Kell Glycoprotein Research Articles

Genetic and functional analyses describe a novel 730delG mutation in the KEL gene causing K0 phenotype in a Taiwanese blood donor

The purpose of this study was to explore the molecular basis of the K0 phenotype of a Taiwanese blood donor found to have anti-Ku alloantibodies. With respect to Kell blood group antigens, almost all Taiwanese have the (K-, k+) phenotype. Alloantibody identification and KEL antigen typing were performed. Enzymatic function assays were carried out to detect the Kell glycoprotein on RBCs. The KEL genes were sequenced to detect genetic variation. To determine the origin of this novel allele, family studies were conducted. The alloantibody was identified as anti-Ku. The donor was typed K0 . The KEL gene-sequencing data revealed that this K0 donor is a compound heterozygote with two different null alleles. He bears a novel 730delG mutation in one allele. Family studies suggested that the donor inherited the 730delG mutation from his father. The endothelin-converting activity assay indicated that his RBCs had no functional Kell glycoprotein. Other family members who had only one null allele with the 730delG mutation had the phenotype (K-, k+). For blood transfusion safety, it is important to establish an effective screening algorithm to identify rare phenotypes, such as the K0 phenotype, and to establish a database of rare blood groups.

Novel Kelnull Allele Detected In a Brazilian Woman with the Kell Null Phenotype.

AbstractAbstract 1111Kell is the most important blood group system after ABO and Rh because all frequently occurring Kell-specific antibodies must be considered clinically significant. The KEL antigens are carried by the human red cell (RBC) membrane Kell glycoprotein (CD238), a proteolytic enzyme encoded by the 19-exon KEL gene on the long arm of chromosome 7 (7q33). Kell null (K0) is a very rare phenotype characterized by the absence of the Kell protein and all KEL antigens on RBC surface caused by different molecular defects on KEL gene. K0 persons, however, may produce anti-KEL5 (anti-Ku) antibody that is associated to severe hemolytic transfusion reaction and perinatal anemia. Although the K0 phenotype has been known since 1957, the first reports of the K0 molecular basis occurred only in 2001. At present, 23 KELnull alleles are recognized to abolish the KEL antigens expression, most of them due to stop codon and alternative splicing caused by single nucleotide mutation. In this study, we report a novel KELnull allele detected in a Brazilian woman with the K0 phenotype. We investigated a 59-year-old Caucasian-Amerindian descent woman (proband) from Northeast Brazil who showed an antibody that reacted against high-prevalence RBC antigens during a pre-transfusion evaluation for orthopedic surgery. We also were able to evaluated three relatives (proband′s sister and two nieces) and normal RBC controls. We performed standard immunohematological methods and PCR-RFLP KEL genotyping as screening tests. To identify the molecular defect, we used sequencing technique covering all 19 coding and exons-introns regions of the KEL gene. To confirm the K0 phenotype and exclude KELel (Kmod) phenotype we employed flow cytometry, a higher sensitive technique, using soroclone anti-KEL1(anti-K), anti-KEL2(anti-k), anti-KEL3(anti-Kpa), anti-KEL4(anti-Kpb) and anti-CD238 antibodies. Proband′s frozen RBCs were washed 3x in 3ml PBS and 2×106/50μl/tube were incubated for 30min at room temperature with monoclonal antibodies (anti-K, -k, -Kpa, -Kpb – 50μl; anti-CD238 1:200,10μl). FITC conjugated polyclonal rabbit anti-human Ig and goat-anti-mouse Ig were used as second antibodies. Data acquisition and analysis in 20,000 events at RBC region were performed using FACscalibur flow cytometer and CellQuest program. Previously known (A) KEL:-1-,2,-3,4 (K-,k+,Kpa-,Kpb+) and (B) KEL:1,2,-3,4 (K+,k+,Kpa-,Kpb+) RBCs samples were used as positive controls. Fresh and thawed RBCs stained with PE conjugated anti-human glycophorin were also tested. RBCs of the proband were phenotyped as KEL:-1,-2,-3,-4,-6,-7 (K-,k-,Kpa-,Kpb-,Jsa-,Jsb-), and the RBC alloantibody was identified as anti-KEL5 (anti-Ku). RBCs of proband's relatives were phenotyped as wild type KEL:-1,2,-3,4,-6,7 (K-,k+,Kpa-,Kpb+,Jsa-,Jsb+). The proband and her relatives had the same KEL*2/KEL*2, KEL*4/KEL*4, KEL*7/KEL*7 wild type genotype determined by PCR-RFLP. The G>A mutation on intron 16, position +1 downstream of exon 16 (IVS16+1G>A mutation) was identified by KEL sequencing technique at homozygous state on the proband and at heterozygous state on her sister. The IVS16+1G>A mutation introduce an alternative splicing that may skip exon 16 and create a premature stop codon (TGA) on exon 17. No mutation was detected in the two proband's nieces. Proband's RBCs did not react against all antibodies by flow cytometry: MFI (mean fluorescent intensity) of CD238=3.7, KEL1(K)=7.6, KEL2(k)=7.6, KEL3(Kpa)=8.5 and KEL4(Kpb)=8.0 against positive results in control A RBCs [MFI of CD238=77, KEL1(K)=8, KEL2(k)=189.4, KEL3(Kpa)=9.3 and KEL4(Kpb)=114.4] and control B RBCs [MFI of CD238=77, KEL1(K)=76, KEL2(k)=50, KEL3(Kpa)=7.8 and KEL4(Kpb)=49.6]. Results of the RBC phenotyping by flow cytometry of the proband's sister were CD238=156, KEL2(k)=51 and KEL4(Kpb)=48. There were no significant differences between results on fresh and frozen samples. The MFI of KEL2 from proband's sister (KEL*2/KEL*2null) RBCs was similar to that observed in control B (KEL*2/KEL*1 heterozygous), 51 and 50, respectively, and lower than the control A (KEL*2/KEL*2 homozygous), 51 and 189.4, respectively, reflecting the single gene dose in the proband's sister and control B, and double gene dose in control A. In conclusion, we report a novel KELnull allele, IVS16+1G>A, detected for the first time in a Brazilian woman with Kell null phenotype. This study was supported by CNPq and FAPESP. Disclosures:No relevant conflicts of interest to declare.

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.

An autoanti‐Kpb immunoglobulin M that simulates antigen suppression

Patients may present with an antibody against a blood group antigen, a negative direct antiglobulin test (DAT), and a null phenotype. Typically, this represents an alloantibody in a null individual. However, on occasion, the antibody disappears coincident with conversion to a positive red blood cell (RBC) phenotype. This has been called antigen loss, antigen suppression, or weakened antigenicity. Herein, a unique serologic profile that mimics this pattern, when in fact antigen loss did not occur, is described. RBCs and serum were analyzed using a gel microtyping system and flow cytometry. Genomic DNA was amplified by polymerase chain reaction and sequenced. Initially, an anti-Kp(b) was detected in MTS gel, RBCs typed K-k-Kp(b-), and the DAT was negative for immunoglobulin G (IgG). Later, the anti-Kp(b) disappeared and RBCs phenotyped as K-k+Kp(b+). Analysis of initial specimens by flow cytometry identified an immunoglobulin M (IgM) anti-Kp(b) with a positive IgM-specific DAT; eluates contained an anti-Kp(b) at immediate spin. Supporting the presence of the Kell glycoprotein, RBCs agglutinated with anti-Js(b). Sequencing showed homozygosity for Kp(b) with no mutations surrounding the Kp(b) polymorphism. In antigen loss, antibody masking is excluded by a negative DAT. However, because typical DAT reagent does not detect IgM, such reasoning was inaccurate in the current case. In addition, an anti-Kp(b) resulted in RBCs typing k-, even though no anti-k was detected. Overall, this case suggests that an IgM may mask adjacent epitopes and illustrates the potential to mistake a non-IgG autoantibody as antigen loss.

Another example of a KEL1 variant red cell phenotype due to a threonine to serine change at position 193 of Kell glycoprotein

Healthy subjects whose red blood cells (RBCs) react variably with anti-KEL1, but strongly express other Kell blood group antigens, have been described and called KEL1 variant. A 53-year-old Caucasian blood donor was identified whose RBCs reacted with three monoclonal and two polyclonal anti-KEL1 and did not react with two monoclonal and one polyclonal anti-KEL1. The molecular basis of this phenotype was investigated. Genomic white blood cell DNA was analyzed for KEL*1/2 genotype by utilizing sequence-specific primers and polymerase chain reaction. In addition, the region of the KEL*1/2 polymorphism at position 578 of KEL was analyzed by DNA sequencing. Genotyping of the donor with the KEL1 variant phenotype revealed that he was KEL*2 homozygous. Sequencing revealed one typical KEL*2 allele and a KEL*2 allele with an adenosine (A) to thymidine (T) substitution at position 577 that predicted a threonine to serine change at position 193. It is not known if this phenotype is clinically relevant, but for at least some genotyping applications probes that identify this polymorphism should be used and anti-KEL1 should be tested for reactivity to this allele.

Association between alloantibody specificity and autoantibodies to red blood cells

Alloantibodies (ALLOs) to red blood cells (RBCs) are frequently associated with autoantibodies (AABs). An association between ALLO specificity and AABs has not yet been described. All patients and healthy blood donors screened for RBC antibodies between 2000 and 2006 were included. The odds ratio (OR) for ALLOs in patients with AABs compared to those without AABs was correlated with the OR of general ALLO prevalence in patients with AABs (normalized OR). ALLOs were found in 4,626 of 204,330 patients and healthy blood donors (2.3%). The ALLOs were associated with AABs in 413 cases (8.9%). Among the specificities, anti-S with a normalized OR of 2.9 was overrepresented. This was most evident in pregnant women who showed a normalized OR of 15.1 for anti-S and AABs. The normalized OR revealed an additional association between Rh antibodies and AABs. No association was found between ALLOs to the Kell glycoprotein, Duffy protein, Lewis, or glycophorin A (M/N) and AABs. The majority of associated ALLOs and AABs are directed against neighboring antigens of the Rh complex and glycophorin B.

A KEL gene encoding serine at position 193 of the Kell glycoprotein results in expression of KEL1 antigen

The KEL2/KEL1 (k/K) blood group polymorphism represents 578C>T in the KEL gene and Thr193Met in the Kell glycoprotein. Anti-KEL1 can cause severe hemolytic disease of the fetus and newborn. Molecular genotyping for KEL*1 is routinely used for assessing whether a fetus is at risk. Red blood cells (RBCs) from a KEL:1 blood donor (D1) were found to have abnormal KEL1 expression during evaluation of anti-KEL1 reagents. Kell genotyping methods, including KEL exon 6 direct sequencing, were applied. KEL cDNA from D1 was sequenced. Flow cytometry was used to assess KEL1 and KEL2 RBC expression. RBCs from the donor, her mother, and an unrelated donor gave weak or negative reactions with some anti-KEL1 reagents. Other Kell-system antigens appeared normal. The three individuals were homozygous for KEL C578 (KEL*2) but heterozygous for a 577A>T transversion, encoding Ser193. They appeared to be KEL*2 homozygotes by routine genotyping methods. Flow cytometry revealed weak KEL1 expression and normal KEL2, similar to that of KEL*2 homozygotes. Ser193 in the Kell glycoprotein appears to result in expression of abnormal KEL1, in addition to KEL2. The mutation is not detected by routine Kell genotyping methods and, because of unpredicted KEL1 expression, could lead to a misdiagnosis.

SI38 Characterisation of Membrane Proteins on Vesicles from Normal Red Cells and on Red Cells of Patients with Membranopathy

Background Red cell vesicles are formed either after prolonged blood storage at 4°C or during incubation in calcium‐loaded medium. Unlike mature red cells, these vesicles are depleted in spectrin but carry band 3, glycophorins, acetylcholinesterase and CD47. Absorption of human anti‐D by vesicles indicated the presence of Rh proteins.Study Design The aims were to identify the membrane components present on red cell vesicles, and to investigate whether red cells after vesiculation in vitro were similar to those from patients with inherited haemolytic anaemias: hereditary spherocytosis (HS) and pyropoikilocytosis (HPP). Vesicles released from fresh normal red cells in Ca2+ loaded medium were purified by filtration through 0.8 μm filters. Specific monoclonal antibodies (Mabs) were used to identify integral membrane components. The analytical techniques used were SDS‐PAGE, Western blotting, confocal microscropy, and rosette formation with magnetic beads (Dynabeads) conjugated with anti‐mouse immunoglobulins. Flow cytometry quantified antibody bound to red cells after vesiculation and from patients with HS and HPP.Results We confirmed depletion of spectrin and the presence of band 3, CD47 and glycophorins in vesicles released from normal red cells. Confocal microscopy demonstrated the coating of Rh(D+) vesicles with human Mab anti‐D. Other membrane molecules detected on the vesicles were Duffy glycoprotein, Kell glycoprotein, glucose transporter, protein 4.2, Rh glycoprotein (RhAG), CD59, and blood group A antigen. Flow cytometry detected reductions in binding of Mabs to band 3, Rh proteins and RhAG on vesiculated red cells as well as on HS red cells. CD47 content was lower on HS than on vesiculated red cells. Greater losses of these membrane components occurred on HPP red cells with a concomitant reduction in CD59, and glycophorins A and C. Confocal microscopy of dual labelled HS and HPP red cells showed that the loss of membrane proteins was not a uniform process but ranged from strong, moderate to dim (or no) fluorescence.Conclusion Shedding a large number of membrane proteins in red cell vesicles is probably a physiological process of cell ageing. The greater loss of band 3, CD47, Rh protein and RhAG on HS and HPP red cells probably reflects an accelerated vesiculation process secondary to their underlying cytoskeletal defect.

Genetic basis of the K0 phenotype in the Swedish population

The absence of all Kell blood group antigens (K(0) phenotype) is very rare. K(0) persons, however, can produce clinically significant anti-Ku (K5) after transfusion and/or pregnancy and require K(0) blood for transfusion. Ten alleles giving rise to the K(0) phenotype have been reported: different populations were studied although none from Scandinavia. Three K(0) samples were identified by blood banks in Sweden (Uppsala, Umeå, and Linköping) during a 20-year period. Kell antigen typing was performed with standard serologic techniques by the respective blood banks and K(0) status was confirmed by the International Blood Group Reference Laboratory in Bristol, England. Polymerase chain reaction and DNA sequencing of the KEL coding region (exons 1-19) was performed on genomic DNA. The Uppsala K(0) was homozygous for a 1540C>T substitution in exon 13, leading to an immediate stop codon. The Umeå K(0) was homozygous for 1023delG in exon 8 that results in a frameshift and a premature stop codon in exon 9. In the Linköping K(0), a previously reported mutation g>a at +1 of intron 3 was found. Two novel and one previously reported null alleles at the KEL locus are described. The identified nonsense mutations abolish expression of the Kell glycoprotein and are thus responsible for the K(0) phenotype in these Swedish families.

Differentialdiagnose hereditärer Chorea-Syndrome

The clinical triad of hereditary chorea syndromes includes (1) choreatiform involuntary movement disorder, (2) psychiatric symptoms, and (3) cognitive impairment. The most frequent hereditary chorea syndrome is Huntington's disease (HD). There are several phenocopies of Huntington's disease, such as the Huntington's disease-like neurodegenerative disorders type 1 and type 2 (HDLD), benign hereditary chorea (BHC), dentato-rubro-pallido-Luysian atrophy (DRPLA), choreoacanthocytosis (CHAC), and McLeod syndrome (MLS). Huntington's disease is caused by an instable CAG trinucleotide expansion in the Huntington disease gene, and onset age and severity of symptoms depend on the number of CAG repeats. The physiological function of the gene product Huntingtin and the disease mechanisms are not fully elucidated yet. However, experimental data strongly suggest that induction of apoptosis through a caspase (cysteine aspartate-specific proteases)-dependent mechanism might be an important factor for the development of the striatal neurodegeneration. The HDLDs are more or less exact phenocopies of Huntington's disease. Two chromosomal localisations are described, and one responsible gene, Junctophilin-3, is identified. The BHC manifests as a pure chorea syndrome, without major psychiatric or cognitive impairment. The disease is located on chromosome 14, but the responsible gene has not yet been identified. Apart from the Huntington's disease-like phenotype, DRPLA may manifest as a spinocerebellar ataxia, a progressive myoclonus epilepsy, or mixed phenotypes. DRPLA is caused by instable CAG expansions in Atrophin-1, whose physiological functions are not yet known. CHAC and MLS belong to the so-called neuroacanthocytosis syndromes. CHAC is an autosomal-recessive disorder characterised by a progressive chorea syndrome, perioral dyskinesias and mutilations, and - less frequently - an akinetic-rigid extrapyramidal syndrome and seizures. The responsible gene is located on chromosome 9, encoding chorein, a protein implicated in intracellular cell sorting. MLS is an X-linked multi-system disorder with haematological, neuromuscular, and CNS involvement. Haematologically, MLS is characterised by absent expression of the Kx erythrocyte antigen, weak expression of Kell antigens, acanthocytosis, and a compensated haemolytic state. Asymptomatic males have elevated serum creatine kinase levels, and are prone to develop neurological symptoms. Neuromuscular manifestations include myopathy, sensory-motor axonal neuropathy, and cardiomyopathy. CNS manifestations comprise a choreatiform movement disorder, neuropsychiatric abnormalities, and - less frequently - generalised seizures. MLS is caused by mutations of the XK gene encoding the XK protein, a putative membrane transport protein containing the Kx erythrocyte antigen. The XK protein is linked to the Kell glycoprotein by a single disulfide bond, probably forming a functional complex. The Kell protein is a member of the metalloproteinase family, and the XK protein has functional similarities to the CED-8 protein in nematodes, in which it controls the timing of apoptosis. These data strongly suggest an important role of the XK-Kell complex in striatal physiology. The advances in the molecular biology of hereditary chorea syndromes offer the possibility for a direct genetic analysis of affected individuals, and presymptomatic testing for individuals at risk.

Kell and XK immunohistochemistry in McLeod myopathy.

The McLeod syndrome is an X-linked neuroacanthocytosis manifesting with myopathy and progressive chorea. It is caused by mutations of the XK gene encoding the XK protein, a putative membrane transport protein of yet unknown function. In erythroid tissues, XK forms a functional complex with the Kell glycoprotein. Here, we present an immunohistochemical study in skeletal muscle of normal controls and a McLeod patient with a XK gene point mutation (C977T) using affinity-purified antibodies against XK and Kell proteins. Histological examination of the affected muscle revealed the typical pattern of McLeod myopathy including type 2 fiber atrophy. In control muscles, Kell immunohistochemistry stained sarcoplasmic membranes. XK immunohistochemistry resulted in a type 2 fiber-specific intracellular staining that was most probably confined to the sarcoplasmic reticulum. In contrast, there was only a weak background signal without a specific staining pattern for XK and Kell in the McLeod muscle. Our results demonstrate that the lack of physiological XK expression correlates to the type 2 fiber atrophy in McLeod myopathy, and suggest that the XK protein represents a crucial factor for the maintenance of normal muscle structure and function.

Molecular Basis of the Kell-null Phenotype: A MUTATION AT THE SPLICE SITE OF HUMAN KEL GENE ABOLISHES THE EXPRESSION OF KELL BLOOD GROUP ANTIGENS

The Kell blood group system is polymorphic, and 23 antigens have been defined to date. The Kell antigens are located on a single red cell transmembrane glycoprotein, encoded by the 19 exons of the KEL gene. The different Kell phenotypes result from point mutations leading to amino acid changes in the Kell glycoprotein. An unusual phenotype, which is defined as the complete lack of all of the Kell antigens, has been identified and designated as the Kell-null or Ko phenotype. The coding region of the KEL gene of the Ko individual showed a normal KEL2/KEL4/KEL7 gene sequence; nevertheless, a G to C mutation at the splice donor site (5' splice site) of intron 3 was found to be present as a homozygote in the individual. The mutation destroys the conserved GT sequence of the splice donor site. Reverse transcription-polymerase chain reaction analysis showed the absence of the complete KEL mRNA. Instead, a major transcript with the exon 3 region skipped was found. The exon 3 of the KEL gene encodes the transmembrane domain of the Kell glycoprotein, and a transcript without exon 3 is predicted to have a premature stop codon that abolishes the translation of C-terminal segment. The segment contains all of the known positions responsible for characterizing different Kell antigens, and this explains the lack of all Kell antigens in Ko red cells.

The mouse Kell blood group gene (Kel): cDNA sequence, genomic organization, expression, and enzymatic function.

The human Kell blood group system is important in transfusion medicine, since Kell is a polymorphic protein and some of its antigens can cause severe reactions if mismatched blood is transfused, while maternal alloimmunization may lead to fetal and neonatal anemia. In humans, Kell is an Mr 93,000 type II membrane glycoprotein with endothelin-3-converting enzyme activity that is linked by a single disulfide bond to another protein, XK, that spans the membrane ten times. An absence of XK leads to clinical symptoms termed the McLeod syndrome. We determined the cDNA sequence of the mouse Kell homologue, the organization of the gene, expression of the protein and its enzymatic function on red cells. Comparison of human and mouse Kell cDNA showed 80% nucleotide and 74% amino acid sequence identity. Notable differences are that the mouse Kell protein has eight probable N-linked carbohydrate side chains, compared to five for human Kell, and that the mouse homologue has one more extracellular cysteine than human Kell protein. The mouse Kell gene (Kel), like its human counterpart, is similarly organized into 19 exons. Kel was located to proximal Chromosome 6. Northern blot analysis showed high expression in spleen and weaker levels in testis and heart. Western blot analysis of red cell membrane proteins demonstrated that mouse Kell glycoprotein has an apparent Mr of 110,000 and, on removal of N-linked sugars, 80,000. As in human red cells, Kell is disulfide-linked to XK and mouse red cells have endothelin-3-converting enzyme activity.

A murine monoclonal antibody against Kx protein which reacts also with beta-spectrin.

Kx is a polytopic membrane protein of human erythrocytes carrying the Kx blood group antigen, which is deficient in rare patients with McLeod syndrome. Kx is disulphide bond linked to the Kell glycoprotein, which is a bitopic type II membrane protein carrying the Kell blood group antigen. Mice immunized with a synthetic peptide predicted to be located on the second external loop of Kx produced a monoclonal antibody called 3E12 which does not recognize red cells with common Kell phenotype by agglutination and flow cytometry. 3E12 recognizes the Kx protein and the spectrin beta-chain on western blots, the affinity for these two proteins being lowered with increasing ionic strength. Linear epitopes recognized by 3E12 are E116EIEKE121 and L484AQELEKE491 on the Kx protein and spectrin beta-chain, respectively. To quantify the relative amount of Kx in Empigen BB extracts of red cell membranes, an ELISA for Kx was set up which showed conclusively that (i) there is less Kx in membranes of K0 individuals (lacking the Kell glycoprotein) than in membranes of common individuals, and (ii) that all common individuals, typed as K+k-, K-k+ and K+k+, have the same amount of Kx on their red cell membranes. When an erythrocyte membrane detergent extract from one K0 individual was chromatographed on an immobilized 3E12 column, a minute amount of authentic Kell glycoprotein was recovered in acid eluted fractions, indicating that at least the K0 individual under study may still produce some Kell protein.

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.

The Expression of Human Blood Group Antigens During Erythropoiesis in a Cell Culture System

Phenotypic analysis of hematopoietic stem and progenitor cells has been an invaluable tool in defining the biology of stem cell populations. We use here flow cytometry to examine the expression of human erythroid-specific surface markers during the maturation of early committed erythroid cells derived from cord blood in vitro. The temporal order of the expression of erythroid specific markers was as follows: Kell glycoprotein (gp), Rh gp, Landsteiner Wiener (LW) gp, glycophorin A (GPA), Band 3, Lutheran (Lu) gp, and Duffy (Fy) gp. The time at which some of these markers appeared suggests possible roles for some of these erythroid-specific polypeptides during the differentiation of these committed progenitors. The early appearance of Kell gp raises the possibility that it may have an important role in the early stages of hematopoiesis or cell lineage determination. Kell gp may also be a useful marker for the diagnosis of erythroleukemia. The late expression of Lu gp suggests it may be involved in the migration of erythroid precursors from the marrow. Fy gp is also expressed late consistent with a role as a scavenger receptor for cytokines in the bone marrow and circulation. Rh c antigen appeared before Rh D antigen, and it is suggested that this may reflect a reorganization of the developing erythroid cell membrane involving the Rh polypeptides and other components, including GPA and Band 3.

The Expression of Human Blood Group Antigens During Erythropoiesis in a Cell Culture System: Presented in part as an abstract at the 39th American Society of Hematology Meeting, December 5-9, 1997 (Blood 90:175b, 1997 [abstr, suppl 1, part 2]).

AbstractPhenotypic analysis of hematopoietic stem and progenitor cells has been an invaluable tool in defining the biology of stem cell populations. We use here flow cytometry to examine the expression of human erythroid-specific surface markers during the maturation of early committed erythroid cells derived from cord blood in vitro. The temporal order of the expression of erythroid specific markers was as follows: Kell glycoprotein (gp), Rh gp, Landsteiner Wiener (LW) gp, glycophorin A (GPA), Band 3, Lutheran (Lu) gp, and Duffy (Fy) gp. The time at which some of these markers appeared suggests possible roles for some of these erythroid-specific polypeptides during the differentiation of these committed progenitors. The early appearance of Kell gp raises the possibility that it may have an important role in the early stages of hematopoiesis or cell lineage determination. Kell gp may also be a useful marker for the diagnosis of erythroleukemia. The late expression of Lu gp suggests it may be involved in the migration of erythroid precursors from the marrow. Fy gp is also expressed late consistent with a role as a scavenger receptor for cytokines in the bone marrow and circulation. Rh c antigen appeared before Rh D antigen, and it is suggested that this may reflect a reorganization of the developing erythroid cell membrane involving the Rh polypeptides and other components, including GPA and Band 3.

Functional aspects of red cell antigens

Over 250 blood group determinants are known and most of these are located on integral red cell proteins and glycoproteins. The functions of some of these structures are known: Diego (band 3) is the red cell anion exchanger; Kidd, a urea transporter; Colton (aquaporin 1), a water channel; Cromer (DAF) and Knops (CRI), complement regulators; Diego (band 3) and Gerbich (glycoprorin C/D) link the red cell membrane and the membrane skeleton. The Duffy glycoprotein is a chemokine receptor that may act as a scavenger for inflammatory mediators in the peripheral blood, but is also exploited as a receptor by Plasmodium vivax merozoites. The functions of some blood group antigens can be speculated upon because of structural similarity to proteins and glycoproteins of known function. For example, the Lutheran, LW and Ok glycoproteins are members of the immunoglobulin superfamily of receptors and signal transducers, the Rh proteins and related glycoproteins show homology to ammonium transporters, and the Kell glycoprotein resembles a family of endopeptidases. Yet most blood groups systems contain null phenotypes associated with no apparent pathology. If these blood group antigens have important functions, other structures must be able to carry out those functions in their absence. Almost nothing is known of the biological significance of blood group polymorphism.

Close Association of the First and Fourth Extracellular Domains of the Duffy Antigen/Receptor for Chemokines by a Disulfide Bond Is Required for Ligand Binding

It has been demonstrated that the promiscuous chemokine binding profile of the Duffy antigen/receptor for chemokines (DARC) is given by its extracellular NH2-terminal region. However, the relationship among the Fy6, Fya/b, and Fy3 epitopes, localized in the first and fourth extracellular domains of DARC, respectively, and the chemokine binding sites remained a matter of controversy. Here, we performed cross-displacement and cross-inhibition experiments indicating that all anti-Fy6, anti-Fya, and anti-Fy3 monoclonal antibodies and interleukin 8 are antagonists for binding to red cells. Biopanning of phage peptide libraries with an anti-Fy6 monoclonal antibody led to the identification of the motif Phe22-Glu23, the mutation of which altered the binding of both anti-Fy6 and chemokines (interleukin 8, MGSA, RANTES (regulated on activation normal T cell expressed)) to DARC transfectants. These results characterized the core of the Fy6 epitope and provided definitive proof of the tight relationship between Fy6 and the chemokine receptor site. Analysis of red cells treated by sulfhydryl group-modifying reagents suggested that the chemokine receptor function of DARC required the integrity of disulfide bond(s) but not that of free sulfhydryl group(s). Accordingly, mutation of cysteines 51 and 276 abolished chemokine binding to DARC transfectants. Altogether, our results suggested that the chemokine binding pocket of DARC included sequences located in the first and fourth extracellular domains which are brought into close vicinity by a disulfide bridge.

Investigation of monoclonal antibodies directed against Fy3, Jra, Lub, Kell or Kell related antigens.

Twenty Mabs of human or murine origin were studied. Serological characterization was achieved by the gel technique with common, rare, and enzyme-modified RBCs. Three clones bound to cell surface proteins on immunoblots: 1 anti-Lub and 2 Mabs raised to a purified Kell glycoprotein. Four anti-K Mabs precipitated the expected 93kD protein and a dimeric form under non-reducing conditions.