Soohee Lee

Protein 4.1R-dependent multiprotein complex: New insights into the structural organization of the red blood cell membrane

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 pull-down 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.

Association of XK and Kell blood group proteins.

A disulfide bond links Kell and XK red cell membrane proteins. Kell, a type II membrane glycoprotein, carries over 20 blood group antigens, and XK, which spans the membrane 10 times, is lacking in rare individuals with the McLeod syndrome. Kell is classified in the neprilysin family of zinc endopeptidases, and XK has structural features that suggest it is a transport protein. Kell has 15 extracellular cysteines, and XK has one in its fifth extracellular loop. Five of the extracellular cysteine residues in Kell are not conserved in the other members of the neprilysin family, and based on the hypothesis that one of the nonconserved cysteines is linked to XK, cysteines 72 and 319 were mutated to serine. The single extracellular cysteine 347 of XK was also mutated. Co-expression of combinations of wild-type and mutant proteins in transfected COS-1 cells showed that Kell C72S did not form a Kell-XK complex with wild-type XK, while wild-type Kell and Kell C319S did. XK C347S was also unable to form a complex with wild-type Kell, indicating that Kell cysteine 72 is linked to XK cysteine 347. Kell C72S was transported to the cell surface, indicating that linkage to XK is not required. In addition, chemical cross-linking of red cell membranes with dithiobispropionimidate indicated that glyceraldehyde-3-phosphate dehydrogenase is a near neighbor of Kell.

Molecular Basis of Kell Blood Group Phenotypes

The molecular basis of different Kell blood group phenotypes is reviewed. Sequence analysis of the Kell gene (KEL) established that single base substitutions, resulting in amino acid changes, are responsible for the different phenotypes. Most of the amino acid substitutions, with the exception of the one responsible for expression of KEL6 (Jsa), occur at the amino‐terminal half of the protein, a domain that has least amino acid homology with a family of zinc endopeptidases, which include neutral endopeptidase 24.11 and endothelin‐converting enzyme‐1. Some of the genes were expressed in transfected cells and typed with alloantibodies to confirm that the identified mutations are responsible for antigen expression. Clinical applications of Kell blood group genotyping which include prenatal diagnosis to monitor hemolytic disease of the newborn are discussed.

The Kell Blood Group System: Kell and XK Membrane Proteins

Two membrane proteins express the antigens that comprise the Kell blood group system. A single antigen, Kx, is carried on XK, a 440-amino acid protein that spans the membrane 10 times, and more than 20 antigens reside on Kell, a 93-kd, type II glycoprotein. XK and Kell are linked, close to the membrane surface, by a single disulfide bond between Kell cysteine 72 and XK cysteine 347. Although primarily expressed in erythroid tissues, Kell and XK are also present in many other tissues. The polymorphic forms of Kell are due to single base mutations that encode different amino acids. Some Kell antigens are highly immunogenic and may cause strong reactions if mismatched blood is transfused and severe fetal anemia in sensitized mothers. Antibodies to KEL1 may suppress erythropoiesis at the progenitor level, leading to fetal anemia. The cellular functions of Kell/XK are complex. Absence of XK, the McLeod phenotype, is associated with acanthocytic red blood cells (RBCs), and with late-onset forms of muscular dystrophy and nerve abnormalities. Kell, by homology, is a member of the neprilysin (M13) family of membrane zinc endopeptidases and it preferentially activates endothelin-3 by specific cleavage of the Trp21-Ile22 bond of big endothelin-3.

Functional and structural aspects of the Kell blood group system.

Two covalently linked proteins, Kell and XK, constitute the Kell blood group system. Kell, a 93-Kd type II glycoprotein, is highly polymorphic and carries all but 1 of the known Kell antigens, and XK, which traverses the membrane 10 times, carries a single antigen, the ubiquitous Kx. The Kell/XK complex is not limited to erythroid tissues and may have multiple physiological roles. Absence of one of the component proteins, XK, is associated with abnormal red cell morphology and late-onset forms of nerve and muscle abnormalities, whereas the other protein component, Kell, is an enzyme whose principal known function is the production of a potent bioactive peptide, ET-3.

Molecular basis of the K:6,-7 [Js(a+b−)] phenotype in the Kell blood group system

BACKGROUND: The Kell blood group system consists of at least 21 antigens. KEL6(Jsa) is a low‐incidence antigen that has an antithetical relationship with the high‐incidence KEL7(Jsb) antigen. The molecular basis of KEL6 that appears in less than 1.0 percent of the general population, but in up to 19.5 percent of African Americans, was unknown.

The value of DNA analysis for antigens of the Kell and Kx blood group systems

K ell is a major human blood group system that is highly polymorphic, and at present it is known to express 28 different alloantigens. After the ABO and Rh systems, Kell is the most important blood group system in transfusion medicine, as some of the antigens are potent immunogens and their antibodies can cause severe transfusion reactions in mismatched blood transfusions and fetal anemia in feto-maternal incompatible pregnancies. The Kx blood group system is composed of a single antigen, Kx, which is carried on the XK protein. This system is important clinically because absence of XK protein, found in rare McLeod phenotypes, leads to red blood cell (RBC) acanthocytosis and to late onset abnormalities, usually commencing about midlife, involving the peripheral and central nervous systems, known as the McLeod syndrome.

Point mutations causing the McLeod phenotype.

BACKGROUND: The McLeod phenotype is defined by absence of Kx, weakening of Kell system antigens, and acanthocytosis. Individuals with the McLeod phenotype usually develop late‐onset neuromuscular abnormalities. Gene deletions, insertions, and point mutations that affect RNA splicing or that lead to premature stop codons have been reported to cause the McLeod phenotype. The McLeod phenotype may also be caused by mutations at a different splice site and by a novel mutation encoding an amino acid substitution that prevents transport to the cell surface.

McLeod phenotype without the McLeod syndrome

BACKGROUND: McLeod neuroacanthocytosis syndrome is a late‐onset X‐linked multisystem disorder affecting the peripheral and central nervous systems, red blood cells (RBCs), and internal organs. A variety of mutations have been found in the responsible gene (XK) including single nonsense and missense mutations, nucleotide mutations at or near the splice junctions of introns of XK, and different deletion mutations. To date no clear phenotype‐genotype correlation is apparent. The clinical details of one case of McLeod phenotype without apparent neuromuscular abnormalities have been reported. Here the clinical details of two additional cases are presented, of which the genetic details have previously been published.

Prenatal diagnosis of Kell blood group genotypes: KEL1 and KEL2

OBJECTIVE Our purpose was to devise diagnostic test(s) that determine fetal KEL1 and KEL2 genotypes. STUDY DESIGN KEL1 and KEL2 polymorphisms are due to a single C to T base substitution at nucleotide 698 of exon 6 of KEL. This allowed us to develop two polymerase chain reaction tests that distinguish KEL1/1 and KEL2/2 homozygotes and KEL1/2 heterozygotes. The first test uses a Bsm I restriction fragment length polymorphism in a genomic deoxyribonucleic acid polymerase chain reaction product containing the single base polymorphism, and the second test uses allele-specific primers to distinguish KEL1 and KEL2 genotypes. These tests were applied in a blind study to 15 amniotic fluid deoxyribonucleic acid samples. The corresponding KEL1 and KEL2 fetal red blood cell phenotypes were determined serologically. The tests were also applied to two families in which the mothers had antibodies to KEL1. RESULTS In all cases results of analysis of Kell genotypes from the amniotic fluid deoxyribonucleic acid samples agreed with the fetal red blood cell Kell phenotypes. The tests were also successfully used to determine fetal Kell genotype by use of peripheral blood deoxyribonucleic acid. CONCLUSION Two polymerase chain reaction-based tests can be used for prenatal diagnosis of KEL1 and KEL2 genotypes; these procedures should prove useful in the proper management of Kell-sensitized pregnancies.