Site‐directed mutagenesis of the human D antigen: definition of D epitopes on the sixth external domain of the D protein expressed on K562 cells

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Abstract
BACKGROUND: The antigens of the human Rh system are of great clinical significance in transfusion medicine and pregnancy. Of the Rh system antigens, D is clinically the most important, being one of the most immunogenic structures arising from human cells. The human D antigen represents a collection of epitopes expressed on a red cell membrane protein that is predicted to have 12 membrane‐spanning segments giving rise to six exofacial domains.STUDY DESIGN AND METHODS: By site‐directed mutagenesis using the method of inverse polymerase chain reaction, cE and D cDNA mutant constructs were generated with changes to the RHD‐specific residues 350, 353, and 354 in the predicted sixth exofacial loop. Each mutant cDNA was subcloned into the pBabe puromycin retroviral vector, and supernatants were used to transduce K562 cells. Puromycin‐resistant K562 clones were screened by flow cytometric analysis using a panel of monoclonal antibodies with specificities to ep (epitope) D1 through epD9.RESULTS: De novo expression of epD3 and epD9 was generated in the K562 cell lines expressing the mutated cE polypeptide (cE‐Asp350His, Gly353Trp, Ala354Asn). Expression of c and E was unaffected. Conversely, the cells expressing the mutated D polypeptide demonstrated loss of expression of epD1, epD2, epD3, epD4, and epD9.CONCLUSION: The data provide strong evidence for the critical involvement of three amino acids, Asp350, Gly353, and Ala354, in the expression of epD3 and epD9 on the predicted sixth external domain of the D protein. This domain also appears to be essential for the expression of epD1, epD2, and epD4, as a loss of expression of these epitopes was observed in K562 cells transduced with the Dmut construct (encoding His350, Trp353, and Asn354). The K562/Dmut, cell line has an identical molecular and serologic profile as the red cell DIVb pheno‐type, which confirms that retroviral gene transfer of Rh cDNA into K562 cells provides us with a powerful means by which to further map epitopes of D.