Molecular configuration of Rh D epitopes as defined by site-directed mutagenesis and expression of mutant Rh constructs in K562 erythroleukemia cells

The Rh D antigen is the most clinically important protein blood group antig en of the erythrocyte. It is expressed as a collection of at least 37 diffe rent epitopes, The external domains of the ph D protein involved in epitope presentation have been predicted based on the analysis of variant ph D pro tein structures inferred from their cDNA sequences and their D epitope expr ession. This analysis can never be absolute because (1) most partial D phen otypes involve multiple amino acid changes in the Rh D protein and (2) defi ciency for 1 or more epitopes may be due to gross structural alteration in the variant Rh D protein structure. We report here the amino acid requireme nts for the majority of D epitopes. They have been defined by generating a series of novel ph mutant constructs by mutagenesis using an Rh cE cDNA as template and mutagenic oligonucleotide primers. When transfected into K562 cells, the D epitope expression of the derived mutant clones was then asses sed by flow cytometry. The introduction of 9 externally predicted ph D-spec ific amino acids on the Rh cE protein was sufficient to express 80% of all tested D epitopes, whereas other clones expressed none. We concluded from o ur data that the D epitope expression is consistent with at least 6 differe nt epitope clusters localized on external regions of the ph D protein, most involving overlapping regions within external loops 3, 4, and 6. (C) 1999 by The American Society of Hematology.