Spherocytosis-Related L1340P Mutation in Ankyrin Affects Its Interactions with Spectrin

Abstract

Previously, we reported a new missense mutation in the ANK1 gene that correlated with the hereditary spherocytosis phenotype. This mutation, resulting in L1340P substitution (HGMD CM149731), likely leads to the changes in the conformation of the ankyrin ZZUD domain important for ankyrin binding to spectrin. Here, we report the molecular and physiological effects of this mutation. First, we assessed the binding activity of human β-spectrin to the mutated ZZUDL1340P domain of ankyrin using two different experimental approaches-the study of association and dissociation responses of the spectrin-ankyrin binding domain and a sedimentation assay. In addition, we documented the changes in morphology caused by the overexpressed ankyrin ZZUD domain in human cell models. Our results prove the key role of the L1340 aa residue for the correct alignment of the ZZUD domain of ankyrin, which results in binding the latter with spectrin within the erythrocyte membrane. Replacing L1340 with a proline residue disrupts the spectrin-binding activity of ankyrin.

Keywords: ankyrin-R; ankyrin–spectrin interaction; erythrocyte membrane skeleton; hereditary spherocytosis; spectrin; spectrin-binding domain.

Figure 1

Schematic representation of the various ankyrin and spectrin fragments used in the assays concerning their length and domains.

Figure 2

Quantification of binding between AnkBD and ZZUD or its mutated form. (A) Binding of ZZUD or ZZUDL1340P mutant to BLI sensor with AnkBD. (B) Saturation binding curve of ZZUD and AnkBD obtained via a sedimentation assay. N = 3; red curve was generated via one site-specific binding fitting.

Figure 3

Effect of the ZZUD domain and its mutant form on the morphology of the resealed erythrocyte ghosts. The ghosts of the erythrocytes loaded with the non-mutated ZZUD domain were characteristically deformed (right panel), while those loaded with the purified mutated ZZUD domain remained spherical (central panel). In the left panel was shown normal morphology of the erythrocyte ghosts. Scale bar 5 µm.

Figure 4

HEL (panel A) and K562 (panel B) cells were transfected with EGFP/RFP ZZUD and EGFP/RFP ZZUDL1340P plasmids and imaged 48 and 72 h after transfection. The cells continue to have a circular outline, but the ankyrin-derived fragments accumulate in a certain region of the cell, leading to markedly changed morphology; such appearance is not present in the wild-type/control cells or those transfected with the ZZUDL1340P bearing vector. This increased number of cells with altered morphology was highly statistically significant as shown in panel (C) (★, p < 0.05; ★★★, p < 0.005).