Mutations in the murine erythroid α-spectrin gene alter spectrin mRNA and protein levels and spectrin incorporation into the red blood cell membrane skeleton

Abstract

Tetramers of ␣-and ␤-spectrin heterodimers, linked by intermediary proteins to transmembrane proteins, stabilize the red blood cell cytoskeleton. Deficiencies of either ␣-or ␤-spectrin can result in severe hereditary spherocytosis (HS) or hereditary elliptocytosis (HE) in mice and humans. Four mouse mutations, sph, sph Dem , sph 2BC , and sph J , affect the erythroid ␣-spectrin gene, Spna1, on chromosome 1 and cause severe HS and HE. Here we describe the molecular alterations in ␣-spectrin and their consequences in sph 2BC /sph 2BC and sph J /sph J erythrocytes. A splicing mutation, sph 2BC initiates the skipping of exon 41 and premature protein termination before the site required for dimerization of ␣-spectrin with ␤-spectrin. A nonsense mutation in exon 52, sph J eliminates the COOHterminal 13 amino acids. Both defects result in instability of the red cell membrane and loss of membrane surface area. In sph 2BC /sph 2BC , barely perceptible levels of messenger RNA and consequent decreased synthesis of ␣-spectrin protein are primarily responsible for the resultant hemolysis. By contrast, sph J /sph J mice synthesize the truncated ␣-spectrin in which the 13-terminal amino acids are deleted at higher levels than normal, but they cannot retain this mutant protein in the cytoskeleton. The sph J deletion is near the 4.1/actin-binding region at the junctional complex providing new evidence that this 13-amino acid segment at the COOH-terminus of ␣-spectrin is crucial to the stability of the junctional complex. IntroductionMice with mutations in genes encoding membrane skeletal proteins are important models for hereditary spherocytosis (HS). 1 Gene mapping and disrupted protein levels 2,3 identified the affected product and became the basis for the subsequent cloning and sequencing of ␣-spectrin (Spna1), ␤-spectrin (Spnb1), and ankyrin (Ank1) from mouse 4-6 and human DNA. 7-10 ␤-Spectrin is a pivotal protein that provides binding sites for ankyrin, the linker between spectrin and the transmembrane protein band 3; ␣-spectrin heterodimerization; tetramerization of heterodimers 11 ; and 4.1, a protein that joins spectrin tetramers to the transmembrane protein glycophorin C at junctional complexes. 12 Anchoring of the spectrinbased cytoskeleton to the transmembrane proteins band 3 and glycophorin C is essential for red blood cell stability. The nucleotide alterations described for the mouse beta spectrin (ja/ ja), 13 ankyrin (nb/nb), 14 and ␣-spectrin (sph/sph, sph Dem /sph Dem ) 4,15 mutations provide an explanation for the instability of red cells in these mutants and clues to the functional consequences of alterations in specific amino acids. Assessing functional effects of mutations in humans is more difficult because the secondary genetic diversity affects results, mutations are heterogeneous, and clinical severity may vary among patients with the same mutation.The murine ␣-spectrin mutations sph, sph 2BC , and sph J result in severe HS, whereas sph Dem is a unique model for hereditary elliptocytosis (HE). 16 ␣-Spect...

Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in α-spectrin–deficient red cells

“…Thus, sph 3J and sph 4J mutations located to novel positions within Spna1 compared with previously described sph alleles ( Figure 1B). 2,6,21,42,43 Severe hemolytic anemia in sph 3J and sph 4J sph 3J and sph 4J homozygotes displayed characteristics of severe hemolytic anemia. The RBC count, hemoglobin, and hematocrit values were significantly decreased (Table 1), whereas the percentage reticulocytes and spleen weights were dramatically increased, indicating a compensatory acceleration of erythropoiesis (Table 2).…”

“…Unlike normal spleens, mutant spleens were devoid of iron ( Figure 3C), likely reflecting extensive use as the result of markedly accelerated erythropoiesis, consistent with other mouse models with severe anemia. 2 Differential alterations in membrane skeletal proteins in sph 3J and sph 4J mice Quantitative SDS-PAGE and Western blot analysis of RBC membrane skeletal proteins in severely anemic mice is complicated by 43 ; sph Dem has an in-frame deletion of exon 11 that deletes 46 amino acids from repeat 5 21 ; sph 2BC has a guanine to thymine substitution in intron 41 that results in a frame shift and premature stop codon in repeat 19 42 ; sph 1J is a deletion of the C-terminal 13 amino acids 42 ; sph 3J is a missense mutation in repeat 19 of the ␣␤-spectrin dimer nucleation site; sph 4J is a missense mutation in the C-terminus within the EF-hand domain just upstream of sph 1J deletion; and sph Ihj is a premature stop codon in repeat 18. (C) The sph 3J mutation creates an RsaI recognition site that was used to genotype mice after PCR amplification.…”

“…39 We can only speculate as to why band 3 deficiency was not detected in previous studies of the sph mutants upon SDS-PAGE. 2,20,21,42 A likely contributing factor is improved SDS-PAGE techniques and, in the past, reliance on discontinuous Laemmli gels for mouse studies, which often fail to clearly delineate band 3 protein upon Coomassie blue staining. 21 In many cases, band 3 was the standard to which everything else was compared.…”

Plasmodium falciparum Erythrocyte Membrane Protein 3 (PfEMP3) Destabilizes Erythrocyte Membrane Skeleton

“…There was no binding to GST or to other spectrin fragments. The ␣17-C construct comprises repeats 17,18,19,20,21, and the C-terminal element with four EF-hands. We next examined the binding of FIa1 to all these parts of the large fragment individually and obtained a positive result for only one of them.…”

“…In contrast to the ␤-spectrin N terminus, very little is known regarding the ␣-spectrin C terminus that is in apposition to the ␤-spectrin N terminus. Studies from sph J mice (where a nonsense mutation in exon 52 of the erythroid ␣-spectrin gene eliminates the C-terminal 13 amino acids) strongly suggested the ␣-spectrin C terminus is also critical to the stability of the junctional complex (21).…”