Surface expression of Rh-associated glycoprotein (RhAG) in nonerythroid COS-1 cells

Abstract

AbstractIn the Rh blood system, RhAG (Rh-associated glycoprotein, or Rh50) is thought to be involved in Rh30 (D, CE) expression by forming a protein complex on the red cell surface. To obtain further insight into the Rh complex, we chose nonerythroid COS-1 cells instead of proerythroblast-like K562 cells, which produce endogenous Rh proteins as cell host, for the expression of both RhAG and RhD. The RhAG cDNA was subcloned into a retroviral vector, and a stable COS-1 cell line was then established via retroviral transduction. Surface expression of RhAG on the COS-1 cells was monitored by flow cytometry using mouse monoclonal anti-RhAG(2D10). Under these conditions, we detected significant expression of RhAG on the cell surface, compared to stable COS-1 cells transduced with the vector alone. To confirm the results, we isolated RhAG by immunoprecipitation from the lysate of the COS-1 cells, which were metabolically labeled with [35S]-methionine. A strong band of the 32 kd on SDS-PAGE was obtained, corresponding to the results obtained from other cultured cells (K562 cell and others), which always produce partially glycosylated RhAG with a molecular weight of 32 kd. Thus, RhAG was expressed without Rh30 and other Rh-related glycoproteins (LW, glycophorin B) in nonerythroid cells. Using the same strategy, however, we could not express RhD epitopes on COS-1 cells even in the presence of RhAG cDNA, suggesting that other factors might be required for the surface expression of RhD antigen. (Blood. 2000;95:336-341)