Molecular Configuration of Rh D Epitopes as Defined by Site-Directed Mutagenesis and Expression of Mutant Rh Constructs in K562 Erythroleukemia Cells

Abstract

The Rh D antigen is the most clinically important protein blood group antigen of the erythrocyte. It is expressed as a collection of at least 37 different epitopes. The external domains of the Rh D protein involved in epitope presentation have been predicted based on the analysis of variant Rh D protein structures inferred from their cDNA sequences and their D epitope expression. This analysis can never be absolute because (1) most partial D phenotypes involve multiple amino acid changes in the Rh D protein and (2) deficiency 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 requirements for the majority of D epitopes. They have been defined by generating a series of novel Rh 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 assessed by flow cytometry. The introduction of 9 externally predicted Rh D-specific 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 our data that the D epitope expression is consistent with at least 6 different epitope clusters localized on external regions of the Rh D protein, most involving overlapping regions within external loops 3, 4, and 6.

Competitive enzyme‐linked immunoassay of monoclonal immunoglobulin G anti‐D preparations

“…The Rh D antigen contains at least 37 different epitopes. Site-directed mutagenesis experiments to study the expression of different D epitopes have identified nine critical amino acids, giving rise to at least six distinct epitope clusters involving one or more of the six external loops of the Rh D polypeptide (Liu et al, 1999) Competitive EIA of monoclonal IgG anti-D 157 4 and 6. Loops 3 and 4 (or all 3 loops in some cases) appear to be required for expression of the most clinically significant epitope, epD6/7, as defined in the original 1-9 epitope model.…”

“…However, just three amino acids located in loop 6 are predicted to give rise to epD3 (although other residues may be necessary to stabilize the configuration). The proposition by Liu et al (1999) that loops 3 and 6 are nonadjacent may account for the very limited ability of the epD3.1reactive MoAbs 1-73 and 1-96 to inhibit the binding of biotinylated BRAD-5, rather than that these MoAbs are poor binders, as their estimated potencies using flow cytometry were 6Á4 and 11Á4 IU mg À1 . Although the four epD6.8-reactive MoAbs, including BRAD-5 itself, had high specific activities, they were not the most potent MoAbs in the study.…”

Reactivity of autoantibodies of autoimmune hemolytic anemia with recombinant rhesus blood group antigens or anion transporter band3

“…Genetic analysis has also found antithetical C/c and E/e core epitopes in the polymorphic amino acid change at the second and fourth loops, respectively [20]. Recently, expression analysis of recombinant Rh polypeptides and band3 glycoprotein was performed on erythroleukemic cell lines, and fine mapping of RhD epitopes became available [21][22][23][24][25]. The use of the recombinant antigen system in analysis is advantageous because of the selective expression of any particular antigen, independent of epitope conformation.…”

Molecular characterization of weak D phenotypes by site‐directed mutagenesis and expression of mutant Rh–green fluorescence protein fusions in K562 cells

“…No epitopes were mapped on loop 5. Site-directed mutagenesis [20] has shown that the external domains of the RhD protein are transduced into the RhcE polypeptide and the expression of D epitopes has been studied. That study revealed loop-loop interactions of the RhD polypeptide.…”