title: Expression of C antigen in transduced K562 cells.
abstract: BACKGROUND
  The Rh blood group system is involved in HDN and transfusion reactions. A retrovirus-expression system was previously used to show that polypeptides carrying the Rh blood group antigens are encoded by the RHD and RHCE genes. This study investigated the structure of the C antigen.
  
  
  METHODS
  K562 cells were transduced with full-length cDNA encoding Ce and CE antigens, and the expression of C, e, and E antigens was examined by flow cytometry using MoAbs. The importance of Cys16 in C antigen expression was examined by utilizing site-directed mutagenesis to convert Cys16 to Trp in cDNA encoding Ce and CE before expression in K562 cells.
  
  
  RESULTS
  When K562 cells were transduced with cDNA that was predicted to encode Ce antigens, clear reactivity with anti-e and anti-C was obtained. In contrast, K562 cells transduced with cDNA that was predicted to encode CE antigens gave strong reactivity with anti-E but failed to react with two examples of anti-C. A third example of anti-C gave weak reactivity. When cDNA encoding Ce antigens was mutated to encode Trp16, one example of anti-C had the same reactivity with the mutated polypeptide as with the wild-type molecule, but reactivity with two other anti-C examples was reduced by 50 percent.
  
  
  CONCLUSIONS
  The nature of polymorphic residue 226 (proline when E is expressed, alanine when e is expressed) has a marked effect on the epitopes recognized by the three C MoAbs studied. The presence of Cys16 in Ce polypeptides influences the presentation of the C epitope recognized by two of the three MoAbs. These experiments provide the first direct demonstration that C and E/e antigens can be expressed on the same polypeptide.
authors.affiliation: International Blood Group Reference Laboratory, Bristol Institute for Transfusion Sciences, Bristol, UK. jon.smythe@nbs.nhs.uk
authors.forename: J S
authors.initials: JS
authors.lastname: Smythe
authors.forename: D J
authors.initials: DJ
authors.lastname: Anstee
mesh_terms: D019943:Amino Acid Substitution; D000906:Antibodies / Q000032:analysis; D000956:Antigens, Viral / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004912:Erythrocytes / Q000276:immunology / Q000502:physiology*; D005434:Flow Cytometry; D015870:Gene Expression; D006513:Hepatitis B e Antigens / Q000276:immunology; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics; D007519:Isoantigens / Q000235:genetics* / Q000276:immunology; D020014:K562 Cells; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012204:Rh-Hr Blood-Group System / Q000276:immunology*; D014161:Transduction, Genetic
chemical_list: D000906:Antibodies; D000956:Antigens, Viral; D018076:DNA, Complementary; D006513:Hepatitis B e Antigens; D017874:Immediate-Early Proteins; D007519:Isoantigens; D010455:Peptides; D012333:RNA, Messenger; D012204:Rh-Hr Blood-Group System; C044582:immediate-early proteins, cytomegalovirus