The LW blood group antigens reside on a 42-kDa erythrocyte membrane glycoprotein that was purified by immunoaffinity and partiafly sequenced. From this information, a specific PCR-amplifled DNA fragment was used to screen a Agtll human bone marrow cDNA library. LW(a-b-). This showed that the protein encoded by these clones was LW gene product and suggested that the N terminus of the LW protein is oriented extracellularly. Most interestingly, the LW protein was found to exhibit sequence similarities (with -30% identity) with intercellular adhesion molecules ICAM-1, -2, and -3, which are the counterreceptors for the lymphocyte functionassociated antigens LFA-1. The extraceflular domain of LW consists, like that of ICAM-2, of two immunoglobulin-like doins, and the critical residues involved in the binding of LFA-1 to ICAMs were partially conserved in LW.The LW and Rh (rhesus) blood group systems were discovered simultaneously and were confused for a long time (reviews, refs. 1-3 Affinity Purification of the LW Protein. Membranes from two units of LW(a+b-) red cells were solubilized with 1% (wt/vol) Triton X-100 in phosphate-buffered saline (PBS) and applied to a specific affinity matrix column, prepared by binding 9 mg of purified murine monoclonal IgG antibody anti-LW (BS46) to 2 ml of protein A-agarose followed by cross-linking of the complex with dimethylpimelimidate (ImmunoPure IgG orientation kit, Pierce). After washing, the LW antigenic material bound was eluted with a glycine buffer (pH 2.8) and immediately brought to near neutrality.
A 37-kDa protein was immunopurified from human erythrocytes as a complex with a monoclonal antibody directed against the Kell blood group protein of 93 kDa. A rabbit antibody raised against the purified complex reacted on a Western blot with the 93-kDa and 37-kDa proteins and was able to immunoprecipitate the 37-kDa component from K0 erythrocytes which express large amount of the Kx antigen, but not from erythrocytes of patients suffering from McLeod syndrome, a X-linked disorder in which the Kx antigen is lacking. Additional studies have shown that the 37-kDa protein is not glycosylated, and permitted the sequence of the 22 first N-terminal amino acids to be established. This sequence was identical to the predicted protein product of the XK gene cloned recently, which is deleted or mutated in McLeod patients [Ho, M., Chelly, J., Carter, N., Danek, A., Crocker, P. & Monaco, A. P. (1994) Cell 77, 869-880]. Our findings provide strong evidence that the 37-kDa red cell membrane protein is identical to the Kx protein produced by the XK structural gene and demonstrate that Kx and Kell proteins are two subunits expressed as a complex hold by disulfide bond(s) at the red cell surface.
A 37-kDa protein was immunopurified from human erythrocytes as a complex with a monoclonal antibody directed against the Kell blood group protein of 93 kDa. A rabbit antibody raised against the purified complex reacted on a Western blot with the 93-kDa and 37-kDa proteins and was able to immunoprecipitate the 37-kDa component from K0 erythrocytes which express large amount of the Kx antigen, but not from erythrocytes of patients suffering from McLeod syndrome, a X-linked disorder in which the Kx antigen is lacking. Additional studies have shown that the 37-kDa protein is not glycosylated, and permitted the sequence of the 22 first N-terminal amino acids to be established. This sequence was identical to the predicted protein product of the XK gene cloned recently, which is deleted or mutated in McLeod patients [Ho, M., Chelly, J., Carter, N., Danek, A., Crocker, P. & Monaco, A. P. (1994) Cell 77, 869-880]. Our findings provide strong evidence that the 37-kDa red cell membrane protein is identical to the Kx protein produced by the XK structural gene and demonstrate that Kx and Kell proteins are two subunits expressed as a complex hold by disulfide bond(s) at the red cell surface.
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