Identification and further characterization of the specific cell binding fragment from sponge aggregation factor.
Monoclonal antibodies (McAbs) were raised against the aggregation factor (AF) from the marine sponge Geodia cydonium. Two clones were identified that secrete McAbs against the cell binding protein of the AF complex. Fab fragments of McAbs: 5D2-D11 completely abolished the activity of the AF to form secondary aggregates from single cells. The McAbs were determined to react with the AF in vitro; this interaction was prevented by addition of the aggregation receptor, isolated and purified from the same species. After dissociation of the AF by sodium dodecyl sulfate and 2-mercaptoethanol, followed by electrophoretical fractionation, a 47-kD protein was identified by immunoblotting which interacted with the McAbs: 5D2-D11. During this dissociation procedure, the sunburst structure of the AF was destroyed. In a second approach, the 47-kD protein was isolated by immunoprecipitation; 12 molecules of this protein species were calculated to be associated with the intact AF particle. The 47-kD AF fragment bound to dissociated Geodia cells with a high affinity (Ka of 7 X 10(8) M-1) even in the absence of Ca++ ions; the number of binding sites was approximately 4 X 10(6)/cell. This interaction was prevented by addition of the aggregation receptor to the 47-kD protein in the homologous cell system. Moreover, it was established that this binding occurs species-specifically. The 47-kD fragment of the AF was localized only extracellularly by indirect immunofluorescence staining in cryostat slices. These data suggest that the 47-kD protein is the cell binding molecule of the AF from Geodia.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000954:Antigens, Surface / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D015815:Cell Adhesion Molecules; D002449:Cell Aggregation; D008970:Molecular Weight; D011161:Porifera / Q000502:physiology*; D011506:Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000954:Antigens, Surface; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D011506:Proteins; C013065:cell aggregation factors