Sponge aggregation factor: In situ localization by fluorescent monoclonal antibody techniques

Abstract

The aggregation factor (AF) from sponges mediates a heterophilic interaction of homologous cells. Applying electron microscopical means, we succeeded only very rarely in identifying the 90 S AF particle in tissue sections from Geodia cydonium. By means of a fluorescent antibody technique, we have now localized the cell binding domain of the AF in situ. Previous studies in this laboratory have led to the identification of the 47-kDa cell binding protein of the AF, using the monoclonal antibody (mab) 5D2-D11 [Gramzow M, Bachmann M, Zahn RK, Uhlenbruck G, Dorn A, Müller WEG, J Cell Biol, 102: 1344-1349, 1986]. This mab and mab 7D5, directed against a 92-kDa protein in the AF complex, were chosen for the fluorescent studies. By using mab 5D2-D11, the plasma membranes of cells from different regions in the sponge could be brightly stained. However, mab 7D5 reacted only very weakly with the sponge surfaces. By applying the immuno-blotting technique it was furthermore demonstrated that the cell binding protein is present both in the associated form with AF complex and in a free state. Moreover, it was established that the 47-kDa binding protein is not present in homologous glycoconjugates, lectin, or collagen; these components are known to be involved in cell-matrix interaction.

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.

“…The proteins were transferred to nitrocellulose sheets (Towbin et al, 1979;Gramzow et al, 1986a) and incubated with 1:250 dilution of topoisomerase II-specific antiserum. The immunocomplexes formed were visualized after an additional incubation with anti-rabbit IgG (Sigma; peroxidase conjugated; 1:50 dilution), applying the 4-chloro-1-naphthol/hydrogen peroxide procedure (Gramzow et al, 1986b). Gel electrophoresis and autoradiography ofpore complex-lamina polypeptides One-dimensional SDS -PAGE was performed according to Laemmli (1970), with the exception that 6 M urea was added.…”