Membrane association and shedding of the GPI-anchored Ca-MOv18 antigen in human ovary carcinoma cells.
The antigen recognized by the MOv18 MAb (Ca-MOv18) was recently shown to be a glycosylphosphatidylinositol (GPI)-linked protein. In this report we show that GPI-anchorage is not limited to IGROVI cells nor to other ovary carcinoma cell lines, but Ca-MOv18 was also found to be sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) treatment on fresh ovarian cancer cells. Furthermore, we found a heterogeneous sensitivity of Ca-MOv18 to PI-PLC cleavage, not only among the different cells studied but also in different experiments performed on the same cell line, during extended periods of time in culture. Sensitivity to PI-PLC cleavage was determined by immunofluorescence on live cells and by double-determinant radioimmunoassay of the antigen released in the supernatant. The specificity of the PI-PLC cleavage was demonstrated as follows: (a) TX114 solubilized Ca-MOv18 shifts from the detergent to the aqueous phase after treatment with PI-PLC; (b) on membrane preparations, PI-PLC specifically released a fraction of the antigen, which is distinct from the weakly associated form released by high-salt treatment; (c) Ca-MOv18 from IGROVI expressed the cross-reacting determinant (CRD), which is characteristic of GPI-linked molecules. The absence of CRD expression on the spontaneously released protein and the possibility of artificially inducing antigen shedding during the resynthesis of Ca-MOv18 which follows bacterial PI-PLC treatment are interesting points which need to be further investigated in order to understand the physiology of the Ca-MOv18 tumor antigen.
Miotti
S
S
Experimental Oncology E, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy.
Alberti
S
S
Facheris
P
P
Mantovani
L
L
Fornaro
M
M
Stella
M
M
Ménard
S
S
Canevari
S
S
Colnaghi
M I
MI
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis*; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000032:analysis*; D005260:Female; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D006801:Humans; D010051:Ovarian Neoplasms / Q000276:immunology*; D010716:Phosphatidylinositols
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D015295:Antigens, Tumor-Associated, Carbohydrate; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D010716:Phosphatidylinositols