Monika Gramzow

Role of phospholipase A2 in the stimulation of sponge cell proliferation by homologous lectin

Using the Geodia cydonium system, we showed that after incubation of competent sponge cells in the presence of lectin, phospholipase A2 was released from the cells. The substrates for this enzyme, phosphatidylethanolamine and phosphatidylcholine, were identified in the extracellular material of sponge tissue. In addition, the phospholipase A2 inhibitor calelectrin was identified by immunobiochemical techniques; this molecule was associated with the aggregation factor. Reconstitution experiments strongly suggested that phospholipase A2 catalyzed the release of arachidonic acid, which is then taken up by the cells. Intracellularly, arachidonic acid was metabolized primarily to prostaglandin E2. Inhibition studies revealed that prostaglandin E2 is involved in the ultimate increase of DNA synthesis. These findings suggest that the phospholipase A2-arachidonic acid system is involved in the matrix-initiated signal transduction pathway in sponges.

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).

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

SummaryThe 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.

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 Mr 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.

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

Abstract 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.