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PubMed1984

Brain spectrin: a review.

Red blood cell spectrin, along with actin and several other proteins, forms a skeletal meshwork on the cytoplasmic surface of the erythrocyte plasma membrane. This structure is thought to maintain red blood cell shape, membrane structural stability, and cellular elasticity, as well as controlling the lateral mobility of integral membrane proteins and the transbilayer movement of phospholipids. It is now clearly established that spectrin-related molecules are ubiquitous structural elements subjacent to the plasma membrane of mammalian and avian nonerythroid cells. In this review, we present the current knowledge concerning brain spectrin. Brain spectrin is an approximately 11S, approximately 1,000,000 molecular weight (alpha beta)2 tetramer containing subunits of 240,000 (alpha) and 235,000 (beta) molecular weight. It is present in the cortical cytoplasm of all neuronal cell bodies and processes, and to a lesser extent in glial cells. Its involvement in the actin-membrane interaction, as well as other proposed functions in the nervous system is discussed.

Actins↗
PubMed1987

Spectrin isoforms in mammalian brain.

In this brief review we discuss the structure, location, developmental expression and potential functions of the spectrin isoforms [spectrin(240/235) and spectrin (240/235E)] within mammalian brain. We also contrast the structure and location of mammalian and avian brain spectrin isoforms.

Animals↗
PubMed2006

Enhanced oxidative cross-linking of hemoglobin E with spectrin and loss of erythrocyte membrane asymmetry in hemoglobin Ebeta-thalassemia.

Oxidative stress to the erythrocytes is associated with formation of large molecular complexes of hemoglobin and the skeletal protein, spectrin. In this work, such complexes are formed with hemoglobin mixtures isolated from patients suffering from HbEbeta-thalassemia with elevated levels of the HbE and purified erythroid spectrin in the presence of hydrogen peroxide. The complexes are separated on 4% SDS-PAGE and analyzed by densitometry. The results indicate enhanced formation of complexes with higher amounts of HbE, the most common hemoglobin variant prevalent in Southeast Asia. The binding affinity of spectrin with hemoglobin, in the absence of hydrogen peroxide, was found to increase with hemoglobin mixtures enriched with HbE. The presence of ATP was also found to decrease the overall yield of such complexes. Flow cytometric measurements of phosphatidylserine on the red cell surface also showed a lower degree of membrane asymmetry in HbEbeta-thalassemic patients than in normal subjects. The present work shows enhanced formation of high molecular weight cross-linked complexes of hemoglobin derivatives with erythroid spectrin in HbEbeta-thalassemia.

Adenosine Triphosphate↗
PubMed2007

betaIV-spectrin forms a diffusion barrier against L1CAM at the axon initial segment.

Axonal and somatodendritic plasma membranes of polarized neurons express distinct sets of functional molecules. It is known that the neuronal polarity can be maintained by a barrier that impedes diffusional mixing of membrane components between the two domains. Using betaIV-spectrin knockout mice, we demonstrate the involvement of this cytoskeletal protein in the formation of a barrier that selectively blocks lateral mobility of L1 cell adhesion molecule (L1CAM) at the axon initial segment of hippocampal neurons. We also show that the betaIV-spectrin-based barrier is required for the axon-specific distribution of L1CAM both in vitro and in vivo. The barrier activity against L1CAM may depend on direct interactions of L1CAM with ankyrinG, a protein binding to betaIV-spectrin, rather than on steric hindrance by other transmembrane proteins clustered at the axon initial segment. Our results highlight the role of betaIV-spectrin and ankyrinG as critical components of a selective barrier against L1CAM.

Analysis of Variance↗
PubMed2004

Signal transduction through the Gal-GalNAc lectin of Entamoeba histolytica involves a spectrin-like protein.

Capping followed by uroid formation in Entamoeba histolytica has been implicated in resistance against the host immune response during development of amoebiasis. The amebic actomyosin cytoskeleton is essential for such a process. A protein from the spectrin family co-localizes with the Gal-GalNAc lectin during capping of this surface protein complex. Co-localization is not observed when capping of the Gal-GalNAc lectin is specifically inhibited by production of the carboxyl-terminal region of its heavy chain that includes the lectin cytoplasmic tail. A peptide encompassing the lectin last 77 amino acids fused to glutathione-S-transferase interacts in vitro with purified spectrin. The spectrin-binding site was narrowed down to a stretch of 21 amino acids within the lectin cytoplasmic domain. This is the first report identifying an amino acid sequence involved in the interaction between the Gal-GalNAc lectin and cytoskeletal spectrin.

Adaptation, Physiological↗
PubMed2003

A protein isolated from Escherichia coli, identified as GroEL, reacts with anti-beta spectrin antibodies.

We found that a protein of molecular weight close to 65kDa, present in Escherichia coli cells, reacts with anti-beta spectrin antibodies. A method of purification of this protein was designed. The method consists of the following: nonionic detergent extraction, gel filtration chromatography, ion-exchange chromatography using DEAE-Servacell, and two FPLC ion-exchange chromatography runs: the first without urea, the second in its presence. This method allowed us to obtain a highly purified protein. The results of mass spectrometry analysis suggest that the investigated protein is GroEL (Hsp60 Class). Using computer programs, by sequence analysis of both proteins we tried to explain why GroEL isolated from E. coli reacts with anti-beta spectrin antibodies. Both proteins may share a single epitope for the antibodies on their surfaces. Additionally, such an assumption is supported by the results of experiments in which antibodies interacting with GroEL were obtained from anti-beta spectrin serum and were shown to react with both GroEL and beta spectrins.

Amino Acid Sequence↗
PubMed2000

Flexibility of the alpha-spectrin N-terminus by EPR and fluorescence polarization.

The structure and flexibility of the biologically important alpha-spectrin amino terminal region was examined by the use of fluorescence and EPR spectroscopy. The region studied has been previously demonstrated to be essential for the alpha-spectrin:beta-spectrin association of the tetramerization site. Appropriate spectroscopic probe moieties were coupled to this region in a recombinant fragment of human erythroid alpha-spectrin. There was good agreement between the EPR and fluorescence techniques in most of this region. Mobility determinations indicated that a portion of the region was relatively immobilized. This is significant, since although predictive methods have indicated that this region should be alpha-helical, previous experimental evidence obtained on smaller synthetic peptides had indicated that this region was disordered. Observed rigidity appears to be incompatible with such a disordered state, and has important ramifications for the flexibility of this molecule that is so integral to its role in stabilizing erythrocyte membranes.

Amino Acid Motifs↗
PubMed1986

Spectrin, human erythrocyte shapes, and mechanochemical properties.

Physical studies of human erythrocyte spectrin indicate that isolated spectrin dimers and tetramers in solution are worm-like coils with a persistence length of approximately 20 nm. This finding, the known polyelectrolytic nature of spectrin, and other structural information about spectrin and the membrane skeleton molecular organization have lead us to the hypothesis that the human erythrocyte membrane skeleton constitutes a two-dimensional ionic gel (swollen ionic elastomer). This concept is incorporated in what we refer to as the protein gel-lipid bilayer membrane model. The model accounts quantitatively for red elastic shear modulus and the maximum elastic extension ratio reported for the human erythrocytes membrane. Gel theory further predicts that depending on the environmental conditions, the membrane skeleton modulus of area compression may be small or large relative to the membrane elastic shear modulus. Our analyses show that the ratio between these two parameters affects both the geometry and the stability of the favored cell shapes and that the higher the membrane skeleton compressibility the smaller the values of the gel tension needed to induce cell shape transformations. The main virtue of the protein gel-lipid bilayer membrane model is that it offers a novel theoretical and molecular basis for the various mechanical properties of the membrane skeleton such as the membrane skeleton modulus of area compression and osmotic tension, and the effects of these properties on local membrane skeleton density, cell shape, and shape transformations.

Erythrocyte Membrane↗
PubMed2000

The domain of brain beta-spectrin responsible for synaptic vesicle association is essential for synaptic transmission.

We have examined the interaction between synapsin I, the major phosphoprotein on the membrane of small synaptic vesicles, and brain spectrin. Using recombinant peptides we have localized the synapsin I attachment site upon the beta-spectrin isoform betaSpIISigmaI to a region of 25 amino acids, residues 211 through 235. This segment is adjacent to the actin binding domain and is within the region of the betaSpIISigmaI that we previously predicted as a candidate synapsin I binding domain based upon sequence homology. We used differential centrifugation techniques to quantitatively assess the interaction of spectrin with synaptic vesicles. Using this assay, high affinity saturable binding of recombinant betaSpIISigmaI proteins was observed with synaptic vesicles. Binding was only observed when the 25 amino acid synapsin I binding site was included on the recombinant peptides. Further, we demonstrate that antibodies directed against 15 amino acids of the synapsin I binding domain specifically blocked synaptic transmission in cultured hippocampal neurons. Thus, the synapsin I attachment site on betaSpIISigmaI spectrin comprises a approximately 25 amino acid segment of the molecule and interaction of these two proteins is an essential step for the process of neurotransmission.

Animals↗
PubMed2000

NMR analysis of secondary structure and dynamics of a recombinant peptide from the N-terminal region of human erythroid alpha-spectrin.

We have studied the nuclear magnetic resonance solution secondary structure of the N-terminal region in human erythroid alpha-spectrin using a recombinant model peptide of alpha-spectrin consisting of residues 1-156. Pulsed field gradient diffusion coefficient measurements show that the model peptide exists as a monomer under the solution conditions used. The first 20 residues are in a random coil conformation, followed by a helix of 25 residues and then a random coil segment before the next helix. The random coil nature of this linker was confirmed by the presence of fast internal motion from (15)N relaxation measurements. The second, third and fourth helices are thought to form the triple helical bundle structural domain, consistent with previous studies. Our study shows that the N-terminal region of alpha-spectrin prior to the first structural domain forms a well behaved helix without its beta-spectrin partner.

Amino Acid Sequence↗
PubMed2001

Identification of the main ubiquitination site in human erythroid alpha-spectrin.

Erythroid spectrin is the main component of the red cell membrane skeleton, which is very important in determining the shape, resistance to mechanical stresses and deformability of red cells. Previously we demonstrated that human erythroid alpha-spectrin is ubiquitinated in vitro and in vivo, and using recombinant peptides we identified on repeat 17 the main ubiquitination site of alpha-spectrin. In order to identify the lysine(s) involved in the ubiquitination process, in the present study we mutated the lysines by site-directed mutagenesis. We found that ubiquitination was dramatically inhibited in peptides carrying the mutation of lysine 27 on repeat 17 (mutants K25,27R and K27R). We also demonstrated that the correct folding of this protein is fundamental for its recognition by the ubiquitin conjugating system. Furthermore, the region flanking lysine 27 showed a 75% similarity with the leucine zipper pattern present in many regulatory proteins. Thus, a new potential ubiquitin recognition motif was identified in alpha-spectrin and may be present in several other proteins.

Amino Acid Sequence↗
PubMed1998

A spectrin membrane skeleton of the Golgi complex.

The existence of a Golgi-localized membrane cytoskeleton has been revealed by the identification of two major components of the spectrin membrane skeleton, spectrin and ankyrin, that associate with the Golgi complex. Golgi spectrin was identified with an antibody specific for the beta-subunit of the erythroid isoform of spectrin (beta1Sigma1). This antibody recognizes a 220 kDa polypeptide that localizes to discrete regions of the Golgi complex and associates with Golgi membranes in a Brefeldin A sensitive manner. Two isoforms of Golgi ankyrin have been identified: a 119 kDa form (AnkG119) which represents a truncated, alternatively spliced isoform of a recently cloned novel ankyrin of the nervous system AnkG, and a larger 195 kDa ankyrin (Ank195) that cross-reacts with antibodies to erythrocyte ankyrin. A Golgi localized membrane skeleton composed of these unique membrane skeleton isoforms could serve a variety of important functions, including the maintenance of Golgi structural organization and the formation of discrete membrane domains within Golgi compartments.

Animals↗
PubMed1999

Interaction of the C-terminal domain of delta glutamate receptor with spectrin in the dendritic spines of cultured Purkinje cells.

The interaction of neurotransmitter receptors with the underlying cytoskeleton via subsynaptic proteins is an important mechanism for the targeting of the receptors to synapses in the central nervous system. We show that delta glutamate receptors (delta receptors), expressed predominantly in the dendritic spines of cerebellar Purkinje cells, directly interact with spectrin, a member of the actin-binding family of proteins. Moreover, the interaction between spectrin and C-terminal domain of the delta receptor is 50% inhibited by 1 microM of Ca2+ in vitro, compared with that in the absence of Ca2+. These results suggest that delta receptors on the postsynaptic membrane of the dendritic spines of cerebellar Purkinje cells are anchored to the actin cytoskeleton via spectrin, and that Ca2+ elevation in the dendritic spines causes delta receptor declustering by dissociation of the receptors from spectrin. This mechanism for receptor anchoring at postsynaptic sites may regulate synaptogenesis and/or synaptic plasticity.

Animals↗
PubMed2000

Development and characterization of antibodies specific to caspase-3-produced alpha II-spectrin 120 kDa breakdown product: marker for neuronal apoptosis.

Alpha II-spectrin (alpha-fodrin) is a demonstrated endogenous substrate for caspase-3 in neurons undergoing unscheduled apoptotic death. We have previously identified the caspase cleavage site that yields the distinctive 120 kDa spectrin breakdown product (SBDP120) as (DSLD(1478)*SVEAL). Here, by using a synthetic peptide (NH(2)-SVEALC) mimicking the neo-N-terminal of SBDP120 as antigen, we report the development of chicken antibodies that specifically recognize the SBDP120 generated by in vitro caspase-3 digestion of bovine alpha-spectrin on Western blot. These anti-SBDP120 antibodies recognize SBDP120 generated by two apoptotic challenges (staurosporine, EGTA) to human neuroblastoma SH-SY5Y cells. Yet they neither react with intact alpha-spectrin nor its other fragments on Western blots. These anti-SBDP120 work equally well in detecting SBDP120 generated in rat cerebellar granule neurons undergoing potassium withdrawal-induced apoptosis. In immunocytochemical studies, these antibodies also specifically stained apoptotic SH-SY5Y or CGN's undergoing apoptosis in a caspase- inhibitor-sensitive manner. These anti-SBDP120s might become powerful markers for apoptotic neurons in various neurological or neurodegenerative conditions in vivo.

Animals↗
PubMed2001

Calpain proteolysis of alpha II-spectrin in the normal adult human brain.

The proteolysis of alphaII-spectrin by calpain may be physiologically involved with synaptic remodeling, long-term potentiation, and memory formation. Calpain activation may also mediate neuronal apoptosis, responses to hypoxic insult, and excitotoxic injury. Surprisingly little is known of the activity of these calpain-mediated processes in the adult human brain. Using an antibody that specifically recognizes calpain-cleaved alphaII-spectrin, we have mapped the topographic distribution of the major alphaII-spectrin break-down product (alphaII-bdp1) in six adult brains examined post-mortem. All brains were from patients without evident neurological disease. Focally positive alphaII-bdp1 was consistently detected in the neuropil of the cortical gray matter, in occasional pyramidal neurons, and in rare reactive astrocytes in the cerebral cortex and hippocampus. Cerebellar Purkinje cells were more frequently, and more intensely, immunopositive. In all fields, staining was most intense in the soma and dendrites of neurons. There was no correlation of the frequency of positive cells with the postmortem interval or clinical condition. While these findings do not rigorously exclude contributions from postmortem calpain activation, they do suggest that a low-level of calpain processing of alphaII-spectrin is likely to be a constitutive process in the adult human brain.

Adult↗
PubMed2002

Calpain activation and alpha-spectrin cleavage in rat brain by ethanol.

Calpain, a calcium-activated cysteine protease, has been implicated in neuronal degeneration and death. In this study, we have characterized calpain activation in adult rat cerebral cortex and cerebellum, using an experimental paradigm of in vivo chronic ethanol exposure. Ethanol treatment increased the calpain activity in cortex and cerebellum, but to a higher extent in the cortex. Western blot analysis revealed a significant decrease in m-calpain levels while calpastatin levels were unaltered. Calpain activation was further monitored by the proteolysis of alpha-spectrin (fodrin) and protein kinase C-alpha (PKC-alpha). Protease specific spectrin breakdown products revealed calpain generated 150- and 145-kDa fragments. In addition, we also observed a 120-kDa fragment characteristic of caspase-3 activation in the cerebellum. PKC-alpha levels were decreased in the cortex and cerebellum by ethanol. Calpain activation, cleavage of alpha-spectrin into calpain specific signature fragments and decreased PKC-alpha protein levels after ethanol treatment provide the evidence of calpain involvement besides caspase-3-mediated cell death in the cortex and cerebellum. Given the role of calpains in cell death, increased calpain activity followed by alpha-spectrin cleavage in this study suggests that calpains are important effectors in ethanol-mediated cell injury and alcoholic neurodegeneration.

Alcohol-Induced Disorders, Nervous System↗
PubMed2001

Alpha-spectrins are major ubiquitinated proteins in rat hippocampal neurons and components of ubiquitinated inclusions in neurodegenerative disorders.

We have demonstrated that alpha-spectrins (alphaSpISigma* and alphaSpIISigma1) are major ubiquitinated proteins in terminally differentiated hippocampal neurons in culture. Western blotting experiments, using alphaSpISigma1, alphaSpIISigma1, and ubiquitin antibodies and lysates of 11-day-old cultured rat hippocampal neurons, have demonstrated that a single band comigrating with alphaSpISigma* and alphaSpIISigma1 in a 5% polyacrylamide sodium dodecyl sulfate gel is recognized by ubiquitin antibodies when (125)I-protein A is used for detection. Immunofluorescence staining of the 7- and 12 -day-old rat hippocampal neuron cultures using ubiquitin, alphaSpISigma1, and alphaSpIISigma1 antibodies demonstrated that all of these antibodies label neurons but not the astrocytes in the cultures. Immunoprecipitation of spectrin subunits in lysates of 12-day-old rat hippocampal neurons under stringent conditions (9.5 M urea) using alphaSpISigma1 and alphaSpIISigma1 antibodies followed by Western blot experiments of the immunoprecipitated spectrin subunits using alphaSpISigma1, alphaSpIISigma1 and ubiquitin antibodies confirmed that both alphaSpISigma* and alphaSpIISigma1 are ubiquitinated in rat hippocampal neurons. Furthermore, we demonstrated by immunohistochemistry that alpha-spectrins are components of the cytoplasmic ubiquitinated inclusions in hippocampal neurons in Alzheimer's and Parkinson's disease patients.

Alzheimer Disease↗
PubMed1998

Ultrastructural localisation of spectrin in sensory and supporting cells of guinea-pig organ of Corti.

Spectrin is a cytoskeletal protein found in the cortex of many cell types. It is known to occur in cochlear outer hair cells (OHCs) with previous immunoelectron microscopical studies showing that it is located in the cuticular plate and the cortical lattice. The latter is a network of filaments associated with the lateral plasma membrane that is thought to play a role in OHC motility. Spectrin has also been found in inner hair cells (IHCs) and supporting cells using immunofluorescent techniques, but its ultrastructural distribution in these cells has not yet been described. This has, therefore, been investigated using a monoclonal antibody to alpha-spectrin in conjunction with pre- and post-embedding immunogold labelling for transmission electron microscopy. Labelling was found in a meshwork of filaments beneath the plasma membranes of both IHCs and supporting cells and, in pillar cells, close to microtubule/microfilament arrays. It was also found in association with the stereocilia of OHCs and IHCs and, as expected, in the cortical lattice and cuticular plate of OHCs. Thus, spectrin is a general component of cytoskeletal structures involved in maintaining the specialised cell shapes in the organ of Corti and may contribute to the mechanical properties of all the cell types examined.

Animals↗