Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in {alpha}-spectrin deficient red cells.
(2010)
Journal - Blood
Abstract :
Five spontaneous, allelic mutations in the alpha-spectrin gene, Spna1, have been identified in mice [spherocytosis (sph), sph(1J), sph(2J), sph(2BC), sph(Dem)]. All cause severe hemolytic anemia. Here, analysis of three new alleles reveals previously unknown consequences of RBC spectrin deficiency. In sph(3J) a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sph(Ihj), a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced red blood cell (RBC) membrane spectrin content, decreased band 3 and absent beta-adducin. Re-evaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph(4J) a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, beta-adducin. The severity of anemia in sph(4J) indicates that the highly conserved cysteine residue at the C-terminus of alpha-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.
Targeted deletion of alpha-adducin results in absent beta- and gamma-adducin, compensated hemolytic anemia, and lethal hydrocephalus in mice.
(2008)
Journal - Blood (United States )
Abstract :
In the red blood cell (RBC), adducin is present primarily as tetramers of alpha- and beta-subunits at spectrin-actin junctions, or junctional complexes. Mouse RBCs also contain small amounts of gamma-adducin. Platelets contain alpha- and gamma-adducin only. Adducin functions as a barbed-end actin capping protein to regulate actin filament length and recruits spectrin to the ends of actin filaments. To further define adducin's role in vivo, we generated alpha-adducin knockout mice. alpha-Adducin is absent in all tissues examined in homozygous null mice. In RBCs, beta- and gamma-adducin are also absent, indicating that alpha-adducin is the limiting subunit in tetramer formation at the spectrin-actin junction. Similarly, gamma-adducin is absent in alpha-null platelets. alpha-Adducin-null mice display compensated hemolytic anemia with features characteristic of RBCs in hereditary spherocytosis (HS), including spherocytes with significant loss of surface area, decreased mean corpuscular volume (MCV), cell dehydration, and increased osmotic fragility. Platelets maintain their normal discoid shape, and bleeding times are normal. alpha-Adducin-null mice show growth retardation at birth and throughout adulthood. Approximately 50% develop lethal communicating hydrocephalus with striking dilation of the lateral, third, and fourth ventricles. These data indicate that adducin plays a role in RBC membrane stability and in cerebrospinal fluid homeostasis.
| ISSN : | 1528-0020 |
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| Mesh Heading : | Actins Anemia, Hemolytic, Congenital Animals Blood Platelets Cytoskeletal Proteins Gene Deletion Hydrocephalus Mice Mice, Knockout Osmotic Fragility Protein Structure, Quaternary Spectrin Spherocytes Spherocytosis, Hereditary genetics metabolism genetics metabolism genetics genetics pathology genetics genetics metabolism pathology genetics metabolism pathology |
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| Mesh Heading Relevant : | metabolism metabolism metabolism metabolism |
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Two New Recessive Mouse Mutations Cause Severe Hemolytic Anemia and Reveal Unexpected Interactions in the C-Terminus of -Spectrin.
(2005)
Journal - ASH Annual Meeting Abstracts
Abstract :
Abstract
The red blood cell (RBC) lipid bilayer is supported by an underlyingmembrane skeleton. Erythroid spectrin, which is composed offlexible alpha and beta subunits, is encoded by the (Spna1)and ß (Spnb1) genes and is the major protein in themembrane skeleton. In mice, five independent autosomal recessivemutations in -spectrin (sph, sph1J, sph2J, sph2BC, sphDem) andone in ß-spectrin (ja) have been identified; all resultin severe hemolytic anemia. We have identified two new mouse-spectrin mutations, sph3J and sph4J, on the NOD.B10 and C57BL/6Jbackground strains, respectively. Linkage analysis in F2 intercrosseslocalized both mutations to the distal portion of mouse chromosome1 near Spna1, an obvious candidate gene. In both sph3J and sph4J,novel mutations distinct from the previously described fivesph alleles were subsequently identified. In sph3J a cytosineto thymine transition in exon 43 causes a histidine to tyrosinesubstitution within the ß nucleation site of -spectrin(H2012Y). Spna1 message levels are significantly reduced insph3J reticulocyte RNA. In sph4J a guanine to adenine transitionin exon 52 results in a cysteine to tyrosine substitution nearthe C-terminus (C2384Y). Spna1 message levels are normal insph4J reticulocytes. Both mutations cause a phenotype of severehemolytic anemia. In homozygous adult sph3J mice, dramatic decreasesin the RBC count (–67%), hemoglobin (–68%), andhematocrit (–65%) are seen. On Wright’s stainedperipheral blood smears and by scanning electron microscopy,large numbers of elliptocytes and spherocytes are evident. Significantlyincreased spleen-to-body weight ratio (+1,200%), bilirubin (+98%),iron (+74%) and circulating reticulocytes are also present.Homozygous adult sph4J mice show similar abnormally shaped RBCsand blood profile changes. SDS-PAGE analysis of sph3J and sph4JRBC membrane skeletons revealed unique changes in membrane skeletonproteins compared to each other and to the five known sph alleles.In sph3J, - and ß-spectrin are significantly decreasedbut ankyrin, protein 4.1 and protein 4.2 levels are normal.Surprisingly, band 3 is reduced to ~30% of normal, and both- and ß-adducin are nearly undetectable in sph3J RBCs.The presence of normal amounts of ankyrin, which binds band3 tetramers, suggests that band 3 dimers are absent in sph3JRBCs. These observations indicate that previously unsuspectedinteractions, direct or indirect, exist between spectrin andband 3 (probably dimers) and between spectrin and adducin withinthe RBC membrane skeleton. In contrast to sph3J, all RBC membraneskeleton proteins appear normal by SDS-PAGE and western blotanalyses of sph4J RBC membranes. Coupled with the severe hemolyticanemia present in these mice, these data suggest that interactionsinvolving the C-terminus of -spectrin, specifically cysteine2384, are critical to RBC membrane integrity. Together, thesph3J and sph4J mouse models provide powerful resources foridentifying critical interactions within the membrane skeletonthat are relevant to the pathogenesis of hereditary elliptocytosisand spherocytosis.