Protein 4.1R (4.1R) is a multifunctional component of the red cell membrane. It forms a ternary complex with actin and spectrin, which defines the nodal junctions of the membrane-skeletal network, and its attachment to the transmembrane protein glycophorin C creates a bridge between the protein network and the membrane bilayer. We now show that deletion of 4.1R in mouse red cells leads to a large diminution of actin accompanied by extensive loss of cytoskeletal lattice structure, with formation of bare areas of membrane. Whereas band 3, the preponderant transmembrane constituent, and proteins known to be associated with it are present in normal or increased amounts, glycophorin C is missing and XK, Duffy, and Rh are much reduced in the 4.1R-deficient cells. The inference that these are associated with 4.1R was borne out by the results of in vitro pulldown assays. Furthermore, whereas Western blot analysis showed normal levels of band 3 and Kell, flow cytometric analysis using an antibody against the extracellular region of band 3 or Kell revealed reduction of these two proteins, suggesting a conformational change of band 3 and Kell epitopes. Taken together, we suggest that 4.1R organizes a macromolecular complex of skeletal and transmembrane proteins at the junctional node and that perturbation of this macromolecular complex not only is responsible for the well characterized membrane instability but may also remodel the red cell surface. macromolecular complex ͉ cytoskeleton

The importance of mobilisation for positive patient outcomes highlights the need to develop methods to ensure that this nursing action is completed on a systematic basis.

The Kell blood group is a major antigenic system in human erythrocytes. Kell antigens reside on a 93-kDa membrane glycoprotein that is surface-exposed and associated with the underlying cytoskeleton. We isolated tryptic peptides and, based on the amino acid sequence of one of the peptides and by using the PCR, prepared a specific oligonucleotide to screen a AgtlO human bone-marrow cDNA library. Four clones were isolated, one containing cDNA with an open reading frame for an 83-kDa protein. Al known Kell amino acid sequences were present in the deduced sequence; moreover, rabbit antibody to a 30-amino acid peptide, prepared from this sequence, reacted on an immunoblot with authentic Kell protein. The Kell cDNA sequence predicts a 732-amino acid protein. Hydropathy analysis indicates a single membranespanning region, suggesting that Kell protein is oriented with 47 of its N-terminal amino acids in the cell cytoplasm, and a 665-amino acid segment, which contains six possible N-glycosylation sites, is located extracellularly. Computer-based search showed that Kell has structural and sequence homology to a family of zinc metalloglycoproteins with neutral endopeptidase activity.The Kell blood group system is important in transfusion medicine. It is a complex system containing at least 24 antigens (1, 2) residing on a 93,000-kDa glycoprotein that is surface-exposed, spans the erythrocyte membrane, and is attached to the underlying cytoskeleton (3,4

Expression of the Kell blood group system is dependent on two proteins, Kell and XK, that are linked by a single disulfide bond. Kell, a type II membrane glycoprotein, is a zinc endopeptidase, while XK, which has 10 transmembrane domains, is a putative membrane transporter. A rare phenotype termed Kell null (Ko) is characterized by the absence of Kell protein and Kell antigens from the red cell membrane and diminished amounts of XK protein. We determined the molecular basis of eight unrelated persons with Ko phenotypes by sequencing the coding and the intron-exon splice regions of KEL and, in some cases, analysis of mRNA transcripts and expression of mutants on the cell surface of transfected cells. Six subjects were homozygous: four with premature stop codons, one with a 5 splice site mutation, G to A, in intron 3, and one with an amino acid substitution (S676N) in exon 18. Two Ko persons with premature stop codons had identical mutations in exon 4 (R128Stop), another had a different mutation in exon 4 (C83Stop), and the fourth had a stop codon in exon 9 (Q348Stop). Two Ko persons were heterozygous for two mutations. One had a 5 splice site mutation (G to A) in intron 3 of one allele that caused aberrant splicing and exon skipping, and the other allele had an amino acid substitution in exon 10 (S363N). The other heterozygote had the same amino acid substitution in exon 10 (S363N) in one allele and a premature stop codon in exon 6 (R192Stop) in the other allele. The S363N and S676N mutants, expressed in 293T cells, were retained in a pre-Golgi compartment and were not transported to the cell surface, indicating that these mutations inhibit trafficking. We conclude that several different molecular defects cause the Kell null phenotype.

No abstract