Rh-associated Glycoprotein Research Articles

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

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)

The rhesus blood group system: Insights from recent advances in molecular biology

O VER A DECADE of intense research has yielded considerable insight into the molecular nature of the Rh antigens. As could be anticipated from the complex serology of the Rh blood group system, the diversity of the Rh proteins at the molecular level is considerable. A combination of gene deletion, genetic rearrangement, and point mutation in the 2 genes at the RH loci has generated a large array of Rh variant phenotypes. This review considers these alterations and summarizes the molecular basis of Rh antigens that have arisen from normal and variant Rh protein structures. Rh protein biosynthesis is totally dependent on the expression of an Rh-associated glycoprotein, RhAG, which together with Rh protein associate to form a tetrameric membrane complex, collectively called the Rh core complex. Despite the fact that Rh proteins are a major component of the erythrocyte membrane, little is known about their functional roles. Recent discoveries of RHAG homologs in primitive life forms (eg, Caenorhabditis elegans) that lack red cells support the concept that the Rh core complex has an important functional role in animal cellular homeostasis. This review considers each of these aspects of the biology of the Rh protein family, and also the molecular requirements for Rh antigen expression. Both the clinical significance of Rh and the rapid advances in understanding of the molecular basis of the Rh blood group system have inspired several reviews by researchers actively involved in this field, of which I refer the reader to 2 recent examples.l,2