Kell, Kx and the McLeod syndrome

BaillieÁre's Clinical Haematology Vol. 12, No. 4, pp. 621±635, 1999

2 Kell, Kx and the McLeod syndrome Colvin M. Redman

PhD

Member, Head of the Laboratory of Membrane Biochemistry

David Russo

DVM, PhD

Research Scientist

Soohee Lee

PhD

Laboratory Member Lindsley F. Kimball Research Institute, The New York Blood Center, 310 East 67th Street, New York, NY 10021, USA

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 di€erent 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 di€erent 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. Key words: Kell; XK; McLeod syndrome; endothelin-3 converting enzyme; fetal anaemia; blood group genotyping.

INTRODUCTION Prominent features of the Kell blood group system are its antigen complexity and its association with disease. Over 23 di€erent blood group antigens have been assigned to the Kell blood group system and the molecular basis for this polymorphic variation has been shown to be due to single base mutations in the KEL gene. This topic has been reviewed previously.1±4 A list of the Kell antigens and the ethnic distribution of the low incidence antigens is presented in Table 1. Some of the Kell antigens are All correspondence to: Colvin M. Redman, PhD. Tel: 1-(212) 570-3059; Fax: 1-(212) 8769-0243; E-mail: [email protected]. 1521±6926/99/040621+15 $12.00/0

c 1999 Harcourt Publishers Ltd *

622 C. M. Redman et al Table 1. Kell antigens. Low incidence

High incidence

KEL1 (K) KEL3 (Kpa) KEL21 (Kpc) KEL6 (Jsa) KEL17 (Wka) KEL24 (K24) ± KEL10 (U1a) ± ± ± ± ± ± ± KEL23 KEL25 (VLAN) ± ±

KEL2 (k) KEL4 (Kpb) KEL7 (Jsb) KEL11 (K11) KEL14 (K14) KEL5 (Ku) ± KEL12 (K12) KEL13 (K13) KEL16 (K16) KEL18 (K18) KEL19 (K19) KEL20 (Km) KEL22 (K22) ± ± KEL26 (TOU) Kx

A. Distribution of low incidence antigens KEL1 9% (Caucasians), 2% (Blacks), 12% (Iranian Jews) KEL3 2% (Caucasians) KEL6 0.01% (Caucasians), 20% (Blacks) KEL10 less than 0.01% (most populations), 2.6% (Finns), 0.46% (Japanese) KEL17 about 0.3% (most populations) KEL21 less than 0.01% (most populations), about 0.32% (Japanese) KEL23 less than 0.01% (most populations) KEL24 less than 0.01% (most populations) KEL25 only found in 1 family The antigens classi®ed as high incidence occur in over 90% of the population.

highly immunogenic, and among blood groups in which antibodies are caused by alloimmunization, KEL1 (K) is second to RhD in its immunizing potential. Most antibodies in the Kell blood group system arise as an immune response to mismatched transfusion, or during pregnancy. The most common Kell antibody encountered is anti-KEL1, but other antibodies, to di€erent Kell antigens, are also clinically important because they also cause haemolytic reactions in mismatched blood transfusions and can also be involved in fetal and neonatal anaemia. Kx is the only known blood group antigen carried by the XK component of the Kell blood group system and its absence from red cells, the McLeod phenotype, is accompanied by a set of clinical symptoms known as the McLeod syndrome, which include red cell acanthocytosis and late onset forms of muscle and neurological abnormalities. KELL MEMBRANE GLYCOPROTEIN Primary structure Kell is a 93 kDa type II membrane glycoprotein, with a short N-terminal intracellular segment, a single transmembrane sequence and a large 665 amino acid extracellular domain.5 Kell has 16 cysteine residues. One of the cysteine residues is in the

Kell, Kx and the McLeod syndrome 623

transmembrane region and the other 15 cysteine residues are in the extracellular domain. One of the cysteine residues in the extracellular domain, Cys72, forms a disulphide bond with XK protein. Presumably the other 14 extracellular cysteine residues are involved in forming seven intramolecular disulphide bonds that stabilize the folded structure of Kell protein. Kell antigens are easily inactivated by treatment of red cells with sulphydryl reducing reagents such as dithiothreitol and 2-amino ethyliso-thio-uronium bromide (AET), suggesting that disruption of the extracellular disulphide bonds unravels Kell protein and disrupts antigenic sites. Kell has ®ve probable N-linked sugar moieties at asparagine residues 94, 115, 191, 345 and 627. The carbohydrate moieties of Kell have not been characterized but it is assumed that they are of the bi-antennary type. Kell protein has several putative phosphorylation sites and has been shown to be a substrate for casein kinases I and II.6 Kell also has a probable tyrosine phosphorylation site, Tyr241, but it is not known if Kell is phosphorylated at this site or whether phosphorylation, in vivo, plays a signi®cant physiological role. A computerassisted search for consensus sequences also indicates that Kell has a matching pattern for two leucine zippers, which occur immediately downstream from its zinc-binding enzymatically active site (H.E.L.L.H.) at amino acid residues 581 to 585. The Cterminus of Kell protein has a consensus sequence, CXXX, that is a possible isoprenylation site. Molecular cloning indicated two possible sites for initiation of translation and since Kell protein, as isolated from red cells, has a blocked N-terminus it has not been possible to determine experimentally the correct N-terminal amino acid residue.5 Based on the ®rst possible initiation methionine, Kell has a 46 amino acid intracellular segment but a shorter segment may occur if methionine at position 20 is the initiation site. For convenience the ®rst possible initiation methionine is included as part of Kell protein, but if necessary this may have to be corrected, when experimental proof is obtained. HOMOLOGY OF KELL WITH THE NEPRILYSIN (M13) FAMILY OF ZINC ENDOPROTEASES The Neprilysin or M13 group of proteins are type II membrane glycoproteins with zinc endopeptidase catalytic activity. Their principal function is the activation of bioactive peptides by proteolytic cleavage of larger inactive polypeptides.7 Currently the M13 family consists of Kell, neutral endopeptidase 24.11 (NEP) also known as CD10, two endothelin-converting enzymes (ECE-1 and ECE-2), the product of the PEX gene and XCE, an enzyme preferentially expressed in the central nervous system. These enzymes are part of a large group of zinc endopeptidases, widely distributed, that share a common zinc-binding catalytic sequence (H.E.X.X.H.)8,9, where the histidines (H) co-ordinate zinc binding and glutamic acid (E) is a nucleophile that acts to hydrolyze the scissile bond of the target peptide.10,11 In the M13 family of proteins, amino acid identity is high in the C-terminal third of the molecule (amino acid residues 550±732 of Kell), and in this segment Kell has 33±36% amino acid identity with human NEP, ECE-1, PEX and XCE. In addition, there are structural similarities in that all M13 membranes have short intracellular N-terminal domains and although there are varying amounts of cysteine residues in the family members, 10 cysteine residues are conserved in similar locations in all of them. Site-directed mutagenesis of NEP and ECE-1 has identi®ed a number of amino

624 C. M. Redman et al

acid residues that are involved in zinc binding and catalysis and most of these amino acids are conserved in all M13 members.12±19 Based on homology with NEP and ECE-1, it is predicted that zinc forms a complex with H581, H585 and E634 of Kell. Glutamic acid residue 582 is essential for catalytic activity, and may act as a nucleophile by polarizing the water molecule. Other amino acids may participate in stabilizing the transition state and in substrate binding. The M13 family of enzymes has di€erent substrate speci®cities. In vitro, NEP has broad speci®city, cleaving peptides at the amino-terminal side of hydrophobic amino acid residues. In vivo, NEP has many physiological substrates, which include the enkephalins, oxytocin, bradykinin and angiotensin. NEP has a wide tissue distribution and its speci®city may depend on substrate availability.20±22 ECE-1 and ECE-2, by contrast, have very speci®c substrates, principally big endothelin-1, which is cleaved at Trp21±Val22 to yield bioactive endothelin-1. ECE-1 and ECE-2 can also cleave big endothelin-2 and big endothelin-3, but to a much lesser extent.23±25 Less is known of the substrate speci®city of PEX and XCE. PEX, in vitro, cleaves parathyroid hormonederived peptides26 but substrates for XCE have not been found and XCE does not activate the endothelins.27 As will be discussed in the following section, Kell preferentially processes big endothelin-3, yielding bioactive endothelin-3. KELL IS AN ENDOTHELIN-3 CONVERTING ENZYME Studies using a recombinant, truncated form of Kell protein, lacking the intracellular and transmembrane domain (sKell) have shown that Kell has endothelin-3 converting enzyme activity.28 sKell has narrow substrate speci®city and does not hydrolyze peptides that are known substrates for NEP. There is overlap, however, with ECE-1 and ECE-2 in that Kell, like the ECEs, processes all three big endothelins yielding bioactive 21 amino acid residue peptides. The di€erences between Kell and ECE-1 and ECE-2 however, are striking. ECE-1 and ECE-2 have a marked preference for big endothelin-1 while Kell prefers big endothelin-3. The Km value of sKell with big endothelin-3 as substrate is 0.33 mM, while those for big endothelin-1 and big endothelin-2 are 60 to 130 times higher. An enzyme that prefers big endothelin-2 as substrate has not yet been identi®ed. ECE-1 and ECE-2 are structurally related enzymes (about 59% amino acid residue identity) that have similar enzymatic speci®city but di€er in that ECE-2 has an acidic pH optimum, 5.5, compared to ECE-1 whose pH optimum is neutral.25 Kell resembles ECE-2, in that it also has an acidic pH optimum, suggesting that Kell and ECE-2 both may have intracellular functions. This raises the question as to what is the function of Kell on red cells since in these tissues Kell only exists as an ecto-enzyme. Similarly to the truncated recombinant protein, Kell on red cells also preferentially activates endothelin-328 and has an acidic pH optimum. Several unrelated Ko(null) persons have been tested and all of them lack endothelin-3 converting enzyme activity on their red cells, demonstrating that Kell is the only red cell membrane protein with endothelin-3 converting enzyme activity. THE ENDOTHELINS Endothelins-1, -2 and -3 are structurally related, 21 amino acid residue peptide isoforms, derived from three separate genes.29±31 The endothelins are initially synthesized as

Kell, Kx and the McLeod syndrome 625

pre-pro-polypeptides of about 200 amino acid residues and are processed intracellularly into inactive intermediate precursors, 37±41 amino acid residues in length, termed big endothelins. In a ®nal step the big endothelins are proteolytically clipped at Trp21±Val22 for big endothelin-1 and -2 and at Trp21±Ile22 for big endothelin-3 to yield the 21 amino acid residue bioactive peptides.23,32,33 The endothelins act on two types of G protein coupled receptors, termed ETA and ETB . At physiological concentrations ETA is activated by endothelin-1 and -2 but not by endothelin-3. On the other hand ETB binds all three endothelins equally well.34±36 The ETA and ETB receptors are present in a wide variety of cells and it is thought that the endothelins act locally, since cells that produce endothelins are in the near vicinity of target cells. Activation of ETA and ETB leads to broad physiological responses depending on the cellular target. In general the endothelins are involved in the regulation of vascular tone by a€ecting contraction and proliferation of smooth muscle cells but they also have vasodilatation e€ects by mediating the release of nitric oxide. Targeted disruption, in mice, of the endothelins and their receptors has shown that the endothelins play multiple physiological roles.37±42 For example, the endothelins are involved in mitogenesis and developmental processes by a€ecting the di€erentiation and migration of neural crest derived cells. The role that Kell plays in these processes is not known. Another protein, present in bovine iris, has been shown also to preferentially process big endothelin-3.43 Thus Kell is not the sole endothelin-3 converting enzyme and this, coupled with the fact that ECE-1 and ECE-2 overlap with Kell in substrate speci®city, indicates that a network of enzymes and substrates in di€erent cellular locations may complement each other to perform a broad set of cellular functions. THE KEL GENE The 93 kDa Kell glycoprotein is the product of a single gene, KEL, whose chromosomal location is 7q33.44,45 KEL consists of 19 exons ranging in size from 63 to 288 bp and spanning approximately 21.5 kb. The size of the introns range from 93 bp to about 6 kb. Exon 1 has an untranslated region containing GATA-1 and Spl binding sites and a single codon for a possible initiation methionine. The upstream region (ÿ176) has a CACCC box and two other putative GATA-1-binding sites. The nucleotide (nt) region, ÿ176 to ÿ1, placed in front of a reporter gene, exhibits promoter activity but a complete analysis of the promoter has not been undertaken. The KEL coding region, with the exception of the possible initiation methionine in exon 1, resides in exon 2 to exon 19. Exon 2 contains another possible initiation methionine at nt 178, amino acid residue 20. Neither of the two putative translation initiation sites in KEL are typical Kozak sequences. The single membrane spanning region of Kell protein is encoded in exon 3 and the zinc-binding catalytic site is in exon 16.46 The base sequences in exons 16 to 19 are the most homologous with the other members of the M13, Neprilysin, family of zinc endopeptidases. MOLECULAR BASIS OF DIFFERENT KELL PHENOTYPES Sequence analysis of the KEL gene demonstrated that single base mutations encoding amino acid residue substitutions are responsible for the di€erent Kell phenotypes.4 A summary of the various phenotypes studied, listing the base mutations encoding the

626 C. M. Redman et al Table 2. Molecular basis of Kell blood group antigens. Antigen

cDNA change

Amino acid change

RFLP

6 8 8 17 17 13 8 10 6

698C4T 961C4T 962G4A 1910T4C 2019A4G 1601A4T 1025T4C 1265A4G 659G4C

193Thr4Met 281Arg4Trp 281Arg4Gln 597Leu4Pro none 494Glu4Val 302Val4Ala 382Gln4Arg 180Arg4Pro

new Bsm I new Nla III new Pvu II Mnl I lost Dde I lost New Acc I new Msc I new Bcn I new Hae III

KEL19 KEL22 KEL26 (TOU)

15 4 4 13 9 11

1763A4G 508C4T 509G4A 1595G4A 1085C4T 1337G4A

548His4Arg 130Arg4Trp 130Arg4Gln 492Arg4Gln 322Ala4Val 406Arg4Gln

Nla III lost new Taq II new Eco57 I none new Tsp 45 I none

C. Weak expression Weak KEL2

11

1388C4T

423Ala4Val

BsoF I lost

A. Group A KEL1 (K) KEL3 (Kpa) KEL21 (Kpc) KEL6 (Jsa) KEL10 (U1a) KEL17 (Wka) KEL23 KEL24 (cls) B. Group B KEL12 KEL18

Exon

Group A: low incidence antigens. Group B: very rare alleles representing absence of high incidence antigens. RFLP, restriction fragment length polymorphism.

amino acid residue substitutions and restriction enzyme sites that are either lost or newly created, is presented in Table 2. It is interesting to note that all of the amino acid substitutions, with the exception of the one encoding expression of KEL6 (Jsa), occur in exon 4 to exon 15, an area that has the least homology with other members of the M13 Neprilysin family of zinc endopeptidases. The base change that encodes KEL6 (Jsa) occurs in exon 17, T1910C, leading to a Leu597Pro substitution.47 This amino acid residue substitution is in close proximity to the zinc-binding site, H.E.L.L.H., at residues 581 to 585 and occurs in a four amino acid residue stretch between two cysteine residues (Cys. Pro. Ala. Cys), therefore probably disrupting the intramolecular disulphide arrangements and tertiary structure and possibly a€ecting enzymatic function. Since KEL6 (Jsa) is more prevalent in persons of African heritage (Table 1) this Kell phenotype may provide a selective advantage. The allelic relationship of KEL3/KEL4/KEL21 (Kpa/Kpb/Kpc) has been con®rmed, since DNA sequencing of appropriate KEL genes showed that two di€erent point mutations occur in the same codon.48 In the common KEL4 (Kpb) phenotype, arginine, a basic amino acid residue is expressed, while in KEL21 (Kpc) a G962A mutation encodes glutamine, a polar uncharged amide and in KEL3 (Kpa) a C961T mutation expresses tryptophan, a non-polar aromatic amino acid. KEL3 (Kpa) red cells are present in only about 2% of the population and when Kpa is expressed it is accompanied by a general depression of all other Kell antigens, due to a reduced amount of Kell protein on the red cell membrane. Recent studies using a recombinant system have shown that the Arg281Trp substitution a€ects intracellular tracking causing an accumulation and proteolytic degradation of nascent KEL3 protein in a preGolgi compartment.49 Thus small changes in primary structure, as indicated by the

Kell, Kx and the McLeod syndrome 627

presence of a non-polar amino acid (tryptophan) instead of a polar amino acid (glutamine) or a charged amino acid (arginine), are sucient to a€ect the amount of Kell protein placed on the cell surface. KELL GENOTYPING Since the di€erent Kell phenotypes are due to single base mutations, many of which either create or abolish a restriction enzyme site, simple genotyping procedures have been developed (see Table 2). Genotyping is particularly useful in the case of KEL1/ KEL2 since KEL1 (K) is the strongest immunogen, and antibodies to KEL1 in pregnant mothers may cause severe fetal and neonatal anaemia. A C698T mutation in exon 6, encoding a Met193Thr substitution accounts for the KEL1 phenotype.50 This amino acid substitution voids an asparagine-linked glycosylation site and KEL1 protein has four instead of ®ve carbohydrate units. Several procedures have been described that identify the KEL1 genotype and these procedures have been shown to be valuable in prenatal determination of KEL1/KEL2 genotypes.51±55 Genotyping is also useful, as an alternative to serology, when there are mixed red cells occurring as a result of transfusion, when good quality antibodies are not available or when shipped red cells are not well preserved. EXPRESSION OF KELL ANTIGENS As determined by the expression of di€erent recombinant Kell proteins in surrogate cells, the single base mutations may be sucient to express the expected cell surface antigen. This has not been shown for all the Kell phenotypes, but several examples support this view. For example, it holds for KEL1, in which the point mutation prevents the formation of an N-linked sugar moiety4; for KEL3 (Kpa) in which a charged amino acid is substituted with a non-polar residue and for KEL6 (Jsa), in which a proline substitution may cause changes in tertiary structure.49 It is not possible to assign Kell epitopes to the points of mutation since amino acid residue substitutions may cause changes in protein conformation, thus creating antigenic sites removed from the points at which mutations cause amino acid residue substitutions. A procedure termed monoclonal antibody immobilization of erythrocyte antigens (MAIEA), which groups antigenic sites based on spatial proximity, has provided information on the neighbouring relationships of di€erent Kell epitopes.56 Thus KEL10 and KEL24/KEL14 appear to be spatially close together but using gene sequencing they are far apart. Also the point mutations that characterize KEL10 and KEL19 are only six base residues apart but these antigens do not appear, using MAIEA, to be in proximity to each other. TRANSIENT LOSS OF KELL ANTIGENS A few patients with autoimmune thrombocytopenic purpura (AITP) have developed transient loss of Kell antigens.57,58 In these rare cases, loss of Kell antigens is due to diminished amounts of Kell protein on the red cell membrane. In one case in which the patient had two consecutive relapses of AITP there was absence of Kell protein during the ®rst relapse and absence of Lutheran protein during the second relapse. On

628 C. M. Redman et al

both occasions normal antigenic expression of Kell and Lutheran was restored once AITP entered remission.58 At present there is no ®rm experimental evidence to explain the transient loss of Kell protein from red cells. This could be due to transient inhibition of expression or to selective shedding of Kell protein. KELL ANTIBODIES AND SUPPRESSION OF ERYTHROPOIESIS Although haemolytic disease of the newborn (HDN) is most common in Rh alloimmunization, about 0.1% of all obstetric patients have maternal antibodies to KEL1.59 Maternal antibodies are usually due to transfusions of mismatched blood, although immunizations from previous pregnancies can occur. Maternal alloimmunization to KEL1 is only clinically important when the fetus, unlike the mother, carries KEL1. This only occurs occasionally since the incidence of KEL1 is about 9% for Caucasians and 2% for persons of African heritage (see Table 1). In contrast to HDN caused by antibodies to RhD, there is a poor correlation between maternal KEL1 antibody titre and the severity of the disease. In addition, increased bilirubin levels are not common in anaemic babies exposed to anti-KEL1. In about 50% of the cases the babies only have mild anaemia and some may require phototherapy. In a few cases, however, the babies are severely a€ected, and can su€er from hydrops faetalis.60±67 It has been suggested that fetal anaemia, due to Kell antibodies, is due mostly to suppression of erythropoiesis rather than to haemolysis.68,69 This view is supported by in vitro studies that have demonstrated that antibodies to Kell inhibit the growth of KEL1 progenitor cells.70 The mechanism by which antibodies to Kell suppress erythropoiesis is not yet known. Recent studies of progenitor cells, cultured to undergo erythropoiesis, demonstrated that Kell surface antigens are expressed ahead of Rh, Landsteiner± Weiner, glycophorin A, band 3, Lutheran and Du€y.71 This raises the possibility that antibodies to Kell may react at an early stage of erythropoiesis when progenitor cells are ®rst committed to erythroid di€erentiation, suppressing further growth. Since the endothelins are mitogenic and may a€ect maturation of erythroid precursors, other possibilities are that the endothelin-3-converting enzyme activity of Kell may be involved in the process and antibodies to Kell may modify its ability to process endothelins. THE MCLEOD GENE The gene for the McLeod syndrome, XK, was mapped by deletion analysis to Xp21.72 XK is a single gene composed of three exons with an open reading frame encoding 444 amino acid residues. XK has a short, 82 bp, 50 untranslated region and an unusually long, 3713 bp 30 untranslated region. Northern blot analysis showed the presence of a 5.2 kb mRNA transcript in several tissues. The highest levels of expression are in erythroid tissues and skeletal muscle, with lower levels of expression in brain, heart and pancreas. Weaker signals were noted in many other tissues.73 Sequencing of XK from unrelated McLeod patients demonstrated mutations in the gene that would abolish mRNA splicing or result in reduced amounts or aberrant XK. In two cases mutations occurred at conserved splice donor and acceptor sites.73 In other McLeod cases mutations occur in the coding region. In one McLeod a nucleotide deletion in exon 2 at codon 90 resulted in premature termination after incorporation

Kell, Kx and the McLeod syndrome 629

of 38 additional amino acid residues not related to normal XK protein. This mutation yields a 128 rather than a 444 amino acid residue XK protein.74 In another McLeod patient a single base deletion in exon 3 at nt 1085 creates a premature stop codon at amino acid residue 408.75 Other McLeod phenotypes are due to gene deletions. These deletions may be large and encompass not only the XK gene but also neighbouring genes such as those involved in chronic granulomatus disease, retinitis pigmentosa and Duchenne muscular dystrophy.72,76,77 XK PROTEIN XK has a predicted molecular weight of 50.9 kDa.73 However, due to its hydrophobic nature XK migrates on sodium dodecyl sulphate-polyacrilamide gel electrophoresis (SDS-PAGE) as a 37 kDa protein.78 DNA sequencing of the XK gene and hydropathy analysis of the encoded protein predicts that XK has 10 trans-membrane segments with a short N-terminal domain (only four amino acid residues including the initiation methionine), and a larger (71 amino acid residues), C-terminal domain, both within the intracellular compartment. The model also predicts a large extracellular hydrophilic loop between the third and fourth transmembrane segments. A single extracellular cysteine residue is present in the ®fth loop between the ninth and tenth transmembrane segments (see Figure 1). Other distinctive features of XK protein are a large number of putative intracellular phosphorylation sites and the fact that six of the 10 transmembrane segments contain charged amino acid residues.73 The presence of charged amino acid residues in the transmembrane domains is unusual and often, when present, they are involved in functional aspects. In vitro studies have determined that XK is phosphorylated by casein kinase II and protein kinase C, but not by casein kinase I.6 XK is palmitoylated and this fatty acid may play a role in anchoring the large C-terminal intracellular fragment to the inner surface of the plasma membrane.79 XK resembles a glutamate transporter in its general structural characteristics, but has very little amino acid residue homology with this group of transporters and its exact cellular function remains to be determined. ASSOCIATION OF KELL AND XK The association of Kell and XK protein on red cells was deduced from early serological studies on Ko(null) and McLeod red cells. On McLeod red cells, which lack the antigen Kx, carried by XK protein, there is a general depression of all Kell antigens. By contrast, Ko(null) red cells, that lack all Kell antigens, have enhanced Kx activity. Also, treatment of normal red cells with sulphydryl reducing reagents, which inactivate the Kell antigens, causes a marked increase in Kx.1,2 Early biochemical studies showed that Kell protein, isolated from red cells in non-reduced conditions, is associated with itself as an oligomer, but is also associated with other red cell membrane proteins.80 Later studies showed that XK protein could be immunopuri®ed from red cells as a complex with Kell protein and that XK, but not the Kell/XK complex, could be isolated from Ko(null) cells.81 These studies showed that Kell and XK were linked by a disulphide bond. Further studies also showed the absence of the Kell/XK complex in McLeod red cells and demonstrated that although Ko(null) red cells have high Kx activity, they contain less XK protein on the membrane than red cells of normal Kell phenotype.82

630 C. M. Redman et al

These studies suggest that lack of Kell protein may expose previously hidden domains of XK that enhance expression of Kx antigen. By site-directed mutagenesis of speci®c cysteine residues in both Kell and XK it was shown that a cysteine residue in Kell protein, Cys72, which is close to the transmembrane region, on the extracellular side of the membrane, is linked to the only extracellular cysteine in XK.83 Kell Cys72 resides in a cluster of ®ve cysteine residues, present within amino acid residues 72 to 108, which are close to the transmembrane region (see Figure 1). Four of the ®ve cysteine residues in this group are conserved in similar locations in the other members of the M13 family of zinc endopeptidases. Kell Cys72, which is not conserved in the other M13 members, forms a disulphide bond with XK. It is not obligatory for Kell and XK to be linked on red cell membranes since both Ko(null) and McLeod red cells exist with only one of the component proteins. It appears however that the Kell/XK complex is more stable than the component proteins, since in the rare phenotypes, Ko(null) and McLeod, absence of one of the component proteins is accompanied by reduced amounts of the other protein component. Thus, on the one hand McLeod red cells that lack XK have greatly reduced levels of Kell protein and, on the other hand, Ko(null) red cells that lack Kell protein have reduced amounts of XK protein, although using serology there is enhanced Kx.1,2,82 Studies using recombinant expressed Kell and XK protein support the idea that either Kell or XK, expressed by themselves, can be transported to the cell surface. In addition, speci®c Kell cDNAs transfected into surrogate cells express well-de®ned Kell antigens, as detected by alloimmune antibodies, indicating that Kell, by itself, can travel to the cell surface and assume its proper con®guration in the absence of XK.4,49,84 The association of Kell and XK, in a recombinant system, commences soon after the two polypeptides are translated and Kell/XK complexes exist in the endoplasmic reticulum. At this nascent stage, the carbohydrate units of the Kell components are not fully processed.85 On rare occasions a combination of gene mutations a€ect the expression of both Kell and Kx antigens. As noted earlier, red cells of the Kpa phenotype have weak expression of all Kell antigens, but express normal levels of Kx. One case has been reported in which a Kpa homozygote had an unusually extensive depression of Kell antigens and weak Kx. This rare phenotype was caused by a single base change at a donor splice consensus sequence in XK intron 2 indicating that a mutation in XK caused further weakening of the Kell antigens.86 EXPRESSION OF KELL AND XK IN NON-ERYTHROID TISSUES XK expression is not con®ned to erythroid tissues, being present in substantial amounts in skeletal muscle, pancreas and brain.46 Kell is primarily expressed in erythroid tissues, but recent studies (our unpublished results) have shown near equal expression in testis and lower, but signi®cant, expression in many other tissues. The Kell/XK complex is present in non-erythroid tissues. THE MCLEOD SYNDROME The McLeod syndrome was ®rst described, using serological analysis, as a red cell with a rare Kell phenotype which expressed low levels of all Kell antigens with no alterations

Kell, Kx and the McLeod syndrome 631

Figure 1. Diagram of Kell/XK complex showing the cysteine residues that link the two protein subunits of the Kell blood group system. HELLH, @A.

632 C. M. Redman et al

of other blood group antigens and lacked an otherwise ubiquitous antigen, termed Kx.87 The McLeod phenotype has an X-linked mode of inheritance, while the KEL gene is autosomal. The McLeod phenotype was one of the ®rst reports of a person with a rare blood group who had red cells with abnormal morphology.88,89 In all cases reported, McLeod red cells have shown variable levels of acanthocytosis, usually with a compensated haemolytic state including an increased reticulocyte count and low haptoglobin.1,2 Although the McLeod phenotype is rare, several cases have been studied and a pattern of clinical symptoms has been described, termed the McLeod syndrome. The clinical symptoms are variable and diverse but in all cases there is acanthocytosis and elevated levels of serum creatine phosphokinase indicating muscle damage.90 In most cases there is late onset, usually starting in the forties, neurological and muscular defects. The McLeod symptoms may include progressive heart disease, cardiomyopathy and neuropathy with choreiform movements. In some cases neuropsychological impairment and seizures occur. Cerebral involvement in the McLeod syndrome is also indicated by mild cerebral atrophy, as determined by neuroimaging.91±93 Glomerular lesions with chronic renal failure have also been reported.94 The clinical symptoms of McLeod syndrome overlap with those associated with neuroacanthocytosis, but can be distinguished in that in neuroacanthocytosis there is normal Kell red cell phenotype. Both McLeod and neuroacanthocytosis, however, share abnormal red cell morphology, dystonic and choreiform movements.95

REFERENCES 1. Marsh WL & Redman CM. Recent developments in the Kell blood group system [review]. Transfusion Medicine Reviews 1987; 1: 4±20. 2. Marsh WL & Redman CM. The Kell blood group system: a review [review]. Transfusion 1990; 30: 158±167. 3. Redman CM & Marsh WL. The Kell blood group system and the McLeod phenotype [review]. Seminars in Hematology 1993; 30: 209±218. * 4. Lee S. Molecular basis of Kell blood group phenotypes. Vox Sanguinis 1997; 73: 1±11. * 5. Lee S, Zambas ED, Marsh WL & Redman CM. Molecular cloning and primary structure of Kell blood group protein. Proceedings of the National Academy of Sciences of the USA 1991; 88: 6353±6357. 6. Carbonnet F, Hattab C, Cartron JP & Bertrand O. Kell and Kx, two disul®de-linked proteins of the human erythrocyte membrane are phosphorylated in vivo. Biochemical and Biophysical Research Communications 1998; 247: 569±575. * 7. Turner AJ & Tanzawa K. Mammalian membrane metallopeptidases: NEP, ECE, KELL, and PEX. FASEB Journal 1997; 11: 355±364. 8. McKerrow JH. Human ®broblast collagenase contains an amino acid sequence homologous to the zincbinding site of Serratia protease. Journal of Biological Chemistry 1987; 262: 5943. 9. Jongeneel CV, Bouvier J & Bairoch A. A unique signature identi®es a family of zinc-dependent metallopeptidases. Federation of European Biochemical Societies Letters 1989; 242: 211±214. 10. Rawlings ND & Barrett NJ. Evolutionary families of metallopeptidases. In Barrett AJ (ed.) Methods in Enzymology, pp 183±228. San Diego: Academic Press, 1995. 11. Vallee BL & Auld DS. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 1990; 29: 5647±5659. 12. Dion N, Le Moual H, Fournie-Zaluski MC et al. Evidence that Asn542 of neprilysin (EC 3.4.24.11) is involved in binding of the P20 residue of substrates and inhibitors. Biochemical Journal 1995; 311: 623±627. 13. Dion N, Le Moual H, Crine P & Boileau G. Kinetic evidence that His-711 of neutral endopeptidase 24.11 is involved in stabilization of the transition state. Federation of European Biochemical Societies Letters 1993; 318: 301±304. 14. Le Moual H, Dion N, Roques BP et al. Asp650 is crucial for catalytic activity of neutral endopeptidase 24± 11. European Journal of Biochemistry 1994; 221: 475±480. 15. Le Moual H, Crine P & Boileau G. Identi®cation of Glu 646 of neutral endopeptidase 24±11 as a zinc binding residue. Matrix Supplement 1992; 1: 100.

Kell, Kx and the McLeod syndrome 633 16. Le Moual H, Beaumont A, Roques BP et al. Identi®cation of two arginine residues involved in the binding of substrate to neutral endopeptidase 24-11. Matrix Supplement 1992; 1: 99. 17. Beaumont A, Barbe B, Le Moual H et al. Charge polarity reversal inverses the speci®city of neutral endopeptidase-24.11. Journal of Biological Chemistry 1992; 267: 2138±2141. 18. Bateman RC Jr, Jackson D, Slaughter CA et al. Identi®cation of the active-site arginine in rat neutral endopeptidase 24.11 (enkephalinase) as arginine 102 and analysis of a glutamine 102 mutant. Journal of Biological Chemistry 1989; 264: 6151±6157. 19. Shimada K, Takahashi M, Turner AJ & Tanzawa K. Rat endothelin-converting enzyme-1 forms a dimer through Cys412 with a similar catalytic mechanism and a distinct substrate binding mechanism compared with neutral endopeptidase-24.11. Biochemical Journal 1996; 315: 863±867. 20. Shipp MA, Richardson NE, Sayre PH et al. Molecular cloning of the common acute lymphoblastic leukemia antigen (CALLA) identi®es a type II integral membrane protein. Proceedings of the National Academy of Sciences of the USA 1988; 85: 4819±4823. 21. Shipp MA & Look AT. Hematopoietic di€erentiation antigens that are membrane-associated enzymes: cutting is the key [review]. Blood 1993; 82: 1052±1070. 22. Turner AJ, Murphy LJ, Medeiros MS & Barnes K. Endopeptidase-24.11 (neprilysin) and relatives. Twenty years on. In Suzuki K & Bond J (eds) Intracellular Protein Catabolism, pp 141±148. New York: Plenum Press, 1996. 23. Turner AJ & Murphy LJ. Molecular pharmacology of endothelin converting enzymes [review]. Biochemical Pharmacology 1996; 51: 91±102. *24. Xu D, Emoto N, Giaid A et al. ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell 1994; 78: 473±485. 25. Emoto N & Yanagisawa M. Endothelin-converting enzyme-2 is a membrane-bound, phosphoramidonsensitive metalloprotease with acidic pH optimum. Journal of Biological Chemistry 1995; 270: 15 262±15 268. 26. Lipman ML, Panda D, Bennett HPJ et al. Cloning of human PEX cDNA ± expression, subcellular localization, and endopeptidase activity. Journal of Biological Chemistry 1998; 273: 13 729±13 737. 27. Valdenaire O, Richards JG, Faull RLM & Schweizer A. XCE, a new member of the endothelin-converting enzyme and neutral endopeptidase family, is preferentially expressed in the CNS. Molecular Brain Research 1999; 64: 211±221. *28. Lee S, Lin M, Mele A et al. Proteolytic processing of big endothelin-3 by the Kell blood group protein. Blood 1999; 94: 1440±1450. 29. Inoue A, Yanagisawa M, Kimura S et al. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proceedings of the National Academy of Sciences of the USA 1989; 86: 2863±2867. 30. Yanagisawa M, Kurihara H, Kimura S et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature, London 1988; 332: 411±415. 31. Yanagisawa M, Inoue A, Ishikawa T et al. Primary structure, synthesis, and biological activity of rat endothelin, an endothelium-derived vasoconstrictor peptide. Proceedings of the National Academy of Sciences of the USA 1988; 85: 6964±6967. 32. Itoh Y, Yanagisawa M, Ohkubo S et al. Cloning and sequence analysis of cDNA encoding the precursor of a human endothelium-derived vasoconstrictor peptide, endothelin: identity of human and porcine endothelin. Federation of European Biochemical Societies Letters 1988; 231: 440±444. 33. Webb DJ, Monge JC, Rabelink TJ et al. Endothelin: new discoveries and rapid progress in the clinic. Trends in Pharmacological Sciences 1998; 19: 5±8. 34. Arai H, Hori S, Aramori I, Ohkubo H & Nakanishi S. Cloning and expression of a cDNA encoding an endothelin receptor. Nature, London 1990; 348: 730±732. 35. Masaki T, Ninomiya H, Sakamoto A & Okamoto Y. Structural basis of the function of endothelin receptor. Molecular and Cellular Biochemistry 1999; 190: 153±156. 36. Sakurai T, Yanagisawa M, Takuwa Y et al. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor [see Comments]. Nature, London 1990; 348: 732±735. 37. Baynash AG, Hosoda K, Giaid A et al. Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell 1994; 79: 1277±1285. 38. Hosoda K, Hammer RE, Richardson JA et al. Targeted and natural (Piebald-Lethal) mutations of endothelin-B receptor gene produce megacolon associated with spotted coat color in mice. Cell 1994; 79: 1267±1276. 39. Kurihara Y, Kurihara H, Suzuki H et al. Elevated blood pressure and craniofacial abnormalities in mice de®cient in endothelin-1. Nature, London 1994; 368: 703±710. 40. Kurihara Y, Kurihara H, Oda H et al. Aortic arch malformations and ventricular septal defect in mice de®cient in endothelin-1. Journal of Clinical Investigation 1995; 96: 293±300.

634 C. M. Redman et al 41. Clouthier DE, Hosoda K, Richardson JA et al. Cranial and cardiac neural crest defects in endothelin-A receptor-de®cient mice. Development 1998; 125: 813±824. 42. Yanagisawa H, Yanagisawa M, Kapur RP et al. Dual genetic pathways of endothelin-mediated intercellular signaling revealed by targeted disruption of endothelin converting enzyme-1 gene. Development 1998; 125: 825±836. 43. Hasegawa H, Hiki K, Sawamura T et al. Puri®cation of a novel endothelin-converting enzyme speci®c for big endothelin-3. Federation of European Biochemical Societies Letters 1998; 428: 304±308. 44. Zelinski T, Coghlan G, Myal Y et al. Genetic linkage between the Kell blood group system and prolactininducible protein loci: provisional assignment of KEL to chromosome 7. Annals of Human Genetics 1991; 55: 137±140. 45. Lee S, Zambas ED, Marsh WL & Redman CM. The human Kell blood group gene maps to chromosome 7q33 and its expression is restricted to erythroid cells. Blood 1993; 81: 2804±2809. *46. Lee S, Zambas E, Green ED & Redman C. Organization of the gene encoding the human Kell blood group protein. Blood 1995; 85: 1364±1370. 47. Lee S, Wu X, Reid M & Redman C. Molecular basis of the K:6,ÿ7[Js(a ‡ bÿ)]phenotype in the Kell blood group system. Transfusion 1995; 35: 822±825. 48. Lee S, Wu X, Son S et al. Point mutations characterize KEL10, the KEL3, KEL4, and KEL21 alleles, and the KEL17 and KEL11 alleles. Transfusion 1996; 36: 490±494. 49. Yazdanbakhsh K, Lee S, Yu Q & Reid ME. Identi®cation of a defect in the intracellular tracking of a Kell blood group variant. Blood 1999; 94: 310±318. 50. Lee S, Wu X, Reid M, Zelinski T & Redman C. Molecular basis of the Kell (K1) phenotype. Blood 1995; 85: 912±916. 51. Lee SH, Bennett PR, Overton T et al. Prenatal diagnosis of Kell blood cell group genotypes: KEL1 and KEL2. American Journal of Obstetrics and Gynecology 1996; 175: 455±459. 52. Avent ND & Martin PG. Kell typing by allele-speci®c PCR (ASP). British Journal of Haematology 1996; 93: 728±730. 53. Murphy MT, Fraser RH & Goddard JP. Development of a PCR-based diagnostic assay for the determination of KEL genotype in donor blood samples. Transfusion Medicine 1996; 6: 133±137. 54. Spence WC, Maddalena A, Demers DB & Bick DP. Prenatal determination of genotypes Kell and Cellano in at-risk pregnancies. Journal of Reproductive Medicine 1997; 42: 353±357. 55. Mifsud NA, Haddad AP, Sparrow RL & Condon JA. Use of polymerase chain reaction-sequence speci®c oligonucleotide technique for the detection of the K1/K2 polymorphism of the Kell blood group system. Blood 1997; 89: 4662±4663. 56. Petty AC, Daniels GL & Tippett P. Application of the MAIEA assay to the Kell blood group system. Vox Sanguinis 1994; 66: 216±224. 57. Vengelen-Tyler V, Gonzalez B, Garratty G et al. Acquired loss of red cell Kell antigens. British Journal of Haematology 1987; 65: 231±234. 58. Williamson LM, Poole J, Redman C et al. Transient loss of proteins carrying Kell and Lutheran red cell antigens during consecutive relapses of autoimmune thrombocytopenia. British Journal of Haematology 1994; 87: 805±812. 59. Caine ME & Mueller-Heubach E. Kell sensitization in pregnancy. American Journal of Obstetrics and Gynecology 1986; 154: 85±90. 60. Mayne KM, Bowell PJ & Pratt GA. The signi®cance of anti-Kell sensitization in pregnancy. Clinical and Laboratory Haematology 1990; 12: 379±385. 61. Leggat HM, Gibson JM, Barron SL & Reid MM. Anti-Kell in pregnancy. British Journal of Obstetrics and Gynaecology 1991; 98: 162±165. 62. Babinszki A, Lapinski RH & Berkowitz RL. Prognostic factors and management in pregnancies complicated with severe Kell alloimmunization: experiences of the last 13 years. American Journal of Perinatology 1998; 15: 695±701. 63. Berkowitz RL, Beyth Y & Sadovsky E. Death in utero due to Kell sensitization without excessive elevation of the delta OD450 value in amniotic ¯uid. Obstetrics and Gynecology 1982; 60: 746±749. 64. Bowman JM, Pollock JM, Manning FA et al. Maternal Kell blood group alloimmunization. Obstetrics and Gynecology 1992; 79: 239±244. 65. Jackson GM & Scott JR. Alloimmune conditions and pregnancy [review]. BaillieÁre's Clinical Obstetrics and Gynaecology 1992; 6: 541±563. 66. Parilla BV & Socol ML. Hydrops fetalis with Kell isoimmunization. Obstetrics and Gynecology 1996; 88: 730. 67. Vaughan JI, Warwick R, Welch CR & Letsky EA. Anti-Kell in pregnancy. British Journal of Obstetrics and Gynaecology 1991; 98: 944±945. 68. Weiner CP & Widness JA. Decreased fetal erythropoiesis and hemolysis in Kell hemolytic anemia. American Journal of Obstetrics and Gynecology 1996; 174: 547±551.

Kell, Kx and the McLeod syndrome 635 69. Vaughan JI, Warwick R, Letsky E et al. Erythropoietic suppression in fetal anemia because of Kell alloimmunization. American Journal of Obstetrics and Gynecology 1994; 171: 247±252. 70. Vaughan JI, Manning M, Warwick RM et al. Inhibition of erythroid progenitor cells by anti-Kell antibodies in fetal alloimmune anemia. New England Journal of Medicine 1998; 338: 798±803. 71. Southcott MJG, Tanner MJA & Anstee DJ. The expression of human blood group antigens during erythropoiesis in a cell culture system. Blood 1999; 93: 4425±4435. 72. Bertelson CJ, Pogo AO, Chaudhuri A et al. Localization of the McLeod locus (XK) within Xp21 by deletion analysis. American Journal of Human Genetics 1988; 42: 703±711. *73. Ho M, Chelly J, Carter N et al. Isolation of the gene for McLeod syndrome that encodes a novel membrane transport protein. Cell 1994; 77: 869±880. 74. Ho MF, Chalmer RM, Davis MB et al. A novel point mutation in the McLeod syndrome gene in neuroacanthocytosis. Annals of Neurology 1996; 39: 672±675. 75. Hanaoka N, Yoshida K, Nakamura A et al. A novel frameshift mutation in the McLeod syndrome gene in a Japanese family. Journal of the Neurological Sciences 1999; 165: 6±9. 76. Francke U, Ochs HD, de Martinville B et al. Minor Xp21 chromosome deletion in a male associated with expression of Duchenne muscular dystrophy, chronic granulomatous disease, retinitis pigmentosa, and McLeod syndrome. American Journal of Human Genetics 1985; 37: 250±267. 77. Frey D, Machler M, Seger R et al. Gene deletion in a patient with chronic granulomatous disease and McLeod syndrome: ®ne mapping of the Xk gene locus. Blood 1988; 71: 252±255. *78. Redman CM, Marsh WL, Scarborough A et al. Biochemical studies on McLeod phenotype red cells and isolation of Kx antigen. British Journal of Haematology 1988; 68: 131±136. 79. Carbonnet F, Hattab C, Callebaut I et al. Kx, a quantitatively minor protein from human erythrocytes, is palmitoylated in vivo. Biochemical and Biophysical Research Communications 1998; 250: 569±574. *80. Redman CM, Avellino G, Pfe€er SR et al. Kell blood group antigens are part of a 93 000-dalton red cell membrane protein. Journal of Biological Chemistry 1986; 261: 9521±9525. 81. Khamlichi S, Bailly P, Blanchard D et al. Puri®cation and partial characterization of the erythrocyte Kx protein de®cient in McLeod patients. European Journal of Biochemistry 1995; 228: 931±934. 82. Carbonnet F, Hattab C, Collec E et al. Immunochemical analysis of the Kx protein from human red cells of di€erent Kll phenotypes using antibodies raised against synthetic peptides. British Journal of Haematology 1997; 96: 857±863. 83. Russo D, Redman C & Lee S. Association of XK and Kell blood group proteins. Journal of Biological Chemistry 1998; 273: 13 950±13 956. 84. Russo DC, Lee S, Reid M & Redman CM. Topology of Kell blood group protein and the expression of multiple antigens by transfected cells. Blood 1994; 84: 3518±3523. 85. Russo D, Lee S & Redman C. Intracellular assembly of Kell and XK blood group proteins. Biochimica et Biophysica Acta 1999; 1461: 10±18. 86. Daniels GL, Weinauer F, Stone C et al. A combination of the e€ects of rare genotypes at the XK and KEL blood group loci results in absence of Kell system antigens from the red blood cells. Blood 1996; 88: 4045±4050. 87. Allen FH Jr, Krabbe SMR & Corcoran PA. A new phenotype (McLeod) in the Kell blood group system. Vox Sanguinis 1961; 6: 555±560. 88. Wimer BM, Marsh WL, Taswell HF & Galey WR. Haematological changes associated with the McLeod phenotype of the Kell blood group system. British Journal of Haematology 1977; 36: 219±224. 89. Symmans WA, Shepherd CS, Marsh WL et al. Hereditary acanthocytosis associated with the McLeod phenotype of the Kell blood group system. British Journal of Haematology 1979; 42: 575±583. 90. Marsh WL, Marsh NJ, Moore A et al. Elevated serum creatine phosphokinase in subjects with McLeod syndrome. Vox Sanguinis 1981; 40: 403±411. 91. Swash M, Schwartz MS, Carter ND et al. Benign X-linked myopathy with acanthocytes (McLeod syndrome). Its relationship to X-linked muscular dystrophy. Brain 1983; 106: 717±733. 92. Danek A, Uttner I, Vogl T et al. Cerebral involvement in McLeod syndrome. Neurology 1994; 44: 117±120. 93. Witt TN, Danek A, Reiter M et al. McLeod syndrome: a distinct form of neuroacanthocytosis. Report of two cases and literature review with emphasis on neuromuscular manifestations [review]. Journal of Neurology 1992; 239: 302±306. 94. Jeren-Strujic B, Jeren T, Thaller N et al. A case of McLeod syndrome with chronic renal failure. Blood Puri®cation 1998; 16: 336±340. 95. Hardie RJ. Acanthocytosis and neurological impairment ± a review. Quarterly Journal of Medicine 1989; 71: 291±306.