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Purification and partial characterization of a cell adhesion molecule (gp150) involved in postaggregation stage
1Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Abstract:
A cell surface glycoprotein of apparent Mr 150,000 (gp150) has been implicated in mediating EDTA-resistant cell-cell adhesion in Dictyostelium discoideum. A simple purification scheme making use of high-performance liquid chromatography has been devised to purify gp150 to near homogeneity. Purified gp150 was capable of neutralizing the effect of a rabbit antiserum raised against gel-purified gp150, which was previously reported to be a potent inhibitor of cell-cell adhesion (Geltosky, J. E., Weseman, J., Bakke, A., and Lerner, R. A. (1979) Cell 18, 391-398). The binding of 125I-labeled gp150 to intact cells was both dose-dependent and saturable, demonstrating the presence of specific cell surface binding sites for gp150. When reassociation of postaggregation stage cells was carried out in the presence of soluble gp150, aggregate formation was strongly inhibited. In contrast, gp150 failed to exert any effect on cells at the aggregation stage. The inhibitory effect of gp150 was sensitive to protease treatment, suggesting that the protein moiety is crucial to gp150 function. These results, taken together, provide direct evidence that gp150 is a cell-cell adhesion molecule involved in cell-cell binding in the postaggregation stage of Dictyostelium development.
Insights
A glycoprotein (gp150) mediates cell-cell adhesion in Dictyostelium discoideum during later development. Purified gp150 inhibits cell aggregation, confirming its role as a key adhesion molecule.
Area of Science:
- Cell Biology
- Developmental Biology
- Biochemistry
Background:
- Cell-cell adhesion is crucial for multicellular development.
- A 150,000 apparent molecular weight cell surface glycoprotein (gp150) was previously implicated in EDTA-resistant cell-cell adhesion in Dictyostelium discoideum.
Purpose of the Study:
- To purify gp150 and provide direct evidence for its function in cell-cell adhesion.
- To investigate the role of gp150 in different developmental stages of Dictyostelium discoideum.
Main Methods:
- Purification of gp150 using high-performance liquid chromatography (HPLC).
- Neutralization assays with rabbit antiserum against gp150.
- Binding assays using 125I-labeled gp150.
- Inhibition assays of cell reassociation in the presence of soluble gp150.
- Protease sensitivity assays.
Main Results:
- gp150 was purified to near homogeneity.
- Purified gp150 neutralized inhibitory antiserum, indicating its role in adhesion.
- Specific binding sites for gp150 were identified on intact cells.
- Soluble gp150 inhibited aggregate formation in postaggregation stage cells but not in aggregation stage cells.
- The inhibitory function of gp150 was sensitive to protease treatment.
Conclusions:
- gp150 is a cell-cell adhesion molecule essential for postaggregation stage Dictyostelium development.
- The protein component of gp150 is critical for its adhesive function.
- gp150 plays a specific role in mediating cell-cell binding during later stages of Dictyostelium development.