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 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.
Liu
W
W
Bristol Institute for Transfusion Sciences, Southmead, Bristol, UK.
Avent
N D
ND
Jones
J W
JW
Scott
M L
ML
Voak
D
D
D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D004915:Leukemia, Erythroblastic, Acute; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012204:Rh-Hr Blood-Group System / Q000737:chemistry* / Q000235:genetics*; D013329:Structure-Activity Relationship
D000939:Epitopes; D012204:Rh-Hr Blood-Group System