occurs. Addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, to quiescent cells resulted in a similar stimulation of phosphorylation of nuclear proteins. Among them we have selected one protein with a polypeptide Mr of 170,000 (pp170) for detailed studies. By immunoblotting pp170 was identified as DNA topoisomerase II. In vitro studies with nuclei and purified, homogeneous protein kinase C together with the required activators of this enzyme also showed a phosphorylation of pp170. After phosphorylation, DNA topoisomerase II activity was found to be 2.5-fold that of the non-phosphorylated enzyme. From these data we conclude that protein kinase C is involved in AF induced transmembrane signalling, ultimately leading to an initiation of DNA synthesis.

Role of the aggregation factor in the regulation of phosphoinositide metabolism in sponges. Possible consequences on calcium efflux and on mitogenesis.

The aggregation factor (AF) of the marine sponge Geodia cydonium recognizes the aggregation receptor (AR) which is inserted in the plasma membrane, under formation of species-specific aggregates. The specific cell-binding fragment of the AF was used to investigate for the first time the phosphoinositide metabolism in a lower avertebrate system. We found that after binding of the cell-binding fragment to the aggregation receptor a strong and rapid stimulation of the phosphate incorporation into phosphatidylinositol occurs followed by an increased turnover of phosphoinositides in the Geodia cells. The consequences of an increased degradation of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol-1,4,5-trisphosphate and diacylglycerol are 2-fold. First, after the addition of the extracellular stimulus the cytosolic Ca2+ concentration rises, resulting in a rapid increased Ca2+ efflux rate. The functional consequence of the increase of the extracellular Ca2+ level is an initiation of the aggregate formation that is mediated by the collagen assembly factor (= primary aggregation factor). Second, some experimental evidences are presented, showing that the other second messenger formed, diacylglycerol, causes a translocation of protein kinase C within the cell. Incubation of Geodia cells with the cell-binding fragment of the AF, or with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, resulted within 5 min after treatment in a 70% decrease in protein kinase C activity in the cytosolic fraction and in a 700% increase in enzyme activity in the membrane fraction. It is proposed that by membrane association protein kinase C becomes activated. As a result of this event a series of cellular proteins are phosphorylated, a process which ultimately leads to an unusually strong induction of DNA polymerase alpha activity. From these data we conclude that inositol trisphosphate and protein kinase C also play a fundamental role in cellular signal transduction in lower eukaryotes.

Evidence for the existence of microtubule protein in the extracellular space of marine sponges

The medulla region of sponges (= mesohyl) is only rarely dispersed with cells (approximately 25% of the total tissue volume). In vivo studies with a series of anti-cytoskeletal drugs revealed that taxol caused a significant contraction of intact specimens. Therefore, we investigated the possibility that microtubules are one component of the dynamic extracellular network in sponges, especially in the mesohyl. Using the sponge Geodia cydonium we found that most cells are not intracellulary stained in indirect immunofluorescence microscopy. However, all cells were brightly stained by the monoclonal antibody (mAb) at their plasma membranes. After incubating cryostat sections with a buffer, containing ATP, large (length up to 75 μm) aster- to filamentous-like structures became visible in the extracellular space of the mesohyl region. By three cycles of assembly and disassembly microtubules could be isolated from the extracellular material which were composed of α- and β-tubulin (M r: 55,000), several polypeptides within the M r range 55,000–82,000 (presumably tau proteins) and one protein of an M r higher than 100,000. Electron microscopical analysis revealed morphologically intact microtubules with typical side projections (very probably composed of microtubule-associated protein). Some aspects of the possible involvement of microtubules in the extracellularly localized displacement and motion network are discussed.

In situ localization of the specific cell-binding fragment on the sponge aggregation factor

Electron microscopical studies revealed that the functionally intact (native) form of the aggregation factor (AF) from the sponge Geodia cydonium has a ball-like structure. A monoclonal antibody (McAb 5132-1311), directed against the 47-kDa cell-binding domain of the AF, was used for the in situ detection of this 47-kDa fragment. After detection of the antigen-antibody reaction with protein A-colloidal gold, three or four molecules of the 47-kDa protein were observed per one AF particle. The 47-kDa protein can be detached from the particle after sodium dodecyl sulfate and Nonidet treatment, releasing the “sunburst” core structure of the AF. The 47-kDa protein, separated from the core AF by gel filtration, has been identified by an immunoblotting procedure. From these experiments we conclude that the 47-kDa cell-binding domain is only present on native AF. It is concluded that the 47-kDa protein binds to the aggregation receptor on the plasma membrane in a polyvalent manner.

Attachment of sponge cells to collagen substrata: Effect of a collagen assembly factor

Collagen, isolated from the sponge Geodia cydonium in the absence of denaturing agents, had the typical amino acid composition and was associated with the carbohydrates galactose and glucose. The resulting individual fibrils with a diameter of 23 nm, displayed a 19.5 nm periodicity with one intraperiod band. A collagen assembly factor (CAF) was identified in and partially purified from the extracellular space. The CAF reacted with antibodies against intact Geodia cells but not with antibodies against Geodia lectin and Geodia aggregation factor. In the presence of the CAF, the collagen fibrils reconstituted collagen bundles in an ordered sequence of events, which were followed by electron-microscopical and biochemical methods. Bundle formation was not dependent on the presence of the homologous lectin, glycoconjugates or aggregation factor. Homologous cells (Geodia archaeocytes) were determined to attach only to those Geodia collagen substrates that contained CAF. The attachment of these cells did not require fibronectin or Geodia lectin. Homologous glycoconjugates or NaOH-treated collagen inhibited cell attachment. Collagen from the sponge Chondrosia reniformis, even in the presence of Geodia CAF, was no appropriate substrate for Geodia cell attachment. Whether collagen is a component of cell-matrix interactions in sponge systems also in vivo is discussed.

Identification and isolation of the primary aggregation factor from the cell membrane of the sponge Geodia cydonium.

The primary aggregation factor (pAF) of sponge cells is a glycoprotein that is firmly associated with the cell membrane. Polyspecific antibodies (anti-GM) prepared from sera raised against membranes of cells from the siliceous sponge Geodia cydonium were found to inhibit initial aggregation of homologous cells. The inhibition of aggregation, caused by anti-GM was neutralized by pAF. The pAF had been successfully solubilized and enriched by affinity chromatography, gel filtration and density gradient centrifugation, if checked by polyacrylamide gel electrophoresis in the presence of urea. The Mr of the native pAF was approximately 40 000 as estimated by gel filtration; under denaturing conditions three protein species (Mr: 16 500, 15 500 and 13 500) were identified in the pAF preparation. The pAF was precipitable by Ca++ and did not cross-react with antisera against homologous purified secondary aggregation factor and lectin. It is mainly composed of protein (48.0%) and carbohydrate (50.2%). The isolated pAF restored the aggregation potency not only of factor-depleted Geodia cells but also of cells from other Demospongiae. However, the pAF displayed no aggregation enhancing effect on urea-treated cells from species belonging to the Calcispongiae or Hexactinellida. We hypothesize that in contrast to the secondary aggregation, the initial aggregation of Geodia cells is mediated by the one-component system, the bivalent and bifunctional pAF.

Further characterization of a lectin and its in vivo receptor from Geodia cydonium

From the results of two-dimensional isoelectric focusing, SDS-gel electrophoresis and from immunochemical data it became evident that lectin I and lectin II (corresponding to fractions Geodia I and Geodia II isolated on immobilized lactose) from the sponge Geodia cydonium are apparently identical mixtures of several isolectins, the pI values of their subunits ranging, in contrast to our previous report, from 4.8–7.5. The hypothetical concept of sugar-mediated, specific lectin-lectin interactions (self-recognition) could not be verified by binding of FITC-labelled isolectins (Geodia I) to the lectin subunits, which had been purified by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The concept should also be dismissed on the basis of carbohydrate analyses revealing in contradiction with previous results the exclusive presence of alkali-labile bound tetraglucose on the purified isolectins (1 mol/mol lectin protein). The combining site of the isolectins was shown by a quantitative microprecipitation inhibition assay to be most complementar to oligosaccharides of the β-galactoside series and to interact specifically with particular structural elements of the subterminal sugar(s). Carbohydrates of the anti aggregation receptor, which are assumed to represent the functional ligand of the Geodia-isolectins in vivo, could be demonstrated to have a high affinity for the lectin combining site, exceeding that of the best disaccharide inhibitor, lactose, by five orders of magnitude. A preliminary chemical characterization of the receptor carbohydrate revealed that D-galactose and D-glucose (each approx. 200 mol/mol receptor) are organized in an oligosaccharide, which could be cleaved from the protein by trifluoroacetolysis.

Characterization of the trimeric, self-recognizing Geodia cydonium lectin I.

A D-galactose-specific lectin I was extracted from the sponge Geodia cydonium and purified by affinity chromatography. The molecular weight of lectin I as determined by high-pressure liquid gel chromatography, was found to be 36500 +/- 1300. Disc gel electrophoresis in the presence and in the absence of sodium dodecyl sulfate showed that lectin I is a trimer composed of three different subunits (Mr: 13800, 13000 and 12200); two of the three subunits are linked by one disulfide bond. Isoelectric focusing gave a pI of 5.6 for the native molecule and a pI of 4.4 and of 7.4 for the subunits. The three subunits carry carbohydrate side chains, composed of D-galactose (94%) and of arabinose (5%). Based on experiments with lectins, the terminal D-galactose residues are bound by beta 1 leads to 6 and/or beta 1 leads to 4 glycosidic linkages. The Geodia lectin I contains, besides two carbohydrate recognition sites, at least one receptor site for a second lectin I molecule.

Partial purification and properties of a chromatin bound endonuclease from the marine sponge Geodia cydonium

1. 1. A chromatin bound endonuclease (M r:107,000) has been extracted and partially purified from the siliceous sponge Geodia cydonium. 2. 2. Disc gel electrophoresis showed that only one enzyme was present in the partially purified preparation which was able to degrade DNA and poly(A). 3. 3. The enzyme liberates oligonucleotides on incubation with poly(A), which are further degraded to yield the 5′-mononucleotide, which has a pI of 6.5 and a pH optimum of 7.5–8.0. 4. 4. Cations are not required for enzymic activity and EDTA does not inhibit the enzyme. 5. 5. Only iodosobenzoic acid was found to completely inhibit the enzyme. 6. 6. The enzyme hydrolysed poly(A), poly(U), poly(C), DNA, poly[d(A-T)], poly [d(G-C)], but not poly (dA) or poly (G).

Chromatin structure from the marine sponge Geodia cydonium

1. 1. The histones isolated from the siliceous sponge Geodia cydonium have been separated using two electrophoretic techniques. 2. 2. A comparison of their mobilities with those of calf thymus and rat liver show that some Geodia histone species (H3, H1 and H1°) exhibit electrophoretic variance. 3. 3. The results show, that as in other eukaryotic systems the sponge chromatin contains the core histones (H2A, H2B, H3 and H4) and the linker histone (H1). 4. 4. ADP-ribosylation of Geodia histones and separation of the individual histones by electrophoresis resulted in four histones being radiolabeled. 5. 5. Digestion of Geodia chromatin with endogenous endonuclease is shown to result in the formation of nucleosome particles containing approximately 200 base pairs of DNA. 6. 6. A major product of endogenous endonuclease digestion is a relatively stable 110 base pair intermediate. 7. 7. Incubation of chromatin with DNase II and separation of the products under denaturing conditions reveals 20 bands migrating at 10 base intervals.

Fibronectin is apparently not involved in species-specific reaggregation of cells from the marine sponge geodia cydonium.

Experiments were carried out to test the hypothesis that fibronectin is involved in reaggregation of dissociated sponge cells. Cells from the siliceous sponge Geodia cydonium were extracted with urea to solubilize fibronectin from cells of higher multicellular organisms. The crude extract was further fractionated by DNA, heparin, and collagen affinity chromatography; they were termed Geodia fibronectin like fractions. The fibronectin like fractions contained a series of proteins with molecular weights different from that of the genuine fibronectin. The Geodia fibronectin like fractions did not react with antiserum, produced against human fibronectin, under formation of a precipitin line. Using this antiserum the sponge cells could not be specifically labeled with FITC-anti-IgG antiserum. Radioimmunoprecipitation experiments revealed that the Geodia fractions contain--if at all--0.1% fibronectin or fibronectin like protein at the most. In the crucial experiments it was shown that the Geodia fibronectinlike fractions, human fibronectin, and antifibronectin antiserum exerted no influence on adhesion of Geodia cells either in the absence or in the presence of the soluble aggregation factor. Based on these findings, we conclude that fibronectin is apparently not present on Geodia cells and does not play a role in aggregation of this biological system.

A mitogenic lactose-binding lectin from the sponge Geodia cydonium.

Two lectins with affinity for hog A + H blood group substance were extracted from the Mediterranean sponge Geodia cydonium. One, termed Geodia lectin I, was purified to near homogeneity by lactose elution from a column of hog A + H coupled to Sepharose 4B. Geodia lectin I was a glycoprotein (carbohydrate content about 14%) with m.w. of about 60,000 and an isoelectric point of pH 4.4. Subunits of about 15,000 daltons were linked by disulfide bonds. The lectin precipitated with various snail galactans composed entirely of DGal and LGal, to a slight degree with guaran, which has DGal alpha 1 leads to 6 in a terminal nonreducing position, and with blood group substances. Of 42 sugars tested, only lactose, dGal beta 1 leads to 4 DGlcNAc, DGal beta 1 leads to 3DGlcNAc, DGalNAc, inhibited precipitation. In view of the relatively high concentration of those disaccharides that inhibited relative to other lectins, the specificity of interaction is thought to be via beta-linked DGal residues that are part of a more complex combining site. Geodia lectin I was mitogenic for human peripheral blood lymphocytes with an optimal concentration of about 5.6 micrograms/ml if serum was omitted for the first 24 hr of culture to allow "triggering." Fetal calf serum apparently contained a high concentration of substances that inhibited mitogenicity and hemagglutination by Geodia lectin I, presumably by specifically interacting with the binding site.

Xenograft Rejection in Marine Sponges

In sponges there exists a graft rejection mechanism in which an inhibitory aggregation factor is involved. The inhibitory aggregation factor has been isolated from a culture medium containing dissociated cells of the sponge Geodia cydonium. Using ion-exchange and gel fractionation the factor was purified and shown to be electrophoretically pure. The factor has a molecular weight of 27000 and was characterized as a glycoprotein. The activity of the inhibitory aggregation factor was not affected by heat treatment, but treatment with trichloroacetic acid resulted in the irreversible loss of activity. The inhibitory aggregation factor affects the aggregation-factor-mediated reaggregation of dissociated sponge cells by extension of the lag phase preceding the aggregation process; the endpoint of the reaggregation process is not changed. The inhibitory aggregation factor competes with the aggregation receptor for the binding site on the aggregation factor. The inhibitory aggregation factor is not synthesized if homologous aggregation factor is added to the dissociated cells. The results indicate that the inhibitory aggregation factor is synthesized in xenografts of the system Geodia cydonium--Geodia rovinjensis after a grafting period of at least 3 days. Following the synthesis of the factor, the concentration of the extractable aggregation factor decreases.

Aggregation of sponge cells: 22. Species-specific reactivity of a lectin from the sponge Geodia cydonium

Single cells of the marine sponge Geodia cydonium aggregate species-specifically in the presence of a soluble aggregation factor to form large cell clumps. A lectin isolated from the same sponge species does not cause agglutination of Geodia cells but agglutinates only cells from heterologous species (e.g. Tethya lyncurium, Hemimycale columella, Pellina semitubulosa, Cacospongia scalaris, Verongia aerophoba). The process of agglutination is independent of divalent cations (they do not affect the agglutination process at concentrations up to 50 mM), occurs at 2°C, causes a reduction in the viability of the cells and results in an inhibition of programmed syntheses. The observed differences between the properties of cell agglutination (effect of a lectin in a heterologous system) and cell aggregation (effect of an aggregation factor in the homologous system) is discussed. Cell aggregation is dependent upon the presence of an aggregation factor, the presence of cations and an incubation temperature 2̃0°C; cell aggregation results in a stimulation of programmed syntheses. Cell agglutination requires a heterologous macromolecule (e.g. lectin), it is independent of divalent cations and causes inhibition of programmed syntheses in the cells.

Aggregation of sponge cells: 23. Interaction of Geodia lectin with Tethya cell-surface glycoprotein

A macromolecule has been isolated from the cell membranes of the sponge Tethyalyncurium. This macromolecule was purified and found to bind to a d-galactose specific lectin from the sponge Geodia cydonium. The lectin receptor was characterized as a glycoprotein with a molecular weight in the region of 155 000. Evidence is presented indicating that the binding of lectin with the lectin receptor is caused by hydrophobic interactions.

Aggregation of sponge cells: a novel mechanism of controlled intercerllular adhesion

From the marine sponge Geodia cydonium a series of macromolecules have been isolated and characterized which are involved in the control of aggregation and separation of sponge cells; these include aggregation factor, aggregation receptor, anti-aggregation receptor, β-glucuronidase, β-glucuronosyltransferase, β-galactosyltransferase, β-galactosidase and a lectin. These components might be linked in the following sequence: (a) activation of the aggregation receptor by its enzymic glucuronylaion; (b) adhesive recognition of the cells, mediated by the aggregation factor and the glucuronylated aggregation receptor; (c) inactivation of the aggregation receptor by its deglucuronylation with the membrane-associated β-glucuronidase; (d) cell separation due either to the loss of the recognition site (glucuronic acid) of the aggregation receptor for the aggregation factor or to an inactivation of the aggregation factor by the anti-aggregation receptor. The activity of the anti-aggregation receptor is probably controlled by the Geodia lectin.