Two New Recessive Mouse Mutations Cause Severe Hemolytic Anemia and Reveal Unexpected Interactions in the C-Terminus of α-Spectrin.

Abstract The red blood cell (RBC) lipid bilayer is supported by an underlying membrane skeleton. Erythroid spectrin, which is composed of flexible alpha and beta subunits, is encoded by the α (Spna1) and β (Spnb1) genes and is the major protein in the membrane skeleton. In mice, five independent autosomal recessive mutations in α-spectrin (sph, sph1J, sph2J, sph2BC, sphDem) and one in β-spectrin (ja) have been identified; all result in severe hemolytic anemia. We have identified two new mouse α-spectrin mutations, sph3J and sph4J, on the NOD.B10 and C57BL/6J background strains, respectively. Linkage analysis in F2 intercrosses localized both mutations to the distal portion of mouse chromosome 1 near Spna1, an obvious candidate gene. In both sph3J and sph4J, novel mutations distinct from the previously described five sph alleles were subsequently identified. In sph3J a cytosine to thymine transition in exon 43 causes a histidine to tyrosine substitution within the αβ nucleation site of α-spectrin (H2012Y). Spna1 message levels are significantly reduced in sph3J reticulocyte RNA. In sph4J a guanine to adenine transition in exon 52 results in a cysteine to tyrosine substitution near the C-terminus (C2384Y). Spna1 message levels are normal in sph4J reticulocytes. Both mutations cause a phenotype of severe hemolytic anemia. In homozygous adult sph3J mice, dramatic decreases in the RBC count (−67%), hemoglobin (−68%), and hematocrit (−65%) are seen. On Wright’s stained peripheral blood smears and by scanning electron microscopy, large numbers of elliptocytes and spherocytes are evident. Significantly increased 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 RBCs and blood profile changes. SDS-PAGE analysis of sph3J and sph4J RBC membrane skeletons revealed unique changes in membrane skeleton proteins compared to each other and to the five known sph alleles. In sph3J, α- and β-spectrin are significantly decreased but ankyrin, protein 4.1 and protein 4.2 levels are normal. Surprisingly, band 3 is reduced to ~30% of normal, and both α- and β-adducin are nearly undetectable in sph3J RBCs. The presence of normal amounts of ankyrin, which binds band 3 tetramers, suggests that band 3 dimers are absent in sph3J RBCs. These observations indicate that previously unsuspected interactions, direct or indirect, exist between spectrin and band 3 (probably dimers) and between spectrin and adducin within the RBC membrane skeleton. In contrast to sph3J, all RBC membrane skeleton proteins appear normal by SDS-PAGE and western blot analyses of sph4J RBC membranes. Coupled with the severe hemolytic anemia present in these mice, these data suggest that interactions involving the C-terminus of α-spectrin, specifically cysteine 2384, are critical to RBC membrane integrity. Together, the sph3J and sph4J mouse models provide powerful resources for identifying critical interactions within the membrane skeleton that are relevant to the pathogenesis of hereditary elliptocytosis and spherocytosis.

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Proteomic Analysis of RBC Ghosts from the Beta-Adducin and Protein 4.2 Knock-Out Mouse Models of Inherited Hemolytic Anemia.

Blood ◽

10.1182/blood.v110.11.1728.1728 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1728-1728

Author(s):

Jason M. Wooden ◽

Greg L. Finney ◽

Michael J. MacCoss ◽

Luanne L. Peters ◽

Diana M. Gilligan

Keyword(s):

Mouse Models ◽

Hemolytic Anemia ◽

Membrane Skeleton ◽

Tandem Mass ◽

Secondary Effects ◽

Knock Out ◽

Rbc Membrane ◽

Protein 4.2 ◽

Knock Out Mice ◽

Rbc Membrane Skeleton

Abstract Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases with an incidence of 1:2500 to 1:5000 in populations of Northern European descent. While it is known that mild to severe inherited hemolytic anemias can arise from defects in the red blood cell (RBC) membrane skeleton, fundamental questions remain unanswered surrounding the clinical variability and non-erythroid effects of known RBC membrane skeleton mutations. To identify proteins that may be involved in disease severity and secondary effects, we used shotgun proteomics to globally profile proteins in RBC ghosts (i.e., RBC membrane skeleton and associated proteins) from well-defined mouse models of inherited hemolytic anemia. A peptide level ‘bottom-up’ analysis was performed on RBCs from normal mice, beta-adducin knock-out mice (Add2-KO, compensated anemia), and protein 4.2 knock-out mice (4.2-KO, mild anemia). For each genotype, whole blood was taken from independent biological replicates and RBCs were purified using cellulose acetate chromatography. The isolated RBCs were lysed to generate RBC ghosts whose protein complements were digested with trypsin. For each biological replicate, five replicate runs utilizing 0.1 ug digested protein were performed via microcapillary liquid chromatography coupled with tandem mass spectrometry. Normal versus diseased comparisons were made using a protein profile found consistently across all independent samples for each genotype. In total, 435 unique proteins were identified for the normal mouse RBC ghost. In contrast, 731 and 848 unique proteins were identified for the Add2-KO and 4.2-KO mice RBC ghosts respectively. Previously identified membrane skeleton proteins were found for all three genotypes with the predicted absence of the knock-out proteins. In addition to well-known membrane proteins, a surprising number of proteins were found involved in processes such as protein repair, protein degradation, Ras oncogene biology, and glycolysis. For both knock-out mice, a large number of proteins involved in translation were identified most likely reflecting their elevated reticulocytosis status. Comparison of the normal and Add2-KO RBC profiles revealed 5 proteins present only in normal the RBC while 53 proteins were present only in the diseased RBC. Likewise, normal vs 4.2 KO comparison revealed 6 proteins present only in the normal RBC while 111 were only in the diseased RBC. Comparison between the two KO mice revealed 34 proteins present only in the Add2-KO and 88 proteins present only in the 4.2 KO. Some of the identified differences are proteins with unknown functions (example, SH3-binding domain glutamic acid-rich protein like). Other differences involve proteins associated with diverse processes such as protein folding (Bcl2-associated athanogene 2), protein modification (magnesium-dependent phosphatase-1), protein transport (RAB35), metabolism (N-acetylneuraminic acid phosphatase), signal transduction (Prohibitin 2), and apoptosis (Rho GTPase activating protein 1). We report that tandem mass spec analysis of disease model RBC ghosts have demonstrated differences in their proteomes and that these identified differences potentially represent candidate proteins involved in disease severity and secondary effects.

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Quantitative Mass Spec Analysis Of RBCs From Patients With Hereditary Spherocytosis

Blood ◽

10.1182/blood.v122.21.4659.4659 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4659-4659

Author(s):

Bhuvaneswari Ramkumar ◽

David Kakhniashvili ◽

Steven Goodman ◽

Diana Gilligan

Keyword(s):

Mass Spectrometry ◽

Hereditary Spherocytosis ◽

Protein Composition ◽

Membrane Skeleton ◽

Mass Spectrometry Analysis ◽

Itraq Labeling ◽

Rbc Membrane ◽

Mass Spec ◽

Sds Page ◽

Rbc Membrane Skeleton

Introduction Spherocytic RBCs have increased fragility due to abnormalities of the components of the RBC membrane skeleton. From various studies about 30-40% of patients showed no molecular defect using standard Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Mass spectrometry analysis (proteomics) would provide a deeper insight into changes in RBC membrane skeleton protein composition in patients with hereditary spherocytosis (HS). The objective of this study is to identify biomarkers that explain phenotypic heterogeneity and severity of hemolysis and variability within the same family using proteomic tools. Methods Blood samples from affected and unaffected family members of patients with hereditary spherocytosis at Upstate Medical University have been collected after obtaining informed consent. The family presented here includes the affected mother (OS2) and affected grandfather (OS3) of the female patient (OS1) with HS. The grandfather underwent splenectomy many years ago. Samples were collected from the three affected members and the unaffected grandmother (OS4). Complete blood count, peripheral smears, reticulocyte percentage (%) and absolute reticulocyte count were performed on those samples. RBC ghosts were prepared by osmotic lysis and were fractionated into membrane and cytosol. Membrane fractions were analyzed by SDS-PAGE. Membrane and cytosol fractions are being analyzed by 4plex Isobaric tags for relative and absolute quantitation (iTRAQ) labeling. The four individual samples are labeled with amine-modifying four different iTRAQ reagents and then combined. The labeled samples are separated by SDS-PAGE, and then regions of the gel are cut out and subjected to in-gel trypsin digest. The labeled peptides are then analyzed by tandem mass spectrometry and relative abundance can be assigned to each of the four labeled samples within the same analysis, corresponding to the three patients and one control. This approach minimizes any differences in sample processing because all four samples are analyzed simultaneously on the mass spectrometer. Preliminary Results Reticulocyte % and absolute retic count were highest for our patient (13.5 %) compared to her mother (9.93%) and her grandfather (3.34%) who was post splenectomy. Hemoglobin and hematocrit were also lower for our patient (12.8/36.0) compared to her mother (13.3/37.1) and grandfather (16.9/48.1). Coomassie blue SDS-PAGE of RBC ghosts did not show any differences in protein composition at this level of detection. Mass spec analysis using 4plex iTRAQ labeling is currently underway. Conclusions Patients with hereditary spherocytosis exhibit a wide range of phenotypes even within the same family. Protein differences are often not detected at the level of SDS-PAGE. In-depth analysis by quantitative mass spectrometry allows the identification of differences that can correlate to severity of illness and aid in choices regarding treatment modalities. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2009-07-232199 ◽

2010 ◽

Vol 115 (9) ◽

pp. 1804-1814 ◽

Cited By ~ 10

Author(s):

Raymond F. Robledo ◽

Amy J. Lambert ◽

Connie S. Birkenmeier ◽

Marius V. Cirlan ◽

Andreea Flavia M. Cirlan ◽

...

Keyword(s):

Missense Mutation ◽

Hemolytic Anemia ◽

Stop Codon ◽

Premature Stop Codon ◽

Band 3 ◽

Cysteine Residue ◽

Normal Band ◽

Junctional Complex ◽

Rbc Membrane ◽

C Terminus

AbstractFive spontaneous, allelic mutations in the α-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph1J, sph2J, sph2BC, sphDem). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph3J, a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sphIhj, a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent β-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph4J, 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, β-adducin. The severity of anemia in sph4J indicates that the highly conserved cysteine residue at the C-terminus of α-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.

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A novel splicing mutation of the α-spectrin gene in the original hereditary pyropoikilocytosis kindred

Blood ◽

10.1182/blood-2005-05-1813 ◽

2005 ◽

Vol 106 (13) ◽

pp. 4367-4369 ◽

Cited By ~ 13

Author(s):

Daniel B. Costa ◽

Larisa Lozovatsky ◽

Patrick G. Gallagher ◽

Bernard G. Forget

Keyword(s):

Premature Termination Codon ◽

Membrane Skeleton ◽

Molecular Defect ◽

Culture Cells ◽

Rbc Membrane ◽

Tissue Culture Cells ◽

Gene Transcripts ◽

Consensus Splice Site ◽

Defective Allele ◽

Rbc Membrane Skeleton

Hereditary pyropoikilocytosis (HPP) is a severe hemolytic anemia due to abnormalities of the red blood cell (RBC) membrane skeleton. In the original HPP kindred, there is compound heterozygosity for an allele encoding a structural variant of α-spectrin (L207P) and an α-spectrin allele associated with a defect in α-spectrin production. To identify the molecular defect in the production-defective allele, reticulocyte α-spectrin cDNA from one of the original HPP patients was analyzed. Transcripts from the production-defective, non-L207P allele demonstrated a pattern of abnormal splicing between exons 22 and 23, resulting in insertion of intronic fragments with an in-frame premature termination codon. A G to A substitution at position +5 of the donor consensus splice site of IVS 22 was identified in the inserts. Following gene transfer into tissue culture cells, there was complete absence of normally spliced α-spectrin gene transcripts derived from a minigene containing the IVS 22 +5 mutation.

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Surface area-to-volume ratio, not cellular rigidity, determines red blood cell traversal through small capillaries

10.1101/2020.07.11.191494 ◽

2020 ◽

Author(s):

Arman Namvar ◽

Adam J. Blanch ◽

Matthew W. Dixon ◽

Olivia M. S. Carmo ◽

Boyin Liu ◽

...

Keyword(s):

Surface Area ◽

Marked Effect ◽

Volume Ratio ◽

Element Model ◽

Membrane Skeleton ◽

Measurable Effect ◽

Rbc Membrane ◽

Nonlinear Mechanical ◽

Small Channels ◽

Rbc Membrane Skeleton

SummaryThe remarkable deformability of red blood cells (RBCs) depends on the viscoelasticity of the plasma membrane and cell contents and the surface area to volume (SA:V) ratio; however, it remains unclear which of these factors is the key determinant for passage through small capillaries. We used a microfluidic device to examine the traversal of normal, stiffened, swollen, parasitised and immature RBCs. We show that dramatic stiffening of RBCs had no measurable effect on their ability to traverse small channels. By contrast, a moderate decrease in the SA:V ratio had a marked effect on the equivalent cylinder diameter that is traversable by RBCs of similar stiffness. We developed a finite element model that provides a coherent rationale for the experimental observations, based on the nonlinear mechanical behaviour of the RBC membrane skeleton. We conclude that the SA:V ratio should be given more prominence in studies of RBC pathologies.

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Human erythrocyte protein 4.2 deficiency associated with hemolytic anemia and a homozygous 40glutamic acid-->lysine substitution in the cytoplasmic domain of band 3 (band 3Montefiore)

Blood ◽

10.1182/blood.v81.8.2155.2155 ◽

1993 ◽

Vol 81 (8) ◽

pp. 2155-2165 ◽

Cited By ~ 57

Author(s):

AC Rybicki ◽

JJ Qiu ◽

S Musto ◽

NL Rosen ◽

RL Nagel ◽

...

Keyword(s):

Sequence Analysis ◽

Hemolytic Anemia ◽

Cytoplasmic Domain ◽

Band 3 ◽

Structural Abnormality ◽

Cell Deformability ◽

Whole Cell ◽

Rbc Membrane ◽

Protein 4.2 ◽

Lysine Substitution

Abstract Red blood cell (RBC) protein 4.2 deficiency is often associated with a moderate nonimmune hemolytic anemia, splenomegaly, and osmotically fragile RBCs resembling, but not identical to, hereditary spherocytosis (HS). In the Japanese type of protein 4.2 deficiency (protein 4.2Nippon), the anemia is associated with a point mutation in the protein 4.2 cDNA. In this report, we describe a patient with moderate and apparently episodic nonimmune hemolytic anemia with splenomegaly, spherocytosis, osmotically fragile RBCs, reduced whole cell deformability, and abnormally dense cells. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis analysis of the proposita's RBC membrane proteins showed an 88% deficiency of protein 4.2 and a 30% deficiency of glyceraldehyde-3-phosphate dehydrogenase (band 6). Structural and molecular analyses of the proposita's protein 4.2 were normal. In contrast, limited tryptic digestion of the proposita's band 3 showed a homozygous abnormality in the cytoplasmic domain. Analysis of the pedigree disclosed six members who were heterozygotes for the band 3 structural abnormality and one member who was a normal homozygote. Direct sequence analysis of the abnormal band 3 tryptic peptide suggested that the structural abnormality resided at or near residue 40. Sequence analysis of the proposita's band 3 cDNA showed a 232G-->A mutation resulting in a 40glutamic acid-->lysine substitution (band 3Montefiore). Allele-specific oligonucleotide hybridization was used to probe for the mutation in the pedigree, showing that the proposita was homozygous, and the pedigree members who were heterozygous for the band 3 structural abnormality were also heterozygous for the band 3Montefiore mutation. The band 3Montefiore mutation was absent in 26 chromosomes from race-matched controls and in one pedigree member who did not express the band 3 structural abnormality. In coincidence with splenectomy, the proposita's anemia was largely corrected along with the disappearance of most spherocytes and considerable improvements of RBC osmotic fragility, whole cell deformability, and cell density. We conclude that this hereditary hemolytic anemia is associated with the homozygous state for band 3Montefiore (40glutamic acid-->lysine) and a decreased RBC membrane content of protein 4.2. We speculate that band 3 structural abnormalities can result in defective interactions with protein 4.2 and band 6, and in particular, that the region of band 3 containing 40glutamic acid is involved directly or indirectly in interactions with these proteins.

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Comprehensive Analysis of Murine and Human RBC Ghost Proteomes.

Blood ◽

10.1182/blood.v108.11.1569.1569 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1569-1569

Author(s):

Jason M. Wooden ◽

Greg Finney ◽

Michael MacCoss ◽

Diana M. Gilligan

Keyword(s):

Mouse Models ◽

Shotgun Proteomics ◽

Membrane Skeleton ◽

Protein Repair ◽

Knock Out ◽

Human Samples ◽

Rbc Membrane ◽

Northern European ◽

Associated Proteins ◽

Rbc Membrane Skeleton

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Tropomodulin 1-null mice have a mild spherocytic elliptocytosis with appearance of Tropomodulin 3 in red blood cells and disruption of the membrane skeleton

Blood ◽

10.1182/blood-2010-02-268458 ◽

2010 ◽

Vol 116 (14) ◽

pp. 2590-2599 ◽

Cited By ~ 59

Author(s):

Jeannette D. Moyer ◽

Roberta B. Nowak ◽

Nancy E. Kim ◽

Sandra K. Larkin ◽

Luanne L. Peters ◽

...

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Membrane Skeleton ◽

Actin Dynamics ◽

Filament Length ◽

Western Blots ◽

Rbc Membrane ◽

Cellular Dehydration ◽

Rbc Membrane Skeleton

Abstract The short actin filaments in the red blood cell (RBC) membrane skeleton are capped at their pointed ends by tropomodulin 1 (Tmod1) and coated with tropomyosin (TM) along their length. Tmod1-TM control of actin filament length is hypothesized to regulate spectrin-actin lattice organization and membrane stability. We used a Tmod1 knockout mouse to investigate the in vivo role of Tmod1 in the RBC membrane skeleton. Western blots of Tmod1-null RBCs confirm the absence of Tmod1 and show the presence of Tmod3, which is normally not present in RBCs. Tmod3 is present at only one-fifth levels of Tmod1 present on wild-type membranes, but levels of actin, TMs, adducins, and other membrane skeleton proteins remain unchanged. Electron microscopy shows that actin filament lengths are more variable with spectrin-actin lattices displaying abnormally large and more variable pore sizes. Tmod1-null mice display a mild anemia with features resembling hereditary spherocytic elliptocytosis, including decreased RBC mean corpuscular volume, cellular dehydration, increased osmotic fragility, reduced deformability, and heterogeneity in osmotic ektacytometry. Insufficient capping of actin filaments by Tmod3 may allow greater actin dynamics at pointed ends, resulting in filament length redistribution, leading to irregular and attenuated spectrin-actin lattice connectivity, and concomitant RBC membrane instability.

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Expression of Tropomodulin1 (Tmod1) in the Heart Rescues Embryonic Lethality of Tmod1 Null Mice and Results in a Mild Hemolytic Anemia Due to Absence of Tmod1 in Red Blood Cells.

Blood ◽

10.1182/blood.v106.11.807.807 ◽

2005 ◽

Vol 106 (11) ◽

pp. 807-807

Author(s):

Velia M. Fowler ◽

Jeannette Moyer ◽

Roberta Nowak ◽

Kimberly Fritz-Six ◽

Carmela Ferreira-Mota ◽

...

Keyword(s):

Hemolytic Anemia ◽

Western Blotting ◽

Actin Filaments ◽

Membrane Skeleton ◽

Yolk Sac ◽

Embryonic Lethality ◽

Mean Corpuscular Hemoglobin ◽

Null Mouse ◽

Distribution Width ◽

Rbc Membrane

Abstract Tropomodulin1 (Tmod1) caps the pointed ends of actin filaments in the red blood cell (RBC) membrane skeleton and is proposed to regulate actin filament lengths and organization in the membrane skeleton. Tmod1 is first expressed at E7.5 of mouse embryogenesis in blood islands of the yolk sac and in the cardiac myocytes of the developing heart tube. Targeted deletion of the Tmod1 gene in mice leads to abnormal cardiac development and defective vasculogenesis and hematopoesis in the yolk sac, followed by embryonic lethality between E9.5–10. To investigate the function of Tmod1 in RBCs we rescued the embryonic lethality of the Tmod1 null mouse by crossing with a TOT mouse that expresses a Tmod1 transgene in the heart under the control of the α-myosin heavy chain promoter. Genotyping of litters from crosses of Tmod1-/+;TOT+ mice with Tmod1+/− mice demonstrates that embryos with no endogenous Tmod1 but expressing the Tmod1 transgene (Tmod1−/−;TOT+) develop to term with no apparent defects in vasculogenesis or hematopoeisis. Western blotting and immunofluorescence staining demonstrates that Tmod1 protein is present in the heart but absent from RBCs in Tmod1−/−;TOT+ rescued embryos or adult mice. Instead, the Tmod3 isoform that is normally present in embryonic RBCs, but not detected in adult RBCs of wild-type mice, is increased on the membranes of Tmod1-deficient adult RBCs. Hematological analyses reveal that these Tmod1-deficient mice exhibit a compensated mild hemolytic anemia, with decreased mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), increased red cell distribution width (RDW), and reticulocytosis. Quantitative western blotting demonstrates that major RBC membrane proteins are not altered in the absence of Tmod1, but that there is about a two-fold increase in levels of actin and tropomyosin on the membrane, suggesting that actin filaments may be longer than in normal RBCs. We conclude that 1) Tmod1 in RBCs is not required for embryonic development or viability, 2) yolk sac and hematopoetic defects in Tmod1 null mice are secondary to cardiac defects, 3) Tmod’s function in embryonic RBCs may be performed by the Tmod3 isoform that is normally expressed in these RBCs, and 4) the absence of Tmod1 in adult RBCs is incompletely compensated by up-regulation of Tmod3, leading to altered actin filaments, decreased membrane stability and a mild hemolytic anemia.

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Erythrocyte membrane changes of chorea-acanthocytosis are the result of altered Lyn kinase activity

Blood ◽

10.1182/blood-2011-05-355339 ◽

2011 ◽

Vol 118 (20) ◽

pp. 5652-5663 ◽

Cited By ~ 45

Author(s):

Lucia De Franceschi ◽

Carlo Tomelleri ◽

Alessandro Matte ◽

Anna Maria Brunati ◽

Petra H. Bovee-Geurts ◽

...

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Neurodegenerative Disorder ◽

Protein Composition ◽

Membrane Skeleton ◽

Band 3 ◽

Diagnostic Feature ◽

Lyn Kinase ◽

Rbc Membrane ◽

Membrane Changes

Abstract Acanthocytic RBCs are a peculiar diagnostic feature of chorea-acanthocytosis (ChAc), a rare autosomal recessive neurodegenerative disorder. Although recent years have witnessed some progress in the molecular characterization of ChAc, the mechanism(s) responsible for generation of acanthocytes in ChAc is largely unknown. As the membrane protein composition of ChAc RBCs is similar to that of normal RBCs, we evaluated the tyrosine (Tyr)–phosphorylation profile of RBCs using comparative proteomics. Increased Tyr phosphorylation state of several membrane proteins, including band 3, β-spectrin, and adducin, was noted in ChAc RBCs. In particular, band 3 was highly phosphorylated on the Tyr-904 residue, a functional target of Lyn, but not on Tyr-8, a functional target of Syk. In ChAc RBCs, band 3 Tyr phosphorylation by Lyn was independent of the canonical Syk-mediated pathway. The ChAc-associated alterations in RBC membrane protein organization appear to be the result of increased Tyr phosphorylation leading to altered linkage of band 3 to the junctional complexes involved in anchoring the membrane to the cytoskeleton as supported by coimmunoprecipitation of β-adducin with band 3 only in ChAc RBC-membrane treated with the Lyn-inhibitor PP2. We propose this altered association between membrane skeleton and membrane proteins as novel mechanism in the generation of acanthocytes in ChAc.

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