Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 336-341
Surface expression of Rh-associated glycoprotein (RhAG) in
nonerythroid COS-1 cells
Kimita Suyama,
Hua Li, and
Alex Zhu
From the Lindsley F. Kimball Research Institute of The New York
Blood Center, New York.
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
[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)
