Use of RhD fusion protein expressed on K562 cell surface in the study of molecular basis for D antigenic epitopes

The human D antigens, one of the most clinically important blood groups, ar e presented by RhD protein with a putative 12 transmembrane topology. To un derstand the molecular basis for the complex antigenic profile of RhD prote in, we expressed a series of RhD fusion proteins using different portions o f Duffy protein as a tag in erythroleukemic K562 cells. Because the reactiv ity of monoclonal anti-RhD antibody, LOR15C9, depends mainly on the sequenc e coded by exon 7 of RhD, we altered DNA sequence corresponding to the amin o acid residues 323-331(A) and 350-354(B) in the exon 7. The mutation in re gion B resulted in a severe reduction in LOR15C9 binding by flow cytometry analysis, suggesting that region B may play an important role in constituti ng antigen epitopes recognized by LOR15C9, On the other hand, a slight decr ease in the antibody binding was observed for the region A mutant, suggesti ng that the intracellularly located region A may elicit a long distance eff ect on the formation of exofacial antigen epitopes, In addition, using vari ous monoclonal. antibodies against RhD, we compared the antigenic profile o f expressed RhD fusion protein with that of endogenous RhD in K562 cells as well as in erythrocytes.