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PubMed1991

A splice site mutation of the beta-spectrin gene causing exon skipping in hereditary elliptocytosis associated with a truncated beta-spectrin chain.

We studied a French kindred with hereditary elliptocytosis associated with a spectrin variant (spectrin LePuy) containing a beta-spectrin chain that is truncated at its C terminus (Dhermy, D., Lecomte, M., Garbarz, M., Bournier, O., Galand, C., Gautero, H., Feo, C., Alloisio, N., Delaunay, J., and Boivin, P. (1982) J. Clin. Invest. 70, 707-715). The structure of the 3' end of the beta-spectrin gene, the region encoding the C terminus of beta-spectrin, was determined. Nucleotide sequencing of amplified genomic DNA revealed a mutation at position +4 (A----G) of the 5' donor consensus splice site of the intron following the third-to-last exon (exon X) in one beta-spectrin allele of a heterozygous patient. Agarose gel electrophoresis of polymerase chain reaction-amplified cDNA revealed an extra band of lower molecular weight, suggesting that the shortened beta-spectrin chain of spectrin LePuy arises from aberrant mRNA splicing. Nucleotide sequencing of the shorter cDNA amplification product revealed that the sequences encoding exon X were absent. Southern blotting of cDNA amplification products confirmed this result. The skipping of exon X causes a shift in the normal reading frame resulting in the encoding of a new amino acid sequence at the C terminus of the mutant beta-spectrin chain. A new in-frame stop codon is encountered following a single residue of this novel sequence.

Amino Acid Sequence↗

PubMed1990

Generation of diversity in nonerythroid spectrins. Multiple polypeptides are predicted by sequence analysis of cDNAs encompassing the coding region of human nonerythroid alpha-spectrin.

Nonerythroid alpha-spectrin (alpha-fodrin) is a major component of the membrane skeleton in diverse cell types. Overlapping cDNAs have been isolated which encompass the coding region of human lung fibroblast nonerythroid alpha-spectrin. The composite sequence of 7,787 nucleotides encodes a polypeptide of 2,472 amino acids (predicted Mr of 283,964). This sequence has 58% amino acid identity with human erythroid alpha-spectrin, which is encoded on a different gene, and 96% amino acid identity with the full-length sequence of chicken brain alpha-spectrin. We previously reported the variable expression in human fibroblast alpha-spectrin of 20 amino acids between repeats 10 and 11 (McMahon, A. P., Giebelhaus, D. H., Champion, J. E., Bailes, J. A., Lacey, S., Carritt, B., Henchman, S. K., and Moon, R. T. (1987) Differentiation 34, 68-78). In this study, we report additional heterogeneity in fibroblast alpha-spectrin near the carboxyl-terminal end. One of the fibroblast cDNAs (clone 3D) has an in-frame deletion of 18 nucleotides within spectrin repeat 21 when compared to an overlapping fibroblast cDNA (clone 7). As this heterogeneity in amino acid sequence occurs near domains of nonerythroid alpha-spectrin suggested to bind calcium or actin, it is possible that fibroblasts express functionally distinct isoforms of nonerythroid alpha-spectrin.

Amino Acid Sequence↗

PubMed1987

Isolation and characterization of sea urchin egg spectrin: calcium modulation of the spectrin-actin interaction.

Sea urchin egg spectrin has been purified from a homogenate of unfertilized Strongylocentrotus purpuratus eggs using standard biochemical procedures. SDS-PAGE analysis of the molecule revealed a closely spaced, high molecular weight doublet at 237/234 kDa (present in an equimolar ratio). Rotary shadowed images of egg spectrin revealed a double-stranded, elongate, flexible rod-shaped contour, measuring 210 nm in length and approximately 4-8 nm in width. Additionally, this molecule is shown to be immunologically related to avian erythroid spectrin, since it crossreacts with antibodies prepared against the chicken erythrocyte alpha-spectrin/240 kDa subunit. The interaction of egg spectrin with actin was examined by sedimentation and falling-ball viscometry assays. The binding and cross linking properties of spectrin to actin demonstrate a unique Ca++-sensitive regulation at micromolar Ca++ concentrations. This observation provides new insight into the way Ca++ may regulate spectrin-actin interactions in vitro and further suggests possible structural and modulatory roles for egg spectrin in the developing sea urchin embryo.

Actins↗

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↗

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↗

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↗

PubMedDate not supplied

Brain spectrin(240/235A): a novel astrocyte specific spectrin isoform.

We have previously demonstrated the existence of two distinct isoforms of spectrin in mammalian brain (23). Brain spectrin(240/235) is found primarily in neuronal axons and presynaptic terminals, and brain spectrin(240/235E) is located in neuronal cell bodies, dendrites and postsynaptic terminals, and oligodendrocytes. These isoforms are thought to play important roles in controlling the early events of synaptic transmission, axonal transport of organelles and vesicles, and lateral mobility of integral membrane proteins. In this study, we have utilized a panel of monoclonal antibodies to identify a novel astrocyte specific isoform(240/235A) with subunits of 240 kDa and 235 kDa in a 1:1 ratio. Double label indirect immunofluorescence has indicated that brain spectrin (240/235A) is distinct from brain spectrin (240/235E). This novel isoform located in the soma and processes of astrocytes may play a role in actin-membrane attachment, cellular architecture, strengthening of the membrane fabric, and translocation of cytoplasmic organelles and vesicles.

Animals↗

PubMed1992

Elliptocytogenic alpha I/36 spectrin Sfax lacks nine amino acids in helix 3 of repeat 4. Evidence for the activation of a cryptic 5'-splice site in exon 8 of spectrin alpha-gene.

Elliptocytogenic alpha I/36 spectrin Sfax is a new variant found in a Tunisian family. The alpha I/36 allele yielded a clinically manifest picture only when occurring in trans to a recently identified, low expression level polymorphism referred to as the alpha V/41 allele. Spectrin dimers were slightly increased in 4 degrees C extracts. On peptide maps, the alpha I domain split into two abnormal fragments of 36 and 33 Kd. The mutated alpha-chain represented 20% and 44% of total alpha-chain in alpha/alpha I/36 and alpha V/41/alpha I/36 heterozygotes, respectively. Peptide sequencing showed that the 36-Kd fragment started at Ala 357 and displayed a deletion extending from amino acids 363 to 371. The corresponding 27-nucleotide deletion was found in alpha-spectrin mRNA. However, exon 8 of spectrin alpha-gene failed to disclose this deletion. Instead, an A to G substitution appeared in position 3 of codon 362, leading to the occurrence of the critical GU dinucleotide within a cryptic 5'-splice site surrounding codon 362. This event would account for the splicing out of codons 363 to 371. The reading frame was preserved and even amino acid 362 (AGG, Arg) remained unaltered. As in most spectrin alpha-chain elliptocytogenic variants, the change involved a helix 3. This is the first elliptocytogenic mutation recorded in repeat alpha 4.

Adult↗

PubMed1991

Molecular defect of truncated beta-spectrin associated with hereditary elliptocytosis. Beta-spectrin Gottingen.

A large German family with autosomal dominantly inherited elliptocytosis, associated with truncated beta-spectrin missing the phosphorylated C-terminal peptide, has been described (Eber, S.W., Morris, S. A., Schroeter, W., and Gratzer, W. B. (1988) J. Clin. Invest. 81, 523-530). We have attempted to delineate the molecular defect of this abnormality at the gene level. Southern blot analyses revealed no evidence of a partial gene deletion or rearrangement. We used polymerase chain reaction (PCR) and amplified several relevant portions of the beta-spectrin gene, using genomic DNA from two different heterozygous patients. No abnormality was found in the last four exons of the beta-spectrin gene produced by PCR. Examination of the introns connecting the last four exons revealed a T to A substitution in the 5' splice site following the exon X in four of eight clones prepared from two affected individuals, but not from a normal subject of this family. To examine the effect of the T to A substitution in these patients, we made cDNA from the reticulocyte mRNA of the patient and examined its composition by PCR. Two distinct PCR fragments produced from the patient's beta-spectrin cDNA were found. One matched the predicted size for normal spectrin, while the other was 197 base pairs shorter. The mutant cDNA sequence revealed that the entire exon preceding the T to A substitution was spliced out, while the two terminal exons were preserved. The deletion of this exon resulted in a frameshift giving a different terminal amino acid (serine instead of leucine) as well as a new termination codon causing a deletion of 129 amino acids including potentially phosphorylated residues.

Base Sequence↗

PubMed1979

Structural studies on human spectrin. Comparison of subunits and fragmentation of native spectrin.

Native spectrin has trypsin-susceptible sites spaced at a constant molecular weight interval. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of spectrin treated with trypsin at low salt concentrations shows a ladder of fragments spaced at approximately 8,000-dalton intervals, from the intact Band 1 (240,000 daltons) and Band 2 (220,000 daltons) down to about 150,000 daltons. The five largest fragments were identified as products of Band 2 using tryptic 125I-peptide mapping of protein from gel slices. Endogenously incorporated [32P]phosphate is absent from the largest fragment, indicating that all phosphorylation sites on spectrin are within 8,000 daltons of a terminal of Band 2. Mapping of both [14C]carboxyamidomethylated cysteine-containing tryptic peptides and 125I-peptides reveals extensive sequence homology between the spectrin subunits. Further, only somewhat over half of the distinct spots expected from the cysteine content are found in both Band 1 and Band 2 peptides. These and the tryptic susceptibility results are interpretable as evidence for a repeating structure in spectrin.

Amino Acids↗

PubMed1979

Purification of two spectrin-binding proteins: biochemical and electron microscopic evidence for site-specific reassociation between spectrin and bands 2.1 and 4.1.

Two peripheral proteins of the human erythrocyte membrane that are capable of forming a stable complex with spectrin have been purified. The proteins, band 2.1 (Mr 210,000) and band 4.1 (Mr 82,000), are water soluble and exist as monomers in solution. Both exhibit strong, specific binding to purified spectrin molecules as determined by cosedimentation in sucrose gradients and both enhance binding to spectrin-depleted, inside-out vesicles that have been stripped of bands 2.1 and 4.1. Rotary replicas of bound material reveal site-specific associations among native, but not heat-denatured, molecules.

Binding Sites↗