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.

Identification, immunomodulatory activity, and immunogenicity of the major helper T-cell epitope on the K blood group antigen.

The K blood group remains an important target in hemolytic disease of the newborn (HDN), with no immune prophylaxis available. The aim was to characterize the Th response to K as a key step in designing specific immunotherapy and understanding the immunogenicity of the Ag. PBMCs from K-negative women who had anti-K Abs after incompatible pregnancy, and PBMCs from unimmunized controls, were screened for proliferative responses to peptide panels spanning the K or k single amino acid polymorphism. A dominant K peptide with the polymorphism at the C terminus elicited proliferation in 90% of alloimmunized women, and it was confirmed that responding cells expressed helper CD3(+)CD4(+) and "memory" CD45RO(+) phenotypes, and were MHC class II restricted. A relatively high prevalence of background peptide responses independent of alloimmunization may contribute to K immunogenicity. First, cross-reactive environmental Ag(s) pre-prime Kell-reactive Th cells, and, second, the K substitution disrupts an N-glycosylation motif, allowing the exposed amino acid chain to stimulate a Th repertoire that is unconstrained by self-tolerance in K-negative individuals. The dominant K peptide was effective in inducing linked suppression in HLA-transgenic mice and can now be taken forward for immunotherapy to prevent HDN because of anti-K responses.

Molecular defects underlying the Kell null phenotype.

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

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.

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.

Applications of molecular biology techniques to transfusion medicine.

Other articles in this issue of Seminars in Hematology have reviewed the results of basic research in relation to the understanding of the genes, the molecular basis of blood group variants, and structural and functional aspects of the proteins carrying blood group antigens. Although molecular techniques are currently being used in a limited fashion in clinical laboratories, their application has far-reaching possibilities and undoubtedly will be soon applied more generally. We focus on two general areas: molecular genotyping for blood group antigens and their expression analysis in heterologous systems.

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.

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.

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.

Kell and Kx, two disulfide-linked proteins of the human erythrocyte membrane are phosphorylated in vivo.

Kell and Kx are two quantitatively minor proteins from the human erythrocyte membrane which carry blood groups antigens and are thought to be a metalloprotease and a membrane transporter, respectively. In the red cell membrane, these proteins form a complex stabilized by disulfide bond(s). Phosphorylation status of these proteins was studied, in the presence or absence of effectors of several kinases, either on intact cells incubated with [32P]-orthophosphate or on ghosts incubated with [gamma-32P]ATP. Purification of Kell-Kx complex, by immunochromatography on an immobilized human monoclonal antibody of Kell blood group specificity allowed to establish that (i) neither protein is phosphorylated on tyrosine; (ii) the Kell protein is a putative substrate for Casein Kinase II (CKII) and Casein Kinase I (CKI) but not for protein kinase C (PKC), whereas Kx protein is phosphorylated by CKII and PKC but not by CKI; (iii) Protein Kinase A neither phosphorylates the Kell nor the Kx proteins.

Detection of Kell blood groups: molecular methods in the diagnostic laboratory.

The introduction of molecular biology techniques to the field of transfusion medicine has allowed more detailed analysis of the basis for the expression of several blood-group antigens. The application of the polymerase chain reaction for the determination of red-cell blood-group genotype is a highly sensitive technique that can be used to determine the likelihood of a fetus being affected by haemolytic disease of the fetuses or newborn. This alone makes polymerase chain reaction based techniques highly desirable for such applications. The determination of the genetic basis for the Kell/Cellano polymorphism has enabled the development of polymerase chain reaction-based techniques for genotyping. Several other red-cell blood-group antigen polymorphisms can also be analysed in this way.

Molecular basis for the high-incidence antigens of the Kell blood group system.

BACKGROUND: The Kell blood group system is complex, containing at least 21 antigens. Some antigens are organized in five allelic sets; other, mostly high-incidence antigens, may be independently expressed. In this study, the molecular basis of five high-incidence antigens in the Kell system are described. STUDY DESIGN AND METHODS: Genomic DNA sequences from K:-12 (KEL:-12), K:-18 (KEL:-18), K:-19 (KEL:-19), K:-22 (KEL:-22), and TOU-(KEL:-26) persons were sequenced and compared to those from persons with a common Kell phenotype. RESULTS: The various Kell phenotypes are due to point mutations that encode amino acid substitutions. In KEL:-18, two mutations in the same codon were noted. In the various phenotypes, the following KEL mutations were noted: in KEL:-12: A1763G, His548Arg; in KEL:-18: C508T and G509A, Arg130Trp and Arg130Gln; in KEL:-19: G1595A, Arg492Gln; in KEL:-22: C1085T, Ala322Val; and in TOU-:G1337A, Arg406Gln. A son of one of the two people with the TOU-phenotype was heterozygous, and he also had the G1337A mutation. CONCLUSION: The high-incidence antigens of the Kell blood group system are characterized by point mutations leading to amino acid substitutions. The KEL:-18 phenotype could be due to either of two point mutations in the same codon replacing arginine with tryptophan or glutamine. TOU was confirmed as a Kell system antigen, and the inheritance of the mutation was demonstrated.

The Kell blood group system.

The Kell blood group system is complex containing over 20 different antigens. Some of the Kell antigens may be organized in 5 sets of paired alleles with opposing high and low incidence antigens while others are independently expressed. Molecular cloning established that Kell antigens are carried on a 93kDa, type II, membrane glycoprotein. The Kell gene (KEL) is located at 7q 32-36 and spans about 21,5 kb. The coding sequence is organized in 19 exons. The promoter region does not contain TATA sequences but has possible transcription binding sites for GATA-1 and Sp1. Kell protein shares a putative enzymatic active amino acid sequence with a large family of zinc endopeptidases and has closest structural and sequence homology with neutral endopeptidase 24,11 (a.k.a. enkephalinase, CALLA) and endothelin converting enzyme (ECE-1). The molecular basis of several important Kell antigens has been determined and all are due to base substitutions causing single amino acid changes. The K1/K2 polymorphism is due to a C to T substitution in exon 6, encoding a threonine to methionine change. This mutation disrupts an N-glycosylation site. Two PCR-based methods, including use of allele-specific primers, have been developed which may be used to determine fetal K1/K2 genotypes. These tests can potentially identify those pregnancies at risk for hemolytic disease of the newborn. The allelic relationship of Kpa, Kpb and Kpc was confirmed, since single base substitutions in the same codon encode 3 different amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)