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

BACKGROUND: The antigens of the human Rh system are of great clinical signi ficance in transfusion medicine and pregnancy. Of the Rh system antigens, D is clinically the most important, being one of the most immunogenic struct ures arising from human cells. The human D antigen represents a collection of epitopes expressed on a red cell membrane protein that is predicted to h ave 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 gen erated with changes to the RHD-specific residues 350, 353, and 354 in the p redicted sixth exofacial loop. Each mutant cDNA was subcloned into the pBab e puromycin retroviral vector, and supernatants were used to transduce K562 cells. Puromycin-resistant K562 clones were screened by flow cytometric an alysis using a panel of monoclonal antibodies with specificities to ep (epi tope) 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, Ala3 54Asn). Expression of c and E was unaffected. Conversely, the cells express ing 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 o f three amino acids, Asp(350), Gly(353), and Ala(354), in the expression of epD3 and epD9 on the predicted sixth external domain of the D protein. Thi s 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 D-mut construct (encoding His(350), Trp(353), and Asn( 354)). The K562/D-mut cell line has an identical molecular and serologic pr ofile as the red cell D-IVb phenotype, which confirms that retroviral gene transfer of Rh cDNA into K562 cells provides us with a powerful means by wh ich to further map epitopes of D.