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PubMed2002

Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin.

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC). In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the beta-subunit, possibly in, or in close proximity of, the ankyrin-binding site. In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.

Ankyrins↗

PubMed1998

Biophysical properties of human erythrocyte spectrin at alkaline pH: implications for spectrin structure, function, and association.

The effects of pH 6-13 on the conformation and assembly of spectrin were studied by means of analytical ultracentrifugation, circular dichroism (CD), 1H NMR, and UV spectrophotometry. Sedimentation velocity analysis showed that spectrin oligomers dissociate cooperatively into component alpha- and beta-subunits above pH 9.5, and that spectrin tetramers, heterodimers, and monomers adopt more extended and/or expanded shapes above this pH. The dissociation to monomers is mostly completed by pH 10.5 and is used as the basis for purifying the subunits [see Fujita et al. (1998) Biochemistry 37, 272-280]. Along with the dissociation, biphasic unfolding of spectrin was observed above pH 9.5 as detected by CD. The first phase of the transition occurred between pH 9.5 and 11, and the second phase between pH 11 and 13. A similar biphasic dependence was observed for the upfield shift of lysine epsilon-CH2 resonances detected by spin-echo 1H NMR and the spectrophotometric titration of the absorbance at 294 nm. These data indicate that deprotonation of tyrosine and lysine residues is closely correlated with (i) the dissociation of spectrin oligomers into heterodimers, (ii) the dissociation of heterodimers into monomers, and (iii) the unfolding of spectrin. Taken together, our data suggest that hydrophobic and electrostatic interactions involving tyrosine and lysine residues play a critical role in the formation of the alpha-helix of spectrin and assembly of physiologically relevant spectrin oligomers from the two component subunits.

Circular Dichroism↗

PubMed2002

Important region in the beta-spectrin C-terminus for spectrin tetramer formation.

Many hereditary hemolytic anemias are due to spectrin mutations at the C-terminal region of beta-spectrin (the betaC region) that destabilize spectrin tetramer formation. However, little is known about the betaC region of spectrin. We have prepared four recombinant beta-peptides of different lengths from human erythrocyte spectrin, all starting at position 1898 of the C-terminal region, but terminating at position 2070, 2071, 2072 or 2073. Native polyacrylamide gel electrophoresis showed that the two peptides terminating at positions 2070 and 2071 did not associate with an N-terminal region alpha-peptide (Spalpha1-156) in the micromolar range. However, the peptides that terminated at positions 2072 and 2073 associated with the alpha-peptide. Circular dichroism results showed that the unassociated helices in both alpha- and beta-peptides became associated, presumably to form a helical bundle, for those beta-peptides that formed an alphabeta complex, but not for those beta-peptides that did not form an alphabeta complex. In addition, upon association, an increase in the alpha-helical content was observed. These results showed that the beta-peptides ending prior to residue 2072 (Thr) would not associate with alpha-peptide, and that no helical bundling of the partial domains was observed. Thus, we suggest that the C-terminal segment of beta-spectrin, starting from residue 2073 (Thr), is not critical to spectrin tetramer formation. However, the C-terminal region ending with residue 2072 is important for its association with alpha-spectrin in forming tetramers.

Amino Acid Sequence↗

PubMed1989

The complete sequence of Drosophila alpha-spectrin: conservation of structural domains between alpha-spectrins and alpha-actinin.

We report the complete sequence of Drosophila alpha-spectrin and show that it is similar to vertebrate nonerythroid spectrins. As in vertebrates, the alpha subunit consists of two large domains of repetitive sequence (segments 1-9 and 11-19) separated by a short nonrepetitive sequence (segment 10). The 106-residue repetitive segments are defined by a consensus sequence of 54 residues. Chicken alpha-spectrin (Wasenius, V.-M., M. Saraste, P. Salven, M. Eramaa, L. Holm, V.-P. Lehto. 1989. J. Cell Biol. 108:79-93) shares 50 of these consensus positions. Through comparison of spectrin and alpha-actinin sequences, we describe a second lineage of spectrin segments (20 and 21) that differs from the 106-residue segments by an 8-residue insertion and by lack of many of the consensus residues. We present a model of spectrin evolution in which the repetitive lineage of spectrin segments and the nonrepetitive lineage of segments found in spectrin and alpha-actinin arose by separate multiplication events.

Actinin↗

PubMed1987

Abnormal oxidant sensitivity and beta-chain structure of spectrin in hereditary spherocytosis associated with defective spectrin-protein 4.1 binding.

Hereditary spherocytosis (HS) is an inherited disorder of erythrocyte shape associated with spectrin deficiency and hemolytic anemia. In a subset of patients with the autosomal dominant form of HS, spectrin displays a reduced capacity to bind protein 4.1 and, therefore, actin; both functions that are critical to the membrane skeleton. A specific structural defect has not been identified in the spectrin from these patients. Chymotryptic digestion of the isolated spectrin chains shows impaired cleavage of the distal peptide of the beta subunit, the beta IV domain. In previous work, we have shown that mild oxidation markedly diminishes the binding capacity of normal spectrin for protein 4.1. Here we observe that chemical reduction of freshly isolated, untreated HS spectrin dramatically improves its function. Thus, a primary structural defect in the beta subunit of spectrin in this subtype of HS may lead to oxidant sensitivity, and secondarily, to a functional defect in the binding of spectrin to protein 4.1 and actin.

Blood Proteins↗

PubMed1991

Sp alpha V/41: a common spectrin polymorphism at the alpha IV-alpha V domain junction. Relevance to the expression level of hereditary elliptocytosis due to alpha-spectrin variants located in trans.

Spectrin alpha-chain mutants associated with hereditary elliptocytosis are highly variable in their level of expression. It has been assumed that the degree of elliptocytosis can be increased when the spectrin alpha chain, encoded by the alpha gene in trans to the variant, is expressed at a low level. We now provide strong evidence for the existence of low-level expression of spectrin alpha chains. This condition is referred to as the alpha V/41 polymorphism. It has been observed in 15 different families or individuals of French, North African, and African ancestry in which seven distinct elliptocytogenic alpha-spectrin variants were co-inherited. Whenever the alpha V/41 polymorphism was present, the severity of the biochemical, morphological, and, sometimes, the clinical phenotype of elliptocytosis was increased. The alpha V/41 polymorphism was also frequently encountered among 36 unrelated control subjects in the heterozygous or homozygous states, and was entirely asymptomatic in both cases. The main biochemical feature was an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction. Alteration of the facing beta IV domain of spectrin was demonstrated by in vitro spectrin dimer reconstitution experiments. It appears that the alpha V/41 polymorphism is often required for alpha-spectrin elliptocytogenic variants to become manifest in the heterozygous state. Thus, alpha-spectrin-related elliptocytosis may be viewed as a bifactorial condition.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1999

Electric birefringence of recombinant spectrin segments 14, 14-15, 14-16, and 14-17 from Drosophila alpha-spectrin.

Members of the spectrin protein family can be found in many different cells and organisms. In all cases studied, the major functional role of these proteins is believed to be structural rather than enzymatic. All spectrin proteins are highly elongated and consist mainly of homologous repeats that constitute rigid segments connected in tandem. It is commonly believed that the details of the spectrin function depend critically on the flexibility of the links between the segments. Here we report on a work addressing this question by studying the transient electric birefringence of recombinant spectrin fragments consisting of segments 14, 14-15, 14-16, and 14-17, respectively, from Drosophila alpha-spectrin. Transient electric birefringence depends sharply on both molecular length and flexibility. We found that the birefringence relaxation time of segment 14 measured at 4 degrees C, but scaled to what is expected at 20 degrees C, equals 16 ns (+/-15%) at pH 7.5 and ionic strength 6 mM. This is consistent with this single segment being rigid, 5 nm long and having an axial ratio equal to about two. Under the same conditions, segments 14-15, 14-16 and 14-17 show relaxation times of 45, 39 and 164 ns (all +/-20%), respectively, scaled to what is expected at 20 degrees C. When the temperature is increased to 37 degrees C the main relaxation time for each of these multisegment fragments, scaled to what is expected at 20 degrees C, increased to 46, 80, and 229 ns (all +/-20%), respectively. When the ionic strength and the Debye shielding is low, the dynamics of these short fragments even at physiological temperature is nearly the same as for fully extended weakly bending rods with the same lengths and axial ratios. When the ionic strength is increased to 85 mM, the main relaxation time for each of these multisegment fragments is reduced 20-50% which suggests that at physiological salt and temperature conditions the links in 2-4-segment-long fragments exhibit significant thermally induced flexing. Provided that the recombinant spectrin fragments can serve as a model for native spectrin, this implies that, at physiological conditions, the overall conformational dynamics of a native spectrin protein containing 20-40 segments equals that of a flexible polymer.

Animals↗

PubMed1988

Direct involvement of spectrin thiols in maintaining erythrocyte membrane thermal stability and spectrin dimer self-association.

Human erythrocytes vesiculate upon exposure to temperatures of 49 degrees C and above. Pretreatment of the cells with the thiol-alkylating agent N-ethylmaleimide (NEM) lowers the temperature needed to produce the same effect. Concomitant with the cells' heat susceptibility, skeletal mechanical instability and an increase in spectrin dissociation have been reported (Smith and Palek (1983) Blood 62, 1190). In the present study, similar results were achieved by preincubation of the cells with diamide, which could be reversed by reduction with dithiothreitol. Another oxidative agent, sodium tetrathionate, could only induce the temperature susceptibility, with little effect on spectrin dissociation. Incubation of spectrin solutions with NEM or diamide caused decreased association of spectrin dimers and increased dissociation of spectrin tetramers. Estimation of membrane and spectrin thiols in the treated cells showed that NEM was effective while blocking less than 20% of the thiols. Diamide and tetrathionate blocked more than 50% of the thiols, but were less effective than NEM. It is suggested that some very defined population of thiols is essential for spectrin self-association and for membrane thermal stability. They are more available to NEM than to diamide and less so to tetrathionate. Other thiols participate in maintaining the membrane thermal stability only.

Diamide↗

PubMed1992

Role of spectrin in Amoeba proteus, as studied by microinjection of anti-spectrin monoclonal antibodies.

Spectrin is a major protein accounting for about 5% of whole-cell proteins in Amoeba proteus, and the precipitation of spectrin by intracellular injection of purified anti-spectrin monoclonal antibodies has a profound effect on cell morphology, motility, and movement-related cell activities in amoebae. Thus, amoebae injected with anti-spectrin antibodies show drastic changes in their shape and movement, suggesting that amoeba spectrin plays an important structural role, unlike nonerythroid spectrins in other cells. However, precipitation of spectrin does not affect the distribution of F-actin in amoebae.

Actins↗

PubMed2003

Fanconi anemia cell lines deficient in alphaII spectrin express normal levels of alphaII spectrin mRNA.

Fanconi anemia is a genetic disorder characterized by hypersensitivity to DNA interstrand cross-linking agents and a defect in the ability to repair this type of damage. This deficiency correlates with reduced levels of alphaII spectrin, a structural protein involved in the repair of DNA interstrand cross-links. The present study addresses the question of whether the reduced levels of alphaII spectrin in FA-A, FA-C, and FA-G cells are due to reduced expression of this protein and/or due to differences in the three regions of alternate splicing of alphaII spectrin mRNA. Relative quantitative RT-PCR showed that levels of alphaII spectrin mRNA in the three FA cell lines were similar to normal as were the sites of alternative mRNA splicing. These results indicate that decreased levels of alphaII spectrin in these FA cell lines are not due to reduced expression of alphaII spectrin mRNA or due to differences in regions of alternate splicing of these transcripts, but rather appear to be related to reduced stability of alphaII spectrin in these cell lines.

Alternative Splicing↗

PubMed1994

Golgi spectrin: identification of an erythroid beta-spectrin homolog associated with the Golgi complex.

Spectrin is a major component of a membrane-associated cytoskeleton involved in the maintenance of membrane structural integrity and the generation of functionally distinct membrane protein domains. Here, we show that a homolog of erythrocyte beta-spectrin (beta I sigma*) co-localizes with markers of the Golgi complex in a variety of cell types, and that microinjected beta-spectrin codistributes with elements of the Golgi complex. Significantly, we show a dynamic relationship between beta-spectrin and the structural and functional organization of the Golgi complex. Disruption of both Golgi structure and function, either in mitotic cells or following addition of brefeldin A, is accompanied by loss of beta-spectrin from Golgi membranes and dispersal in the cytoplasm. In contrast, perturbation of Golgi structure without a loss of function, by the addition of nocodazole, results in retention of beta-spectrin with the dispersed Golgi elements. These results indicate that the association of beta-spectrin with Golgi membranes is coupled to Golgi organization and function.

Animals↗

PubMed1990

Point mutation in the beta-spectrin gene associated with alpha I/74 hereditary elliptocytosis. Implications for the mechanism of spectrin dimer self-association.

alpha I/74 hereditary elliptocytosis (HE) is a subgroup of HE in which patients exhibit an impaired self-association of spectrin dimers and an abnormal proteolytic cleavage of the alpha I domain of spectrin. We studied a family in which the proband presented with a severe neonatal hemolytic anemia with poikilocytosis. Biochemical analysis of erythrocytes from the proband and his family members allowed us to ascertain a diagnosis of homozygosity for alpha I/74 HE in the proband and heterozygosity in his parents and several of their offspring. Results of polymorphism linkage analysis suggested that the defect in this family was located in beta rather than alpha spectrin. We analyzed the 3' end of the beta-spectrin gene of the proband and detected a mutation that changes a codon for alanine to one for proline. Allele-specific oligomer hybridization on slot blots of DNA from other family members confirmed the presence of the mutation only in members heterozygous for the disorder. This is the first example of a point mutation in the beta-spectrin chain that is associated with defective spectrin dimer self-association and an abnormal proteolytic cleavage of the alpha chain. Based on this finding, we propose a model for the mechanism of interaction between the alpha- and beta-spectrin chains.

Amino Acid Sequence↗

PubMed2006

Pathogenic proline mutation in the linker between spectrin repeats: disease caused by spectrin unfolding.

Pathogenic mutations in alpha and beta spectrin result in a variety of syndromes, including hereditary elliptocytosis (HE), hereditary pyropoikilocytosis (HPP), and hereditary spherocytosis (HS). Although some mutations clearly lie at sites of interaction, such as the sites of spectrin alpha-betatetramer formation, a surprising number of HE-causing mutations have been identified within linker regions between distal spectrin repeats. Here we apply solution structural and single molecule methods to the folding and stability of recombinant proteins consisting of the first 5 spectrin repeats of alpha-spectrin, comparing normal spectrin with a pathogenic linker mutation, Q471P, between repeats R4 and R5. Results show that the linker mutation destabilizes a significant fraction of the 5-repeat construct at 37 degrees C, whereas the WT remains fully folded well above body temperature. In WT protein, helical linkers propagate stability from one repeat to the next, but the mutation disrupts the stabilizing influence of adjacent repeats. The results suggest a molecular mechanism for the high frequency of disease caused by proline mutations in spectrin linkers.

Elliptocytosis, Hereditary↗

PubMed2000

Identification of a novel C-terminal variant of beta II spectrin: two isoforms of beta II spectrin have distinct intracellular locations and activities.

It is established that variations in the structure and activities of betaI spectrin are mediated by differential mRNA splicing. The two betaI spectrin splice forms so far identified have either long or short C-terminal regions. Are analogous mechanisms likely to mediate regulation of betaII spectrins? Thus far, only a long form of betaII spectrin is reported in the literature. Five human expressed sequence tags indicated the existence of a short splice variant of betaII spectrin. The occurrence and DNA sequence of the short C-terminal variant was confirmed by analysis of human and rat cDNA. The novel variant lacks a pleckstrin homology domain, and has 28 C-terminal residues not present in the previously recognized longer form. Transcripts of the short C-terminal variant (7.5 and 7. 0 kb) were most abundant in tissues originating from muscle and nervous system. Antibodies raised to a unique sequence of short C-terminal variant recognized 240 kDa polypeptides in cardiac and skeletal muscle and in nervous tissue; in cerebellum and forebrain, additional 270 kDa polypeptides were detected. In rat heart and skeletal muscle, both long and short C-terminal forms of betaII spectrin localized in the region of the Z line. The central region of the sarcomere, coincident with the M line, was selectively labeled with antibodies to the short C-terminal form. In cerebellum, the short form was not detectable in parallel fibers, structures in which the long form was readily detected. In cultured cerebellar granule neurons, the long form was dominant in neurites, with the short form being most abundant in cell bodies. In vitro, the short form was found to lack the binding activity for the axonal protein fodaxin, which characterizes the C-terminal region of the long form. Subcellular fractionation of brain revealed that the short form was scarcely detectable in post-synaptic density preparations, in which the long form was readily detected. We conclude that variation in the structure of the C-terminal regions of betaII spectrin isoforms correlates with their differential intracellular targeting.

Alternative Splicing↗

PubMed1992

A deletional frameshift mutation of the beta-spectrin gene associated with elliptocytosis in spectrin Tokyo (beta 220/216).

A novel spectrin variant carrying a truncated beta-chain and designated Spectrin Tokyo (beta 220/216) is presented. It was associated with elliptocytosis and moderate uncompensated hemolysis. The dimer self-association was reduced. An increase of the alpha I 74-Kd fragment was detected upon partial trypsin digestion. Analysis of cDNA and genomic DNA showed a 1-base deletion in codon 2059 (GCC AGC-->GCA GCT; Ala-Ser-->Ala-Ala) that belongs to exon X of spectrin beta-gene. A missense sequence extended down to (new) codon 2075. Serine 2060, a potential phosphorylation site, was replaced by alanine. The shortened beta-chain failed to undergo phosphorylation in vitro. Spectrin Tokyo shared the same stop codon, overlapping normal codons 2076 and 2077 (CTG AAA), as Spectrin Nice (beta 220/216), which is caused by a dinucleotide insertion in codon 2046 and contains 2076 amino acids. However, for some reason, Spectrin Tokyo had a lower incorporation level into the membrane than Spectrin Nice.

Base Sequence↗

PubMed2004

Ubiquitination of erythrocyte spectrin regulates the dissociation of the spectrin-adducin-f-actin ternary complex in vitro.

We demonstrate that ubiquitinated red blood cell (RBC) spectrin dissociates more rapidly from the spectrin-adducin-actin ternary complex, than non-ubiquitinated spectrin. Homozygous (SS) sickle cell spectrin has substantially diminished ubiquitination of alpha-spectrin resulting in slower dissociation from the spectrin-adducin-actin ternary complex, than normal (AA) cell spectrin. These results supply a partial explanation of the slow dissociation of the irreversible sickle cell (ISC) membrane skeleton, which leads to the inability of the ISC to change shape.

Actins↗

PubMed1987

Brain spectrin(240/235) and brain spectrin(240/235E): differential expression during mouse brain development.

Mouse brain contains at least 2 distinct spectrin subtypes: brain spectrin(240/235) and brain spectrin(240/235E) (Riederer et al., 1986). In this study, we demonstrate that these subtypes are differentially expressed during mouse brain development. Brain spectrin(240/235) can be detected in fetal tissue and increases 2-fold during brain development. This subtype is enriched in the cortical cytoplasm of germinative neural cells and is also found in fibers resembling axons as early as fetal life. Brain spectrin(240/235E), which is specifically detected with antibodies to red blood cell spectrin, is below the limits of detection in fetal and neonatal brain but rapidly increases in concentration during the second postnatal week. Brain spectrin(240/235E) is confined to the cell body and dendrites of differentiating neurons and to glial cells but is not expressed in mitotic cells. This subtype is most prominent in granule cells of the cerebellum and dentate gyrus in the hippocampus.

Animals↗

PubMed1985

Sequencing of the chicken non-erythroid spectrin cDNA reveals an internal repetitive structure homologous to the human erythrocyte spectrin.

Immunological screening of a chicken gizzard cDNA expression library was used to isolate two clones encoding a part of the non-erythroid spectrin-like protein. Clones were identified by immunoblotting of the polypeptides synthesized in Escherichia coli cells transformed with cDNA cloned in the pUC8 plasmid vector using polyclonal rabbit antibodies raised against bovine non-erythroid spectrin. The sequence of an approximately 1.5-kb cDNA insert of one clone was determined. Analysis of the predicted amino acid sequence reveals that, despite differences in immunological cross-reactivity and peptide maps, the chicken non-erythroid and the human erythrocyte spectrins are highly homologous proteins. Like the human erythrocyte spectrin, the chicken smooth muscle spectrin appears also to be constructed from repeated, homologous structures of 106 amino acid residues. This is probably a universal structure motif of spectrins.

Amino Acid Sequence↗