Detection and characterization of mouse mammary tumor virus cell surface antigens: estimation of antigen abundance by protein A assay

Abstract

Antisera against the following mouse mammary tumor virus (MMTV) structural proteins were used to detect MMTV cell surface antigens: (i) the 27,000-dalton nucleoid protein, p27; (ii) the 36,000-dalton envelope glycoprotein, gp36; and (iii) the 52,000-dalton exterior envelope glycoprotein, gp52. We report here the development of an adherent-cell isotopic staphylococcal protein A (SPA) test (ISPAT) for MMTV structural proteins which allows for the detection of an MMTV membrane-associated antigen as well as an estimate of its relative abundance on the cell surface. This test demonstrated that the gp52 was the predominant MMTV cell surface antigen detected on both C3H and GR mouse mammary tumor cells. In a comparative study with anti-gp52 and anti-gp36 sera, SPA-specific binding with anti-gp36 serum was found to be only 5 to 6% of that obtained for the external virion glycoprotein, gp52. Both direct and indirect ISPAT indicated the presence of a low but detectable number of gp36 determinants on GR-MMTV cells; however, these gp36 determinants, unlike gp52 determinants, appeared to be exposed by the fixation procedure used. Only 0.9 to 1.1% of the gp52-specific binding was detected when anti-gp36 serum was allowed to react with viable cells. The binding of [125I]SPA achieved with anti-p27 serum was even less than that detected with gp36-directed reagents, indicating that p27 is not a cell surface antigen. The use of fluoresceinated SPA further demonstrated that p27 and gp36 reactivity was only associated with a small number of cells in each of the mammary cultures tested. When N-[4-(5-nitro-2-furyl)-2-thiazoly]-formamide-induced C3H bladder tumor cells were subjected to a gp52-directed ISPAT, the failure to detect gp52-specific binding demonstrated the specificity of this assay for MMTV gp52-expressing cells. In addition to detecting and characterizing MMTV cell surface antigens, the newly developed adherent cell assay could measure changes in the abundance of cell surface gp52. When dexamethasone-treated and untreated GR cells were compared, measurements of gp52-specific SPA binding indicated that dexamethasone stimulation leads to a 12.2-fold increase in the amount of cell surface gp52 detected.