PubMed · 6622498
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M M Kay. 1983. Antigenic changes associated with cellular aging.. https://pubmed.ncbi.nlm.nih.gov/6622498/
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The human erythrocyte anion exchanger (band 3) contains a cytoplasmic domain (cdb3) that exists in a reversible, pH-dependent structural equilibrium among three native conformations. To understand how this conformational equilibrium might influence the association state of band 3, we have incubated stripped erythrocyte membranes in solutions ranging from pH 6.0 to pH 10.5 and have examined the oligomeric state of the protein by size exclusion high performance liquid chromatography. We demonstrate that incubation of membranes in slightly acidic conditions favors dimer formation, whereas extended incubation at higher pHs (pH>9) leads to irreversible formation of an oligomeric species larger than the tetramer. Since the pH dependence of the conformational state of the cytoplasmic domain exhibits a similar pH profile, we suggest that the conformation of the cytoplasmic domain can modulate the self-association of band 3. Importantly, this modulation would appear to require the structural interactions present within the intact protein, since the isolated membrane-spanning domain does not display any pH dependence of association. The irreversible nature of the alkali-induced aggregation further suggests that a secondary reaction subsequent to band 3 association is required to stabilize the high molecular weight aggregate. Although we were able to eliminate covalent bond formation in this irreversible aggregation process, the exact nature of the secondary reaction remains to be elucidated.
The cytoplasmic domain of erythrocyte membrane band 3 (cdb3) serves as a center of membrane organization, interacting with such proteins as ankyrin, protein 4.1, protein 4.2, hemoglobin, several glycolytic enzymes, and a tyrosine kinase, p72syk. cdb3 exists in a reversible, pH-dependent conformational equilibrium characterized by large changes in Stokes radius (11 A) and intrinsic fluorescence (2-fold). Based on the crystallographic structure of the cdb3 dimer, we hypothesized that the above conformational equilibrium might involve the movement of flanking peripheral protein binding domains away from a shared dimerization domain. To test this hypothesis, we have mutated both donor (W105L) and acceptor (D316A) residues of a prominent H bond that bridges the above two domains and have examined the effect on the resulting conformational equilibrium. Analysis of the intrinsic fluorescence, Stokes radius, thermal stability, urea stability, and segmental mobility of these mutants reveals that the above H bond is indeed present in the low pH conformation of cdb3 and broken in a higher pH conformation. The data further reveal that cdb3 exists in three native pH-dependent conformations and that rupture of the aforementioned H bond occurs only during conversion of the low pH conformation to the mid-pH conformation. Conversion of the mid-pH conformation to the high pH conformation would now appear to involve structural changes primarily in the peripheral protein binding domain. Because ankyrin associates avidly with the low pH conformation of cdb3, ankyrin occupancy should strongly influence this structural equilibrium and thereby affect band 3 and perhaps global membrane properties.
K562 cells were stably transfected with cDNAs encoding the band 3 found in Southeast Asian ovalocytosis (B3SAO, deletion of residues 400-408), band 3 with a transport-inactivating E681Q point mutation (B3EQ), or normal band 3 (B3). Flow cytometric analysis and quantitative immunoblotting revealed that B3SAO expressed alone was translocated to the plasma membrane, at levels similar to B3 or B3EQ. Nine monoclonal antibodies that reacted with extracellular loops of B3 also reacted with B3SAO, although the affinity of most antibodies for the mutant protein was reduced. Both known Wr(b) epitopes were expressed on K562/B3SAO cells, demonstrating that B3SAO interacts with glycophorin A. The growth rates of K562 clones expressing equivalent amounts of B3 and B3EQ were the same, suggesting that the potentially toxic transport function of band 3 may be regulated in K562 cells. The band 3-mediated enhancement of Rh antigen reactivity and the depression of Rh epitopes on SAO erythrocytes were investigated by comparing the coexpression of B3, B3SAO, or B3EQ in K562 clones expressing exogenous RhcE or RhD polypeptides. The results are consistent with an interaction between band 3 and the Rh polypeptide-Rh glycoprotein (RhAG) complex, which may enhance translocation of the complex or affect its conformation in the plasma membrane. The data suggest that the interaction between band 3 and the RhD-RhAG complex is weaker than it is between band 3 and the RhCcEe-RhAG complex.