Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 3986-3996
Molecular Configuration of Rh D Epitopes as Defined by Site-Directed
Mutagenesis and Expression of Mutant Rh Constructs in K562
Erythroleukemia Cells
Wendy Liu,
Neil D. Avent,
Jeffrey W. Jones,
Marion L. Scott, and
Douglas Voak
From the Bristol Institute for Transfusion Sciences, Southmead,
Bristol, UK; the National Blood Service, Liverpool, Liverpool, UK; and
the National Blood Service, Cambridge, Cambridge, UK.
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.
