Identification and further characterization of the specific cell binding fragment from sponge aggregation factor.

Abstract

Abstract. 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-D 11. 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 108 M -j) even in the absence of Ca ÷÷ ions; the number of binding sites was ~4 × 106/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.

Intracellular Distribution of the La Antigen in CV-1 Cells after Herpes Simplex Virus Type 1 Infection Compared with the Localization of U Small Nuclear Ribonucleoprotein Particles

“…PAGE was performed in 10~ slab gels in the presence of 0.1 ~ SDS as described by Laemmli (1970). The separated proteins were transferred to nitrocellulose sheets according to the method described by Towbin et al (1979) with the modifications given by Gramzow et al (1986). The sheets were incubated with anti-La MAb or monospecific anti-La antibodies; the immunocomplexes that formed were visualized by incubation with a 1 : 500 dilution of either peroxidase-conjugated anti-mouse IgG or peroxidaseconjugated anti-human IgG using the 4-chloro-1-naphthol/hydrogen peroxide procedure (Nakane, 1968).…”

Association of La and Ro antigens with intracellular structures in HEp-2 carcinoma cells.

“…The homogeneity of the antigen preparations was proven by NaDodSO4/gel electrophoresis, and their specificities, by immunoblotting (12) with standardized reference antisera (anti-Ro and anti-La; Centers for Disease Control, Atlanta). General procedures for preparation of mAbs (12,13) …”

“…The homogeneity of the antigen preparations was proven by NaDodSO4/gel electrophoresis, and their specificities, by immunoblotting (12) with standardized reference antisera (anti-Ro and anti-La; Centers for Disease Control, Atlanta). General procedures for preparation of mAbs (12,13) (12). In a further step, the antibodies were purified by immunoaffinity column chromatography using normal anti-mouse IgG coupled to Sepharose 4B (14).…”

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

“…This finding was our first evidence that calelectrin is present extracellularly in this organism. Next, we studied whether calelectrin is associated with G. cydonium AF, which is known to be composed of a series of polypeptides among which is the cell binding fragment (Gramzow et al, 1986). Then it was shown that the AF particles also contained a protein with an M, of 32,000 that was recognized in the immunoblot by antibodies against calelectrin (Figure 3 For further proof that the 32 kd polypeptide in the AF particle that reacted with anticalelectrin is presumably calelectrin, inhibition studies were performed.…”