C.L. Johnson

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

Summary 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.

Isolation of Kell‐active protein from the red cell membrane

Kell blood‐group‐active protein has been isolated by labeling red cell surface proteins with 125I, sensitizing intact cells with anti‐K1, anti‐ K2, anti‐K7, or anti‐K22, solubilizing the cell membranes, isolating immune complexes, and separating their components by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Each antibody separated a protein of approximately 93,000 daltons. Periodic‐acid Schiff (PAS) staining of Kell protein showed that it was glycosylated. When separated under non‐reducing conditions, Kell protein had different SDS‐PAGE characteristics with protein bands of approximately 85,000 daltons and 115,000 daltons. This suggests that in the red cell membrane Kell protein is complexed with other proteins. Quantitative experiments made with anti‐K7, anti‐K22, and a mixture of anti‐K7 and anti‐K22 indicate that both antigen specificities are present in the same molecule. These biochemical data support serological studies which indicate that K22 is part of the Kell system.

Acquired loss of red cell Kell antigens

Summary. A 19‐year‐old patient with a long history of idiopathic thrombocytopenic purpura developed a potent antibody against a high‐incidence antigen in the Kell blood group system. The direct antiglobulin test on his red cells was negative. His cells exhibited profound depression of Kell blood group antigens, but antigens of other blood groups were normal. Transfusion of incompatible blood was well tolerated and differential agglutination tests, using selected Rh antisera, showed in vivo survival of the transfused red cells for more than 8 weeks. However, the transfused red cells also showed acquired loss of Kell antigens. Five months after the initial findings, Kell‐related antibody disappeared and Kell antigens reappeared on his red cells. The patients serum stored from the initial investigation now reacted with his freshly collected red cells. These data suggest that an environmental agent in the patients plasma was responsible for the temporary loss of Kell antigens from red cells in his circulation.

An individual with McLeod syndrome and the Kell blood group antigen K(K1)

McLeod syndrome is an X‐linked condition in which individuals of McLeod blood group phenotype have weak Kell antigens, acanthocytic red cells, and a muscular disorder. We now report a family in which two brothers have McLeod syndrome. One is K:‐1, while the other is the first known K:1 person with McLeod syndrome. The K1 gene in the latter is expressed weakly and was inherited from the father, in whom it is expressed normally. The brothers have the same clinical and laboratory manifestations of McLeod syndrome but have different Kell genes. Therefore, the Kell gene is unlikely to have any positive input into development of McLeod syndrome; its role is one of passive involvement in which its expression is modified.

The first example of autoanti-Kx

An IgG autoantibody with Kx specificity was found in the blood of a 61‐ year‐old white man. The antibody did not cause hemolysis of his own or transfused Kx‐positive red cells. The patient is of common Kell blood type and does not exhibit any clinical or hematologic features of McLeod syndrome.

Antibodies associated with the Kell blood group system.

The Kell blood group presently includes 23 antigens [1], a null (K 0 ) phenotype, a number of phenotypes characterized by weak Kell antigen activity, and an independent antigen, designated Kx, which appears to play a role in Kell antigen expression. Kell blood-group antigens are markers on the surface-exposed domain of a 93,000 daltons (93 kD) membrane glycoprotein [2]. Intrachain disulfide linkages [3], and probably the proper intra-membrane milieu, are important for the antigenic integrity of Kell 93 kD protein. Separated 93 kD Kell protein does not react by Western blot analysis with the Kell antibody used for its initial immuno-precipitation [1]. Incubation of intact red cells with solutions containing papain/DTT mixture [4] or 2-aminoethylisothiouronium bromide (AET) inactivates all antigens of the Kell complex except for Kx [5]. Five monoclonal antibodies have been examined for serological specificity within the Kell system and for their ability to recognize epitopes on a 93 kD red cell membrane protein by Western blot analysis.