No abstract

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The Wright (Wr) blood group antigens, Wra and Wrb, have been suggested to be determined by alleles of the same gene. The Wrb antigen appears to involve both red blood cell (RBC) band 3 and glycophorin A (GPA). We have examined the cDNA sequences of the band 3 and GPA of one of the two known Wr(a+b-) individuals. We show that this individual is homozygous for the mutation Glu658--<Lys in band 3, but has normal GPA. Putative heterozygotes with Wr(a+b+) RBCs have both Glu and Lys at residue 658 of band 3, whereas the common Wr(a-b+) RBC phenotype only have band 3 with Glu658. The Wra and Wrb antigens are determined by the amino acid at residue 658 of band 3 and are antithetical. Examination of the amino acid sequence and Wrb antigen expression of GPA-related hybrid glycophorins suggests that Arg61 of GPA interacts with Glu658 of band 3 to form the Wrb antigen. We suggest that the interaction is stabilized by the presence of anti-Wrb antibodies and that this site of association between GPA and band 3 may be responsible for the previously reported ability of anti-GPA antibodies to decrease the deformability of RBCs.

We describe three mutations of the red-cell anion exchangerband 3 (AE1, SLC4A1) gene associated with distalrenal tubular acidosis (dRTA) in families from Malaysia and Papua NewGuinea: Gly(701)-->Asp (G701D), Ala(858)-->Asp(A858D) and deletion of Val(850) (DeltaV850). The mutationsA858D and DeltaV850 are novel; all three mutations seem to berestricted to South-East Asian populations. South-East Asianovalocytosis (SAO), resulting from the band 3 deletion of residues400-408, occurred in many of the families but did not itselfresult in dRTA. Compound heterozygotes of each of the dRTA mutationswith SAO all had dRTA, evidence of haemolytic anaemia and abnormal red-cell properties. The A858D mutation showed dominant inheritance and therecessive DeltaV850 and G701D mutations showed a pseudo-dominantphenotype when the transport-inactive SAO allele was also present. Red-cell and Xenopus oocyte expression studies showed that theDeltaV850 and A858D mutant proteins have greatly decreased aniontransport when present as compound heterozygotes (DeltaV850/A858D,DeltaV850/SAO or A858D/SAO). Red cells with A858D/SAO had only 3% ofthe SO(4)(2-) efflux of normal cells, thelowest anion transport activity so far reported for human red cells. The results suggest dRTA might arise by a different mechanism for eachmutation. We confirm that the G701D mutant protein has an absoluterequirement for glycophorin A for movement to the cell surface. Wesuggest that the dominant A858D mutant protein is possibly mis-targetedto an inappropriate plasma membrane domain in the renal tubular cell,and that the recessive DeltaV850 mutation might give dRTA because ofits decreased anion transport activity.

The anion transport activity of the human erythrocyte anion transporter (band 3; AE1) has been examined in both normal and glycophorin A (GPA)- deficient (MkMk) human red blood cells (RBCs). The sulfate transport activity of MkMk cells (from two ethnically diverse sources) was approximately 60% that of normal erythrocytes under the transport assay conditions used. However, MkMk and normal RBCs contained similar amounts of band 3. The reduction in sulfate transport activity was shown to be caused by an increase in the apparent Km for sulfate in MkMk RBCs, suggesting the band 3 in the MkMk RBCs has a lowered binding affinity for sulfate anions. The size of the N-glycan chain on band 3 of the MkMk cells was larger than that on band 3 from normal RBCs. In contrast, the size of the N-glycan chain on the glucose transporter (GLUT1) from MkMk cells was smaller than that on GLUT1 from normal cells. The possible role of GPA in the biosynthesis and anion transport activity of band 3 in normal RBCs is discussed.

1. We have shown that the Dia antigen of the Diego blood group system is associated with the presence of red cell band 3 Memphis, but not all band 3 Memphis samples carry the Dia antigen. 2. The band 3 Memphis associated with the Dia antigen was covalently labelled by 4,4'-di-isothiocyanato-1,2-diphenylethane-2,2'-disulphonic acid (H2DIDS) more readily than was normal band 3 or band 3 Memphis not associated with the Dia antigen. This altered reactivity with H2DIDS has previously been noted for a band 3 Memphis sub-type designated variant 2. 3. This is the first example of a band 3 polymorphism associated with an antigenic change in the extracellular region of the band 3 polypeptide and with altered H2DIDS binding.

The Wright (Wr) blood group antigens, Wra and Wrb, have been suggested to be determined by alleles of the same gene. The Wrb antigen appears to involve both red blood cell (RBC) band 3 and glycophorin A (GPA). We have examined the cDNA sequences of the band 3 and GPA of one of the two known Wr(a+b-) individuals. We show that this individual is homozygous for the mutation Glu658--<Lys in band 3, but has normal GPA. Putative heterozygotes with Wr(a+b+) RBCs have both Glu and Lys at residue 658 of band 3, whereas the common Wr(a-b+) RBC phenotype only have band 3 with Glu658. The Wra and Wrb antigens are determined by the amino acid at residue 658 of band 3 and are antithetical. Examination of the amino acid sequence and Wrb antigen expression of GPA-related hybrid glycophorins suggests that Arg61 of GPA interacts with Glu658 of band 3 to form the Wrb antigen. We suggest that the interaction is stabilized by the presence of anti-Wrb antibodies and that this site of association between GPA and band 3 may be responsible for the previously reported ability of anti-GPA antibodies to decrease the deformability of RBCs.

Normal human RBCs have a very low basal permeability (leak) to cations, which is continuously corrected by the Na,K-ATPase. The leak is temperature-dependent, and this temperature dependence has been evaluated in the presence of inhibitors to exclude the activity of the Na,K-ATPase and NaK2Cl transporter. The severity of the RBC cation leak is altered in various conditions, most notably the hereditary stomatocytosis group of conditions. Pedigrees within this group have been classified into distinct phenotypes according to various factors, including the severity and temperature-dependence of the cation leak. As recent breakthroughs have provided more information regarding the molecular basis of hereditary stomatocytosis, it has become clear that these phenotypes elegantly segregate with distinct genetic backgrounds. The cryohydrocytosis phenotype, including South-east Asian Ovalocytosis, results from mutations in SLC4A1, and the very rare condition, stomatin-deficient cryohydrocytosis, is caused by mutations in SLC2A1. Mutations in RHAG cause the very leaky condition over-hydrated stomatocytosis, and mutations in ABCB6 result in familial pseudohyperkalemia. All of the above are large multi-spanning membrane proteins and the mutations may either modify the structure of these proteins, resulting in formation of a cation pore, or otherwise disrupt the membrane to allow unregulated cation movement across the membrane. More recently mutations have been found in two RBC cation channels, PIEZO1 and KCNN4, which result in dehydrated stomatocytosis. These mutations alter the activation and deactivation kinetics of these channels, leading to increased opening and allowing greater cation fluxes than in wild type.