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At least 775 records · Page 43Linked to original sources
PubMed2000

Binding of anti-spectrin antibodies to red blood cells and vesiculation in various in vivo and in vitro ageing conditions in the rat.

In this study, the binding of naturally occurring antibodies as well as of induced anti-spectrin antibodies to red blood cells (RBC), in relation with different ageing conditions, was investigated in the rat. RBC from aged animals, or from rats whose RBC were age-induced either by means of hypertransfusion (which blocks erythropoiesis) or by treatment with clodronate-containing liposomes (which reduces RBC removal from circulation), were used. Attainment of RBC ageing was demonstrated by MCV reduction and by an increase of both RBC density and 4.1a/4.1b RBC membrane protein ratio. The results demonstrate an augmented anti-spectrin antibody binding to RBC in relation with their ageing condition, especially when induced by hypertransfusion. The vesiculation process was also investigated and correlated with antibody binding: vesicles were found only in the plasma of clodronate-treated rats, whose RBC showed the lowest level of anti-spectrin antibody binding with respect to the other groups. In addition, RBC preserved in vitro in different media showed a binding of anti-spectrin antibody, which inversely correlated with the vesiculation process. On the whole, the latter results suggest a protective effect of vesicles towards IgG opsonization of aged RBC.

Aging↗
PubMed1996

Spectrin: on the path from structure to function.

New structural analyses of the spectrin family of actin cross-linking proteins are providing molecular explanations for both the interchain binding between the alpha and beta chains of spectrin and the intermolecular associations between spectrin and other proteins. Additionally, the analyses bring into focus a conformation which may explain aspects of spectrin's interaction with lipids.

Protein Structure, Tertiary↗
PubMed2002

Pathways and intermediates in forced unfolding of spectrin repeats.

Spectrin repeats are triple-helical coiled-coil domains found in many proteins that are regularly subjected to mechanical stress. We used atomic force microscopy technique and steered molecular dynamics simulations to study the behavior of a wild-type spectrin repeat and two mutants. The experiments indicate that spectrin repeats can form stable unfolding intermediates when subjected to external forces. In the simulations the unfolding proceeded via a variety of pathways. Stable intermediates were associated to kinking of the central helix close to a proline residue. A mutant stabilizing the central helix showed no intermediates in experiments, in agreement with simulation. Spectrin repeats may thus function as elastic elements, extendable to intermediate states at various lengths.

Animals↗
PubMed1991

Temperature and pH dependence of the self-association of human spectrin.

The self-association of human spectrin between 21 and 35 degrees C and between pH 6.5 and 9.5 has been studied at sedimentation equilibrium. For a given set of solution conditions between pH 6.5 and 8.5, coincidence of omega function plots as a function of total spectrin concentration (0-2 g/L) indicated that equilibrium was attained and that no significant concentration of solute was incapable of participating in the self-association reaction. Above pH 8.5, however, irreversible aggregation occurred, inferred from a failure of overlap in the omega function and molecular weight distributions. The behavior of spectrin can best be described by a cooperative isodesmic model, in which the promoter for association is the heterodimer and for which K12 is between 10(6) and 10(7) M-1 (depending on pH and temperature) and all other K are approximately 10(6) M-1. The returned values of the second viral coefficient for this model fall within the range calculated from the charge and Stokes radius of spectrin. Association appears to be favored slightly by decreased temperature and by decreased pH. The pH dependence resides only in K12 and is consistent with the presence of a single group, possibly histidine, displaying a slightly higher pKa value in the tetramer than in the dimer. The association reaction appears to be driven by the loss of enthalpy associated with release of strain in the heterodimer. The association sites appear to be conserved in the association reactions, consistent with the images from electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗
PubMed2001

Human alpha spectrin II and the FANCA, FANCC, and FANCG proteins bind to DNA containing psoralen interstrand cross-links.

Repair of DNA interstrand cross-links is a complex process critical to which is the identification of sites of damage by specific proteins. We have recently identified the structural protein nonerythroid alpha spectrin (alphaSpIISigma) as a component of a nuclear protein complex in normal human cells which is involved in the repair of DNA interstrand cross-links and have shown that it forms a complex with the Fanconi anemia proteins FANCA, FANCC, and FANCG. Using DNA affinity chromatography, we now show that alphaSpIISigma, present in HeLa cell nuclei, specifically binds to DNA containing psoralen interstrand cross-links and that the FANCA, FANCC, and FANCG proteins are bound to this damaged DNA as well. That spectrin binds directly to the cross-linked DNA has been shown using purified bovine brain spectrin (alphaSpIISigma1/betaSpIISigma1)2. Binding of the Fanconi anemia (FA) proteins to the damaged DNA may be either direct or indirect via their association with alphaSpIISigma. These results demonstrate a role for alpha spectrin in the nucleus as well as a new function for this protein in the cell, an involvement in DNA repair. alphaSpIISigma may bind to cross-linked DNA and act as a scaffold to help in the recruitment of repair proteins to the site of damage and aid in their alignment and interaction with each other, thus enhancing the efficiency of the repair process.

Animals↗
PubMed1984

Functional characterization of human erythrocyte spectrin alpha and beta chains: association with actin and erythrocyte protein 4.1.

Human erythrocyte spectrin alpha and beta chains were purified by preparative sodium dodecyl sulfate gel electrophoresis and also by DEAE-cellulose chromatography in the presence of urea. The purified chains behaved as individual monomers on sucrose gradients and did not form homodimers. Recombination of the chains led to the formation of alpha-beta heterodimers with sedimentation characteristics identical with native alpha-beta dimers. The binding of 125I-labeled band 4.1 to alpha and beta chains was measured by sucrose gradient rate zonal sedimentation and by quantitative immunoassay. It was found that both alpha and beta chains associated with 125I-labeled band 4.1 in a nearly identical manner over the range of band 4.1 concentration studied. The association was abolished by heat denaturation of the spectrin chains or by denaturation of band 4.1 with a 40-fold molar excess of N-ethylmaleimide. As expected, purified beta chains but not alpha chains bound to 125I-labeled ankyrin as measured by a quantitative radioimmunoassay. The binding of purified alpha chains, beta chains, and recombinant alpha-beta heterodimers to F-actin was measured in the presence of band 4.1. We found that alpha or beta chains separately exhibited no band 4.1 dependent association with F-actin but that alpha-beta heterodimers formed by recombination of the chains did. We conclude that spectrin binding to F-actin in the presence of band 4.1 requires the participation of both of spectrin's polypeptide chains.

Actins↗
PubMed1987

Electrostatic coupling of spectrin dimers to phosphatidylserine containing lipid lamellae.

We studied the interaction of spectrin dimers from human erythrocytes with (bilayer and monolayer) model membranes of mixtures of dimyristoylphosphatidylethanolamine, dimyristoylphosphatidylcholine, and dimyristoylphosphatidylserine (DMPS) by densitometric evaluation of phase transitions and phase boundaries, film balance experiments, and microfluorescence. We demonstrate that spectrin readily adsorbs to mixed bilayers and monolayers even in the presence of small DMPS concentrations (30 mol %) whereas no appreciable interaction with lamellae containing zwitterionic lipids alone is observed. The selectivity of the DMPS/spectrin interaction is established by quantitative evaluation of the shifts of the phase boundaries (liquidus and solidus line) caused by the lipid/protein interaction as a function of the composition of the binary lipid mixtures. Quantitative information about the free energy of the lipid/protein interaction is obtained by computer simulation of the phase diagram of the lipid mixture in the absence or in the presence of a very small molar fraction of the protein and comparison of calculated and measured shifts. A binding energy of about 10(-17) J per spectrin molecule is found. The present perturbation method can be generalized to study selective lipid/protein interaction mechanisms in ternary or higher component mixtures. The present results provide evidence that in addition to the binding to band III, spectrin may also couple directly to the lipid moiety of the inner monolayer of erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Dimyristoylphosphatidylcholine↗
PubMed1977

Structural study of spectrin from human erythrocyte membranes.

Human erythrocyte spectrin prepared from fresh blood is a mixture of different association states. Depending on the manner of preparation, the two-chain dimer or the tetramer predominates. These forms are not in rapid thermodynamic equilibrium. The molecular weight of the dimer by sedimentation and diffusion and by light scattering is about 5 X 10(5). The frictional properties indicate a low or moderate asymmetry (axial ratio in the range 2-10), and from the angular dependence of light scattering intensity an upper limit of about 80 A can be set for the radius of gyration. The tetramer similarly has a moderate asymmetry. Electron microscopy reveals that the dimer is a compact, slightly elongated molecule, and that the tetramer probably consists of two parallel dimers. On increasing the concentration of solutions containing spectrin dimers, oligomers are formed, which are not rapidly dissociated on dilution. At very low protein concentrations (below about 0.05 mg/mL) there is evidence of the onset of a rapid dissociation equilibrium between dimers and single chains. Other physical properties of the spectrin have been measured. The size and shape of the spectrin molecule would seem to rule out any major physical resemblance to myosin.

Erythrocyte Membrane↗
PubMed2001

Role of terminal nonhomologous domains in initiation of human red cell spectrin dimerization.

Human erythrocyte spectrin is an antiparallel heterodimer comprised of a 280 kDa alpha subunit and a 246 kDa beta subunit which further associates into tetramers in the red cell membrane cytoskeleton. Lateral association of the flexible rodlike monomers involves a multiple-step process that is initiated by a high affinity association near the actin-binding end of the molecule (dimer nucleation site). In this study, recombinant alpha and beta proteins comprising two or four "spectrin type" motifs with and without adjacent, terminal nonhomologous domains were evaluated for their relative contributions to dimer initiation, and the thermodynamic properties of these heterodimer complexes were measured. Sedimentation equilibrium studies showed that in the absence of the heterologous subunit, individual recombinant proteins formed weak homodimers (K(d) > 0.3 mM). When 2-motif (alpha20-21 and beta1-2) and 4-motif (alpha18-21 and beta1-4) recombinants lacking the terminal nonhomologous domains were paired with the complementary protein, high affinity heterodimers were formed in sedimentation equilibrium analysis. Both the alpha20-21/beta1-2 complex and the alpha20-21EF/betaABD1-2 complex showed stoichiometric binding with similar binding affinities (K(d) approximately 10 nM) using isothermal titration calorimetry. The alpha20-21/beta1-2 complex showed an enthalpy of -10 kcal/mol, while the alpha20-21EF/betaABD1-2 complex showed an enthalpy of -13 kcal/mol. Pull-down assays using alpha spectrin GST fusion proteins showed strong associations between all heterodimer complexes in physiological buffer, but all heterodimer complexes were destabilized by the presence of Triton X-100 and other detergents. Complexes lacking the nonhomologous domains were destabilized to a greater extent than complexes that included the nonhomologous domains. The detergent effect appears to be responsible for the apparent essential role of the nonhomologous domains in prior reports. Taken together, our results indicate that the terminal nonhomologous domains do not contribute to dimer initiation nor are they required for formation of high affinity spectrin heterodimers in physiological buffers.

Amino Acid Motifs↗
PubMed2001

alpha beta Spectrin coiled coil association at the tetramerization site.

On the basis of sequence homology studies, it has been suggested that the association of human erythrocytes alpha and beta spectrin at the tetramerization site involves interactions between helices. However, no empirical details are available, presumably due to the experimental difficulties in studying spectrin molecules because of its size and/or its structural flexibility. It has been speculated that erythrocyte tetramerization involves helical bundling rather than coiled coil association. We have used recombinant spectrin peptides to model alpha and beta spectrin to study their association at the tetramerization site. Two alpha peptides, Sp alpha 1-156 and Sp alpha 1-368, and one beta peptide, Sp beta 1898-2083, were used as model peptides to demonstrate the formation of the alpha beta complex. We also found that the replacement of R28 in Sp alpha 1-368 to give Sp alpha 1-368R28C abolished complex formation with the beta peptide. Circular dichroism techniques were used to monitor the secondary structures of the individual peptides and of the complex, and the results showed that both Sp alpha 1-156 and Sp beta 1898-2083 peptides in solution, separately, included helices that were not paired with other helices in the absence of their binding partners. However, in a mixture of Sp alpha 1-156 and Sp beta 1898-2083 and formation of the alpha beta complex, the unpaired helices associated to form coiled coils. Since the sequences of these two peptides that are involved in the coiled coil association are derived from a native protein, the information obtained from this study also provides insight toward a better understanding of naturally occurring coiled coil subunit-subunit association.

Amino Acid Sequence↗
PubMed2005

Identification and functional characterization of protein 4.1R and actin-binding sites in erythrocyte beta spectrin: regulation of the interactions by phosphatidylinositol-4,5-bisphosphate.

The ternary complex of spectrin, F-actin, and protein 4.1R defines the erythrocyte membrane skeletal network, which governs the stability and elasticity of the membrane. It has been shown that both 4.1R and actin bind to the N-terminal region (residues 1-301) of the spectrin beta chain, which contains two calponin homology domains, designated CH1 and CH2. Here, we show that 4.1R also binds to the separate CH1 and CH2 domains. Unexpectedly, truncation of the CH2 domain by its 20 amino acids, corresponding to its N-terminal alpha helix, was found to greatly enhance its binding to 4.1R. The intact N terminus and the CH1 but not the CH2 domain bind to F-actin, but again, deletion of the first 20 amino acids of the latter exposes an actin-binding activity. As expected, the polypeptide 1-301 inhibits the binding of spectrin dimer to actin and formation of the spectrin-actin-4.1R ternary complex in vitro. Furthermore, the binding of 4.1R to 1-301 is greatly enhanced by PIP(2), implying the existence of a regulatory switch in the cell.

Actins↗
PubMed1979

The membrane attachment protein for spectrin is associated with band 3 in human erythrocyte membranes.

Ankyrin, the membrane attachment protein for human erythrocyte spectrin, is tightly linked in a 1:1 molar ratio with band 3 in detergent extracts of spectrin-depleted membranes. Ankyrin-linked band 3, which represents 10--15% of the total band 3, spans the membrane, and is nearly identical to the major band 3 by peptide analysis. Spectrin binds to solubilised ankyrin-linked band 3, but not to free band 3. A portion of band 3 remains firmly associated with detergent-extracted cytoskeletal proteins. It is concluded that a fraction of band 3 is attached to the erythrocyte cytoskeleton through association with ankyrin, which in turn is bound to spectrin.

Chromatography, Gel↗
PubMedDate not supplied

Erythrocyte spectrin is comprised of many homologous triple helical segments.

Spectrin is an alpha beta heterodimeric protein (molecular weight (Mr) = 460,000) which is a major component of the erythrocyte membrane skeleton. The membrane skeleton also includes actin (band 5) and is attached to the membrane via non-covalent associations with two linking proteins. Recently we have reported the amino acid sequence of a peptide of molecular weight 80,000 which comprises the NH2-terminal one-third of the alpha subunit. This alpha-subunit peptide contains multiple homologous non-identical sequences with a periodicity of 106 amino acids and an approximate molecular weight of 12,000. It was also established that spectrin is not related to any other proteins whose sequence was known. We now report additional amino acid sequence of peptides representative of other domains of both spectrin subunits. The results suggest that most of the human erythrocyte spectrin molecule is comprised of homologous segments with a 106 amino acid (Mr 12,000) length per segment. Each homologous 106-amino acid segment may be folded into a triple helical structure with a short non-helical region connecting adjacent units.

Amino Acid Sequence↗
PubMed2006

Spectrin mutations cause spinocerebellar ataxia type 5.

We have discovered that beta-III spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 (SCA5) in an 11-generation American kindred descended from President Lincoln's grandparents and two additional families. Two families have separate in-frame deletions of 39 and 15 bp, and a third family has a mutation in the actin/ARP1 binding region. Beta-III spectrin is highly expressed in Purkinje cells and has been shown to stabilize the glutamate transporter EAAT4 at the surface of the plasma membrane. We found marked differences in EAAT4 and GluRdelta2 by protein blot and cell fractionation in SCA5 autopsy tissue. Cell culture studies demonstrate that wild-type but not mutant beta-III spectrin stabilizes EAAT4 at the plasma membrane. Spectrin mutations are a previously unknown cause of ataxia and neurodegenerative disease that affect membrane proteins involved in glutamate signaling.

Adolescent↗
PubMed1999

Elf3 encodes a novel 200-kD beta-spectrin: role in liver development.

beta-spectrins are crucial for the maintenance of cell shape, the establishment of cell polarity, and the formation of distinct membrane domains. Our strategy for identifying genes important for hepatocyte polarity has been to utilize subtractive hybridization of early embryonic mouse cDNA liver libraries. As a result, we have cloned three isoforms of a novel beta-spectrin elf (embryonic liver beta-fodrin), and here we report the analysis of elf3, the longest isoform (8172 nt). ELF3 comprises 2154 residues with an overall similarity of 89.0% and 95.3% to mouse beta-spectrin (betaSpIIsigma1) at the nucleotide and amino acid level, respectively. ELF3 is characterized by an actin-binding domain, a long repeat domain, and a short regulatory domain remarkable for the absence of a PH domain. Linkage analysis reveals that elf3 maps to mouse chromosome 11 between D11Bir6 and D11Xrf477, a different chromosomal locus from that of the other four spectrin genes. Northern blot analysis utilizing an elf3 3'-UTR probe demonstrates an abundant 9.0-kb transcript in brain, liver, and heart tissues. Western blot with a polyclonal antibody against ELF identifies a 200 kD protein in mouse liver, brain, kidney, and heart tissues. Immunohistochemical studies demonstrate ELF labeling of the basolateral or sinusoidal membranes surface as well as a granular cytoplasmic pattern in hepatocytes. Antisense studies utilizing cultured liver explants show a vital role of elf3 in hepatocyte differentiation and intrahepatic bile duct formation. The differential expression, tissue localization, and functional studies demonstrate the importance of elf3 in modulating interactions between various components of the cytoskeleton proteins controlling liver and bile duct development.

Amino Acid Sequence↗
PubMed1992

Fluorescence quenching of spectrin and other red cell membrane cytoskeletal proteins. Relation to hydrophobic binding sites.

The intrinsic fluorescence of spectrin is strongly quenched by low concentrations of 2-bromostearate. This results from binding at a series of hydrophobic sites. Analysis of dynamic fluorescence quenching by acrylamide, iodide and caesium ions, separately and in conjunction with 2-bromostearate, leads to the conclusion that most of the tryptophan side-chains are exposed to solvent. The sites at which the fatty-acid-quenched tryptophans are located apparently interact with the lipid bilayer in the cell, as judged by quenching by bromostearate dissolved in the lipid phase. A minor proportion of the side-chains in native spectrin give rise to sharp proton magnetic resonance signals, indicative of segmental mobility; these chain elements contain some tryptophan residues, as revealed by weak downfield signals from the heterocyclic ring protons. These signals are not appreciably perturbed by stearic acid or by phosphatidylserine liposomes, suggesting that the hydrophobic binding sites are not in mobile chain elements. By contrast with a series of globular proteins which, with the exception of serum albumins, show little or no quenching by 2-bromostearate, the peripheral red cell membrane skeletal proteins ankyrin (and its spectrin-binding domain), protein 4.1 and (to a lesser extent) actin show evidence of a high affinity for the hydrophobic ligand and may, like spectrin, interact directly with the bilayer in situ.

Binding Sites↗
PubMedDate not supplied

Spectrin extractability in blood storage.

The extractability of spectrin from red blood cell membranes was measured both by dialysis against 1 mM EDTA for 44 hours and by incubation with 1 mM EDTA for 60 minutes. We confirmed that the extractability of spectrin is somewhat impaired when membranes are prepared from metabolically depleted erythrocytes, but the extent of impairment was considerably less than has been previously reported. Red blood cells which had been rendered nonviable by prolonged storage in citrate phosphate dextrose solution manifested normal spectrin extractability. We conclude that the loss of spectrin extractability is an insensitive manifestation of loss of red blood cell viability, and that it is not suitable for the prediction of loss of viability during red blood cell storage.

Blood Glucose↗
PubMed1977

Induction of spectrin in erythroleukemic cells transformed by Friend virus.

The presence and accumulation in murine erythroleukemic cells transformed by Friend virus of the erythrocyte membrane-associated protein spectrin has been investigated. Spectrin was present in the uninduced cells and was induced 10- to 20-fold in dimethyl sulfoxide-treated differentiating cells. The intracellular concentration of spectrin reached a peak on the third day of dimethyl sulfoxide treatment, after which it fell to levels found in mouse erythrocytes. We also found that the small subunit of spectrin was phosphorylated in the cells.

Animals↗