Previously, we reported a new missense mutation in the ANK1 gene correlated with the HS 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. In this report, we have shown the molecular and physiological effects of this mutation. First, we assessed the binding activity of human beta-spectrin to the mutated ZZUDL1340P domain of ankyrin using two different experimental approaches: the study of association and dissociation responses of spectrin ankyrin binding domain and sedimentation assay. In addition, we demonstrated changes in morphology caused by the overexpressed ankyrin ZZUD domain in human cell models. Our results prove the key role of L1340 aa residue in the UPA domain for the correct alignment of the ZZUD domain of ankyrin, which results in binding the latter with spectrin within the erythrocyte membrane. Replacing the L1340 with a proline residue disrupts the spectrin binding activity of ankyrin.

中文翻译：

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锚蛋白中与球形红细胞增多症相关的 L1340P 突变影响其与血影蛋白的相互作用

以前，我们报道了 ANK1 基因中与 HS 表型相关的新错义突变。这种突变导致 L1340P 取代 (HGMD CM149731)，可能导致锚蛋白 ZZUD 结构域的构象发生变化，这对于锚蛋白与血影蛋白的结合很重要。在这份报告中，我们展示了这种突变的分子和生理效应。首先，我们使用两种不同的实验方法评估了人 β-血影蛋白与锚蛋白突变 ZZUDL1340P 结构域的结合活性：血影蛋白锚蛋白结合域的结合和解离反应研究以及沉降测定。此外，我们展示了人类细胞模型中过表达的锚蛋白 ZZUD 结构域引起的形态学变化。我们的结果证明了 UPA 结构域中 L1340 aa 残基对于正确对齐锚蛋白的 ZZUD 结构域的关键作用，这导致后者与红细胞膜内的血影蛋白结合。用脯氨酸残基替换 L1340 会破坏锚蛋白的血影蛋白结合活性。