Identification and Characterization of α-Spectrin Mutations Associated with Inherited Hemolytic Anemia.
Authors: Tolpinrud Whitney , Gaetani Massimiliano , Maksimova Yelena , Mootien Sara , Harper Sandra , Forget Bernard G. , Speicher David W. , Gallagher Patrick G. 
Published: April 2020

Abstract
Spectrin, the major structural component of the erythrocyte membrane skeleton, is composed of α and β chains that self-associate to form tetramers. These tetramers provide the structural integrity and flexibility critical for erythrocyte stability and shape. Mutations of α spectrin have been associated with hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary pyropoikilocytosis (HPP). The large size of the spectrin molecule has complicated its study. We developed a high-throughput capillary nucleotide sequencing strategy to identify mutations of the α-spectrin gene in a group of patients with spectrin-linked HS, HE, or HPP. We identified several variants including 8 nonsense, 4 splice junction, and 4 deletion/insertion mutations.(Mutations in &gt;1 patient are counted only once.) We were interested in the identification of missense mutations, as we hypothesize that defects in α-spectrin occur in regions of structural and functional importance and their identification and characterization will provide important information about spectrin and the membrane skeleton. We identified 16 missense mutations in the region encoding the spectrin self-association site; 6 were proline substitutions and 2 were glycine substitutions, both predicted to disrupt the triple helical configuration of spectrin. Outside the self-association site, excluding 3 common protein polymorphisms, we identified 13 missense mutations; 3 were proline substitutions. To begin to study the functional significance of these mutations, we prepared 15 recombinant spectrin-GST fusion peptides containing residues 1–158 of α spectrin, the self-association contact site, representing wild type (WT) or 14 different missense mutations. After expression and purification, purity was assured by SDS-PAGE, absence of aggregation was verified by analytical HPLC gel filtration, and mass confirmed by MS analyses. Analyses by circular dichroism demonstrated that none of the missense mutations significantly modified secondary structure of the recombinant peptide. WT and mutant peptides exhibited a helical content of ∼65%. Ultracentrifugation studies verified that all peptides were monomeric at 4 and 30°C. Differential scanning calorimetry showed that the WT peptide was very stable with a single reversible 2-state transition with a Tm of 54.6°C. All mutations, except R34W, showed transitions similar to WT. R34W unfolded at a much lower Tm, 49.1°C, with a broader single peak transition. Analysis of spectrin tetramerization between α-spectrin peptides and a recombinant β-spectrin peptide (repeats 16, 17 and COOH-terminus) was performed using an analytical HPLC gel filtration assay. A wide range of binding affinities was observed: WT binding Kd=0.43μM at 23°C; group I: I24S, R28C, R28H, R28L, R28S, R45S, no binding; group II: I24T, R41W, L49F, much weaker binding than WT; group III: V31A, R45T, G46V, binding weaker than WT, and R34W and K48R, binding equal to WT. Quantitative thermodynamic analyses of spectrin tetramerization site formation between α and β spectrin peptides were assessed by isothermal calorimetry. These results were essentially comparable to the gel filtration data except the R34W mutant bound β-spectrin more avidly than WT. The identification and characterization of variants associated with HS, HE and HPP continues to extend our understanding and knowledge of both normal membrane biology and human disease pathogenesis.