Surface expression of Rh-associated glycoprotein (RhAG) in nonerythroid COS-1 cells.
In 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 [(35)S]-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)
Suyama
K
K
Lindsley F. Kimball Research Institute of The New York Blood Center, New York 10021, USA. ksuyama@nybc.org
Li
H
H
Zhu
A
A
D000818:Animals; D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D008358:Mannose / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D015514:Oncogene Proteins, Fusion / Q000096:biosynthesis / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012190:Retroviridae; D012204:Rh-Hr Blood-Group System; D014162:Transfection
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D008562:Membrane Glycoproteins; D015514:Oncogene Proteins, Fusion; C498523:RHAG protein, human; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012204:Rh-Hr Blood-Group System; C117760:RhD fusion protein, human; C401210:Rhag protein, mouse; C026410:Rho(D) antigen; D008715:Methionine; D008358:Mannose