An 11-amino acid beta-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes

Abstract

The best-studied cytoskeletal system is the inner surface of the erythrocyte membrane, which provides an erythrocyte with the structural support needed to be stable yet flexible as it passes through the circulation. Current structural models predict that the spectrin-actin-based cytoskeletal network is attached to the plasma membrane through interactions of the protein ankyrin, which binds to both spectrin and the cytoplasmic domain of the transmembrane protein band 3. The crystal structure of the cytoplasmic domain of band 3 predicted that the ankyrin binding site was located on a beta-hairpin loop in the cytoplasmic domain. In vitro, deletion of this loop eliminated ankyrin affinity for band 3 without affecting any other protein-band 3 interaction. To evaluate the importance of the ankyrin-band 3 linkage to membrane properties in vivo, we generated mice with the nucleotides encoding the 11-aa beta-hairpin loop in the mouse Slc4a1 gene replaced with sequence encoding a diglycine bridge. Mice homozygous for the loop deletion were viable with mildly spherocytic and osmotically fragile erythrocytes. In vitro, homozygous ld/ld erythrocytes were incapable of binding ankyrin, but contrary to all previous predictions, abolishing the ankyrin-band 3 linkage destabilized the erythrocyte membrane to a lesser degree than complete deficiencies of either band 3 or ankyrin. Our data indicate that as yet uncharacterized interactions between other membrane proteins must significantly contribute to linkage of the spectrin-actin-based membrane cytoskeleton to the plasma membrane.

Fig. 1.

Homologous recombination to delete an 11-amino acid β-hairpin loop in the cytoplasmic domain of band 3. Strategy for targeting the murine Slc4a1 locus (Upper). The amino acid sequence of the β-hairpin loop and the amino acid sequence after the loop deletion (Lower).

Fig. 2.

Homozygous ld/ld erythrocytes have altered morphology. (Upper) Comparison of whole erythrocytes under a light microscope from wild-type (+/+) and homozygous loop-deleted (ld/ld) mice. (Lower) Comparison of erythrocytes from +/+ and ld/ld mice by using SEM.

Fig. 3.

RBCs from ld/ld mice are osmotically fragile. Erythrocytes from +/+, heterozygous (+/ld), and ld/ld mice were exposed to increasing amounts of saline (x axis). The percent of cells lysed is shown on the y axis.

Fig. 4.

ld/ld erythrocytes are not deficient in band 3. (a) SDS/PAGE of erythrocyte ghost membranes from +/+, +/ld, and ld/ld mouse blood. (b and c) Western blot of whole RBCs and ghosts from +/+, +/ld, and ld/ld mutant mice. Ctl, human erythrocyte ghosts. Double antibody (anti-band 3 and anti-actin) staining was used.

Fig. 5.

Band 3 retention in membrane skeletons is reduced in ld/ld erythrocytes. SDS/PAGE of whole RBCs extracted with 2% Triton X-100. Bands were quantified with densitometry scanning. One milliliter of packed RBCs was washed three times with PBS and solubilized with 1 ml of 2% Triton X-100 containing 20 μg/ml pepstatin A, 40 μg/ml PMSF, 20 μg/ml leupeptin, 0.5 mM DTT, and 1 mM EDTA, pH = 6.5. The experiment was repeated three times and the average ratio of band 3 to actin from these experiments is shown.

Fig. 6.

KI-IOVs from ld/ld mice do not bind ankyrin. KI-IOVs (30 μg/ml) were incubated for 3 h on ice with increasing amounts of D3/D4 ¹²⁵I-ankyrin (a) or intact ¹²⁵I-ankyrin (b). Membranes were pelleted, washed, and assayed in γ counter for radioactive ankyrin (n = 5). The resulting graph is a plot with subtracted heat-denatured controls. Mutant mice KI-IOVs exhibit negligible binding to either the D3/D4 ankyrin fragment of full-length ankyrin.