Protein-4.2 association with band 3 (AE1, SLCA4) in Xenopus oocytes: effects of three natural protein-4.2 mutations associated with hemolytic anemia

Abstract

IntroductionProtein 4.2 is a major constituent of the red blood cell (RBC) membrane skeletal network, present at about 200 000 copies per RBC. 1 The protein-4.2 gene EPB42 contains 13 exons. 2,3 There are two isoforms of protein 4.2, a minor 74-kDa isoform obtained when all these exons of the gene are expressed and a 72-kDa major isoform of protein 4.2 that lacks 30 of the 33 amino acids that are encoded by exon 1. 4,5 The exact role of protein 4.2 in RBCs has not been elucidated, but protein 4.2 binds to the N-terminal cytoplasmic domain of the band-3 anion exchanger (AE1) and also interacts with ankyrin in RBCs. 6,7 The presence of band 3 is critical for the stable incorporation of protein 4.2 into the RBC membrane, since human, 8 mouse, 9 and cow 10 RBCs deficient in band 3 are also completely deficient in protein 4.2. The functional significance of the association of protein 4.2 with band 3 in RBCs is currently unclear. In liposomes containing reconstituted band 3, anion transport activity decreased in the presence of increasing amounts of protein 4.2, 11 which suggested that protein 4.2 was a negative modulator of band-3 anion exchange activity. However, band-3-mediated anion transport activity has been reported to be unaffected 12 or increased 13 in human RBCs with protein-4.2 deficiency. In contrast, the absence of protein 4.2 slightly decreased anion transport activity in protein-4.2-null mouse RBCs. 14 Protein 4.2 also associates with spectrin, 15,16 ankyrin, 7 and protein 4.1 7 in solution, although the significance of these associations in vivo has yet to be demonstrated. Recently, protein 4.2 has been shown to interact with CD47, which probably contributes to the anchoring of the Rh complex to the RBC skeleton. 17,18 Protein 4.2 can probably interact with the inner leaflet of the lipid bilayer since it is fatty acylated with myristoyl and palmitoyl chains. 19,20 The complete or nearly complete absence of protein 4.2 is associated with an atypical form of hereditary spherocytosis (HS), highlighting the important role 4.2 plays in maintaining the stability and flexibility of RBCs. To date, 9 protein-4.2 mutations have been found associated with HS. Five lead to the premature termination of translation. 17,[21][22][23][24] Other protein-4.2 variants result from missense mutations that yield amino acid substitutions: protein 4.2 Nippon (GCTϾACT; A142T 25 ; occurs sporadically in the Japanese population and has been encountered once in whites), 26 protein 4.2 Tozeur (CGAϾCAA; R310Q), 27 protein 4.2 Shiga (CGCϾTGC; R317C), 28 and protein 4.2 Komatsu (GATϾTAT; D175Y). 29 Although there is a strict correlation between the occurrence of HS and the presence of these mutations (in homozygous or compound heterozygous states), the fact that these mutations are the direct cause of the absence of protein 4.2 has not been formally established. One possibility is that these point mutations affect the binding of protein 4.2 to band 3 and therefore its function. This has proved difficult to study using biochemical We h...

Molecular physiology and genetics of Na+-independent SLC4 anion exchangers

“…The extreme N-terminal sequence of human AE1 binds multiple glycolytic enzymes (Campanella et al, 2008;Chu and Low, 2006) and hemoglobin under the control of hemoglobin oxygenation. Other parts of the N-terminal cytoplasmic domain provide binding sites for the erythroid cytoskeletal proteins ankyrin-1 (Chang and Low, 2003;Stefanovic et al, 2007), protein 4.2 (Toye et al, 2005), the ERM protein 4.1R (Salomao et al, 2008), and integrin-linked kinase (Keskanokwong et al, 2007). Solution of a 2.6 Å X-ray structure of the dimeric human erythroid AE1 cytoplasmic domain (amino acids 1-379) required crystallization at pH 4.8 and resolved residues 55-201 and 212-356 within a globular domain (Zhang et al, 2000).…”

Investigating the key membrane protein changes during in vitro erythropoiesis of protein 4.2 (-) cells (mutations Chartres 1 and 2)

“…40,41 However, an earlier simultaneous expression profile is in keeping with the known dependence of protein 4.2 on band 3 expression. 8,[10][11][12] The level and association of band 3 and protein 4.2 remains constant after 72h (normoblast stage) suggesting that the majority of protein 4.2 may already be associated with band 3 early in differentiation.…”

“…[14][15][16] To date, nine mutations in EPB42 have been associated with hereditary spherocytosis, 6,[17][18][19][20][21][22] some of these mutations influence protein 4.2 stability and band 3-ankyrin-1 binding. 8,23 Protein 4.2 deficiency in humans causes a severe reduction in the marker of self, 24 CD47 (by approximately 80%), 6,25,26 suggesting an association between protein 4.2 and CD47. Furthermore, protein 4.2 deficiency causes elevated expression of the ankyrin binding protein CD44 27 and increased RhAG glycosylation.…”

“…6 The peripheral membrane protein, protein 4.2, shares significant homology to transglutaminases but lacks the catalytic triad residues required for transglutaminase activity. [7][8][9] Stable expression of protein 4.2 in erythrocytes depends on its interaction with the band 3 and ankyrin-1.…”

Anion Exchanger 1 Interacts with Nephrin in Podocytes

“…Rather, at low concentrations of injected kAE1 cRNA, nephrin coexpression slightly reduced kAE1 transport activity, probably as a result of competition between cRNAs for the translational machinery, as has been previously observed in some other types of coexpression experiments. 38 Nephrin did not rescue activity of any tested dRTA-or hereditary spherocytosis-associated kAE1 mutants. These findings suggest that nephrin is not a nonspecific chaperonin protein for kAE1.…”