Biochemical studies on McLeod phenotype red cells and isolation of Kx antigen

Red cells of the McLeod blood group phenotype have weak Kell antigens, lack Kx antigen and have acanthocytic morphology. We have immunoprecipitated Kell antigens from McLeod red cells and show that they are markers on the same 93 kDa membrane protein that carries Kell antigens on normal red cells. However, as determined by Western immunoblotting, McLeod red cells have a marked deficiency of this protein. We have also studied the near-neighbour relationship of McLeod and common Kell red-cell membrane proteins by cross-linking intrinsic sulphydryl groups by oxidation, catalysed with orthophenanthroline and copper, or by cross-linking amino groups with dimethyl-3,3'-dithiobispropionimidate. Results were analysed by diagonal mapping in two-dimensional gels. No abnormalities of membrane protein inter-relationship were detected in McLeod red cells. We have isolated Kx antigen from K0 red cells by immunoprecipitation with human alloimmune anti-Kx serum, isolation of immune complexes from detergent-solubilized cell membranes with protein A-Sepharose and analysis of the eluted immune complex by SDS-PAGE under reducing conditions. Kx antigen is a marker on a red-cell membrane protein of approximately 37 kDa. Ko (Knull) red cells have about twice the amount of Kx antigen as do red cells of common Kell type. McLeod red cells have no detectable Kx antigen by serological tests or by immunoprecipitation.