Kell, Kx and the McLeod syndrome

The antigens of the Kell blood group system are carried on a 93 kDa type II glycoprotein encoded by a single gene on chromosome 7 at 7q33. XK is a 50.9 kDa protein that traverses the membrane ten times and derives from a single gene on the X chromosome at Xp21. A single disulphide bond, Kell Cys 72-XK Cys 347, links Kell to XK. The Kell component of the Kell/XK complex is important in transfusion medicine since it is a highly polymorphic protein, carrying over 23 different antigens, that can cause severe reactions if mismatched blood is transfused and in pregnant mothers antibodies to Kell may elicit serious fetal and neonatal anaemia. The different Kell phenotypes are all caused by base mutations leading to single amino acid substitutions. By contrast the XK component carries a single blood group antigen, termed Kx. The physiological functions of Kell and XK have not been fully elucidated but Kell is a zinc endopeptidase with endothelin-3-converting enzyme activity and XK has the structural characteristics of a membrane transporter. Lack of Kx, the McLeod phenotype, is associated with red cell acanthocytosis, elevated levels of serum creatine phosphokinase and late onset forms of muscular and neurological defects.     

Expression of Kell blood group protein in nonerythroid tissues

The Kell blood group protein is a zinc endopeptidase that yields endothelin-3, a potent bioactive peptide, by cleavage of big endothelin-3, a larger intermediate precursor. On red cells, Kell protein is linked by a single disulfide bond to XK, a protein that traverses the membrane 10 times and whose absence, as occurs in the McLeod phenotype, is associated with a set of clinical symptoms that include nerve and muscle disorders and red cell acanthocytosis. Previous studies indicated that Kell is primarily expressed in erythroid tissues, whereas XK has a wider tissue distribution. The tissue distribution of Kell protein has been further investigated by Northern blot analysis, PCR-screening of tissue complementary DNAs (cDNAs), and Western immunoblots. Screening of an RNA dot-blot panel confirmed that Kell is primarily expressed in erythroid tissues but is also expressed in a near equal amount in testis, with weaker expression in a large number of other tissues. PCR-screening of cDNAs from different tissues and DNA sequencing of the products gave similar results. In 2 of the nonerythroid tissues tested, testis and skeletal muscle, Kell protein was detected by Western immunoblotting. In skeletal muscle, isolation of XK with a specific antibody coisolated Kell protein. These studies demonstrate that Kell is expressed in both erythroid and nonerythroid tissues and is associated with XK.     

Changes in red cell ion transport,reduced intratumoral neovascularization,and some mild motor function abnormalities accompany targeted disruption of the Mouse Kell gene (Kel)

Kell (ECE‐3), a highly polymorphic blood group glycoprotein, displays more than 30 antigens that produce allo‐antibodies and, on red blood cells (RBCs), is complexed through a single disulfide bond with the integral membrane protein, XK. XK is a putative membrane transporter whose absence results in a late onset form of neuromuscular abnormalities known as the McLeod syndrome. Although Kell glycoprotein is known to be an endothelin‐3‐converting enzyme, the full extent of its physiological function is unknown. To study the functions of Kell glycoprotein, we undertook targeted disruption of the murine Kel gene by homologous recombination. RBCs from Kel(–/–) mice lacked Kell glycoprotein, Kell/XK complex, and endothelin‐3‐converting enzyme activity and had reduced levels of XK. XK mRNA levels in spleen, brain, and testis were unchanged. In Kel(–/–) mice RBC Gardos channel activity was increased and the normal enhancement by endothelin‐3 was blunted. Analysis of the microvessels of tumors produced from LL2 cells indicated that the central portion of tumors from wild‐type mice were populated with many mature blood vessels, but that vessels in tumors from Kel(–/–) mice were fewer and smaller. The absence of Kell glycoprotein mildly affected some motor activities identified by foot splay on the drop tests. The targeted disruption of Kel in mouse enabled us to identify phenotypes that would not be easily detected in humans lacking Kell glycoprotein. In this regard, the Kell knockout mouse provides a good animal model for the study of normal and/or pathophysiological functions of Kell glycoprotein. Am. J. Hematol., 2009. © 2009 Wiley‐Liss, Inc.     

A combination of the effects of rare genotypes at the XK and KEL blood group loci results in absence of Kell system antigens from the red blood cells

The 22 antigens of the Kell blood group system are located on a red blood cell (RBC) membrane glycoprotein that shows sequence homology with a family of metalloendopeptidases. Expression of the Kell system antigens is partially governed by XK, an X-linked gene that encodes the Kx protein; absence of Kx results in reduced Kell antigen expression. Almost total absence of Kell antigens from the RBCs of a German man with no symptoms of neuroacanthocytosis could not be due to the Kell- null phenotype, Ko, because his RBCs had very weak expression of Kx antigen and his three children were Kp(a + b+). Kell antigens were normal on the RBCs of his son but weak on those of his two daughters. An Nla III restriction fragment-length polymorphism within the KEL gene showed the Kpa/Kpa genotype in the propositus. Sequencing of his XK gene showed a single base change within the donor splice consensus sequence of intron 2. A BsaAl restriction fragment-length polymorphism showed the mutation in both of his daughters but not in his son. The extreme depression of the Kell antigens of the propositus must be due to a combination of effects, ie, homozygosity for Kpa and deficiency of Kx protein, each of which is capable of causing some degree of weakening of Kell antigens.     

Identification of a defect in the intracellular trafficking of a Kell blood group variant.

Blood group polymorphisms have been used as tools to study the architecture of the red blood cell (RBC) membrane. Some blood group variants have reduced antigen expression at the cell surface. Understanding the underlying mechanism for this reduced expression can potentially provide structural information and help to elucidate protein trafficking pathways of membrane proteins. The Kp(a+) phenotype is a variant in the Kell blood group system that is associated with a single amino acid substitution (R281W) in the Kell glycoprotein and serologically associated with a weakened expression of other Kell system antigens by an unknown mechanism. We found by immunoblotting of RBCs that the weakening of Kell antigens in this variant is due to a reduced amount of total Kell glycoprotein at the cell surface rather than to the inaccessibility of the antigens to Kell antibodies. Using a heterologous expression system, we demonstrate that the Kpa mutation causes retention of most of the Kell glycoprotein in a pre-Golgi compartment due to differential processing, thereby suggesting aberrant transport of the Kell protein to the cell surface. Furthermore, we demonstrated that single nucleotide substitutions into the coding region of the common KEL allele, as predicted by the molecular genotyping studies, was sufficient to encode three clinically significant low incidence antigens. We found that two low incidence antigens can be expressed on a single Kell protein, thus showing that the historical failure to detect such a variant is not due to structural constraints in the Kell protein. These studies demonstrate the power of studying the molecular mechanisms of blood group variants for elucidating the intracellular transport pathways of membrane proteins and the requirements for cell surface expression.     

Autoimmune hemolytic anemia and the Kell blood groups

Approximately one in 250 people with autoimmunity involving their red cells have IgG autoantibodies with specificity in the Kell blood groups. Red cells of these individuals have an acquired temporary weakening of their Kell antigens. Some of the patients also have allo-anti-K in their serum. This report presents a case in which an IgG autoantibody may define a new high-incidence red cell antigen related to the Kell blood groups. The patient's Kell blood group antigens are depressed, and his serum contains allo-anti-K. It is postulated that reduced red cell Kell antigenicity is caused by enzymatic degradation, possibly of bacterial origin, and that the acquired loss of Kell antigens, the Kell-specific autoimmune state, and the serum all0-anti-K, are all related aspects of one phenomenon.     

Transfusion support for a patient with McLeod phenotype without chronic granulomatous disease and with antibodies to Kx and Km

Background and Objectives   Kell antigens are encoded by the KEL gene on the long arm of chromosome 7. Kx antigen is encoded by the XK gene on the short arm of the X chromosome. Kell and Kx proteins in the red cell membrane are covalently linked by a disulphide bond. The McLeod phenotype is characterized by weakened expression of antigens in the Kell blood group system, absence of Km and Kx antigens, and acanthocytosis. It has an X-linked mode of inheritance with transmission through carrier females. Some males with the McLeod syndrome also have chronic granulomatous disease (CGD). It is generally believed that patients with non-CGD McLeod may develop anti-Km but not anti-Kx, but that those with CGD McLeod can develop both anti-Km and anti-Kx.
Materials and Methods   We present serological data, DNA genotyping and gene sequencing, monocyte monolayer assay and neutrophil oxidative burst test from a patient with the McLeod phenotype without clinical evidence of CGD.
Results   We report here the second example of a patient with non-CGD McLeod who developed anti-Kx in addition to anti-Km. Sequencing of our patient's XK gene confirmed the presence of a mutation resulting in a premature stop codon and lack of Kx protein in the red cell membrane, which is consistent with the diagnosis of McLeod syndrome. Neutrophil oxidative burst test was normal, indicating that our patient did not have CGD. The challenge of providing 10 compatible blood units for multiple surgeries was met.
Conclusion   The second case of a rare entity, a patient with non-CGD McLeod who developed anti-Kx and anti-Km, was managed successfully with a combination of autologous donations and procurement of compatible units from national and international sources.     

Inactivation of Kell blood group antigens by 2-aminoethylisothiouronium bromide

Human red cells incubated with a solution containing 6% 2-aminoethyl-isothiouronium bromide (AET) lose activity of antigens that are part of, or related to, the Kell blood group system. However, Kx antigen is not inactivated. Studies on a wide range of other blood-group antigens show no other evidence of changes and AET appears to react specifically with red-cell membrane structures that have Kell activity. The AET procedure produces an artificial K₀ red cell that can be used in blood group serology, and allows easy recognition of antibodies that are associated with the Kell system.
AET has been used by other workers to produce a red cell that has many serological and biochemical characteristics of a PNH cell. Our studies on red cells from PNH patients have not shown any changes in Kell blood-group antigens.     

Topology of Kell blood group protein and the expression of multiple antigens by transfected cells

Kell is one of the major blood group systems in human red blood cells (RBCs). The Kell antigens are carried on a 731 amino acid glycoprotein that is thought to span the erythrocyte membrane once. Rabbit antibodies to three synthetic peptides, derived from different parts of the Kell protein, were used to determine the topology of Kell protein on the RBC. Antibodies to a C-terminal peptide and to a peptide derived from amino acid residues 410 to 439 reacted with RBCs treated with 0.2 mol/L dithiothreitol. An antibody to the N-terminal peptide reacted with inside-out RBC vesicles but not with right-side-out vesicles nor with intact RBCs, showing that Kell is a type II membrane protein and that the extracellular portion of the protein is folded by disulfide bonds. By transfection, Kell protein was expressed on the cell surface of surrogate cells, and the transfected cells expressed similar antigenic properties as native RBCs. Kell protein was expressed in COS- 1 and K562 cells and in Sf9 cells infected by the Baculovirus system. Transfected K562 cells expressed several high-incidence antigens but not the low-incidence antigen K1.     

Ablation of the Kell/Xk complex alters erythrocyte divalent cation homeostasis

XK is a putative transporter of unknown function that is ubiquitously expressed and linked through disulfide bonds to Kell protein, an endothelin-3 (ET-3)-converting enzyme. We generated three knockout (KO) mice that lacked either Xk, Kell or both proteins and characterized erythrocyte cation levels, transport and hematological parameters. Absence of Xk or Kell was accompanied by changes in erythrocyte K⁺, Mg^2 +, Na⁺ and Ca^2 + transport that were associated with changes in mean cellular volume and corpuscular hemoglobin concentration mean. Baseline Ca^2 +-ATPase activity was undetected in erythrocytes from all three mouse types but was restored upon pre-incubation with ET-3. Consistent with these alterations in Ca^2 + handling, we observed increased Gardos channel activity in Kel and Xk KO mice. In addition Kel deletion was associated with increased Mg^2 + permeability while Xk deletion blocked Na/Mg exchanger activity. Our results provide evidence that cellular divalent cation regulation is functionally coupled to the Kell/XK system in erythrocytes and loss of this complex may contribute to acanthocytosis formation in McLeod syndrome.     

Disulfide bonds are a requirement for Kell and Cartwright (Yta) blood group antigen integrity

S ummary . We have investigated the effect of dithiothreitol (DTT) upon the Kell blood group system and other red cell antigens. All Kell blood group antigens studied (K, k, Kp^a, Kp^b, Js^a, Js^b and Ku) as well as the Cartwright (Yt^a) antigen were completely denatured after treatment with DTT. The Gerbich antigen was substantially weakened but not completely denatured. The Js^a and Js^b antigens appear to have an exquisite sensitivity to treatment with DTT and can be completely denatured using very low concentrations (≤2 m m ) whereas other Kell system antigens require much higher concentrations of DTT for their denaturation (100–200 m m ). Of 38 other blood group antigens investigated, only the Yt^a antigen was completely denatured using 200 m m DTT. Furthermore, the Yt^a antigen was denatured within the same concentration range as Kell and one can speculate that this indicates some biochemical relationship between these two blood group systems. From our results, we conclude that: (1) at least two distinct disulfide (S–S) bonds are required for maintenance of the Kell blood group antigen system; (2) Js^a and Js^b antigens are distinctly different from other Kell system antigens based upon sensitivity to treatment with DTT; these antigens may be located on a different antigenic domain; and (3) the Yt^a antigen requires at least one disulfide bond for its maintenance of antigen integrity. Although the Gerbich antigen was not completely denatured, results indicate that disulfide bonds may also be important structural determinants for these antigens.     

Molecular cloning and primary structure of Kell blood group protein.

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 lambda gt10 human bone-marrow cDNA library. Four clones were isolated, one containing cDNA with an open reading frame for an 83-kDa protein. All 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 membrane-spanning 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.     

McLeod syndrome resulting from a novel XK mutation

McLeod Syndrome (MLS) is a rare X-linked disorder characterized by haemopoietic abnormalities and late-onset neurological and muscular defects. The McLeod blood group phenotype is typically associated with erythrocyte acanthocytosis, absence of the Kx antigen and reduced expression of Kell system antigens. MLS is caused by hemizygosity for mutations in the XK gene. We describe a patient with MLS who first showed symptoms in 1989 (aged 51 years). As the disease progressed, the patient developed a slight dementia, aggressive behaviour and choreatic movements. A cardiomyopathy was also diagnosed. An electroneuromyography showed neuropathic and myopathic changes. Liver enzymes were elevated and a blood smear showed acanthocytes. MLS was confirmed by serological analysis of the Kell antigens. Analysis of red blood cells by flow cytometry revealed the patient and his grandson to have reduced Kell antigen expression. The patient's daughters had two populations of red cells, consistent with them being heterozygous for an XK0 allele. The molecular basis of MLS in this family is a novel mutation consisting of a 7453-bp deletion that includes exon 2 of the XK gene. This confirms that the patient's 7-year-old grandson, who is currently asymptomatic, also has the XK0 allele and is therefore likely to develop MLS.     

The human Kell blood group binds the erythroid 4.1R protein: new insights into the 4.1R‐dependent red cell membrane complex

Protein 4.1R plays an important role in maintaining the mechanical properties of the erythrocyte membrane. We analysed the expression of Kell blood group protein in erythrocytes from a patient with hereditary elliptocytosis associated with complete 4.1R deficiency (4.1(?) HE). Flow cytometry and Western blot analyses revealed a severe reduction of Kell. In vitro pull down and co‐immunoprecipitation experiments from erythrocyte membranes showed a direct interaction between Kell and 4.1R. Using different recombinant domains of 4.1R and the cytoplasmic domain of Kell, we demonstrated that the R⁴⁶R motif in the juxta‐membrane region of Kell binds to lobe B of the 4.1R FERM domain. We also observed that 4.1R deficiency is associated with a reduction of XK and DARC (also termed ACKR1) proteins, the absence of the glycosylated form of the urea transporter B and a slight decrease of band 3. The functional alteration of the 4.1(?) HE erythrocyte membranes was also determined by measuring various transport activities. We documented a slower rate of HCO₃^?/Cl^? exchange, but normal water and ammonia transport across erythrocyte membrane in the absence of 4.1. These findings provide novel insights into the structural organization of blood group antigen proteins into the 4.1R complex of the human red cell membrane.     

Haematological Changes Associated with the McLeod Phenotype of the Kell Blood Group System

The McLeod phenotype is inherited as an X-linked characteristic. The red cells have weak antigenicity in the Kell blood group and lack Kx, a precursorlike substance that appears to be necessary for proper biosynthesis of Kell antigens. Kx antigen is also required for establishment of normal cell morphology. Absence of Kx antigen causes a membrane abnormality, in which the most prominent feature is acanthocytosis, and a compensated haemolytic state.
The X-linked gene that determines normal Kx production is called X¹ k . Inheritance of a variant allele at the Xk locus is responsible for lack of Kx synthesis and the McLeod phenotype. The Xk locus is inactivated by the Lyon effect, and female carriers of the variant gene exhibit blood group mosaicism in the Kell system and have a dual red cell population of acanthocytes and discocytes.     

Chronic Granulomatous Disease and the Kell Blood Groups

Fifteen antigenic determinants are known to be related to the Kell blood group. Some boys with X-linked chronic granulomatous disease have the very rare McLeod or Ko phenotype on their red cells. Serological studies of the McLeod type suggest that the weak Kell antigens that are present differ qualitatively and quantitatively from those on red cells of common Kell type. A new antigen, Kx, has been characterized and shown to be present on red cells and neutrophil leucocytes. Lack of red-cell Kx is associated with the McLeod phenotype, lack of leucocyte Kx is associated with chronic granulomatous disease.     

Active amino acids of the Kell blood group protein and model of the ectodomain based on the structure of neutral endopeptidase 24.11

In addition to its importance in transfusion, Kell protein is a member of the M13 family of zinc endopeptidases and functions as an endothelin-3-converting enzyme. To obtain information on the structure of Kell protein we built a model based on the crystal structure of the ectodomain of neutral endopeptidase 24.11 (NEP). Similar to NEP, the Kell protein has 2 globular domains consisting mostly of alpha-helical segments. The domain situated closest to the membrane contains both the N- and C-terminal sequences and the enzyme-active site. The outer domain contains all of the amino acids whose substitutions lead to different Kell blood group phenotypes. In the model, the zinc peptidase inhibitor, phosphoramidon, was docked in the active site. Site-directed mutagenesis of amino acids in the active site was performed and the enzymatic activities of expressed mutant Kell proteins analyzed and compared with NEP. Our studies indicate that Kell and NEP use the same homologous amino acids in the coordination of zinc and in peptide hydrolysis. However, Kell uses different amino acids than NEP in substrate binding and appears to have more flexibility in the composition of amino acids allowed in the active site.     

Molecular basis of the Kell (K1) phenotype

K1 (K, Kell) is a strong immunogen; its antibodies can cause severe reactions if incompatible blood is transfused and may cause hemolytic disease of the newborn in sensitized mothers. K1 is a member of the Kell blood group system, which is complex, containing over 20 different antigens. Some of the antigens are organized in allelic pairs of high and low prevalence whereas others are independently expressed. K1, which is present in 9% of the population, is antithetical to the high- prevalence K2 (k) antigen. We have determined the molecular basis of the K1/K2 polymorphism by sequencing the 19 exons of the Kell gene (KEL) of a K1/K1 person. Polymerase chain reaction was performed on genomic DNA isolated from peripheral blood and the amplified products were either directly sequenced or subcloned and sequenced. Comparisons of K1/K1 and K2/K2 DNA showed a C to T base substitution in exon 6 that predicts a threonine to methionine change at amino acid residue 193. This amino acid substitution occurs at a consensus N-glycosylation site (Asn. X. Thr) and probably prevents N-glycosylation, leading to a change in phenotype. The C to T substitution creates a Bsm I restriction enzyme site, which was tested in 42 different samples to confirm that this base change identifies the K1/K1 genotype. This test differentiates genotypes, K1/K1, K2/K2, and the K1/K2 heterozygote and should prove useful in the prenatal diagnosis of K1-related hemolytic disease of the newborn.     

Anti-K14: an antibody specificity associated with Kell blood group system.

Abstract. The San serum (anti-K14) recognizes a high-frequency red-cell antigen that is related to the Kell blood group system. The antibody reacts with the red cells of all of more than 600 random blood donors, but red cells of K_o phenotype are nonreactive. Cells having null phenotypes in other blood group systems are incompatible. The reactive antigen is thus related to the Kell system, but our investigation has established it to be different from the known high-frequency antigens related to this blood group. The antibody caused mild hemolytic disease of the newborn in two of the proposita's children.     

Unmasking of Kx antigen by reduction of disulphide bonds on normal and McLeod red cells

We have investigated the effect of dithiothreitol (DTT) treatment of human red cells upon the Kx blood group antigen. At low concentrations of DTT (less than or equal to 2 microM) there is enhancement of the Kx antigen concomitant with the complete denaturation of the Jsa and Jsb antigens of the Kell blood system. This unmasking of the Kx antigenic site is near maximal using 2 microM DTT. At this concentration of DTT, only the Jsa and Jsb antigens are completely denatured; all other Kell system antigens tested (K, k, Kpb, Ku) are essentially unaffected. These results argue against the Kx antigen serving strictly as a carbohydrate precursor substance involved in a sequential biosynthetic pathway of Kell blood group antigens. Also, McLeod red cells, after treatment with DTT, were found to contain Kx antigen, although in much lower density than normal red cells, indicating that, although not a typical carbohydrate precursor substance, Kx may, nevertheless, be essential for the serological expression of Kell related antigens. It is hypothesized that the Kx structure and the Kell blood group antigen structure are two separate subunits associated in a quaternary conformation involving at least one interchain S-S bond. Our results should allow for a clearer understanding of the relationship between the serological expression of the Kx antigen and the serologically observed reactivity of the Kell blood group antigens of individuals having normal, Ko and McLeod phenotypes.