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PubMed1978

Binding of protoporphyrin and haemin to human spectrin.

Haemin and protoporphyrin IX, but not bilirubin, are extensively bound by human spectrin. The absorption spectrum of the bound haemin is indicative of coordination of the iron by nitrogenous ligands in the protein. The protoporphyrin IX generates difference spectra on binding, which change with ligand:protein ratio, showing the existence of at least two structurally distinct types of site. The binding of both ligands is complex, and may be cooperative. Binding isotherms, based on spectrophotometric titrations, are given. Haemin and protoporphyrin IX also bind strongly to erythrocyte ghosts. At ionic strengths near physiological we can find no evidence of binding of haemoglobin to spectrin, as judged by sedimentation velocity, and it appears that reported interactions of this nature represent only non-specific binding at low ionic strength.

Bilirubin↗
PubMed1993

Low expression allele alpha LELY of red cell spectrin is associated with mutations in exon 40 (alpha V/41 polymorphism) and intron 45 and with partial skipping of exon 46.

The alpha V/41 polymorphism of erythroid alpha-spectrin has been characterized initially by an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction (Alloisio N., L. Morlé, J. Maréchal, A.-F. Roux, M.-T. Ducluzeau, D. Guetarni, B. Pothier, F. Baklouti, A. Ghanem, R. Kastally, et al. 1991. J. Clin. Invest. 87:2169-2177). Until now, it has been found associated invariably with a low expression level of the corresponding alpha chain. Among 61 chromosomes investigated in French and North African individuals or kindreds, we observed 19 chromosomes with the alpha V/41 polymorphism. With no single exception, the latter displayed a point mutation in exon 40 (Leu-->Val; CTA-->GTA) at position alpha 1857. According to the triple helical model of spectrin structure, this change accounts for the peptide maps' abnormalities. Sequencing the entire alpha V domain cDNA disclosed, in addition, a partial skipping of exon 46. At the gene level, a substitution (C-->T) was evidenced at nucleotide -12 of intron 45. This mutation appeared linked to the exon 40 mutation in 17 chromosomes, again with no single exception, among 53 examined chromosomes. We hypothesized that the lack of exon 46 would hamper the nucleation process and eventually account for the low expression feature. The present doubly mutated allele was renamed allele alpha LELY (low expression, Lyon).

Alleles↗
PubMed1981

Identification of abnormally [32P]-phosphorylated cyanogen bromide cleavage product of erythrocyte membrane spectrin in Duchenne muscular dystrophy.

We measured [32P]-phosphorylation of erythrocyte membrane spectrin band 2 peptides from patients with Duchenne muscular dystrophy. Erythrocyte ghosts were prepared and subjected to [32P]-phosphorylation by endogenous protein kinase incubations. Purified spectrin was then cleaved by cyanogen bromide, and the resulting peptides were analyzed by electrophoresis on 5%/15% SDS polyacrylamide stacking slab and tube gel systems. More than 50% of the incorporated [32P] was associated with a single band, CN-A, with an apparent molecular weight of 23 kilodaltons. The Coomassie blue-stained peptides were identical in patients and controls. Band CN-A represented approximately 2% of the total peptide protein but was more [32P]-phosphorylated in patients than in controls.

Autoradiography↗
PubMed2001

Nesprins: a novel family of spectrin-repeat-containing proteins that localize to the nuclear membrane in multiple tissues.

In search of vascular smooth muscle cell differentiation markers, we identified two genes encoding members of a new family of type II integral membrane proteins. Both are ubiquitously expressed, and tissue-specific alternative mRNA initiation and splicing generate at least two major isoforms of each protein, with the smaller isoforms being truncated at the N-terminus. We have named these proteins nesprin-1 and -2 for nuclear envelope spectrin repeat, as they are characterized by the presence of multiple, clustered spectrin repeats, bipartite nuclear localization sequences and a conserved C-terminal, single transmembrane domain. Transient transfection of EGFP-fusion expression constructs demonstrated their localization to the nuclear membrane with a novel C-terminal, TM-domain-containing sequence essential for perinuclear localization. Using antibodies to nesprin-1, we documented its colocalization with LAP1, emerin and lamins at the nuclear envelope, and immunogold labeling confirmed its presence at the nuclear envelope and in the nucleus where it colocalized with heterochromatin. Nesprin-1 is developmentally regulated in both smooth and skeletal muscle and is re-localized from the nuclear envelope to the nucleus and cytoplasm during C2C12 myoblast differentiation. These data and structural analogies with other proteins suggest that nesprins may function as 'dystrophins of the nucleus' to maintain nuclear organization and structural integrity.

Amino Acid Sequence↗
PubMed1990

Localization of actin, beta-spectrin, 43 x 10(3) Mr and 58 x 10(3) Mr proteins to receptor-enriched domains of newly formed acetylcholine receptor aggregates in isolated myotube membranes.

I have examined the possible involvement of specific cytoskeletal and peripheral membrane proteins in the early stages of acetylcholine receptor (AChR) aggregation in rat myotubes in culture by immunofluorescence localization of these proteins on the cytoplasmic face of isolated plasma membranes. A culture procedure utilizing selective replating of myoblasts and subsequent treatment with cytosine arabinoside was devised to obtain large, multipolar myotubes with extensive upper surfaces that are free of fibroblasts. These cultures were exposed for 4-6 h to embryonic pig brain extract (EBX) to induce AChR aggregate formation on the upper cell surface, and the AChRs were labeled with TRITC-conjugated alpha-bungarotoxin. Large sheets of plasma membranes from the upper cell surface were isolated by adhesion to a coverslip coated with a polypeptide adhesive (Cell-Tak) that was pressed on top of the culture. The membranes were labeled by indirect immunofluorescence with monoclonal antibodies against the 43 x 10(3) Mr and 58 x 10(3) Mr proteins, originally identified in the AChR-enriched membranes of Torpedo electroplaques, and with monoclonal antibodies against isoforms of actin and beta-spectrin. The labeling patterns showed that all four of these proteins are concentrated in the punctate AChR-enriched domains within the aggregates, suggesting that they may be involved in the early stages of AChR aggregation. Immunofluorescence labeling with monoclonal antibodies against vinculin and clathrin, and with an antiserum to talin, showed that these proteins are also associated with AChR aggregates; however, their labeling patterns did not correspond closely to the AChR-enriched domains. Furthermore, vinculin and talin dissociated from most of the membrane during isolation. The concentration of beta-spectrin and actin isoforms on the cytoplasmic fact of the AChR-enriched domains is consistent with the formation, early in the aggregation process, of a membrane-cytoskeleton association similar to that of erythrocytes.

Actins↗
PubMed2001

A novel isoform of beta-spectrin II localizes to cerebellar Purkinje-cell bodies and interacts with neurofibromatosis type 2 gene product schwannomin.

We report the identification of a full-length novel beta-spectrin II gene (betaSpIIsigma2) in human brain. The betaSpIIsigma2 gene has 32 exons encoding an actin-binding domain, followed by 17-spectrin repeats, and a short COOH-terminal regulatory region that lacks the Pleckstrin homology (PH) domain. Pair-wise sequence analysis showed an additional 36 and 28 amino acids located at the NH2 and COOH-terminal regions of betaSpIIsigma2, respectively. Northern-blot analysis showed an abundant expression of betaSpIIsigma2 transcripts in brain, lung, and kidney. Western-blot analysis confirmed the predicted approximately 225 kD molecular size of betaSpIIsigma2 protein in these same tissues. In brain, immunofluorescent staining revealed that betaSpIIsigma2 was enriched in cerebellar neurons, with specific enrichment in Purkinje cell bodies, but not in dendrites. Of considerable interest, neurofibromatosis type 2 (NF2) gene product schwannomin was found to co-immunoprecipitate with betaSpIIsigma2 in cultured Purkinje cells. These results suggest that betaSpIIsigma2 may play an important role in the assembly of the specialized plasma membrane domain of Purkinje neurons and that schwannomin may be involved in actin-cytoskeleton organization by interacting with betaSpIIsigma2.

Amino Acid Sequence↗
PubMed2002

Selective release of calpain produced alphalI-spectrin (alpha-fodrin) breakdown products by acute neuronal cell death.

Activation of calpain results in the breakdown of alpha II spectrin (alpha-fodrin), a neuronal cytoskeleton protein, which has previously been detected in various in vitro and in vivo neuronal injury models. In this study, a 150 kDa spectrin breakdown product (SBDP150) was found to be released into the cell-conditioned media from SH-SY5Y cells treated with the calcium channel opener maitotoxin (MTX). SBDP150 release can be readily quantified on immunoblot using an SBDP150-specific polyclonal antibody. Increase of SBDP150 also correlated with cell death in a time-dependent manner. MDL28170, a selective calpain inhibitor, was the only protease inhibitor tested that significantly reduced MTX-induced SBDP150 release. The cell-conditioned media of cerebellar granule neurons challenged with excitotoxins (NMDA and kainate) also exhibited a significant increase of SBDP150 that was attenuated by pretreatment with an NMDA receptor antagonist, R(-)-3-(2-carbopiperazine-4-yl)-propyl-1-phosphonic acid (CPP), and MDL28170. In addition, hypoxic/hypoglycemic challenge of cerebrocortical cultures also resulted in SBDP150 liberation into the media. These results support the theory that an antibody-based detection of SBDP150 in the cell-conditioned media can be utilized to quantify injury to neural cells. Furthermore, SBDP150 may potentially be used as a surrogate biomarker for acute neuronal injury in clinical settings.

Analysis of Variance↗
PubMed1989

The influence of experimental conditions on the spectrin-hemoglobin interaction.

Human spectrin, when isolated, purified and stored in such conditions that preserve its tetrameric form, is able to associate with human hemoglobin as it is clearly shown by gel filtration. However, this hemoglobin-spectrin association does not seem to have a significant effect on hemoglobin oxygenation as indicated by equilibrium and rapid kinetics measurements.

Adhesiveness↗
PubMedDate not supplied

[Possible role of spectrin in thermohemolysis of erythrocytes].

The activation energy of thermodenaturation of erythrocyte spectrin was defined by two different methods as Esp = 218 +/- 4 kcal/mole. This value was more than two times greater than that of thermohemolysis activation energy (Eth = 99 +/- 5 kcal/mole). The conclusion is made that spectrin does not play a significant role in thermohemolysis of erythrocytes.

Hemolysis↗
PubMed1988

Spectrin Tunis (alpha I/78): a new alpha I variant that causes asymptomatic hereditary elliptocytosis in the heterozygous state.

Spectrin Tunis (alpha 1/78) was found in the heterozygous state in a young white North-African man and his mother. Both of them presented with mild elliptocytosis. Using one-dimensional electrophoresis, a sharp 78 kd fragment was present with a reciprocal decrease of the alpha I 80 kd domain. Kinetic analysis unambiguously confirmed that the 78 kd fragment developed at the expense of the alpha I 80 domain. The alpha I 74 kd peptide was not flanked with a peptide lacking a 2 kd fragment. From this fact, it could be inferred that the site for additional proteolysis is located upstream from arginyl residue 39 and, more precisely, should lie 10 to 20 amino-acid residues (-2 kd) from the alpha-chain N-terminus. The percentage of spectrin dimers in 4 degrees C extracts was high (over 40%), contrasting with the absence of clinical symptoms related to elliptocytosis. This is the first mutation responsible for elliptocytosis found in Tunisia.

Adult↗
PubMed1988

Role of spectrin in cross bonding of the red cell membrane.

Membrane cross bonding--an adhesion between opposing areas of the cytoplasmic face of the red cell membrane--was achieved by treating red cells with heat, diamide, N-ethymaleimide, urea, or by ATP depletion in conjunction with cell shrinking. Membrane cross bonding could be recognized by the shape of the cells upon swelling. Quantitated by the percentage of cross-bonded red cells the effectivity of the treatments decreased in the order given above. Cross bonding was hardly reversible by reducing the diamide-induced S-S bonds with dithioerythritol. The effect of heat and urea treatment as well as ATP depletion was partly reversible. Transmission electron micrographs of the cross-bonded region showed basically parallel membranes. The distance between the respective phospholipid bilayers varied between 40 and 120 nm from cell to cell. Hb-free ghosts prepared from diamide-treated red cells could also be cross bonded. The following conclusions are drawn: spectrin provides the molecular cross link in membrane cross bonding. Aggregation and enrichment of spectrin in the cross-bonded region are probably involved in membrane cross bonding.

Adenosine Triphosphate↗
PubMed1986

Structure of the spectrin-actin binding site of erythrocyte protein 4.1.

The complete primary structure of the functional site of erythrocyte protein 4.1 involved in spectrin-actin associations has been determined. The sequence of this domain, which contains 67 amino acids and has a molecular mass of 8045 daltons, has been obtained by NH2-terminal sequence analysis of an 8-kDa chymotryptic peptide, three endoproteinase lysine C-cleaved peptides and two peptides obtained by Staphylococcus aureus protease V8 cleavage. All peptides including the 8-kDa domain peptide were purified by reverse-phase high performance liquid chromatography. Antibodies against two different synthetic peptides of the 8-kDa domain are able to inhibit the association between protein 4.1, spectrin, and F-actin, corroborating that the 8-kDa domain is responsible for the formation of a ternary complex. A computer search of the 8-kDa sequence with the National Biomedical Research Foundation database did not detect any significant homologies to known sequences. Protein 4.1 is not related to any known proteins and may represent a new protein superfamily.

Actins↗
PubMed1987

Hydrophobic labelling of spectrin in erythrocytes using arylisothiocyanates.

Labelling of spectrin with the hydrophobic probes 2-naphthylisothiocyanate and 4-[125I]-iodophenylisothiocyanate dispersed in lipid vesicles was studied. Although spectrin is a peripheral protein of the erythrocyte membrane, it was found to be labelled as a result of incubation of washed red blood cells with hydrophobic arylisothiocyanates.

1-Naphthylisothiocyanate↗
PubMed1986

Identification of the functional site of erythrocyte protein 4.1 involved in spectrin-actin associations.

Peptides produced by mild chymotryptic digestion of human erythrocyte protein 4.1 mimic the ability of intact 4.1 to promote the binding of spectrin to F-actin. This complex-promoting activity was found to reside in an 8-kDa peptide which was fully functional when dissociated from other protein 4.1-derived peptides, indicating that noncovalent complexes of multiple peptides were not essential for activity. The 8-kDa peptide was incorporated into a ternary complex with spectrin and F-actin in approximately stoichiometric amounts. Amino acid composition and two-dimensional peptide mapping show that the 8-kDa active peptide is located within the 10-kDa region of protein 4.1 which contains a cAMP-dependent phosphorylated site.

Actins↗
PubMed1983

Spectrin: structure, function, and abnormalities.

A number of proteins that make up the membrane skeleton have been identified, and we have a rough but tantalizing picture of the ways in which they interact to maintain its integrity. An approximate molecular model of spectrin has been proposed, and many new analytic procedures have been developed to search for biochemical variants that may be related to membrane defects. Even at this rudimentary stage of molecular description we have been able to identify structural changes in spectrin that are correlated with pathophysiologic states. The prospects for some genuine insights into the pathogenesis of some congenital hemolytic anemias seem good.

Anemia, Hemolytic, Congenital↗
PubMed1983

[Genetic polymorphism of the alpha chain of spectrin].

On 80 black or white subjects, spectrin subunits were purified by SDS-polyacrylamide gel electrophoresis and subjected to limited alpha-chymotryptic digestion according to the Cleveland method. A genetic polymorphism exists in spectrin alpha chain and is not associated with a membranous disease. Up to now, this variant has only been detected in the black population.

Black People↗
PubMed1984

Structural and functional features of the alpha-1 domain from human erythrocyte spectrin.

The complete sequence of the alpha-I domain of human erythrocyte spectrin has been determined. It contains a single type of internal homology comprised of multiple 106 amino acid repeats consistent with the occurrence of multiple gene duplications during the course of spectrin evolution. The only portion of the alpha-I sequence that does not appear to contain the sequence repeat is a segment containing the amino terminal 17 residues which may contain all or part of the alpha subunit portion of the self-association site. Secondary structural predictions and modeling suggest that each repeat unit contains a triple helical segment approximately 50 A in length. The alpha-I sequence is apparently unrelated to any other known protein sequence and apparently represents the first structural elucidation of a new class of proteins.

Amino Acid Sequence↗
PubMed1983

Structure of human erythrocyte spectrin. I. Isolation of the alpha-I domain and its cyanogen bromide peptides.

The alpha-I domain of human erythrocyte spectrin was produced by a mild tryptic digestion of the intact molecule and purified by a single step affinity chromatography procedure using a monoclonal antibody. A tryptic peptide representing the alpha-I domain, which migrated on polyacrylamide gels as an 80,000-dalton peptide, was subjected to automated Edman-Begg degradation. Products from automated sequencing were identified by reverse-phase high performance liquid chromatography. Two smaller proteolytic products of the alpha-I domain (T74 and T50) were also subjected to automated sequence analysis. CNBr cleavage of the alpha-I domain produced nine unique peptides which were separated by gel filtration on a high performance liquid chromatograph. Peptides were further purified by reverse-phase chromatography and characterized by amino acid analysis. Partial sequences were determined by automated NH2-terminal sequence analysis. A single aspartate-proline bond, which was partially hydrolyzed during the cyanogen bromide cleavage reaction, was also identified. These sequence data include the first 86 residues of the alpha-I domain, and the spectrin oligomer binding site has been tentatively localized within the first 39 residues. The sequence of 293 residues of a total 633 residues in the alpha-I domain is presented and represents the first structural information for this protein.

Amino Acid Sequence↗