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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↗

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↗

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↗

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↗

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↗

PubMed1984

Spectrin deficient inherited hemolytic anemias in the mouse: characterization by spectrin synthesis and mRNA activity in reticulocytes.

We have investigated spectrin synthesis and mRNA activity in mice homozygous and heterozygous for six mutations occurring at three distinct loci (nb, ja, sph). When homozygous, these mutations cause severe hemolytic anemias that are characterized by specific spectrin deficiencies. Our results indicate that the primary effect of the nb mutation is a deficiency of another erythrocyte membrane skeletal protein, ankyrin. The severe deficiency of spectrin in the red blood cells of ja/ja mice is the result of a beta spectrin defect. Analysis of spectrin synthesis in mice homozygous and heterozygous for several alleles of sph indicates that the sph locus is the structural gene locus for alpha spectrin. We have mapped the sph locus to mouse Chromosome 1.

Anemia, Hemolytic↗

PubMed1982

Binding of an 80 000 dalton trypsin fragment of spectrin to intact spectrin.

A specific and saturable interaction of an 80 000 dalton tryptic fragment of spectrin with intact spectrin has been detected. When spectrin was incubated with 125I-labelled 80 kDa fragment at 37 degrees C in the presence of 0.1 M NaCl, acrylamide gradient gel electrophoresis showed the presence of bands in addition to the usual spectrin dimer and tetramer and the 80 kDa fragment. These bands correspond to 5.6 X 10(5) daltons (dimer + fragment), 1.04 X 10(6) daltons (tetramer + fragment) and 1.52 X 10(6) daltons (hexamer + fragment). Measurement of radioactivity showed that these additional bands contained the labelled fragment. Maximal binding capacity of the 80 kDa fragment was approximately 170 micrograms fragment per mg spectrin, corresponding to 1 mol fragment per mol spectrin dimer.

Electrophoresis, Polyacrylamide Gel↗

PubMed1994

A deletional frameshift mutation in spectrin beta-gene associated with hereditary elliptocytosis in spectrin Napoli.

We studied a clinically manifest, dominantly transmitted elliptocytosis in an Italian family. We found a new spectrin variant, designated spectrin Napoli. Its beta-chain was truncated in its C-terminal region (apparent MW 216 kD). It displayed a low expression level (15%). There was a 8 nt deletion: CTTTTGAGAAGT-->CTGT (nt 6255-6262), starting after codon 2053. This deletion was followed by a 54 nt (18 amino acids) missense sequence and terminated by the TGA triplet which normally overlaps codons 2074 and 2075 (CTTGAG). The overall length of the mutated beta-chain was comparable to that found in spectrin Nice, spectrin Tokyo and spectrin Tandil, which are other variants with truncated beta-chains; however, a distinct nonsense codon was used in spectrin Napoli.

Adult↗

PubMed1992

Spectrin Jendouba: an alpha II/31 spectrin variant that is associated with elliptocytosis and carries a mutation distant from the dimer self-association site.

Spectrin Jendouba (alpha II/31) was found in a Tunisian family. In the heterozygous state, it is associated with asymptomatic elliptocytosis and a minimal defect in spectrin dimer self-association. On partial digestion of spectrin with trypsin, an abnormal cleavage appeared following Lys 788. Peptide and DNA sequencing indicated that the responsible mutation is alpha 791 Asp----Glu (GAC----GAA). As in most alpha-spectrin variants associated with elliptocytosis, the change alters helix 3 of the proposed triple helical model of spectrin structure. Modified helix 3 in repeat alpha 8 is the most distant from the N-terminus of alpha-spectrin in known variants associated with elliptocytosis.

Alleles↗

PubMed1984

Interaction of spectrin with hemin disaggregates spectrin associations.

Crude spectrin preparations were extracted from red cell membranes either in dimeric or tetrameric forms and incubated at 4 degrees C with hemin. The mixtures were subjected immediately or after 18 hours to nondenaturing electrophoresis. It was found that immediately after addition of 0.3 mM hemin, the fraction of spectrin complexed with other skeletal proteins, disaggregated to tetramer and dimer forms. After incubation for 18 hours at 4 degrees C most of the spectrin appeared in two additional bands which contained more hemin and migrated on the gels as molecular weight forms smaller than the dimers. Since SDS electrophoresis showed that spectrin subunits retained their integrity in these mixtures, it was concluded that hemin bound spectrin dissociates with time into monomers. It is suggested that there are pathophysiological implications to the disaggregation of spectrin complexes in the cytoskeleton by hemin.

Erythrocyte Membrane↗

PubMed1995

Hemoglobin-spectrin complexes: interference with spectrin tetramer assembly as a mechanism for compartmentalization of band 1 and band 2 complexes.

The irreducible complexation of hemoglobin with spectrin is a natural phenomenon of red blood cell aging, positively correlating with increasing cell density and decreasing cell deformability. The current study begins to address the role of these complexes in the disruption of membrane skeletal physiology and structure. The effect of bound hemoglobin on spectrin dimer self-association was investigated in vitro. The extent of conversion of isolated spectrin dimers to tetramers was evaluated as a function of peroxide-induced globin complexation before the conversion incubations. The incremental accumulation of tetramer was observed to decrease with increasing peroxide concentration used in the globin complexation step. The role of oxidized heme in this process was made apparent by the inability of carboxyhemoglobin to inhibit tetramer accumulation. A Western blot analysis of naturally formed globin-spectrin conjugates demonstrated irreducible complexes of globin with both bands 1 and 2. The complexes are tentatively designated "h1" and "h2". This analysis also demonstrated that h1 is completely extractable from cell ghosts, whereas h2 is only 50% extractable. These findings are incorporated into a hypothesis linking globin-spectrin complexation and the consequent inhibition of spectrin dimer self-association to the clustered band 3 senescence antigen (Low et al, Science 227:531, 1985).

Carboxyhemoglobin↗

PubMed1994

Quantitative assessment of the association of the alpha-I fragment of spectrin with oligomers of intact spectrin.

The alpha-I fragment of human spectrin that carries the binding site on the alpha-chain of spectrin for the beta-chain has been purified from limited trypsin digests of spectrin by means of FPLC. The self-association of spectrin and the binding of the alpha-I fragment to spectrin heterodimers and to tetramers have been quantified through the use of gel electrophoresis, staining with Coomassie Blue, and quantification of the bound dye following elution with pyridine. The parameters of self-association were found to be consistent with those estimated from sedimentation equilibrium analysis. The data were consistent with a model in which both self-association and the binding of the alpha-I fragment are considered to occur through an intermediate in which the alpha-beta interface is initially dissociated. The alpha-beta interface in the heterodimer was found to be less stable than that of higher oligomers by approx. 3 kJ/mol.

Binding Sites↗

PubMed1983

Fodrin is the general spectrin-like protein found in most cells whereas spectrin and the TW protein have a restricted distribution.

Spectrin and related proteins are made up of a common calmodulin-binding subunit tightly associated with a variant subunit. We have analyzed the distribution of the variant subunits in various cell types using subunit-specific antibodies in immunofluorescence as well as western blotting and in some cases have compared the subunits by two-dimensional peptide mapping. We have found that in the majority of cell types (lymphocytes, hepatocytes, neurons, fibroblasts) fodrin 235 K is present in the absence of the other two variant subunits, spectrin 220 K and TW260. Two cell types were found (skeletal muscle and erythrocytes) which contained only the spectrin variant. Two cell types display two distinct variant subunits. Both fodrin 235 K and spectrin 220 K are detected in cardiac muscle whereas TW260 is present in addition to fodrin 235 K in intestinal epithelial cells. During the early stages of embryonic development of the chicken intestine, fodrin 235 K is expressed in the epithelial cells whereas TW260 and spectrin are not detectable. TW260 is expressed relatively late in development (15-16 days) and is inserted only in the apical (brush border) membrane compartment whereas fodrin 235 K is present in these same cells and underlies the entire plasma membrane. These results suggest that fodrin provides the general linkage system between microfilaments and the membrane in nonerythroid and nonmuscle cells.

Animals↗