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PubMed1980

Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane.

Binding of F-actin to spectrin-actin-depleted erythrocyte membrane inside-out vesicles was measured using [3H]F-actin. F-actin binding to vesicles at 25 degrees C was stimulated 5-10 fold by addition of spectrin dimers or tetramers to vesicles. Spectrin tetramer was twice as effective as dimer in stimulating actin binding, but neither tetramer nor dimer stimulated binding at 4 degrees C. The addition of purified erythrocyte membrane protein band 4.1 to spectrin-reconstituted vesicles doubled their actin-binding capacity. Trypsinization of unreconstituted vesicles that contain < 10% of the spectrin but nearly all of the band 4.1, relative to ghosts, decreased their F-actin-binding capacity by 70%. Whereas little or none of the residual spectrin was affected by trypsinization, band 4.1 was significantly degraded. Our results show that spectrin can anchor actin filaments to the cytoplasmic surface of erythrocyte membranes and suggest that band 4.1 may be importantly involved in the association.

Actins↗
PubMed1997

Segregation of two spectrin isoforms: polarized membrane-binding sites direct polarized membrane skeleton assembly.

Spectrin isoforms are often segregated within specialized plasma membrane subdomains where they are thought to contribute to the development of cell surface polarity. It was previously shown that ankyrin and beta spectrin are recruited to sites of cell-cell contact in Drosophila S2 cells expressing the homophilic adhesion molecule neuroglian. Here, we show that neuroglian has no apparent effect on a second spectrin isoform (alpha beta H), which is constitutively associated with the plasma membrane in S2 cells. Another membrane marker, the Na,K-ATPase, codistributes with ankyrin and alpha beta spectrin at sites of neuroglian-mediated contact. The distributions of these markers in epithelial cells in vivo are consistent with the order of events observed in S2 cells. Neuroglian, ankyrin, alpha beta spectrin, and the Na,K-ATPase colocalize at the lateral domain of salivary gland cells. In contrast, alpha beta H spectrin is sorted to the apical domain of salivary gland and somatic follicle cells. Thus, the two spectrin isoforms respond independently to positional cues at the cell surface: in one case an apically sorted receptor and in the other case a locally activated cell-cell adhesion molecule. The results support a model in which the membrane skeleton behaves as a transducer of positional information within cells.

Animals↗
PubMed1985

A case of elliptocytosis associated with a truncated spectrin chain.

A case of haemolytic anaemia with elliptocytosis is described, in which a large part of the smaller (beta) subunit of the spectrin is truncated, and has an apparent molecular weight of about 214 000 compared with about 230 000 for the normal chain. It is shown that this is not a product of adventitious proteolysis during lysis or extraction. At the same time about 35% of the total spectrin in the cells is liberated from the membrane as the dimer (which is present in normal cells to the extent of less than 10%). The truncated (beta') chain appears exclusively in this dimer fraction. The beta'-chain is incapable of phosphorylation by the endogenous cAMP-independent membrane kinase, and it may be inferred that the deleted segment of the chain contains both the spectrin self-association site and the residues normally phosphorylated. The alpha beta'-dimer is active with respect to participation in a ternary complex with its partnering proteins in the membrane cytoskeleton, F-actin and 4.1, confirming that the phosphorylation sites are not involved in the primary interaction with the other cytoskeletal proteins at the network junctions. The spectrin alpha-chain generates the terminal tryptic fragment of molecular weight 80 000 characteristic of normal spectrin, rather than the 74 000 molecular weight peptide derived from the alpha-chain in cases of hereditary elliptocytosis and pyropoikilocytosis, associated with anomalous self-association of spectrin dimer. Membrane cytoskeletons, extracted from the patient's red cells, undergo normal gelation on incubation with cAMP-independent kinase and ATP, and thus do not resemble those derived from hereditary spherocytosis cells. The properties of the anomalous spectrin resemble in most respects that described in a French family by Dhermy et al (1982).

Adolescent↗
PubMed1992

Elliptocytosis-associated spectrin Rouen (beta 220/218) has a truncated but still phosphorylatable beta chain.

Spectrin Rouen (beta 220/218) is a novel variant, carrying a shortened beta chain with an apparent molecular weight of 218 kDa. It was detected in a French family. All affected members suffered from haemolytic hereditary elliptocytosis. As other shortened beta chain variants described before, the beta Rouen chain is truncated at its carboxyl terminus. Spectrin Rouen is associated with a defect in spectrin dimer self-association and with an abnormally high amount of the alpha I 74 kDa peptide following partial tryptic digestion. Dimer reconstitution experiments from normal and abnormal purified Sp subunits indicated that the increased alpha I 74 kDa fragment is induced by the altered beta chain. However, spectrin Rouen is different from other mutants with a truncated beta chain in several respects: its amount is low (less than 10%) and the spectrin dimer self-associated defect is mild. Critically, the beta Rouen chain has retained the ability of undergoing phosphorylation, even though it is modified in its C-terminal region. These results, compared to those obtained with beta 220/214 spectrin Le Puy and beta 220/216 spectrin Nice, allowed better localization of the beta chain sites that can be phosphorylated by a membrane-bound casein kinase.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

cDNA cloning, sequencing and chromosome mapping of a non-erythroid spectrin, human alpha-fodrin.

Several overlapping cDNA clones encompassing 2760 nucleotides of the alpha-subunit of a human non-erythroid spectrin (termed fodrin) were isolated from a human lung fibroblast cDNA library. DNA and RNA blot analyses indicated that a single copy alpha-fodrin gene encodes a 9-kb transcript. The cDNA clones were sequenced, and all were found to contain long open reading frames. The overlapping regions were identical except for a 60-nucleotide inframe insertion at position 1133 in the composite sequence. This result suggests that at least two distinct transcripts exist in fibroblast cells. The chromosomal location of human alpha-fodrin was assigned to 1p34-1p36.1 by hybridization to somatic cell hybrids, and it is thus distinct from that of human alpha-spectrin which has been mapped to 1q22-1q25. Alignment of the composite 919 amino acids of the predicted protein sequence of human alpha-fodrin with that of human alpha-spectrin indicated that alpha-fodrin has a similar 106-amino-acid repeating structure, which is homologous with alpha-spectrin repeats 7-15. Repeats 10 and 11 are anomalous in sequence and structure from other repeats. A comparison of nucleic acid and amino acid homologies between alpha-spectrin and the alpha-fodrin of several vertebrates indicated that human non-erythroid alpha-fodrin and the common alpha-subunit of erythroid and non-erythroid cells of non-mammalian vertebrates are closely related (90%-96% amino acid homology), whereas alpha-fodrin is only distantly related to the erythroid-specific alpha-spectrin subunit of mammals (55%-59% amino acid homology). These data suggest that mammalian erythroid alpha-spectrin evolved by duplication and rapid divergence from an ancestral alpha-fodrin-like gene.

Amino Acid Sequence↗
PubMed1984

Chromaffin granule membrane-F-actin interactions and spectrin-like protein of subcellular organelles: a possible relationship.

The membrane of chromaffin granule, the secretory vesicle of adrenal medullary cells storing catecholamines, enkephalins, and many other components, interacts with F-actin. Using low shear falling ball viscometry to estimate actin binding to membranes, we demonstrated that mitochondrial and plasma membranes from chromaffin cells also provoked large increases in viscosity of F-actin solutions. Mitochondrial membranes also had the capacity to cause complete gelation of F-actin. In addition, vasopressin-containing granules from neurohypophysial tissue were shown to bind F-actin and to increase the viscosity of F-actin solutions. Using an antibody directed against human erythrocyte spectrin, it was found that a spectrin-like protein was associated with secretory granule membrane, mitochondrial membrane, and plasma membrane. The chromaffin granule membrane-associated spectrin-like protein faces the cytoplasmic side, is composed of two subunits (240 kD and 235kD ), the alpha-subunit (240 kD, pHi5 .5) being recognized by the antibody. Nonionic detergents such as Triton X-100 or Nonidet P40 failed to release fully active spectrin-like protein. In contrast, Kyro EOB , a different nonionic detergent, was found to release spectrin-like protein while keeping intact F-actin binding capacity, at least below 0.5% Kyro EOB concentration. Chromaffin cells in culture were stained with antispectrin antibody, showing the presence of spectrin-like protein in the cell periphery close to the cell membrane but also in the cytoplasm. We conclude that in living cells the interaction of F-actin with chromaffin granule membrane spectrin observed in vitro is important in controlling the potential function of secretory vesicles.

Acetylcholinesterase↗
PubMed1981

Tryptic digestion of spectrin in variants of hereditary elliptocytosis.

Spectrin, either in the form of unfractionated low ionic strength extracts of erythrocyte membranes or purified by chromatography on Sepharose (CL)4B, was subjected to tryptic digestion at 0 degrees C. Four patients, each with a different variant of hereditary elliptocytosis, were studied. In one patient, whose erythrocytes showed significant fragmentation on heating on 45 degrees C, such preparations generated a remarkably different pattern of polypeptide fragments on tryptic digestion at low ionic strength. In this patient 32P was released at a slower rate on tryptic digestion of labeled band 2, and an unusual 32P-labeled peptide fragment was also generated, in contrast to control preparations in which such a peptide could not be easily distinguished. There was increased susceptibility of this patient's spectrin to tryptic digestion at physiological ionic strength, but the qualitative pattern of polypeptide fragments was normal. Phosphorylation of spectrin by membrane protein kinase was markedly impaired in this patient, whereas phosphorylation of casein ws unimpaired. However, the phosphorylation of spectrin in her intact erythrocytes was normal. Our findings suggest an abnormality of spectrin structure which we postulate is causally related to the predisposition to hemolysis in this patient, but do not distinguish whether this is a primary abnormality or a post-translational modification of the spectrin molecule. The other three patients showed normal tryptic digestion of spectrin.

Elliptocytosis, Hereditary↗
PubMed1981

Altered spectrin dimer-dimer association and instability of erythrocyte membrane skeletons in hereditary pyropoikilocytosis.

Hereditary pyropoikilocytosis (HPP) is a hemolytic anemia characterized by microspherocytosis, poikilocytosis, and an unusual thermal sensitivity of erythrocytes. We have investigated the contribution of abnormal membrane skeletal assembly to these abnormal HPP erythrocyte properties. Skeletons prepared from fresh HPP ghosts with Triton X-100 were considerably more fragile than skeletons from control erythrocytes. Spectrin, the major skeleton component, extracted at 0 degrees C from normal erythrocytes, was present primarily as tetramers and high molecular weight complexes. In contrast, spectrin extracted from HPP erythrocytes under identical conditions contained a significant amount of dimers with a concomitant decrease of tetramers. Furthermore, spectrin dimers from HPP erythrocytes differed from normal spectrin dimers in their failure to reassociate into tetramers both in solution and in the membrane. Presumptive HPP carriers (asymptomatic mothers of the two patients) exhibited a mild but reproducible increase of spectrin dimers in 0 degrees C extracts and a defective reassociation of spectrin dimers of tetramers both in solution and in the membrane. We conclude that in HPP, self-association of spectrin dimers into tetramers is defective, which accounts for the instability of membrane skeletons.

Cytoplasm↗
PubMed1987

Mutant forms of spectrin alpha-subunits in hereditary elliptocytosis.

Two variant spectrins have been described in hereditary elliptocytosis (HE) and pyropoikilocytosis (HPP). Both are characterized by increased susceptibility of the alpha I (N-terminal) 80-kD domain to mild tryptic digestion, yielding peptides of 46-50 or 65-68 kD (T50a and T68 in our terminology). In this report we add a third unstable spectrin alpha I domain found in three kindreds with HE; alpha IT80 in this type of spectrin is cleaved by mild tryptic digestion to a 50-kD peptide (T50b) distinguished from T50a by its more basic isoelectric point. All three spectrins show impaired self-association to form oligomers. Intermediate tryptic peptides of the three unstable alpha I domains from HE spectrins were characterized by monoclonal immunoblotting and I125 limit peptide mapping and affinity purified using polyclonal anti-alpha IT80. Partial amino acid sequences of alpha I domain peptides were obtained from two unrelated patients for each of the three variant spectrins. T50a results from cleavage at arginine 250 or lysine 252 of alpha IT80; a proline replaced the normal leucine or serine at residues 254 and 255, respectively. T50b and a 19-kD peptide result from cleavage at arginine 462 or arginine 464; a proline replaced the normal residue 465 (in T19b) in one of the two patients studied. T68 results from cleavage at arginine 131. In both 68-kD peptides examined, a leucine is inserted at residue 150. The relationship of the sequence changes to the new tryptic cleavages, to the current model of alpha I domain structure, and to defective spectrin self-association is discussed.

Amino Acid Sequence↗
PubMed1997

Mutation of a highly conserved residue of betaI spectrin associated with fatal and near-fatal neonatal hemolytic anemia.

We studied an infant with severe nonimmune hemolytic anemia and hydrops fetalis at birth. His neonatal course was marked by ongoing hemolysis of undetermined etiology requiring repeated erythrocyte transfusions. He has remained transfusion-dependent for more than 2 yr. A previous sibling born with hemolytic anemia and hydrops fetalis died on the second day of life. Peripheral blood smears from the parents revealed rare elliptocytes. Examination of their erythrocyte membranes revealed abnormal mechanical stability as well as structural and functional abnormalities in spectrin. Genetic studies revealed that the proband and his deceased sister were homozygous for a mutation of betaIsigma1 spectrin, L2025R, in a region of spectrin that is critical for normal function. The importance of leucine in this position of the proposed triple helical model of spectrin repeats is highlighted by its evolutionary conservation in all beta spectrins from Drosophila to humans. Molecular modeling demonstrated the disruption of hydrophobic interactions in the interior of the triple helix critical for spectrin function caused by the replacement of the hydrophobic, uncharged leucine by a hydrophilic, positively charged arginine. This mutation must also be expressed in the betaIsigma2 spectrin found in muscle, yet pathologic and immunohistochemical examination of skeletal muscle from the deceased sibling was unremarkable.

Anemia, Hemolytic, Congenital↗
PubMed1994

Abnormalities in the expression of beta-spectrin in Duchenne muscular dystrophy.

We studied beta-spectrin using an immunologic probe in muscle samples from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), other disease controls, and normal controls. By immunohistochemistry in DMD samples, beta-spectrin showed reduced or interrupted staining of the entire cell surface or only patches of bright staining at the cell periphery. There were also alterations of beta-spectrin immunostain in fibers that were not degenerating or regenerating. By immunoblotting, the amount of beta-spectrin in muscle was reduced and varied from 52% to 78% of the normal controls. We found normal values of beta-spectrin in BMD and disease controls. These observations indicate that the expression of beta-spectrin in DMD is abnormal and that beta-spectrin immunolabeling is not a good marker for monitoring membrane integrity in DMD muscle.

Adolescent↗
PubMed2004

BetaIV spectrins are essential for membrane stability and the molecular organization of nodes of Ranvier.

High densities of sodium channels at nodes of Ranvier permit action potential conduction and depend on betaIV spectrins, a family of scaffolding proteins linked to the cortical actin cytoskeleton. To investigate the molecular organization of nodes, we analyzed qv(3J)"quivering" mice, whose betaIV spectrins have a truncated proline-rich "specific" domain (SD) and lack the pleckstrin homology (PH) domain. Central nodes of qv(3J) mice, which lack betaIV spectrins, are significantly broader and have prominent vesicle-filled nodal membrane protrusions, whereas axon shape and neurofilament density are dramatically altered. PNS qv(3J) nodes, some with detectable betaIV spectrins, are less affected. In contrast, a larger truncation of betaIV spectrins in qv(4J) mice, deleting the SD, PH, and ankyrinG binding domains, causes betaIV spectrins to be undetectable and causes dramatic changes, even in peripheral nodes. These results show that quivering mutations disrupt betaIV spectrin retention and stability at nodes and that distinct protein domains regulate nodal structural integrity and molecular organization.

Action Potentials↗
PubMed2005

Erythrocyte spectrin's chimeric E2/E3 ubiquitin conjugating/ligating activity.

Spectrin is important for the shape and the physical properties of the red blood cell, such as deformability and resistance to mechanical stress. Previous findings from our laboratory indicated that human erythrocyte alpha-spectrin can facilitate formation of ubiquitin-spectrin adducts and conjugates. Computer analysis revealed domains that contained significant homologies to known consensus catalytic E2 and E3 sequences, and allowed us to develop a model for alpha-spectrin ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3) enzymatic activities. In order to identify the precise E2/E3 site(s), in the present study, a GST-fusion alpha-spectrin (2005-2415) recombinant protein was tested using a cell free in vitro ubiquitination assay. We found that cysteine 2071 and cysteine 2100 are critical for alpha-spectrin (2005-2415) E2/E3 activity. Furthermore, together with testing an additional 13 site-specific mutants, we also demonstrated that both Cys2071 and Cys2100 are capable of transferring ubiquitin from an E1 enzyme to target sites within alpha-spectrin (2005-2415).

Blotting, Western↗
PubMed1992

[Abnormalities of beta spectrin with hereditary elliptocytosis in mother and child].

It is generally considered that abnormality of the erythrocyte membrane skeleton co elliptocytes. There are, however, few reports of beta spectrin variants. We found a new variant of beta spectrin in a child and her mother. This report is the first case of abnormality of beta spectrin in Japan. The propositus was an 8 month-old girl who was first examined by us in 1988. On laboratory findings, she showed anemia, increased reticulocyte count and decreased haptoglobin concentration. Both peripheral blood smears of patient and her mother showed typical elliptocytosis and they were diagnosed as hereditary elliptocytosis. SDS-PAGE patterns of the red cell membranes of the propositus and her mother were characterized by the presence of an abnormal component migrating immediately below the spectrin chains. We confirmed that the abnormal spectrin appeared clearly at the expense of normal beta chain. The abnormal spectrin (M.W. 216,000d) makes up 16% of the total beta chain. The inheritance of our case was autosomal dominant. The present case is considered as a new spectrin variant.

Adult↗
PubMed1990

The identification and sequence of the actin-binding domain of human red blood cell beta-spectrin.

The junctions of the red blood cell membrane skeleton are formed by interactions between spectrin and actin protofilaments. A spectrin tryptic peptide of 16.5-kDa apparent molecular mass (based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis) which binds to F-actin in cosedimentation experiments has been identified. The peptide has been partially purified by gel filtration, anion-, and cation exchange chromatography. Intact spectrin heterodimer causes half-maximal inhibition of the 16.5-kDa peptide/F-actin interaction at a concentration of 5 microM. Comparison of the two-dimensional iodopeptide maps of the 16.5-kDa peptide with maps of alpha- and beta-spectrin, demonstrate that the peptide is generated from the beta subunit. It shows no significant relationship to the peptide maps of the beta-spectrin domains I-IV. Protein sequencing indicated that this actin-binding domain represents a stretch of amino acids at the N terminus of the beta subunit from alanine 47 probably through lysine 186. The sequence derived molecular weight of this actin-binding domain is 16,290 g/mol. The sequence presented represents the region of greatest homology among the spectrin supergene family (spectrin, dystrophin, alpha-actinin).

Actins↗
PubMed1988

Brain adducin: a protein kinase C substrate that may mediate site-directed assembly at the spectrin-actin junction.

Erythrocyte adducin is a membrane skeletal protein that binds to calmodulin, is a major substrate for protein kinase C, and associates preferentially with spectrin-actin complexes. Erythrocyte adducin also promotes association of spectrin with actin, and this activity is inhibited by calmodulin. This study describes the isolation and characterization of a brain peripheral membrane protein closely related to erythrocyte adducin. Brain and erythrocyte adducin have at least 50% antigenic sites in common, each contains a protease-resistant core of Mr = 48,000-48,500, and both proteins are comprised of two partially homologous polypeptides of Mr = 103,000 and 97,000 (erythrocytes) and Mr = 104,000 and 107,000-110,000 (brain). Brain and erythrocyte adducin associate preferentially with spectrin-actin complexes as compared to spectrin or actin alone, and both proteins also promote binding of spectrin to actin. Brain adducin binds calmodulin in a calcium-dependent manner, although the Kd of 1.3 microM is weaker by 5-6-fold than the Kd of erythrocyte adducin for calmodulin. Brain adducin is a substrate for protein kinase C in vitro and can accept up to 2 mol of phosphate/mol of protein. Adducin provides a potential mechanism in cells for mediating site-directed assembly of additional spectrin molecules and possibly other proteins at the spectrin-actin junction. Brain tissue contains 12 pmol of adducin/mg of membrane protein, which is the most of any tissue examined other than erythrocytes, which have 50 pmol/mg. The presence of high amounts of adducin in brain suggests some role for this protein in specialized activities of nerve cells.

Actins↗
PubMed1989

[Research on genetic abnormality in the hemolytic form of hereditary elliptocytosis with homozygosity for the spectrin alpha I/74 variant].

We have undertaken to identify the spectrin gene mutation in a patient with a severe hemolytic form of Hereditary Elliptocytosis with homozygosity for the spectrin alpha I/74 variant. This variant corresponds to the presence of a 74,000 peptide which is produced during mild tryptic digestion of spectrin by cleavage at the Arginine-39 of the alpha I/80,000 domain of the spectrin alpha chain (595 amino acids). We hypothesized that the alpha I/74 mutation would be closed to the cleavage site Arg-39. A genomic library built with the patient's DNA was screened with a probe corresponding to a fragment of the alpha spectrin gene. Two clones were isolated, one being of paternal, the other of maternal origin. The subclones obtained contained the alpha spectrin gene exons 2 and 3 which encode for the first 88 amino-acids of the spectrin alpha I domain. The sequences obtained did not show any abnormality. The implications of these results are discussed.

Anemia, Hemolytic, Congenital↗
PubMed1985

A regional analysis of alpha-spectrin in the isolated Mauthner neuron and in isolated axons of the goldfish and rabbit.

Isolated dendrites, somata, and desheathed axons of the goldfish Mauthner neuron (M-cell), in addition to other isolated myelin sheath-free axons of the goldfish spinal cord and of rabbit lumbar ventral roots, were shown by immunochemical and immunofluorescence techniques to contain alpha-spectrin (fodrin). alpha-Spectrin appeared to be organized as a randomly distributed reticular network, localized to the surface of isolated neuronal cellular structures. In addition, alpha-spectrin was also distributed nonrandomly at specialized cellular sites. These sites included synaptic junctions and morphologically differentiated nodes of Ranvier (i.e., rabbit axons, but not goldfish axons). At the latter sites, it is possible to demonstrate that alpha-spectrin is co-localized with F-actin, as indicated by a striking correspondence of fluorescent images due to double labeling, using the indirect immunofluorescence technique with alpha-spectrin antiserum, and direct binding of F-actin by rhodamine-conjugated palloidin. However, the spectrin-actin network at synaptic junctions appears to be distributed over the entire area of junctional contact and is not just restricted to postsynaptic densities. The possibility of a duality of roles of spectrin in membrane-related motile and anchorage functions is discussed.

Animals↗