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Towards a complete atomic structure of spectrin family proteins.

The spectrin family of proteins represents a discrete group of cytoskeletal proteins comprising principally alpha-actinin, spectrin, dystrophin, and homologues and isoforms. They all share three main structural and functional motifs, namely, the spectrin repeat, EF-hands, and a CH domain-containing actin-binding domain. These proteins are variously involved in organisation of the actin cytoskeleton, membrane cytoskeleton architecture, cell adhesion, and contractile apparatus. The highly modular nature of these molecules has been a hindrance to the determination of their complete structures due to the inherent flexibility imparted on the proteins, but has also been an asset, inasmuch as the individual modules were of a size amenable to structural analysis by both crystallographic and NMR approaches. Representative structures of all the major domains shared by spectrin family proteins have now been solved at atomic resolution, including in some cases multiple domains from several family members. High-resolution structures, coupled with lower resolution methods to determine the overall molecular shape of these proteins, allow us for the first time to build complete atomic structures of the spectrin family of proteins.

Actins↗
PubMed1978

Phospholipid composition of human erythrocyte spectrin.

Human erythrocyte spectrin, which has been extracted at low ionic strength and precipitated at pH 5.1, contains 0.025 mumoles phospholipid/mg protein. The composition of the spectrin-phospholipids differs from that of the erythrocyte membrane. A remarkable similarity was found between the composition of the "available" phospholipids of the inner leaflet of the membrane bilayer and the spectrin-associated phospholipids. Rebinding studies with 125I-labeled spectrin show that the labeled spectrin binds preferentially to the cytoplasmic side of the membrane and that this binding is influenced by protein perturbations.

Erythrocyte Membrane↗
PubMed1977

The alteration of the functional properties of human haemoglobin by spectrin.

Kinetic investigation by means of stopped flow techniques showed the rate of deoxygenation of haemoglobin to be slower in the presence of spectrin. At pH 7.15, the kinetic constant was 27.2 sec-1 in presence of spectrin instead of 34.3 sec-1 for haemoglobin alone. Also, equilibrium studies have revealed that the oxygen pressure for half-saturated haemoglobin decreased when spectrin was added to the reaction medium. At pH 7.35, the log (pO2)1/2 was 0.88 for haemoglobin in presence of spectrin instead of 0.93 for haemoglobin alone. From these results, an interaction between spectrin and haemoglobin may be suspected.

Hemoglobins↗
PubMed1983

A dynamical study on the interactions between the cytoskeleton components in the human erythrocyte as detected by saturation transfer electron paramagnetic resonance of spin-labeled spectrin, ankyrin, and protein 4.1.

Isolated human erythrocyte spectrin, ankyrin, and protein 4.1 have been labeled with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl, and studied by saturation transfer electron paramagnetic resonance spectroscopy. The presence of the labels does not affect the reassociation of these proteins with erythrocyte membranes selectively depleted of either spectrin-actin or of all the extrinsic proteins. When maleimide spin-labeled spectrin is reassociated with the erythrocyte membrane in presence of all the cytoskeleton components, including endogeneous or purified muscle actin, spectrin still preserves its flexible character. The rotational mobilities of maleimide spin-labeled ankyrin and maleimide spin-labeled protein 4.1 are of the same order of magnitude (tau c (L"/L) approximately 5 X 10(-5) and 8 X 10(-5) s, respectively, at 2 degrees C), while protein 4.1 is almost three times smaller in size than ankyrin. This result indicates that the movements of membrane-bound maleimide spin-labeled protein 4.1 are more restricted than those of ankyrin. This suggests that their respective binding sites have different structural properties. The rotational movements of both proteins are slowed down on the addition of spectrin indicating that protein 4.1 as well as ankyrin also represents one of the links of the cytoskeleton to the membrane.

Actins↗
PubMed1978

Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane.

After treatment of intact human erythrocytes with SH-oxidizing agents (e.g. tetrathionate and diamide) phospholipase A2 cleaves approx. 30% of the phosphatidylserine and 50% of the phosphatidylethanolamine without causing hemolysis (Haest, C.W.M. and Deuticke, B (1976) Biochim. Biophys. Acta 436, 353--365). These phospholipids are scarcely hydrolysed in fresh erythrocytes and are assumed to be located in the inner lipid layer of the membrane (Verkleij, A.J., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and van Deenen, L.L.M. (1973) Biochim. Biophys Acta 323, 178--193). The enhancement of the phospholipid cleavage is now shown to be accompanied by a 50% decrease of the membrane SH-groups and a cross-linking of spectrin, located at the inner surface of the membrane, to oligomers of less than 10(6) dalton. Blocking approx. 10% of the membrane SH groups with N-ethylmaleimide suppresses both the polymerization of spectrin and the enhancement of the phospholipid cleavage. N-Ethylmaleimide, under these conditions, reacts with three SH groups per molecule of spectrin, 0.7 SH groups per major intrinsic 100 000 dalton protein (band 3) and 1.1 SH groups per molecule of an extrinsic protein of 72 000 daltons (band 4.2). Blocking studies with iodoacetamide demonstrate that the SH groups of the 100 000-dalton protein are not involved in the effects of the SH-oxidizing agents. It is suggested that a release of constraints imposed by spectrin enables phosphatidylserine and phosphatidylethanolamine to move from the inner to the outer lipid layer of the erythrocyte membrane and that spectrin, in the native erythrocyte, stabilizes the orientation of these phospholipids to the inner surface of the membrane.

Diamide↗
PubMed1985

Labelling of erythrocyte spectrin in situ with phenylisothiocyanate.

The labelling of erythrocyte spectrin in situ with the hydrophobic reagent phenylisothiocyanate (Sigrist, H. and Zahler, P. (1978) FEBS Lett. 95, 116-120) is studied. Spectrin isolated from erythrocytes which have been incubated with phenylisothiocyanate is covalently modified by the probe. The modification in the spectrin molecule is stable under an excess of nucleophile in alkaline conditions. The labelling is very little or not affected by preincubation of erythrocytes of membranes with the polar, structural analogue of phenylisothiocyanate, p-sulfophenylisothiocyanate. When erythrocyte ghosts are subjected to labelling, a substantial increase in the degree of spectrin modification is observed. Subunits of labelled spectrin separated electrophoretically show similar amounts of attached label.

Affinity Labels↗
PubMed1989

Rotational dynamics of erythrocyte spectrin.

The rotational diffusion of erythrocyte spectrin has been measured using time-resolved phosphorescence anisotropy. The anisotropy of the spectrin dimer decays to zero with a time constant of 3 microseconds at 21 degrees C. The results are compared with the correlation times predicted for the anisotropy decay of an equivalent sphere and rigid rod. The data indicate that the ribbon-like spectrin molecule possesses considerable torsional and segmental flexibility. These motions are restricted, but not abolished, when spectrin is reconstituted into cross-linked cytoskeletal protein networks, or bound to spectrin-actin depleted erythrocyte membrane vesicles.

Actins↗
PubMed1989

Mechanism of spectrin degradation induced by phenylhydrazine in intact human erythrocytes.

The exposure of human erythrocytes to phenylhydrazine results in the degradation of both monomers of spectrin, a major cytoskeleton membrane protein. The degradative process, characterized by a loss of spectrin without the appearance of high-molecular-weight products, either under reducing conditions or not, is almost complete in 10 min when a 5% erythrocyte suspension is treated with 1 mM phenylhydrazine. Under these conditions, we found a loss of 62.3 and 48.5% for the alpha and beta monomer, respectively. A similar degradative extent was obtained when the membrane ghost plus cellular free extracts, were dialyzed, and the membrane ghost plus hemoglobin was exposed to 1 mM phenylhydrazine for 10 min. The presence of different proteinase inhibitors and effectors, such as EDTA, diethylenetriaminepentaacetic acid, EGTA, leupeptin, aprotinin, phenylmethylsulfonyl fluoride, pepstatin, Ca2+ and ATP plus Mg2+, in the membrane ghost plus cellular free extract system (undialyzed) did not affect the degree of the spectrin-degradative process induced by phenylhydrazine. In addition, a purified spectrin tetramer preparation exposed to 1 mM phenylhydrazine in the presence of hemoglobin was degraded to an extent comparable to that with intact cells. Our data suggest that the initial degradative step of spectrin induced by phenylhydrazine in intact erythrocytes may be ascribed more to a direct oxidative breakdown, probably involving main-chain cleavage and side-chain cleavage processes, than to an eventual proteolytic system.

Erythrocyte Membrane↗
PubMed1978

Human spectrin. I. A classical light scattering study.

Human spectrin heterodimers were analyzed in solutions containing different amounts of salt employing the classical light scattering technique. 1. At 22 degrees C the radius of gyration of isolated human spectrin heterodimers in 0.1 M NaCl aqueous solution (pH 7.3) was found to be about 22 nm. 2. The radius of gyration of isolated human spectrin heterodimers was found to increase to about 40 nm as the ionic strength of the spectrin solution (pH 7.3) was reduced to about 1 mM. 3. The light scattering study indicates that the isolated human spectrin heterodimers were highly expanded and flexible molecules with a contour length exceeding about 140 nm.

Adult↗
PubMed1988

Lesions of entorhinal cortex produce a calpain-mediated degradation of brain spectrin in dentate gyrus. II. Anatomical studies.

Lesions of the various afferents to the hippocampus have been widely used to investigate the mechanisms underlying growth and degeneration in adult mammalian CNS. It has been proposed that disturbances in intracellular calcium and activation of calcium-dependent proteases represent key steps in producing come of the consequences of the lesions. In this study, we show that lesions of the entorhinal cortex or of the commissural pathway result in profound changes in the distribution of brain spectrin. At 2 days after lesions of the entorhinal cortex, immunoreactivity to spectrin is markedly increased in the outer molecular layer (OML) of the dentate gyrus; conversely at 2 days after commissural lesions, immunoreactivity to the same antigen is increased in the inner molecular layer. The increase in immunoreactivity to spectrin varies with survival time after lesions of the entorhinal cortex. By 24 h post lesion, the increase is homogeneous across the OML, and becomes more intense by 48 h. Between 1 and 3 weeks the increase is much less than at 48 h and is concentrated at the inner border of the OML. Pretreatment of the animals with the calpain inhibitor leupeptin reduces the increase in spectrin immunoreactivity normally seen 48 h after the lesion of the entorhinal cortex. Changes in the pattern of immunoreactivity to GFAP are very different to that seen with spectrin antibodies and are consistent with the known modifications in astrocytes that follow lesions of hippocampal afferents.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Crude spectrin extraction from reticulocyte-rich blood samples.

Crude spectrin was extracted from the isolated red cell ghosts with low ionic strength buffer at 37 degrees C for 30 min. No significant alterations in crude spectrin extractability in wide range of patients with various hematologic diseases were observed. However, blood samples characterized by elevated reticulocytosis provided crude extracts with increased amount of non-heme membrane skeletal proteins. The presence of ribose in the crude spectrin extracts obtained from reticulocyte-rich blood samples indicates also the presence of nucleic acids which causes the shift of protein peak in the extract from 280 nm towards lower wavelengths. A model experiment with a normal crude spectrin extract mixed with various amounts of RNA allowed us to obtain the correction curve which served for determination of non-heme protein (crude spectrin) extractability.

Blood Protein Electrophoresis↗
PubMed1990

Sea urchin spectrin in oogenesis and embryogenesis: a multifunctional integrator of membrane-cytoskeletal interactions.

Using indirect immunofluorescence microscopy on semithin cryosections of maturing ovarian tissue, eggs, and developing embryos, we have mapped the cellular distribution and dynamic redistribution of spectrin in oogenesis and early embryogenesis. During oogenesis, spectrin is initially found in the cortex of oogonia and previtellogenic oocytes, and later accumulates in the cytoplasm of vitellogenic oocytes on the surfaces of cortical granules, pigment granules/acidic vesicles, and yolk platelets. Following egg activation, spectrin undergoes a rapid redistribution coincident with three major developmental events including: (1) restructuring of the cell surface, (2) translocation of pigment granules/acidic vesicles to the cortex during the first cell cycle, and (3) amplification of the embryo's surface during the rapid cleavage phase of early embryogenesis. The synthesis and storage of spectrin during oogenesis appears to prime the egg with a preestablished pool of membrane-cytoskeletal precursor for use during embryogenesis. Results from this study support the hypothesis that spectrin may function as a key integrator and modulator of multiple membrane-cytoskeletal functions during embryonic growth and cellular differentiation.

Animals↗
PubMed1983

Properties of brain spectrin (fodrin).

Fodrin, a protein from bovine brain, immunologically related to spectrin, is shown, unlike some other proteins of generally similar appearance in the electron microscope, to resemble spectrin closely in its most distinctive structural characteristic, the very high alpha-helix content. Like spectrin, it is also insoluble below pH 5. One of the subunits only is phosphorylated by the cAMP-independent red cell membrane kinase, that phosphorylates the smaller subunit of spectrin. Fodrin also forms a ternary complex with F-actin and the third constituent of the red cell membranes cytoskeleton, protein 4.1. In the presence of 4.1 the interaction between fodrin and F-actin is enhanced. It is surmised that fodrin plays an analogous functional role in neuronal cells to that of spectrin in the red cell.

Actins↗
PubMed1984

Calmodulin binding to human spectrin.

Calmodulin is shown to interact with human spectrin dimer. The binding was highly calcium-dependent and observed in two different kinds of experiments. Firstly, affinity chromatography of calmodulin on a Sepharose 4B column with immobilized spectrin, and secondly, partition in aqueous two-phase polymer systems. In the column experiments stoichiometric amounts of calmodulin were retained on the spectrin-Sepharose column when micromolar concentrations of calcium were present. The calmodulin bound could be eluted with EGTA. The partition coefficient of calmodulin in an aqueous two-phase polymer system containing calcium was changed upon addition of spectrin, indicating an association between the two proteins. In the absence of calcium, spectrin did not cause any change in the partition behaviour of calmodulin, thus showing that the association requires calcium.

Animals↗
PubMed1987

Brain spectrin fragments and crosslinks actin filaments.

The effect of brain spectrin (fodrin) on actin has been studied using viscometry and fluorimetry. Brain spectrin resembles erythrocyte spectrin tetramer in its action on actin. Both proteins crosslink actin filaments giving rise to a large increase in the viscosity but fluorimetry shows that neither affects actin polymerization significantly. In addition, brain spectrin as well as erythrocyte spectrin fragments preformed actin filaments. Actin filaments incubated in the presence of either of the two proteins incorporate actin monomers at a much higher rate showing that more filament ends are generated.

Actins↗
PubMed1989

Weak interaction of spectrin with phosphatidylcholine-phosphatidylserine multilayers: a 2H and 31P NMR study.

Spectrin from human erythrocytes binds to bilayer dispersions of both DMPC and DMPS:DMPC (1:1, w/w). However, no effect of bound spectrin on the conformation of the lipid head groups, as measured from the deuterium quadrupolar splittings of DMPC or DMPS specifically deuterated in the polar head groups, was detected in 1:1 mixtures of the two lipids containing either deuterated DMPC or DMPS. Neither the phase transition of the DMPS:DMPC mixtures, nor the spin-lattice relaxation time (T1) of the deuterated DMPS head group, was affected by spectrin. These results argue against any strong interaction of spectrin with phosphatidylserine and rule out the possibility that spectrin is responsible for the maintenance of PS in the inner monolayer of the erythrocyte membrane during the whole life-span of this cell.

Deuterium↗
PubMed1993

The self-association of ovine erythrocyte spectrin.

1. Spectrin extracted from ovine erythrocyte membranes at low temperature shows association behaviour similar to that reported for human and bovine erythrocytes. 2. The spectrin tetramer is the predominant oligomer, the dimer is well represented, and smaller amounts of hexamer and higher oligomers are present. 3. The estimates of parameters describing the self-association of purified ovine spectrin studied by sedimentation equilibrium analysis were found to be indistinguishable from those obtained for human spectrin under the same conditions, within the precision of the measurements. 4. The data suggest that the cooperative isodesmic model may be general for spectrin, and not a peculiarity of the human.

Animals↗
PubMed1989

Quantitative detection of rapid motions in spectrin by NMR.

Previous high resolution proton NMR data on human erythrocyte spectrin molecules has indicated the existence of regions exhibiting rapid internal motions within the intact molecules [L. W.-M. Fung, H.-Z. Lu, R. P. Hjelm, jr, M. E. Johnson, FEBS Lett., 197, 234 (1986)]. We have extended the studies by developing quantitative NMR methods to determine the fraction of spectrin protons exhibiting rapid internal motions, in both the isolated molecule and within the spectrin-actin network. Using both one-pulse and spin echo pulse sequences, we find that the fraction of the protons in rapid motion is about 15% of the total protons in the spectrin molecule at 37 degrees C in phosphate buffer with 150 mM NaCl at pH 7.4. Quantitative information on these rapid motions will be important in understanding the structural, mechanical and functional properties of spectrin molecules, as well as in understanding filamentous protein structures in general.

Erythrocyte Membrane↗