Novel KEL allele associated with loss of Kpb identified in a white blood donor.

The importance of identifying variant alleles among blood donors is significant to the safety of transfusion for recipients. Molecular methods have become more prominent in the routine process of antigen typing donor units. Some variant antigens cannot be detected using only serologic methods. Molecular testing allows the determination of nucleotide sequences that are used to predict a phenotype. Antigens of the Kell blood group system are known for being highly immunogenic and causing adverse reactions upon antibody formation. A female white blood donor who typed Kp(b-) using serologic methods on multiple donations since 2005 was the subject of a typing discrepancy investigation. Routine genotyping using a commercial genotyping kit (HemoID DQS Panel; Agena Bioscience, San Diego, CA) predicted the donor to type Kp(a+b+). Investigation of the discrepancy between these two results identified a rare single nucleotide variant in the KEL gene at nucleotide position c.948G>T that alters amino acid residue 316 from tryptophan (Trp) to cysteine (Cys). After discovery of the novel allele, adsorption and elution studies were performed to see if there was weakened Kpb expression. The elution studies yielded negative results, which indicated that Kpb is not expressed. The KEL transcripts expressed by the donor were determined using cDNA analysis, and the predicted amino acid sequence of the novel allele was modeled to investigate the impact of the amino acid sequence on the structure of the KEL polypeptide. Both SWISS-MODEL and Robetta software were used to evaluate the impact of the p.Trp316Cys on the three-dimensional protein structure. There was no conformational change noted with SWISS-MODEL, whereas the Robetta software showed a significant conformational change compared with the normal Kp(b+) reference sequence. Because the donor is homozygous for variants associated with k and Jsb expression, it was not possible to determine whether the novel allele is associated with loss of Kpb only or loss of all Kell antigens.

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 R(46) R 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.

Novel alleles at the Kell blood group locus that lead to Kell variant phenotype in the Dutch population.

BACKGROUND: Alloantibodies directed against antigens of the Kell blood group system are clinically significant. In the Netherlands, the KEL1 antigen is determined in all blood donors. In this study, after phenotyping of KEL:1-positive donors, genotyping analysis was conducted in KEL:1,-2 donors to identify possible KEL*02 variant alleles. STUDY DESIGN AND METHODS: A total of 407 donors with the KEL:1,-2 phenotype were genotyped for the KEL*01/02 polymorphism, followed by direct sequencing of the KEL gene if the KEL*02 allele was detected. Two K0 patients were also included. Transcript analysis was conducted in two probands with the KEL*02. M05 allele defined by a synonymous mutation (G573G). Flow cytometry analysis to determine the expression of Kell antigen was performed. RESULTS: Thirty KEL:1,-2 individuals (30/407, 7.4%) with discrepant KEL*01/02 genotype were identified. Seven novel alleles were identified: KEL*02(R86Q, R281W)mod, KEL*02(L133P)null, KEL*02(436delG)null, KEL*02(F418S)null, KEL*02(R492X)null, KEL*02(L611R)null, and KEL*02(R700X)null. Nine variant alleles described before were detected: KEL*02N.06, KEL*02N.15, KEL*02N.17, KEL*02N.19, KEL*02N.21, KEL*02M.02, KEL*02M.04, KEL*02M.05, and KEL*02(Q362K)mod. A transcript lacking Exon 16 was identified in two probands with the KEL*02M.05 allele as described before. Finally, flow cytometry analysis showed a decreased total Kell expression and a relatively increased KEL1 expression in individuals with the KEL:1,2null or KEL:1,2mod phenotype, compared to KEL:1,2 controls. CONCLUSION: In 7.4% of a group of tested KEL:1,-2 Dutch donors, a KEL*02null or KEL*02mod allele was found. A relatively increased KEL1 antigen expression in KEL:1,2null and KEL:1,2mod individuals suggest that the expression of Kell-XK complexes depends on the availability of the XK protein.

Resolution of translation start site for the human Kell glycoprotein.

BACKGROUND: The human Kell blood group system currently contains 35 antigens determined by allelic polymorphisms in the Kell glycoprotein, a single-pass Type II transmembrane protein. The Kell glycoprotein was initially cloned through screening of a cDNA library; however, direct amino acid sequencing of most of the Kell glycoprotein has not been reported. The N-terminus of the Kell glycoprotein contains two potential translational start sites, which result in differences in the cytoplasmic tail. STUDY DESIGN AND METHODS: Protein extracts were isolated from human red blood cell membranes and were digested with trypsin. The resulting peptides were subjected to liquid chromatography-tandem mass spectrometry, allowing resolution of peptides from the N-terminus of the Kell glycoprotein. RESULTS: Peptides were isolated and sequenced that correspond to the upstream methionine start site predicted by the full cDNA sequence. No evidence of internal translation initiation at Methionine 20 was detected. CONCLUSIONS: These findings identify the translational start site and define the full cytoplasmic tail of the human Kell glycoprotein.

Possible suppression of fetal erythropoiesis by the Kell blood group antibody anti-Kp(a).

Antibodies to antigens in the Kell blood group system are usually immunoglobulin G, and, notoriously, anti-K, anti-k, and anti-Kp(a) can cause severe hemolytic transfusion reactions, as well as severe hemolytic disease of the fetus and newborn (HDFN). It has been shown that the titer of anti-K does not correlate with the severity of HDFN because, in addition to immune destruction of red blood cells (RBCs), anti-K causes suppression of erythropoiesis in the fetus, which can result in severe anemia. We report a case involving anti-Kp(a) in which one twin was anemic and the other was not. Standard hemagglutination and polymerase chain reaction (PCR)-based tests were used. At delivery, anti-Kp(a) was identified in serum from the mother and twin A, and in the eluate prepared from the baby's RBCs. PCR-based assays showed twin A (boy) was KEL*841T/C (KEL*03/KEL*04), which is predicted to encode Kp(a+b+). Twin B (girl) was KEL*841C/C (KEL*04/KEL*04), which is predicted to encode Kp(a­b+). We describe the first reported case of probable suppression of erythropoiesis attributable to anti-Kp(a). One twin born to a woman whose serum contained anti-Kp(a) experienced HDFN while the other did not. Based on DNA analysis, the predicted blood type of the affected twin was Kp(a+b+) and that of the unaffected twin was Kp(a­b+). The laboratory findings and clinical course of the affected twin were consistent with suppression of erythropoiesis in addition to immune RBC destruction.

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.

A novel KEL*1,3 allele with weak Kell antigen expression confirming the cis-modifier effect of KEL3.

BACKGROUND: KEL1 (K) is the most immunogenic red blood cell antigen of the Kell blood group system. The frequently occurring anti-KEL1 alloantibodies may cause hemolytic transfusion reactions as well as severe hemolytic disease of the fetus and newborn. So far, reports on weak KEL phenotypes are scarce. STUDY DESIGN AND METHODS: Blood samples of two unrelated Central European propositi with faint reactions in routine KEL1 typing were analyzed by extended serologic phenotyping, flow cytometry, genotyping by polymerase chain reaction with sequence-specific priming, and genomic DNA sequencing of separated parental KEL alleles. RESULTS: Both propositi exhibited an unusual KEL:1,2,3,4 phenotype: markedly weakened and negative reactions were observed in serologic KEL1 typing in gel and tube technique, respectively. No KEL1 epitope loss was detected using five different monoclonal anti-KEL1 reagents. KEL genotyping confirmed KEL*1/2 and KEL*3/4 (Kpa/Kpb) heterozygosity of both individuals. Importantly, DNA sequencing of the two separated parental alleles of both propositi revealed a KEL*1-specific coding nucleotide T578 and a KEL*3-specific T841 on the same allele. This novel KEL*1,3 (KKpa)allele was associated with an approximately 80 percent reduction in KEL1 expression, compared to KEL:1,2,-3,4 controls. The low KEL1 density was attributed to acis-modifier effect of KEL3, so far only reported in association with weakened expression of KEL2 (k). CONCLUSION: This is the first description of the KEL*1,3 allele encoding KEL1 and KEL3 on the same molecule. In individuals with weakened KEL1 because of KEL3 in cis, very sensitive serologic or molecular genetic techniques may be required for reliable Kell typing.

The Kell and XK proteins of the Kell blood group are not co-expressed in the central nervous system.

The Kell blood group is constituted by two covalently linked antigens at the surface of red blood cells, Kell and Kx. Whereas Kell is a metalloprotease with demonstrated in vitro enzymatic activity, the role of Kx thereon, and/or alone, remains unknown, although its absence is linked to the McLeod syndrome, a neuroacanthocytosis. In the central nervous system, the expression of Kell and XK has been suggested, but their expression patterns remain uncharacterized, as are the post-translational pathogenic mechanisms involved in the development of the McLeod syndrome. The distributions of Kell and XK were thus studied by in situ hybridization as well as immunohistochemistry in rodent and human brain. The results reveal an independent localization of the two constituents of the Kell blood group, XK (Kx) being expressed throughout this tissue, whereas Kell expression is restricted to red blood cells in cerebral vessels. The XK protein is shown to be neuronal, located mainly in intracellular compartments, suggesting a cell specific trafficking pattern, possibly associated with specific physiological functions.

Onset of expression of the components of the Kell blood group complex.

BACKGROUND: Kell and XK, two distinct red blood cell membrane proteins, are linked by a disulfide bond and form the Kell blood group complex. Kell surface antigens are expressed early during erythropoiesis but the onset of expression of XK which carries the Kx antigen is unknown. STUDY DESIGN AND METHODS: To determine whether Kell and XK are synchronously expressed, sorted human hematopoietic progenitor cells and mouse progenitor cells of defined lineage were studied. To determine the onset of expression, human marrow and cord blood cells were sorted into three subpopulations, representing stem, multipotent, and erythroid progenitor cells, and the expression of Kell and XK was determined by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorting (FACS) analysis. Mouse Kell and XK transcripts were determined by cDNA blotting of progenitor cells of defined lineage. RESULTS: By RT-PCR, human peripheral blood progenitor cells had weak expression of Kell and XK transcripts but FACS analysis did not detect surface antigens. Kell and XK transcripts are expressed in multipotent progenitor cells and these cells express Kell surface antigens. The expression of Kx antigen in progenitor cells was not determined owing to nonspecific reactions with the antibody. By cDNA blotting, mouse Kell expression was detected in bipotential megakaryocytes-erythroid cells and in colony-forming units-erythroid (CFU-E) and burst-forming units-erythroid (BFU-E), whereas XK was only detected in CFU-E and BFU-E. CONCLUSION: Both Kell and XK transcripts occur early during erythropoiesis; however, expression may not be coincident because, in mice, Kell transcripts, but not XK, occur in bipotential megakaryocytes-erythroid progenitor cells.

Kell expression on myeloid progenitor cells.

Kell is one of the major human red blood cell groups and comprises 22 antigens. These antigens are produced by alleles located on chromosome 7, including sets of antithetical antigens such as Kell (K, K1) and cellano (k, K2), which differ in a single amino acid change (T193M). It consists of a 93-Kd transmembrane glycoprotein that is surface-exposed and shares sequence and structural homology with zinc endopeptidases, which are involved in regulating bioactive peptides. Anti-Kell antibodies have been shown to suppress fetal erythropoiesis. Recently published data indicate a similar effect on myeolopoiesis and megakaryopoiesis. Substantial thrombocytopenia in fetuses affected with HDN due to anti-K antibodies led to the discovery of the inhibitory effect of Kell-related antibodies on CFU-MK growth. In addition to its inhibitory effect on BFU-E growth, anti-Kell antibodies significantly reduced CFU-GM colony formation from haematologically normal individuals. Moreover, anti-cellano and anti-Kp(b) antibodies also inhibited the growth of CFU-GM from antigen positive MNC. These data indicate that Kell is not restricted to erythroid blood cells, but is expressed on a broader spectrum of haematopoietic cells than previously believed.

Proteolytic processing of big endothelin-3 by the kell blood group protein.

Kell blood group protein shares a consensus sequence (H.E.X.X.H) with a large family of zinc-dependent endopeptidases. Kell has closest homology with neutral endopeptidase 24.11, endothelin converting enzyme-1 (ECE-1), and the PEX gene product that, as a group, comprise the M13 subfamily of mammalian neutral endopeptidases. The proteolytic activity of the M13 members, but not of Kell, has been previously demonstrated. A secreted form of wild-type Kell protein (s-Kell), devoid of the intracellular and transmembrane domains, was expressed in sf9 cells. As a negative control, an inactive mutant Kell protein (E582G) was expressed. As determined by N-terminal amino acid sequencing and mass spectrometry of the cleaved products, wild-type s-Kell, but not the control mutant protein, specifically cleaved big endothelin-3 (ET-3) at Trp(21)-Ile(22), yielding ET-3, and, to a much lesser extent, also cleaved big ET-1 and big ET-2 at Trp(21)-Val(22), yielding ET-1 and ET-2. Enzymatic activity was partially inhibited by phosphoramidon. s-Kell has an acidic pH optimum (pH 6.0 to 6.5). Like the recombinant protein, red blood cells of common Kell phenotype also preferentially process big ET-3, in contrast to Ko (null) cells that do not. These data demonstrate that the Kell blood group protein is a proteolytic enzyme that processes big ET-3, generating ET-3, a potent bioactive peptide with multiple biological roles.

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.

Kx, a quantitatively minor protein from human erythrocytes, is palmitoylated in vivo.

Kx is a quantitatively minor blood group protein of human erythrocytes which is thought to be a membrane transporter. In the red cell membrane, Kx forms a complex stabilized by a disulfide bond with the Kell blood group membrane protein which might function as a metalloprotease. The palmitoylation status of these proteins was studied by incubating red cells with [3H] palmitic acid. Purification of the Kell-Kx complex, by immunochromatography on an immobilized human monoclonal antibody of Kell blood group specificity demonstrated that the Kx but not the Kell protein is palmitoylated. Six cysteines in Kx are predicted to be intracytoplasmic and might be targets for palmitoylation. Three of these cysteines are present in a portion of sequence which is predicted to form an amphipathic alpha helix. Palmitoylation of one or several of these cysteines might contribute to anchor the cytoplasmic portion of the Kx protein to the inner surface of red cell membrane.