Site directed mutagenesis of the human Rh D antigen: molecular basis of D epitopes.
Previous attempts to define the molecular configuration of D epitopes has been confined to the analysis of the serological profile and Rh D molecular structure of partial D phenotypes. There are numerous drawbacks in this approach, most fundamental of which is that with the exception of RoHar, partial D phenotypes are defined by the loss of D epitope expression, and is thus difficult to directly correlate a specific amino acid to a particular D epitope. Furthermore, most partial D phenotypes are associated with multiple amino acid changes in the mutant Rh protein species associated with partial D expression. In our study we have applied site directed mutagenesis to introduce RhD amino acids in a stepwise manner to a Rh cE cDNA. This cDNA was introduced into K562 cells using retroviral mediated gene delivery, and D epitope expression analysed by flow cytometry. Our study provides evidence for at least six different epitope clusters on the external face of the Rh D protein. The relative predicted positions of these epitope clusters has resulted in us presenting a model for the hypothetical arrangement of external Rh D protein loops.
Department of Biological & Biomedical Sciences, University of the West of England, UK. neil.avent@uwe.ac.uk
N D
ND
Avent
W
W
Liu
M L
ML
Scott
J W
JW
Jones
D
D
Voak
D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D020014:K562 Cells; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012204:Rh-Hr Blood-Group System / Q000235:genetics*; D014162:Transfection
D000939:Epitopes; D012204:Rh-Hr Blood-Group System