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

Kimita Suyama, Hua Li, Alex Zhu

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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 immuno-precipitation 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.

Original languageEnglish (US)
Pages (from-to)336-141
Number of pages196
JournalBlood
Volume95
Issue number1
StatePublished - Jan 1 2000
Externally publishedYes

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COS Cells
Glycoproteins
K562 Cells
Cells
Complementary DNA
Glycophorin
Erythroblasts
Membrane Glycoproteins
Flow cytometry
Methionine
Epitopes
Polyacrylamide Gel Electrophoresis
Cultured Cells
Flow Cytometry
Proteins
Molecular Weight
Blood
Molecular weight
Antigens
Cell Line

ASJC Scopus subject areas

  • Hematology

Cite this

Surface expression of Rh-associated glycoprotein (RhAG) in nonerythroid COS-1 cells. / Suyama, Kimita; Li, Hua; Zhu, Alex.

In: Blood, Vol. 95, No. 1, 01.01.2000, p. 336-141.

Research output: Contribution to journalArticle

Suyama, Kimita ; Li, Hua ; Zhu, Alex. / Surface expression of Rh-associated glycoprotein (RhAG) in nonerythroid COS-1 cells. In: Blood. 2000 ; Vol. 95, No. 1. pp. 336-141.
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