Erythroid band 3 variants and disease

BaillieÁre's Clinical Haematology Vol. 12, No. 4, pp. 637±654, 1999

3 Erythroid band 3 variants and disease Lesley J. Bruce

PhD

Research Associate

Michael J. A. Tanner

PhD

Professor Department of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UK

This review describes some of the naturally occurring band 3 (AE1) variants and their association with disease. Southeast Asian Ovalocytic (SAO) band 3, an inactive and misfolded protein, is probably only maintained in certain populations because it provides protection against the cerebral form of malaria. Many mutations that cause instability of band 3, either at the mRNA or protein level, result in hereditary spherocytosis (HS). Some polymorphisms alter amino acid residues in the extracellular loops of band 3 and are associated with blood group antigens. A truncated form of AE1 is expressed in kidney cells and certain AE1 mutations are associated with distal renal tubular acidosis (dRTA). The molecular basis of these variants and their e€ect on the structure and function of band 3 are discussed. The association between band 3 and glycophorin A (GPA) and the structure/function changes of band 3 in the absence of GPA are also described. Key words: polymorphism; anion exchanger; glycophorin A; red cell membrane; Southeast Asian ovalocytosis; hereditary spherocytosis; blood group antigen; renal tubular acidosis.

INTRODUCTION Band 3, the red cell anion exchanger (AE1), is a member of a widely distributed family of proteins present in many tissues. These play a part in many cellular processes that involve the exchange transport of anions across membranes (for reviews, see1,2). The anion exchanger (AE) gene family members show strong sequence similarity in the membrane-associated domain and di€er most in their N-terminal cytoplasmic domains. Recently, the AE family has been shown to belong to a superfamily of proteins whose membrane domains probably all share the same protein fold (for a review, see3). Genes that are members of this superfamily are also found in the recently sequenced genomes of the yeast, Saccharomyces cerevisiae, and the nematode, Caenorhabditis elegans. Although the functional activity of these proteins and some of their mammalian homologues has not been established, their common feature may be to act as bicarbonate transporters. While the AE family of proteins carries out the All correspondence to: Dr LJ Bruce. Tel: (‡) 44 (0) 117-9288271; Fax: (‡) 44 (0) 117-9288274; email: [email protected] 1521±6926/99/040637+18 $12.00/0

c 1999 Harcourt Publishers Ltd *

638 L. J. Bruce and M. J. A. Tanner

obligatory electroneutral exchange transport of bicarbonate and other anions, other members of the superfamily carry out the electrogenic co-transport of Na ‡ and HCO3ÿ , which is important in processes such as HCO3ÿ reclamation from the kidney glomerular ®ltrate (see3). Band 3 (human gene designation SLC4A1) is only known to be expressed in red cells and the intercalated cells of the distal and collecting tubules of the kidney, and has also recently been suggested to be expressed in mouse heart.4 It functions in the red cell to enhance the ability of blood to carry carbon dioxide from the tissues to the lungs. The bicarbonate formed from carbon dioxide within the red cell by carbonic anhydrase enters the plasma in exchange for chloride, so that the cell and plasma phases can be used to carry bicarbonate. Bicarbonate is much more water-soluble than carbon dioxide, and without this system blood would have a very limited capacity to move carbon dioxide from the tissues to the lungs. Band 3 is also important in maintaining the stability and integrity of the red cell by anchoring the sub-membrane protein skeleton to the lipid bilayer and so forms part of the structure that gives the red cell its special mechanical properties. The domain structure of band 3 re¯ects its bifunctional nature. The amino-terminal 40 kDa of the protein is located in the cytoplasm and binds the red cell skeleton and other cytoplasmic proteins, including glycolytic enzymes, while the glycosylated 55 kDa C-terminal membrane domain carries out the obligatory exchange of anions. The two domains can be readily separated by proteolysis or expressed from recombinant constructs and appear to function independently. In addition the short Cterminal cytoplasmic tail of band 3 binds carbonic anhydrase II, localizing this protein to the membrane and directly coupling bicarbonate supply to its transport out of the cell.5 An isoform of AE1, which lacks the amino-terminal 65 residues of erythroid band 3 is expressed in the distal nephron of human kidney, where it has a role in acid secretion into the tubular lumen. Band 3 is also thought to have a role in the turnover of red cells by acting as a senescence antigen. There have been several recent reviews of band 3.6±8 In addition, three collections of review papers on di€erent aspects of band 3 have recently been published.9±11 BAND 3 STRUCTURE The human band 3 membrane domain extends from around Tyr359Lys360 to the C-terminus of the protein, since the former are the sites of mild proteolysis from the cytoplasmic surface which separates the protein into two fragments. This domain is active in anion transport either when produced by proteolysis in red cell membranes12,13 or when expressed from a recombinant.14,15 Hydropathy analysis of the amino acid residue sequence combined with experimental evidence derived from covalent labelling studies using impermeant reagents, proteolysis and antibody epitope mapping have suggested models of the protein which contain 14 membrane spans.16±18 These models di€er in detail, particularly around the predicted transmembrane segments 9 and 10 in the C-terminal portion of the protein, mainly because of the ambiguity of the hydropathy pro®le and the lack of experimental data on the structure in this region. More recent topological analyses using N-glycosylation cassette mutagenesis19,20 and scanning cysteine mutagenesis, coupled with chemical modi®cation using impermeant reagents21, have suggested models containing 12 (Figure 119,20) or 13 membrane spans respectively. These models re¯ect di€ering conclusions about the topology in the region of spans 9±12 of the protein. The lack of

Figure 1. A folding model for the membrane domain of band 3 showing the glycosylation site (Asn642), the external chymotrypsin cleavage sites (C) and the anion transport inhibitor (DIDS and H2DIDS) binding sites. The position of the SAO deletion and the amino acid substitutions associated with the Wright and Diego antigens, band 3 HT and dRTA are shown. Adapted with permission from Popov et al (1997).19

Band 3 variants 639

640 L. J. Bruce and M. J. A. Tanner

consensus about the structure in this part of the protein may re¯ect the presence of topologically mobile sequences in this region. It may also result from the limitations of the methods available for studying the topology of multispanning membrane proteins like band 3. Cysteine scanning mutagenesis has also been used to study the environment around span 8, which is suggested to form part of an aqueous pore.22,23 Although a three-dimensional map of the structure of the band 3 membrane domain has been determined using electron microscopy of two-dimensional crystals24,25, the low resolution of the map (20 AÊ) has limited its utility in understanding the molecular details of the structure of the band 3 membrane domain. Indirect approaches have been used to gain an insight into the structure of band 3. Nuclear magnetic resonance (NMR) derived solution structures have been obtained for the ®rst and second membrane spans of the protein26,27 and for a large cytoplasmic loop.28 Complementation studies suggest that the band 3 membrane domain divided into two fragments, at most (but not all) surface loops, will reassemble into a transportactive structure.29±32 A chloride-transporting structure could be induced in Xenopus oocytes when the protein was reassembled from up to four di€erent fragments. Unexpectedly, a stilbene disulphonate-sensitive chloride-transporting structure was obtained even when spans 6 and 7 were absent.32 This suggests that these spans do not form part of the chloride transport mechanism but may have other roles, for example in targeting of the protein to the appropriate membrane in the polarized intercalated cells of the kidney distal tubule (see below). Other recent studies suggest that some individual membrane spans of band 3 are unable to insert into the membrane with the correct orientation. These spans require interactions with other membrane spans in the protein in order to attain their correct orientation in the membrane indicating the presence of subclusters of membrane spans in the protein.33±35 This conclusion has been strengthened by proteolysis studies on band 3 in the red cell membrane.36 A study of the association of fragments of the membrane domain has suggested a model for the organization of the membrane spans in which a symmetrical dimer of subdomains containing spans 1±5 and 9±12 (a 14 span model) forms the core of the protein.37 This chapter will describe some of the known band 3 variants and their association with disease. The structural and functional consequences of these mutations can also give valuable information on the structure of the band 3 protein.

BAND 3 MOLECULAR GENETICS AND NATURAL BAND 3 MUTATIONS The AE1 gene is located on chromosome 17, contains 20 exons and spans about 20 kb.38,39 Exons 2±19 code for the erythroid AE1 (eAE1) protein sequence. The 50 non-coding promoter region of the gene is in exon 1 and contains a GATA box and other transcription factor binding sites.38,39 Intron 3 contains an additional promoter region for the truncated kidney AE1 (kAE1), which consists of part of intron 3 together with exons 4±20.40 Band 3 (eAE1) protein is intrinsic to the stability of the red cell membrane, not only through its attachment to the cytoskeleton (via ankyrin and protein 4.2) but also because band 3 makes up 20% of the red cell membrane protein and is therefore fundamental to the organization of the membrane layer. Band 3 variants that a€ect the morphology and stability of the red cell membrane include Southeast Asian

Band 3 variants 641

Ovalocytosis (band 3 SAO) and the numerous band 3 mutations that result in hereditary spherocytosis (HS). SOUTHEAST ASIAN OVALOCYTOSIS Southeast Asian Ovalocytosis (SAO) occurs mainly in Melanesian populations in areas where malaria is endemic.41 However SAO has also been found in a Mauritian family of Indian descent42, an African±American family43, two South African kindreds44 and a white family.45 SAO red cells are oval and have a pronounced stoma. SAO band 3 results from the deletion of amino acid residues 400±408 from band 3 and is generally found linked to the common polymorphism band 3 Memphis (Lys56 ! Glu).42,46±48 Despite this deletion of nine amino acid residues, SAO band 3 is not degraded in the internal membranes of the cell, but inserts into the erythrocyte membrane. SAO red cells are much more rigid than normal red cells49,50 and it has been suggested that the deletion removes a ¯exible region of the protein causing a non-speci®c interaction of the cytoplasmic domain of SAO band 3 with the cytoskeleton.48,51±53 For many years it was thought that SAO red cells were protected against the invasion of the malarial parasites Plasmodium falciparum and Plasmodium vivax. Experiments in vitro showed SAO red cells to be resistant to invasion by these parasites and it was hypothesized that this resistance was due to the increased rigidity of these cells.49,50 However an in vivo study, conducted in malarial regions of Papua New Guinea, showed that the susceptibility of SAO individuals to parasitaemia is only slightly reduced.54 Furthermore, a more recent in vitro study has shown that SAO red cells can be substantially invaded by malarial parasites (up to 50%) and that loss of invasion correlates with the depletion of intracellular ATP.55 SAO red cells are more leaky to sodium and potassium ions than control red cells in vitro and when stored in the cold this leak becomes pronounced.56 The Na‡ ,K‡ -ATPase is more active in SAO red cells, probably in order to compensate for the above leak, and cold-stored SAO red cells are rapidly depleted of intracellular ATP, ADP and AMP.56 The sodium and potassium ion concentrations of SAO red cells and plasma are almost normal when fresh blood samples are assayed56 suggesting that the previously reported apparent resistance to malarial invasion detected in these cells was an artefact of storage. However, a recent study has shown that SAO band 3 is associated with protection against cerebral malaria in children in the Madang area of Papua New Guinea.57 A further study in Papua New Guinea showed that none of 68 children with cerebral malaria had SAO whereas SAO was present in 8.8% of 68 matched community control children.58 As cerebral malaria is normally fatal this would explain why the SAO band 3 gene has been maintained in regions where malaria is endemic. The structure of SAO band 3 is altered, most noticeably in the membrane domain. NMR studies of the ®rst transmembrane domain of SAO band 3 suggest that this helix is elongated and di€erent residues may lie within the lipid bilayer.27 Circular dichroism studies have shown that the transmembrane region retains its overall helical structure but the mutant protein has altered thermal denaturation suggesting it has modi®ed tertiary structure.52 SAO band 3 does not transport anions or bind the anion transport inhibitors 4,40 -diisothiocyanatostilbene-2,20 -disulphonate (DIDS), eosin maleimide (EM), 5-([4,6-dichlorotriazinyl]amino)¯uorescein (5-DTAF) or 4-benzamido-40 -aminostilbene-2,20 -disulphonate (BADS).42,52,59 The structure of the loop carrying the N-glycan chain may also be changed because SAO band 3 does not have a polylactosaminyl oligosaccharide.59 Alternatively, since SAO band 3 has a tendency to form

642 L. J. Bruce and M. J. A. Tanner

higher oligomers, the protein may move more quickly through the endoplasmic reticulum (ER) and Golgi apparatus, not allowing time for the addition of these structures. Expression of SAO band 3 in Xenopus oocytes does not require normal band 3 or glycophorin A (GPA) and SAO band 3 is translocated to the oocyte plasma membrane more readily than normal band 3, supporting the view that movement through the ER and Golgi apparatus may also be enhanced in the red cell.60 Freezefracture electron microscopy has shown SAO red cells to contain linear arrangements of intramembranous particles, probably formed by band 3 aggregates, which do not occur in normal red cells.61 This altered membrane organization may be responsible for the selective depression of certain blood group antigens on SAO red cells.62,63 SAO band 3 forms heterodimers64,65 and thereby seems to a€ect the normal population of band 3 in these cells. Anion transport in SAO red cells has been reported to be about 40% that in normal cells.42 Eosin maleimide does not bind SAO band 3; however the lateral di€usion of band 3, detected by this probe, is reduced in SAO red cells. This must re¯ect a reduced mobility of the normal band 3 population in SAO cells.52 In order for SAO band 3 to form heterodimers with normal band 3, the dimer interface of SAO band 3 must be substantially unchanged. Not surprisingly, all the known individuals with SAO are heterozygous for band 3 SAO. Individuals homozygous for band 3 SAO have never been detected and this condition is probably lethal.66 HEREDITARY SPHEROCYTOSIS Hereditary spherocytosis (HS) is characterized by spherocytic red cells with increased osmotic and mechanical fragility and results in haemolytic anaemia. The spherocytic shape of these cells results from loss of membrane surface area relative to intracellular volume. HS has been associated with abnormalities in the red cell membrane proteins spectrin, ankyrin, band 3 and protein 4.2.6,67,68 The band 3 mutations associated with HS have recently been reviewed7,69±71 and some of these mutations are listed in Table 1. Certain band 3 mutations associated with HS a€ect the protein 4.2 or ankyrin binding sites on band 3 and may disrupt the cytoskeleton. Band 3 attaches the red cell membrane to the cytoskeleton via ankyrin, and this interaction is stabilized by protein 4.2. Band 3 Monte®ore (Glu40 ! Lys) produces an 88% reduction in protein 4.2.72 The protein 4.2 binding site for band 3 contains two adjacent arginine residues and it is suggested that these interact with the acidic N-terminal domain of band 3.93 Band 3 Fukuoka (Gly130 ! Arg), when present as a homozygous mutation, results in a slight reduction of band 3 (9%) and a substantial reduction in protein 4.2 (45%).74 Band 3 Tuscaloosa (Pro327 ! Arg) is also de®cient in protein 4.2 (29% reduction).76 Since Pro327 is in a highly conserved region of band 3, its substitution with Arg may well disrupt the secondary and tertiary structure of the cytoplasmic domain of band 3, which may lead to the altered binding of protein 4.2.76 In band 3 Nachod ®ve highly conserved amino acid residues are deleted from the putative ankyrin binding site (amino acid residues 117±121) disrupting ankyrin binding.77 Other HS associated band 3 mutations a€ect the amount of band 3 in the red cell membrane. These mutants fall into two groups: where the band 3 mutations result in an unstable mRNA and where the mRNA is present but the mutation protein is either degraded or not inserted into the red cell membrane (Table 1). In both cases the result is a reduction in the amount of band 3 in the red cell membrane, and when band 3 is reduced the red cells also have a proportional reduction in protein 4.2. In all

Band 3 variants 643 Table 1. Band 3 mutations associated with hereditary spherocytosis. Band 3 variant

Mutation

A. A€ects protein 4.2 binding Monte®ore Glu40 ! Lys Fukouka Gly130 ! Arg Tuscaloosa Pro327 ! Arg (Lys56 ! Glu)

Reference no. (72,73) (74,75) (76)

B. A€ects ankyrin binding Nachod (Hradec Kralove II) Aberrant splicing: deletion of codons 117±121

(77)

C. mRNA instability Genas Neapolis

(78) (79)

Kagoshima Fukayama I Foggia Fukayama II Bohain Hodonin (Prague IV) Napoli I Osnabruck I (Lyon) Worcester Campinas Princeton Noirterre Bruggen Bicetre II Pribam (Prague VI) Evry Smichov (Prague VII) Trutnov Hobart Osnabruck II

G89 ! A (exon 2)a Aberrant splicing: insertion of intron 2: stop codon (‡19) after exon 2 or deletion of exon 2: truncated protein using alternative ATG Deletion A in codon 19 Deletion AC in codon 38 Deletion ACCCAC ! ACCAC (codon 54 or 55) Insertion A in codon 61 Deletion T in codon 81 Trp81 ! Stop Insertion TCT ! TTCT (codon 100) Arg150 ! Stop Insertion G into codons 170±172 Aberrant splicing: stop codon (‡13) after exon 7 Insertion C into codons 273±275 Gln330 ! Stop Deletion C in codon 419 Deletion G in codons 454±456 Aberrant splicing: stop codon (‡7) after exon 12 Deletion T in codon 496 Deletion C in codon 616 Tyr628 ! Stop Deletion G in codons 646±647 Deletion of codon 663 or 664

D. Band 3 protein instability Boston Ala285 ! Asp Benesov (Prague V) Gly455 ! Glu Coimbra Val488 ! Met Bicetre I Arg490 ! Cys Milano Insertion of 23 amino acid residues at codon 498 Dresden Arg518 ! Cys Most (Prague VIII) Leu707 ! Pro Okinawa Gly714 ! Arg Kumamoto (Prague II) Arg760 ! Gln Hradec Kralove Arg760 ! Trp Chur Gly771 ! Asp Napoli II Ile783 ! Asn Jablonec Arg808 ! Cys Nara Arg808 ! His Prague Duplication (nucleotides 2455±2464) Birmingham His834 ! Pro Philadelphia Thr837 ! Met Tokyo Thr837 ! Ala Prague III Arg870 ! Trp Vesuvio Deletion ACC ! AC in codon 894

Amino acid and nucleotide numbering as described by Tanner et al 1988.92 Nucleotide numbering from the ®rst residue of exon 1.

a

(80) (80) (81) (80) (82) (77) (81) (73,78) (77) (83) (77) (84) (73) (82) (77) (82) (77) (77) (77) (73) (77) (77) (85) (82) (86) (73) (77) (75) (80,87) (87) (88) (81) (87) (80) (89) (77) (77,80,82) (90) (87) (91)

644 L. J. Bruce and M. J. A. Tanner

these cases the reduction in band 3 probably destabilizes the lipid bilayer resulting in blebbing, the loss of microvesicles and HS.6 Of interest is a case where band 3 de®cient HS was found in conjunction with b-thalassemia (b8 codon 39 (C to T mutation)) and the HS was alleviated.94 b-thalassemic red cells have normal surface area with a reduced cell volume and the cytoskeleton is thought to be disrupted. Miraglia del Giudice et al proposed that the two conditions complement each other and therefore relieve the symptoms of HS.94 Complete absence of band 3 has been described in a baby homozygous for the Coimbra mutation (Val488 ! Met).95 The baby had severe haemolytic anaemia that required repeated blood transfusions and the kidney function was severely impaired (see below). Animal studies show that the targeted disruption of the band 3 gene in mice results in the complete absence of red cell band 3, protein 4.2 and GPA and causes severe haemolytic anaemia despite the assembly of a nearly normal membrane skeleton.96,97 The total absence of band 3 has also recently been reported in variant cattle and results in de®cient spectrin, ankyrin, actin and protein 4.2.98 Both band 3 SAO and band 3 mutants associated with HS demonstrate the amount of redundancy found in physiological systems. In SAO red cells only about 40% of the band 3 is functional and in HS red cells the amount of band 3 present can be reduced by 20±40%, and yet transport of CO2 by the red cells and excretion of acid by the kidney cells does not seem to be unduly a€ected. At the other end of the spectrum is a band 3 mutant (band 3 HT) that increases the activity of band 3 without apparently a€ecting red cell or kidney cell function. BAND 3 HIGH TRANSPORT The molecular basis for band 3 high transport (band 3 HT) is a homozygous mutation Pro868 ! Leu.99 Pro868 is located within transmembrane span 12 of band 3 (Figure 1) and is conserved throughout the AE gene family. Sulphate transport is increased in band 3 HT red cells, characterized by a marked increase in Vmax , which suggests that this region of the protein is involved in the anion transport translocation step.99 The anion transport inhibitor, 4,40 -diisothiocyanato-2,20 -dihydrostilbene disulphonate (H2DIDS), is less readily bound to band 3 HT, indicating that the mutation a€ects the conformation of the ®nal extracellular loop of band 3 and therefore the H2DIDS binding pocket.99,100 The H2DIDS binding pocket is thought to involve Lys851 of band 3. Band 3 HT has an elongated N-glycan chain and a reduced number of high anity ankyrin binding sites.99,101 As ankyrin may be involved in the movement of band 3 out of the endoplasmic reticulum102 this weakened association with ankyrin may cause band 3 HT to be retained for longer in the endoplasmic reticulum and Golgi apparatus, allowing the extension of the N-glycan chain. Band 3 HT red cells are also acanthocytic, although the cause of this morphological change is not known. Despite all the structural and functional changes this mutation causes, band 3 HT is not known to produce a disease phenotype or indeed to confer any bene®t to the a€ected individual. BAND 3 MUTATIONS ASSOCIATED WITH ALTERED BLOOD GROUP ANTIGENS Band 3 Memphis variant I is a common polymorphism (6±7% of random bloods).103 This polymorphism (Lys56 ! Glu)104,105 is often found as a background mutation

Band 3 variants 645

(note: linked to SAO, above) and is therefore an `old' polymorphism in terms of evolution. It is asymptomatic except that the red cells have a slightly reduced phosphoenolpyruvate transport.106 Of interest is the observation that the Memphis polymorphism gives anomalous mobility of the N-terminal chymotryptic fragment of band 3 on an SDS-PAGE gel. The Memphis N-terminal chymotryptic fragment migrates with an apparent Mr of 63 kDa instead of the 60 kDa found with normal band 3.107 This property allowed detection of a subtype of band 3 Memphis in which the Memphis 63 kDa N-terminal chymotryptic band was more readily labelled by H2DIDS than either normal band 3 or Memphis variant I band 3.108 This subtype, named band 3 Memphis variant II, was found to be associated with the expression of the Dia antigen.109 The Diego blood group consists of two allelic antigens (Dia and Dib). The Dia antigen is common in the indigenous peoples of America and south-east Asia, but rare in other populations.110 The presence of the Dia antigen is of clinical signi®cance, since anti-Dia is known to cause haemolytic disease of the newborn.111,112 The Dib and Dia antigens are associated, respectively, with the presence of Pro854 and Leu854 of band 3.113 Leu854 is found in cis with the Memphis I mutation Glu56.113 Amino acid residue 854 of band 3 is in the ®nal extracellular loop of band 3 (see Figure 1), close to Lys851, which is thought to form part of the H2DIDS binding pocket. The mutation Pro854 ! Leu probably alters the conformation of this loop and therefore the structure of the H2DIDS binding pocket.113,114 However, since the anion transport activity of band 3 Memphis variant II is unchanged, modi®cation of the membrane domain of band 3 is not likely to be extensive. There is evidence that glycophorin A (GPA) and band 3 interact during their biosynthesis and processing. In rare red cells that lack GPA (En(aÿ) and MkMk red cells) the average length of the N-glycan chain attached to band 3 is increased115±117 and the anion transport activity of band 3 is decreased, suggesting that band 3 is dependent on GPA for ecient translocation to the red cell membrane and for some ®nal processing in order to adopt an active con®rmation.117 Studies in Xenopus oocytes show that GPA facilitates the translocation of band 3 to the oocyte surface14 and studies in band 3 knockout mice show that GPA is not expressed in the red cell membrane in the absence of band 3.97 Studies using anti-GPA antibodies have shown that band 3 and GPA interact in the red cell membrane. In the presence of anti-GPA antibodies the rotational di€usion of band 3 decreases118,119, the rigidity of the red cell membrane is altered120 and band 3 and GPA are co-immunoprecipitated by anti-Wrb antibodies.121,122 The Wright blood group consists of two allelic antigens, the high incidence antigen Wrb and the low incidence antigen Wra.123 Wr(a‡bÿ) individuals are extremely rare: currently only two individuals are known to have this phenotype.124,125 Studies on naturally occurring hybrid glycophorins showed the Wrb antigen to be associated with residues 59±70 of GPA126 but the Wrb and Wra antigens are associated with the presence of Glu658 and Lys658 of band 3 respectively.127 The Glu658 ! Lys mutation does not a€ect the anion transport activity of band 3.127 Although the Wright antigens depend on this polymorphism in band 3, the importance of GPA to the structure of the Wrb antigen was highlighted in two further reports of red cells with abnormal expression of Wrb.128,129 These red cells contained normal band 3 but had mutations in the GPA gene, Ala65 ! Pro128, Gln63 ! Lys.129 The Ala65 ! Pro mutation was also associated with a new low incidence antigen (HAG) and an antibody to a high incidence antigen (ENEP).128 The Gln63 and Lys63 of GPA were associated with the high incidence antigen (ENAV) and the low incidence antigen (MARS), respectively.129

646 L. J. Bruce and M. J. A. Tanner Table 2. Amino acid residues associated with antigens of the Diego blood group system. Historical name Redelberger Taversu Warrior Van Vugt Waldner Bowyer Newfoundland Wulfsberg Nunhart Bishop Swann Hughes Moen Fritz Wright Luebano Diego

Antigen name

Associated amino acida

Normal amino acidb

ELO Rba Tra WARR Vga Wda BOW NFLD Wu Jna KREP Bpa Swa Hga Moa Wrb Wra Dib Dia

Trp432 Leu548 Asn551 Ile552 His555 Met557 Ser561 Ala561/Asp429 Ala565 Ser566 Ala566 Lys569 Gln646 Cys656 His656 Glu658e Lys658 Pro854 Leu854

Arg432 Pro548 Lys551 Thr552 Tyr555 Val557 Pro561 Pro561/Glu429 Gly565 Pro566 Pro566 Asn569 Arg646 Arg656 Arg656 Glu658 Glu658 Pro854 Pro854

Loopc

Reference no.

1 3 3 3 3 3 3 3/1 3 3 3 3d 4 4 4 4 4 5 5

(130,131) (132) (132) (133) (131) (133,134) (131,135) (135) (131,136) (137) (137) (131) (138) (131) (131) (127) (127) (113) (113)

a

Amino acid residue associated with the expression of the antigen. Amino acid residue found in the common form of band 3.92 loop number refers to the extracellular loops of band 3 shown in Figure 1, loop 5 being the ®nal loop. dNote that the model of band 3 in Figure 1 has amino acid residue 569 as part of transmembrane span 6; the substitution of Lys569 for Asn569 may disrupt this part of the helix. e The Wrb antigen also requires the presence of glycophorin A. b

cThe

Amino acid residue 685 of band 3 is at the top of the eighth putative membrane span, in the fourth extracellular loop of the protein (Figure 1). Amino acid residues 59±70 of GPA are thought to form an a-helical structure close to the outer membrane of the red cell. In order for the Wrb antigen to be formed, GPA probably packs close to membrane span 8 of band 3. Other blood group antigens have been assigned to band 3 (Table 2). These have been found mainly in extracellular loops 3 and 4 (Figure 1). None of these polymorphisms have been found to a€ect the anion transport activity of band 3. They do, however, help to con®rm which portions of the protein are extracellular. BAND 3 MUTATIONS ASSOCIATED WITH DISTAL RENAL TUBULAR ACIDOSIS The main symptom of patients with familial distal renal tubular acidosis (dRTA) is an inability to acidify their urine. Other symptoms include metabolic acidosis, hypokalemia, nephrocalcinosis, kidney stones and metabolic bone disease (for review, see139,140). Inheritance of dRTA can be both autosomal dominant and autosomal recessive. The recessive form is sometimes linked to sensorineural deafness. The severity of the disease can vary widely and there is a symptomless form known as `incomplete dRTA', which is diagnosed by the inability to acidify the urine after an acid loading challenge, either frusemide and ¯udrocortisone or ammonium chloride. Acid

Band 3 variants 647

Figure 2. Diagram of an a-intercalated cell of the distal tubule and collecting duct. These cells secrete acid into the lumen of the kidney tubule. Hydration of carbon dioxide by carbonic anhydrase produces hydrogen ions. These are pumped out of the cell by the H‡ -ATPase. The remaining bicarbonate ions are exchanged for chloride ions via band 3 in the basolateral membrane. Reprinted by permission from Nature 382: 209±210, copyright 1996 Macmillan Magazines Ltd.142.

secretion takes place in the a-intercalated cell of the distal tubule and collecting ducts where carbonic anhydrase produces carbonic acid, which dissociates to hydrogen and bicarbonate ions (for a review, see141). The hydrogen ions are pumped out of the cell via the H‡ -ATPase in the apical membrane, while the bicarbonate ions are exchanged for chloride ions via the kidney AE1 (kAE1) in the basolateral membrane (Figure 2). In patients with dRTA one or more of these proteins is malfunctioning. A number of di€erent band 3 mutations have been found to be associated with the autosomal dominant form of dRTA. The mutations Arg589 ! His, Arg589 ! Cys or Arg589 ! Ser have all been associated with dRTA and suggest that this amino acid residue is a `hot spot' for mutation.140,143±145 Red cell studies show the Arg589 ! His and Arg589 ! Cys mutants reduce the sulphate transport activity of band 3 to 80% and increase the length of the N-glycan chain attached to the mutant band 3.143 Another band 3 mutation associated with autosomal dominant dRTA, Ser613 ! Phe, increases the sulphate transport activity of the mutant band 3 about twofold.143 Each of these mutations produce a similar disease phenotype, so the disease cannot be due to changes in the anion transport properties of band 3. However amino acid residues 589 and 613 are clustered in the region of membrane spans 6 and 7 of the band 3 protein and it may be that this region is involved in targeting the kAE1 to the correct membrane of the intercalated cell. A dominant disease phenotype would be seen if kAE1 were mistargeted to the apical membrane of the intercalated cell where it would excrete bicarbonate, which would neutralize acid secretion.143 Another autosomal dominant mutation associated with dRTA involves a 13 bp duplication in the region of Arg901.144 This duplication results in the replacement of Arg901 with a stop codon.144 We are currently assessing the properties of this truncated band 3 but it is interesting to note that a recent report shows that carbonic anhydrase II binds to the C-terminal domain of band 3.5 In this dRTA band 3 mutant it is possible that the C-terminal deletion a€ects the activity of the carbonic anhydrase and this exacerbates dRTA.

648 L. J. Bruce and M. J. A. Tanner

Preliminary studies suggested that band 3 was not involved in the autosomal recessive form of dRTA144 and indeed autosomal recessive dRTA with sensorineural deafness has been shown to result from mutations in the H‡ -ATPase.146 However, a recent report described a family from Thailand with a band 3 mutation (Gly701 ! Asp) and homozygous inheritance of this mutation was associated with dRTA.147 Expression studies in Xenopus oocytes showed that the Gly701 ! Asp band 3 mutant protein is not translocated to the oocyte membrane unless GPA is present.147 Intercalated cells of the kidney are not known to contain GPA so it is likely that the Gly701 ! Asp band 3 mutant causes dRTA because translocation of the mutant protein is impaired. Without a means of excreting bicarbonate ions, hydrogen ion excretion from the a-intercalated cell would be severely a€ected.

CONCLUDING COMMENTS Study of naturally occurring band 3 mutations helps in the understanding of the diseases associated with them. These mutations can also give us valuable information on the structure and function of band 3. Acknowledgements Work carried out in this laboratory was supported by the Wellcome Trust.

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