Spectrin Dimer Research Articles

PS1205 META‐ANALYSIS USING DATA OF RED BLOOD CELL ENZYME ASSAY AND PROTEOME IN PATIENTS WITH DIAMOND‐BLACKFAN ANEMIA

Background:Diamond‐Blackfan anemia (DBA) is a form of congenital bone marrow failure syndrome, which is characterized by RBC aplasia with varied malformation in infants. Elevated enzymatic activity of adenosine deaminase (ADA) is known to be a valuable biomarker of DBA. The ADA assay has been shown to be more sensitive than the gene test of ribosome subunit genes. We recently reported that reduced glutathione (GSH) in red blood cells (RBCs) is another biomarker of DBA, and simultaneous assessment of ADA and GSH enabled us to discriminate DBA patients from normal subjects as well as unaffected family members (Blood Cells Mol Dis 59:31–36, 2016).Aims:This study was performed to investigate the detailed changes in enzymatic activity and enzyme protein mass in DBA erythrocytes. We reanalyzed the enzymatic activity and GSH in 14 DBA patients and compared the results with those obtained from simultaneously assayed 14 normal subjects. Furthermore, we performed a meta‐analysis using proteome data as recently reported by Pesciotta, et al. (PLoS ONE 10:e0140036, 2015).Methods:The gene mutations of the enrolled patients were previously confirmed. We estimated the activity of RBC enzymes and GSH content by a standard method using a spectrophotometer. We calculated the relative expression of both enzymatic activity and protein content. The statistical analyses were performed by Wilcoxon rank‐sum test and a P value of <0.001 was considered statistically significant.Results:The combined analyses of proteomic and enzymatic data revealed that ADA, PGD, and HK‐1 (proteome)/HK (activity) were elevated, while PFKL (proteome)/PFK (activity) and PKLR (proteome)/PK (activity) were decreased. The G6PD enzyme activity, but not its protein level, was characteristically increased.Summary/Conclusion:We speculate that the discrepancy between the activity and protein level of G6PD was attributable to the decreased inhibition of G6PD activity by NADPH. In other words, G6PD activity was supposedly activated by a lower NADPH level in DBA. To maintain the affordable level of GSH in RBCs, reduction of GSSG was necessarily maximized through the reaction catalyzed by glutathione reductase, which used NADPH as a cofactor. As a result, NADPH content was presumably exhausted, resulting in decreased inhibition of G6PD. In this study, we found that HK activity and HK‐1 content were elevated significantly. On the other hand, the activity and protein content of two important rate‐limiting enzymes (PFK/PK and PFKL/PKLR) involved in glycolysis were found to be decreased. Higher demand for NADPH production through the pentose phosphate pathway leads to a reduction in glucose consumption in the glycolytic pathway, and/or activation of p53 leads to inhibition of glycolysis, as previously reported. The present study shows the inhibition of glycolysis in the RBCs in DBA. It needs to be elucidated whether impaired glycolysis partly accounts for premature cell death of erythroblasts in DBA.Abbreviations: DBA: Diamond‐Blackfan anemia; RBC: red blood cell; ADA: adenosine deaminase; GSH: reduced glutathione; PGD: 6‐phosphogluconate dehydrogenase; HK: hexokinase; PFK (L): 6‐phosphofructokinase (liver type); PK (LR): pyruvate kinase (liver and erythrocytes type); G6PD: glucose‐6‐phosphate dehydrogenase; NADPH: reduced nicotinamide adenine dinucleotide phosphate; GSSG: glutathione disulfide.

Unfolding Transitions and Interdomain Coupling in Human Dystrophin Spectrin Repeats

Dystrophin is a 427 kDa rod-shaped protein encoded by one of the largest mammalian genes. The protein is formed by 27 domains, with 24 of them being single spectrin repeats interposed by two hinge regions. Single point and missing domain mutations within Dystrophin have been linked to Becker's Muscular Dystrophy (BMD), which is a degenerative muscle disease. This suggests that one role for Dystrophin is to reduce mechanical force between the sarcolemma membrane and the cytoskeleton. The mechanism for this function is unknown. The observation that single point mutations within Dystrophin are correlated with BMD suggests there is communication between each amino acid within each Dystrophin domain and that this communication extends to its neighboring domains. This led to the hypothesis that as force is transduced through the spectrin repeats, this results in the partial unfolding of the repeats which then dissipates force. If this partial unfolding of a given domain is accentuated when another repeat domain is in tandem, then this is consistent with negative coupling. Conversely, stabilization of one domain by another is positive coupling. Molecular dynamics (MD) forced unfolding experiments were consistent with negative coupling through non-additive force trajectories for tandem domains compared to individual domains. Therefore, we designed an experimental approach to test the computational results.Using monomeric spectrin 17, monomeric spectrin 18, and dimeric spectrin 17-18, we experimentally defined the thermodynamic and structural basis of energetic coupling between the spectrin domains through differential scanning calorimetry (DSC), fluorescence lifetime (FLT), and Overhauser dynamic nuclear polarization (ODNP). A combined approach utilizing these methods will provide insight into how coupling between spectrin repeats affects signal propagation and mechanical force dissipation.

Binding of hemin, hematoporphyrin, and protoporphyrin with erythroid spectrin: fluorescence and molecular docking studies.

Free heme has toxic effects, for example lipid peroxidation, DNA damage, and protein aggregation. In severe hemolysis, which occurs during pathological states, for example sickle cell disease, ischemia reperfusion, and malaria, levels of free heme increase inside erythrocytes. The purpose of this study was to investigate whether spectrin, the major erythroid cytoskeleton protein, is involved as an acceptor of free heme. We compared the interactions of three heme derivatives, hemin chloride, hematoporphyrin, and protoporphyrin-IX, with dimeric and tetrameric spectrin. The dissociation constants (K d) for binding to spectrin dimer and tetramer were 0.57 and 1.16 µM respectively. Thermodynamic data associated with this binding revealed the binding to be favored by a positive change in entropy. Although molecular docking studies identified the SH3 domain as the unique binding site of these heme derivatives to erythroid spectrin, experimental results indicated a binding stoichiometry of 1 heme attached to both dimeric and tetrameric spectrin, indicating the common self-associating domain to be the unique binding site. We also noticed heme-induced structural changes in the membrane skeletal protein. Erythroid spectrin could thus act as a potential acceptor of heme, particularly relevant under disease conditions.

Probing conformational stability and dynamics of erythroid and nonerythroid spectrin: effects of urea and guanidine hydrochloride.

We have studied the conformational stability of the two homologous membrane skeletal proteins, the erythroid and non-erythroid spectrins, in their dimeric and tetrameric forms respectively during unfolding in the presence of urea and guanidine hydrochloride (GuHCl). Fluorescence and circular dichroism (CD) spectroscopy have been used to study the changes of intrinsic tryptophan fluorescence, anisotropy, far UV-CD and extrinsic fluorescence of bound 1-anilinonapthalene-8-sulfonic acid (ANS). Chemical unfolding of both proteins were reversible and could be described as a two state transition. The folded erythroid spectrin and non-erythroid spectrin were directly converted to unfolded monomer without formation of any intermediate. Fluorescence quenching, anisotropy, ANS binding and dynamic light scattering data suggest that in presence of low concentrations of the denaturants (up-to 1M) hydrogen bonding network and van der Waals interaction play a role inducing changes in quaternary as well as tertiary structures without complete dissociation of the subunits. This is the first report of two large worm like, multi-domain proteins obeying twofold rule which is commonly found in small globular proteins. The free energy of stabilization (ΔGu H 2 0) for the dimeric spectrin has been 20 kcal/mol lesser than the tetrameric from.

Novel exon 2 spectrin mutation and intragenic crossover: three morphological phenotypes associated with four distinct spectrin defects

Hereditary pyropoikilocytosis is a severe hemolytic anemia caused by spectrin deficiency and defective spectrin dimer self-association, typically found in African populations. We describe two Utah families of northern European ancestry including 2 propositi with atypical non-microcytic hereditary pyropoikilocytosis, 7 hereditary elliptocytosis members and one asymptomatic carrier. The underlying molecular defect is a novel mutation in the alpha(α) spectrin gene, SPTA(R34P) that impairs spectrin tetramer formation. It is inherited in trans to the hypomorphic SPTA(αLELY) in the 2 propositi and 5 of 7 hereditary elliptocytosis individuals indicating that SPTA(αLELY) is not the sole determinant of the variable clinical expression. α Spectrin mRNA was mildly decreased in all hereditary elliptocytosis subjects, whereas both hereditary pyropoikilocytosis propositi had a severe decrease to ~10% of normal. Genotyping identified a unique SPTA intragenic crossover and uniparental disomy in one hereditary elliptocytosis individual. Two additional crossover events demonstrated the susceptibility of SPTA gene to rearrangement and revealed a novel segregation of the two SPTA(αLELY) mutations. We conclude that the profound phenotypic heterogeneity in these families can be attributed to the SPTA(R34P) mutation in combination with: 1) inheritance in trans of either SPTA(αLELY); or 2) the wild-type SPTA; 3) a decrease of α spectrin mRNA; and 4) SPTA intragenic crossover.

Identification of a Novel Role for Dematin in Regulating Red Cell Membrane Function by Modulating Spectrin-Actin Interaction

The membrane skeleton plays a central role in maintaining the elasticity and stability of the erythrocyte membrane, two biophysical features critical for optimal functioning and survival of red cells. Many constituent proteins of the membrane skeleton are phosphorylated by various kinases, and phosphorylation of β-spectrin by casein kinase and of protein 4.1R by PKC has been documented to modulate erythrocyte membrane mechanical stability. In this study, we show that activation of endogenous PKA by cAMP decreases membrane mechanical stability and that this effect is mediated primarily by phosphorylation of dematin. Co-sedimentation assay showed that dematin facilitated interaction between spectrin and F-actin, and phosphorylation of dematin by PKA markedly diminished this activity. Quartz crystal microbalance measurement revealed that purified dematin specifically bound the tail region of the spectrin dimer in a saturable manner with a submicromolar affinity. Pulldown assay using recombinant spectrin fragments showed that dematin, but not phospho-dematin, bound to the tail region of the spectrin dimer. These findings imply that dematin contributes to the maintenance of erythrocyte membrane mechanical stability by facilitating spectrin-actin interaction and that phosphorylation of dematin by PKA can modulate these effects. In this study, we have uncovered a novel functional role for dematin in regulating erythrocyte membrane function.

An unexpectedly bizarre blood film in hemoglobin H disease

A 3-year-old boy from Saudi Arabia had been followed up with a diagnosis of hemoglobin H disease, identified in a neonatal screening programme. His parents were second cousins. Alpha globin genotyping at birth had revealed homozygosity for the non-deletional Saudi-T mutation (αTα/αTα), both parents being carriers. He had remained clinically asymptomatic. Red cell indices at the age of three years were: RBC 3.84 x 1012/l, hemoglobin concentration 80 g/l, MCV 61 fl, MCH 20.9 pg, MCHC 34 g/l, red cell distribution width 34.8 (normal range 11.8-14.3) and reticulocyte count 447 x109/l. His blood film, in addition to microcytosis, showed gross poikilocytosis with the abnormal red cells including elliptocytes and many bizarre poikilocytes, some very small (image). Although poikilocytosis can be prominent in hemoglobin H disease, we have not seen a case with this degree of abnormality. The film was reminiscent of hereditary pyropoikilocytosis. For this reason red cell membrane studies were initiated. An eosin-5 maleimide (EMA)-binding assay [1] gave a twin peak fluorescence profile: 57% of phenotypically normal red cells (MCF = 64.7 units) and 42% having reduced fluorescence (MCF = 32.4 units). Spectrin (Sp) analysis showed a Sp dimer content of 25.6 %, compared with 7.4 % for a normal control within the same assay. In limited trypsin digestion of spectrin, SpαI/50 variant was detected in the child, and absent in the mother. Neither had an increase of SpαV/41 tryptic peptide, thus excluding the presence of the polymorphic low expression allele, SpαLELY. Maternal red cells were not elliptocytic and had normal spectrin dimer content and normal EMA binding. A paternal sample was not available for testing. Although the EMA-binding assay has high specificity and sensitivity for hereditary spherocytosis, it can also be abnormal in hereditary pyropoikilocytosis, Southeast Asian ovalocytosis, cryohydrocytosis, and some cases of congenital dyserythropoietic anemia type II [2]. Hereditary elliptocytosis usually gives a single fluorescence peak within the range for normal red cells but occasionally twin peaks are observed. In this child with co-inheritance of hemoglobin H disease and hereditary elliptocytosis, the severity of the hemolytic anaemia is that expected in uncomplicated hemoglobin H disease but the bizarre blood film reflects an interaction of the hemoglobinopathy and the membrane defect. 1

A genomic deletion causes truncation of α-spectrin and ellipto-poikilocytosis

We report on a truncated α-spectrin chain, spectrinExeter, associated with ellipto-poikilocytosis. Analysis of erythrocyte membranes of affected individuals revealed a truncated α-spectrin chain with normal amounts of spectrin dimer. In the proband and her father, one haploid set of α-spectrin cDNA lacked exons 11 and 12, leading to partial deletion of repeats α4 and α5 (83 amino acids) of the α-spectrin chain. In one allele of genomic DNA, a 3567bp deletion starting in intron 10 and ending in intron 12 of the SPTA1 gene was found. The common polymorphic SPTA1 αLELY allele was found in trans to the SPTA1αExeter allele in the proband. The proband had inherited the SPTA1Exeter allele from her father and the αLELY allele from her healthy, asymptomatic mother. This is the first report of an interstitial deletion in the SPTA1 gene associated with ellipto-poikilocytosis.

Control of Erythrocyte Membrane-Skeletal Cohesion by the Spectrin-Membrane Linkage

Spectrin tetramer is the major structural member of the membrane-associated skeletal network of red cells. We show here that disruption of the spectrin-ankyrin-band 3 link to the membrane leads to dissociation of a large proportion of the tetramers into dimers. Noncovalent perturbation of the linkage was induced by a peptide containing the ankyrin-binding site of the spectrin beta-chain, and covalent perturbation by treatment with the thiol reagent, N-ethylmaleimide (NEM). This reagent left the intrinsic self-association capacity of the spectrin dimers unaffected and disturbed only the ankyrin-band 3 interaction. The dissociation of spectrin tetramers on the membrane into functional dimers was confirmed by the binding of a spectrin peptide directed against the self-association sites. Dissociation of the tetramers resulted, we infer, from detachment of the proximal ends of the constituent dimers from the membrane, thereby reducing their proximity to one another and thus weakening their association. The measured affinity of the interaction of the peptides with the free dimer ends on the membrane permits an estimate of the equilibrium between intact and dissociated tetramers on the native membrane. This indicates that in the physiological state the equilibrium proportion of the dissociated tetramers may be as high as 5-10%. These findings enabled us to identify an additional important functional role for the spectrin-ankyrin-band 3 link in regulating spectrin self-association in the red cell membrane.

Crystal Structure of the Nonerythroid α-Spectrin Tetramerization Site Reveals Differences between Erythroid and Nonerythroid Spectrin Tetramer Formation

We have solved the crystal structure of a segment of nonerythroid alpha-spectrin (alphaII) consisting of the first 147 residues to a resolution of 2.3 A. We find that the structure of this segment is generally similar to a corresponding segment from erythroid alpha-spectrin (alphaI) but exhibits unique differences with functional significance. Specific features include the following: (i) an irregular and frayed first helix (Helix C'); (ii) a helical conformation in the junction region connecting Helix C' with the first structural domain (D1); (iii) a long A(1)B(1) loop in D1; and (iv) specific inter-helix hydrogen bonds/salt bridges that stabilize D1. Our findings suggest that the hydrogen bond networks contribute to structural domain stability, and thus rigidity, in alphaII, and the lack of such hydrogen bond networks in alphaI leads to flexibility in alphaI. We have previously shown the junction region connecting Helix C' to D1 to be unstructured in alphaI (Park, S., Caffrey, M. S., Johnson, M. E., and Fung, L. W. (2003) J. Biol. Chem. 278, 21837-21844) and now find it to be helical in alphaII, an important difference for alpha-spectrin association with beta-spectrin in forming tetramers. Homology modeling and molecular dynamics simulation studies of the structure of the tetramerization site, a triple helical bundle of partial domain helices, show that mutations in alpha-spectrin will affect Helix C' structural flexibility and/or the junction region conformation and may alter the equilibrium between spectrin dimers and tetramers in cells. Mutations leading to reduced levels of functional tetramers in cells may potentially lead to abnormal neuronal functions.

A Fused α-β “Mini-spectrin” Mimics the Intact Erythrocyte Spectrin Head-to-head Tetramer

Head-to-head assembly of two spectrin heterodimers to form an actin-cross-linking tetramer is a physiologically dynamic interaction that contributes to red cell membrane integrity. Recombinant beta-spectrin C-terminal and alpha-spectrin N-terminal peptides can form tetramer-like univalent complexes, but they cannot evaluate effects of the open-closed dimer interactions or lateral associations of the two-spectrin strands on tetramer formation. In this study we produced and characterized a fused "mini-spectrin dimer" containing the beta-spectrin C-terminal region linked to the alpha-spectrin N-terminal region. This fused mini-spectrin mimics structural and functional properties of intact, full-length dimers and tetramers, including lateral association of the alpha and beta subunits in the dimer and formation of a closed dimer. High performance liquid chromatography gel filtration analyses of this mini-spectrin provide the first direct non-imaging experimental evidence for open and closed spectrin dimers and show that dimer-tetramer-oligomer interconversion is slow at low temperatures and accelerated at 30 degrees C, analogous to full-length spectrin. This protein exhibits wild type dimer-tetramer dissociation constants of approximately 1 mum at 30 degrees C, independent of initial oligomeric state. Conformational states of the mini-spectrin dimer were probed further using chemical cross-linking, which identified distinct groups of cross-links for "open" and "closed" dimers and confirmed the N-terminal region of alpha-spectrin remains highly flexible in the complex, exhibiting closely analogous structures to those observed for the isolated alpha-spectrin N-terminal using NMR (Park, S., Caffrey, M. S., Johnson, M. E., and Fung, L. W. (2003) J. Biol. Chem. 278, 21837-21844). This fusion protein should serve as a useful template for structural and functional studies of the divalent tetramer site.

Canine elliptocytosis due to a mutant β‐spectrin

A 5-year-old, spayed female, mixed-breed dog with persistent elliptocytosis was evaluated at the Veterinary Medical Teaching Hospital at Kansas State University. The elliptocytosis was asymptomatic and was detected during the evaluation of lameness. When subjected to shear stress in an ektacytometer, the dog's erythrocytes had reduced cellular deformability and erythrocyte membranes had decreased mechanical stability. Analysis of erythrocyte membrane spectrin by nondenaturing gel electrophoresis revealed an increased amount of spectrin dimers, indicating a defect in spectrin self-association. DNA analysis detected a beta-spectrin mutation in codon 2110 in which threonine was replaced by methionine. This mutation likely altered the molecular structure of the erythrocyte membrane, leading to impaired spectrin self-association and elliptocyte formation.

Adhesive activity of Lu glycoproteins is regulated by interaction with spectrin

AbstractThe Lutheran (Lu) and Lu(v13) blood group glycoproteins function as receptors for extracellular matrix laminins. Lu and Lu(v13) are linked to the erythrocyte cytoskeleton through a direct interaction with spectrin. However, neither the molecular basis of the interaction nor its functional consequences have previously been delineated. In the present study, we defined the binding motifs of Lu and Lu(v13) on spectrin and identified a functional role for this interaction. We found that the cytoplasmic domains of both Lu and Lu(v13) bound to repeat 4 of the α spectrin chain. The interaction of full-length spectrin dimer to Lu and Lu(v13) was inhibited by repeat 4 of α-spectrin. Further, resealing of this repeat peptide into erythrocytes led to weakened Lu-cytoskeleton interaction as demonstrated by increased detergent extractability of Lu. Importantly, disruption of the Lu-spectrin linkage was accompanied by enhanced cell adhesion to laminin. We conclude that the interaction of the Lu cytoplasmic tail with the cytoskeleton regulates its adhesive receptor function.

Novel Exon 2 Alpha Spectrin Mutation in a Caucasian Family with Pyropoikilocytosis, Elliptocytosis, and Normal Morphology: Three Phenotypes Due to Decreased Spectrin mRNA and Intragenic Crossover

Mutations of alpha spectrin involving the heterodimer self-association site represent a common group of membrane skeletal defects resulting in hereditary elliptocytosis (HE) and a closely related disorder, hereditary pyropoikilocytosis (HPP). HPP is characterized by extreme anisocytic microcytosis, hemolytic anemia, severe spectrin dimer self-association defect, and spectrin deficiency. The conventional explanation for the different phenotypes is that HPP subjects are compound heterozygotes for a functional alpha spectrin defect that interferes with tetramer assembly due to missense mutations (commonly present in exon 2), and a second defect that results in decreased synthesis of normal alpha spectrin. In contrast, HE subjects have normal spectrin content and a less severe self-association defect. The modifying factor accounting for decreased synthesis of alpha spectrin is a hypomorphic mutation termed alpha spectrin LELY. LELY is a low-expression allele of the alpha spectrin gene characterized by a C>G mutation in codon 1857 of exon 40 and a C>T mutation in intron 45 resulting in decreased assembly of alpha/beta spectrin dimers. Here, we describe a family of Northern European descent in which six members in three generations were heterozygous for a novel mutation in alpha spectrin exon 2, codon 34: CGG->CCG (Arg>Pro) and for alpha spectrin LELY inherited in trans. Affected members harboring this novel exon 2 mutation exhibit three different RBC phenotypes: atypical HPP (1/6), HE (4/6), and normal (1/6) (see figure). Quantitation of RBC membrane proteins in subjects with abnormal RBC morphology revealed an increase in spectrin dimers due to an increase in the mutant 74kD alpha spectrin peptide. There was a significant association of abnormal morphology with the levels of dimers and mutant 74kD peptide (N<HE<HPP). In addition, HPP and HE subjects had a greater expression of mutant exon 2 allele compared to wild type (by allele specific PCR) at a ratio approximating 3:1. Moreover, the HPP subject had a decreased amount of reticulocyte total alpha spectrin mRNA (~10% of normal) while HE individuals had only a slight decrease in reticulocyte total alpha spectrin mRNA (~ 85% of normal). One individual (II-6), who had an exon 2 missense mutation and homozygous intron 45 LELY mutation exhibiting normal RBC morphology, was investigated in greater detail. After ruling out non-paternity by standard testing, genotyping of his alpha spectrin gene was performed and an intragenic crossover detected, resulting in homozygosity for the intron 45 LELY mutation and wild type exon 40 (see figure). We conclude that the phenotype of individual II-6 is ameliorated by this de novo mitotic event. The HPP phenotype of the propositus is likely due to decreased transcription of both alpha spectrin alleles; however, its molecular basis remains to be determined. # Equal contribution. [Display omitted]

Identificação bioquímica e molecular das variantes de eliptocitose hereditária: importância da digestão tríptica da espectrina

Abstract Hereditay elliptocytosis (HE) is a relatively common,clinically and genetically heterogenous disorder, characterized bythe presennce of a large number of ellypticlly-shaped red bloodcells in the peripheral blood.From the biochemical point of view,several alterations of the major membrane skeleton proteins suchas spectrin (Sp) and protein 4.1 have been observed, influencingthe structure or quantity of these proteins in affected erythrocytes.Defects within the Sp molecule include shortening of the α or β-chain and, in most cases, alterations in the Sp-dimer self-associationprocess.The N terminus of α -spectrin and the C terminus of βspectrin are known to play important roles in the self-associationof spectrin dimers into tetramers and larger oligomers, aphenomenon that confers stability to the membrane skeleton. SpαI domain mutants have been detected after limited tryptic digestionof Sp, which shows the presence of peptides derived from thenormal αI domain of 80 kDa. Each variant has been classifiedaccording to the molecular weight and isoelectric point of theabnormal peptide: Sp αI/78, Sp αI/74, Sp αI/65, Sp αI/46, Sp αI/50b and Sp αI/43. In this study, six propositi and eleven familymembers with elliptocytic red cells were evaluated. Limited trypticdigestion of spectrin, followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) revealed both adecrease in the αI domain (80 kDa) and the presence of abnormalpeptides with molecular weights of 65 kDa and 46 kDa. In onestructural variant, there was an increase in a 74 kDa peptide, withoutalterations in the αI domain. The v¤ 41 polymorphism, with a low-expression, was also encountered in one propositus and her mother.All of these spectrin variants inhibit self-association to formtetramers and higher oligomers, a process that is known to bedependent upon an intact αI domain, and one which is thought tobe important in the formation of the spectrin-actin-4.1 network onthe inner surface of the red blood cell membrane.

Dematin and Adducin Provide a Novel Link between the Spectrin Cytoskeleton and Human Erythrocyte Membrane by Directly Interacting with Glucose Transporter-1

Dematin and adducin are actin-binding proteins located at the spectrin-actin junctions, also called the junctional complex, in the erythrocyte membrane. Here we propose a new model whereby dematin and adducin link the junctional complex to human erythrocyte plasma membrane. Using a combination of surface labeling, immunoprecipitation, and vesicle proteomics approaches, we have identified glucose transporter-1 as the receptor for dematin and adducin in the human erythrocyte membrane. This finding is the first description of a transmembrane protein that binds to dematin and adducin, thus providing a rationale for the attachment of the junctional complex to the lipid bilayer. Because homologues of dematin, adducin, and glucose transporter-1 exist in many non-erythroid cells, we propose that a conserved mechanism may exist that couples sugar and other related transporters to the actin cytoskeleton.

A Novel Mutation of the α Spectrin Gene in a Family of Northern European Descent Is Associated with Three Different Phenotypes.

Mutations of α spectrin (Sp) involving the Sp heterodimer self-association site (the α I domain of Sp) represent the most common group of membrane skeletal defects in hereditary elliptocytosis (HE) and a closely related disorder, hereditary pyropoikilocytosis (HPP). HPP is characterized by extreme anisocytic microcytosis, a severe spectrin dimer self-association defect and spectrin deficiency. The conventional explanation for the different phenotypes is that HPP subjects are compound heterozygotes for an α sp defect that interferes with sp tetramer assembly and a second defect which results in decreased synthesis of functional α sp. In contrast, HE subjects have normal spectrin content and a less severe sp self-association defect. The clinical expression of HE/HPP is influenced by the inheritance of modifying factors such as the α Sp hypomorphic mutation, α LELY. α LELY is a low expression allele of the α Sp gene characterized by a C>G mutation in codon 1857 of exon 40 and a C>T-12 mutation in intron 45 that is responsible for partial skipping of exon 46, which is essential for the functional assembly of α/b sp dimers. Here we describe a family of northern European descent in which members had different erythrocyte morphology ranging from atypical HPP to HE to normal and a novel αSp mutation. Quantitation of RBC membrane proteins of the propositus with atypical non-microcytic HPP revealed 48% spectrin dimers (control 10%) due to a marked increase in the 74kD αI Sp peptide. There was only a slight decrease in the spectrin/band 3 ratio, which correlated with the normocytic morphology. There was also an abnormal α Sp peptide at 41kD suggesting presence of α LELY. Sequencing of his α Sp gene revealed heterozygosity for a novel mutation in exon 2, codon 34: CGG->CCG (Arg>Pro) and heterozygosity for α LELY. These mutations were also present in his brother and daughter who have HE, while another son, who had Arg34Pro, but not α LELY has repeatedly confirmed normal morphology (data in pedigree, figure 1). Ongoing clinical, α Sp peptide, DNA and quantitative Real Time PCR mRNA studies of extended family reveal a complex interaction of codon 34: G>C mutation with α LELY and different levels of transcripts of α Sp alleles. #equal contribution. [Display omitted]

Dematin and Adducin Provide a Critical Link between the Spectrin Cytoskeleton and the Erythrocyte Membrane Via Glucose Transporter-1.

There is considerable interest in the elucidation of the mechanism that governs the linkage of elongated spectrin molecules to the erythrocyte plasma membrane. The mechanism by which the “head” region of the spectrin dimer, which participates in tetramer formation, binds to the membrane via ankyrin and band 3 has been reasonably well characterized. However, the mechanism by which the tail end of the spectrin dimer is anchored to the plasma membrane is not completely understood. Dematin and adducin are actin binding proteins located at the spectrin-actin junctions or “junctional complex” in the erythrocyte membrane. Individual suppression of their function in mice by the gene deletion exerts a modest effect on erythrocyte shape and membrane stability. In contrast, the combined deletion of dematin and adducin genes results in severe defects of erythrocyte shape, membrane instability, and hemolysis. Based on these findings, we proposed a model whereby dematin and adducin could function as a molecular bridge linking the junctional complex to the plasma membrane. Using a combination of cell surface labeling, immunoprecipitation, and vesicle proteomics, we have identified glucose transporter-1 as the receptor for dematin and adducin in the human erythrocyte membrane. This finding is the first description of a transmembrane protein that binds to dematin and adducin, thus providing a rationale for the attachment of the cytoskeletal junctional complex to the lipid bilayer via glucose transporter-1. Since homologues of dematin, adducin, and glucose transporter-1 exist in many non-erythroid cells, we propose that a conserved mechanism may exist that couples sugar and other related transporters to the actin cytoskeleton.

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

Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) is a parasite-derived protein that appears on the cytoplasmic surface of the host cell membrane in the later stages of the parasite's development where it associates with membrane skeleton. We have recently demonstrated that a 60-residue fragment (FIa1, residues 38-97) of PfEMP3 bound to spectrin. Here we show that this polypeptide binds specifically to a site near the C terminus of alpha-spectrin at the point that spectrin attaches to actin and protein 4.1R in forming the junctions of the membrane skeletal network. We further show that this polypeptide disrupts formation of the ternary spectrin-actin-4.1R complex in solution. Importantly, when incorporated into the cell, the PfEMP3 fragment causes extensive reduction in shear resistance of the cell. We conjecture that the loss of mechanical cohesion of the membrane may facilitate the exit of the mature merozoites from the cell.

Initiation and Propagation of Spectrin Heterodimer Assembly Involves Distinct Energetic Processes

Red cell spectrin alpha and beta subunits consist primarily of many tandem homologous motifs with very similar three- helix-bundle structures and similar dimer interfaces. Although misassembled homodimers can form under some conditions, correctly aligned heterodimers consistently assemble provided a small "dimer initiation" site near the actin binding domain is present. The dimer initiation site has been characterized to some extent, but little is known about the subsequent, low-affinity lateral interactions of the remaining motifs along the length of this ropelike molecule or the forces involved in these two steps of the dimerization process. In this study, we used isothermal titration calorimetry to deduce the mechanism and energetics of the two heterodimer assembly phases. The high-affinity initiation of dimerization is primarily enthalpically driven, which is consistent with initial alignment and docking of specific complementary alpha and beta motifs in the dimer initiation site driven by long-range electrostatic interactions followed by tight binding stabilized by hydrogen bonds and other hydrophilic interactions. In contrast, the subsequent weak lateral associations of additional motifs are primarily entropically driven, suggesting binding primarily involves weak hydrophobic interactions. Although initial docking is largely electrostatic, the only lateral interaction within the first four pairs of motifs that involves a net change in protons is the interaction of the alpha18 and beta4 repeats. This substoichiometric uptake of protons could be due to a pKa shift of a histidine in the alpha18 motif located near the dimer interface in a proposed homology-based model. On the basis of this analysis of heterodimer thermodynamics, a detailed model of spectrin dimer assembly is proposed.