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PubMed1992

Properties of human red cell spectrin heterodimer (side-to-side) assembly and identification of an essential nucleation site.

The antiparallel side-to-side association of spectrin alpha and beta monomers is a two-step process which occurs in seconds even at 0 degrees C and at low concentrations. Assembly involves initial contact of complementary nucleation sites on each subunit, which are located near the actin binding end of the long, flexible heterodimer rod. The minimum nucleation sites are comprised of approximately four contiguous 106-residue homologous segments or repeats. Three repeats in the nucleation site contain an 8-residue insertion and have the highest homology to the four spectrin-like repeats in alpha-actinin. The adjacent actin binding domain on the beta subunit and the adjacent EF hand motifs on the alpha subunit are not required for heterodimer assembly. The nucleation sites probably have a specific lock and key structure which defines the unique side-to-side pairing of the many homologous segments in both subunits. Assembly of spectrin heterodimers is probably most analogous to a zipper. After initial nucleation site binding, the remainder of the subunits quickly associate along their full lengths to reconstitute a normal dimer by supercoiling around each other to form a rope-like, flexible rod. Assembly is terminated if either polypeptide is interrupted by a protease cleavage. Heterozygotic mutations involving either nucleation site are predicted to affect allele incorporation into the mature membrane skeleton.

Amino Acid Sequence↗
PubMed1991

[Spectrin extractabilities from erythrocyte membranes in patients with myotonic dystrophy].

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectrin extractabilities (SE) were determined after spectrins were removed from erythrocyte membranes by low ionic strength and chelating agent. The SE from red cell membranes was significantly reduced with respect to matched controls in 13 myotonic dystrophy patients, 81.3 +/- 7.2% and 54.2 +/- 9.1% (m +/- SD), respectively (P less than 0.01). There were no significant differences between the patients and controls in molecular weights and amounts of spectrin as well as Band3 protein. Our results are consistent with the concept of membrane defect affecting erythrocytes in this disorder.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1991

Distribution of actin, actin-binding proteins, 43K protein, and spectrin in D. tschudii electrocytes.

With a view to establishing the location of proteins related to the nicotinic cholinergic receptor (AChR), immunocytochemical studies were carried out in the electric tissue of the South American ray Discopyge tschudii. The receptor protein was localized at the innervated, ventral region of the electrocyte in cryostat sections using Texas Red-labelled alpha-bungarotoxin, a specific AChR marker. A series of antibodies against actin and actin-associated proteins (alpha-actinin, vinculin, tropomyosin and spectrin) were also employed in indirect immunofluorescence assays. Furthermore, with the aid of the monoclonal antibody 1234A, the peripheral, nonreceptor 43K protein was found to be colocated with the AChR and spectrin at the ventral surface of the electrocyte. NBD-phallacidin was used to detect the filamentous form of actin or F-actin. The anti-actin antibody recognized epitopes throughout the electrocyte cytoplasm. F-actin was mainly distributed at the dorsal, non-innervated region, where alpha-actinin, vinculin and tropomyosin were also localized. The results indicate a) the existence of a close structural relationship between the 43K protein, spectrin, and the AChR at the ventral, innervated face of the electrocyte; b) the occurrence of an actin isoform probably mono or oligomeric-throughout the electrocyte's cytoplasm, and c) the possible association of F-actin, localized at the dorsal region of the electrocyte, with alpha-actinin, tropomyosin and vinculin, as in other cellular systems.

Actins↗
PubMed1978

[Studies of spin-labeled spectrin].

Spectrin isolated from human erythrocytes has been spin-labeled with five maleimide nitroxides. The mobility of the labels is strongly dependent on their size, and on the temperature. A thermal transition of spectrin is shown to occur above 30 degrees C. Calcium and magnesium provoke a strong immobilization of the labels. This effect is yet more pronounced when spectrin is allowed to reassociate with the cytoplasmic surface of the membrane.

Electron Spin Resonance Spectroscopy↗
PubMed1990

Cloning and chromosomal localization of the human cytoskeletal alpha-actinin gene reveals linkage to the beta-spectrin gene.

We report the cloning and characterization of a full-length cDNA encoding the human cytoskeletal isoform of alpha-actinin (alpha A), a ubiquitous actin-binding protein that shares structural homology with spectrin and dystrophin. The gene encodes 891 amino acids with 96%-98% sequence identity at the amino acid level to chicken nonskeletal muscle alpha A. Transient expression in COS cells produces a protein of approximately 104 kD that comigrates on SDS-PAGE with native alpha A. This alpha A gene is localized to chromosome 14q22-q24 by somatic cell hybrid and in situ hybridization analyses. Pulsed-field gel analysis of human genomic DNA revealed identically sized fragments when cDNA probes for alpha A and erythroid beta-spectrin were used; the latter gene has been previously localized to chromosome 14, band q22. These observations indicate that the genes for cytoskeletal alpha A and beta-spectrin are, in all likelihood, closely physically linked and that, in accordance with their similar structural features, they arose by partial duplication of an ancestral gene.

Actinin↗
PubMed1989

Molecular basis of Sp alpha I/65 hereditary elliptocytosis in North Africa: insertion of a TTG triplet between codons 147 and 149 in the alpha-spectrin gene from five unrelated families.

Hereditary elliptocytosis in North Africa is frequently associated with the alpha I/65 spectrin variant, characterized by an abnormal alpha I 65-kD instead of the normal alpha I 80-kD peptide following limited trypsin digestion of whole spectrin. A similar variant (although it yielded a 68-kD fragment) has been shown recently, in two black patients, to result from the insertion of a leucyl residue at position 148 (Marchesi et al: J Clin Invest 80:191, 1987). In order to determine if the underlying molecular defect was the same in North Africans and blacks (who originate from both sides of the Sahara Desert), we performed analysis directly at the DNA level. Starting from the DNA of an Algerian alpha I/65 heterozygote in whom the mutation was associated with identifiable RFLPs, we cloned and sequenced the alpha-spectrin gene region, which includes the mutation. We thus identified an extra leucine codon (TTG) between codons 147 and 149, the coding sequence becoming CAG TTG TTG CTG instead of CAG TTG CTG. We then used the polymerase chain reaction (PCR) method and dot-blot hybridization of the amplified DNA with mutant and normal allele-specific oligonucleotides to screen the DNA from four other unrelated North African subjects with Sp alpha I/65 hereditary elliptocytosis. In all families we studied, these subjects were heterozygous for the TTG insertion. These results demonstrate that Sp alpha I/65 hereditary elliptocytosis has the same molecular basis in North Africans and blacks.

Africa, Northern↗
PubMed1989

Sp alpha I/78: a mutation of the alpha I spectrin domain in a white kindred with HE and HPP phenotypes.

Limited tryptic digestion of spectrin (Sp) from seven related individuals manifesting hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP) phenotypes revealed the presence of a novel peptide with a molecular weight of 78 Kd and a concomitant decrease in the alpha I domain (80-Kd peptide), which is the domain involved in the dimer self-association process. Sp from the normal members of this white family exhibited a normal peptide pattern, as compared with controls. The abnormal peptide pattern was associated with a decreased ability of Sp dimer to self-associate. In this kindred in which three generations were available for study, the clinical manifestations were quite variable and ranged from the asymptomatic HE carrier state to hemolytic HE or to severe anemia requiring splenectomy. The severity of the disease appeared to be correlated both with the amount of mutant spectrin (31% to 69%) and with the excess of the Sp dimer found in the membrane (26% to 60%, compared with a normal value of 5.6% +/- 2.2%). Partial amino acid sequencing showed that the alpha I/78-Kd peptide resulted from cleavage at lysine residue 10 of the alpha I/80-Kd domain. Knowledge of the exon/intron structure of cloned genomic DNA encoding the alpha I domain allowed us to amplify in vitro a DNA fragment containing the third exon of the alpha-spectrin gene. The amplified fragment was subcloned and sequenced. A G to T transversion was found in the 39th codon (AGT for AGG), which changed the normal arginine to a serine. Hybridization of amplified DNAs with allele-specific oligonucleotides corresponding to the normal and mutant sequences confirmed the presence of the mutation in three other HE members of the family (the propositus mother, brother, and sister).

Adult↗
PubMed1979

Parallel decrease of erythrocyte membrane deformability and spectrin solubility at low pH.

Reduction of pH over the range 6.0-4.5 results in a decrease of erythrocyte deformability in parallel with the induced progressive sphericity of cells. At low deformation rates employed, increase of hemoglobin viscosity was not significant. A decrease of membrane elasticity was detected in cells when sphering, the major determinant of cellular deformability, was prevented at pH 5.0 by hyperosmotic medium. The pronounced change of deformability and the reduced elasticity occurred at calculated intracellular pH values at which solubility in vitro of extracted erythrocyte spectrin is also markedly reduced. The parallel decrease of deformability and spectrin solubility supports the hypothesis that spectrin aggregation may contribute to regulation of erythrocyte deformability through effects on shape and membrane elastic characteristics.

Erythrocyte Membrane↗
PubMed1988

[Hereditary elliptocytosis in West Africa: frequency and repartition of spectrin variants].

Hereditary Elliptocytosis (HE) is a hemolytic disorder inherited as autosomal-dominant trait and characterized by elliptically shaped erythrocytes. Preliminary studies in France have showed a high proportion of HE patients of black extraction (West Africa and Antilles). In order to confirm this prevalence, we made a systematic search for HE in West Africa: Benin, Burkina Faso, Ivory Coast, Togo. The diagnosis of elliptocytosis was established by the observation of a high percentage (greater than 70%) of characteristic regular and symmetric elliptic red cells after fixation in 0.3% glutaraldehyde saline buffer. The diagnosis of HE was confirmed by cytological studies of related members and/or the discovery of a well defined molecular variant of spectrin, the main protein of erythrocyte membrane skeleton. We found: in Abidjan centre 6 HE out of 1,000 subjects representative of main ethnic groups; in Lome Centre 6 cases out of 750 subjects originated from the South or Central areas of Togo; in Cotonou Centre 5 cases out of 1,000 subjects originated from the South area of Benin; in Bobo Dioulasso centre 6 HE out of 700 subjects. From this multicentre studies HE appears roughly 10 times more frequent in West Africa than in Europe or USA where incidence was estimated at between 2.5 and 5 cases per 10,000. Tryptic digestion of spectrin revealed that: 10 patients from different ethnic groups have the most frequent variant found in our laboratory (21 kindreds) and named spectrin alpha I/65. Five cases originated from limited areas in the South of Benin and Togo and related to closed ethnic groups have the variant Sp alpha I/46.

Africa, Western↗
PubMed1986

A potential role for spectrin during neurulation.

An actin-myosin complex located in apical regions of the neurectoderm has been postulated to play a role in neurulation. Numerous studies have documented the presence of microfilaments in this area and confirmed their composition as actin. By necessity, if such a contractile system is to exert a force, these filaments must be anchored in some way to the cell membrane. In this study, the presence of the actin-binding protein, spectrin (fodrin), is demonstrated in the neurectoderm of neurulating mouse embryos using antispectrin antibodies and indirect immunofluorescent techniques. The patterns of spectrin localization correlate with the previously reported regions of increased numbers of microfilaments and also with the morphology of the neural folds. Thus, during the initial stages of cranial fold elevation, a process reportedly dependent on increased glycosaminoglycan synthesis, little spectrin is present in the neuroepithelial cells. Later as the folds begin to converge toward the midline, deposition of the protein, as demonstrated by the intensity of fluorescence, is increased in the apices of these cells, and is most prominent in regions of greatest bending in the neural folds. Caudal neural fold regions show a similar pattern of staining. Thus, the hypothesis that a cytoskeletal system assists in neurulation is supported by these results, which for the first time demonstrate the presence of a putative actin-membrane attachment protein in a morphogenetically active system.

Animals↗
PubMed1979

Identification by peptide analysis of the spectrin-binding protein in human erythrocytes.

One-dimensional and two-dimensional peptide-mapping techniques are used to identify the protein which gives rise to the 72,000 dalton alpha-chymotryptic fragment previously shown to be the membrane attachment site for spectrin. Peptide maps of the 72,000 dalton fragment are very different from maps of Bands 1, 2, 2.9, 3, 3.1, 4.1, and 4.2 and very similar to maps of the apparently closely homologous polypeptides, Bands 2.1, 2.2, 2.3, and 2.6. Limited proteolysis of erythrocyte membranes is shown to generate Band 3', another polypeptide which has been associated with spectrin-binding activity. Peptide maps of Band 3' are very similar to maps of Band 2.1, suggesting that Band 3' is also a proteolytic fragment of Band 2.1. It is concluded that Band 2.1 and possibly some or all of the other, related polypeptides which electrophorese in the 2 region is (are) the spectrin-binding protein(s) of the human erythrocyte.

Carrier Proteins↗
PubMed1981

Calmodulin-dependent spectrin kinase activity in human erythrocytes.

Membrane protein phosphorylation has been studied in intact human erythrocytes and in resealed erythrocyte ghosts by measuring the incorporation of 32P into band 2 of spectrin. alpha-Adrenergic agonists and Ca+2 stimulate 32P-phosphate incorporation, an effect inhibited by trifluoperazine and diminished in resealed ghosts depleted of calmodulin. Ghosts prepared with endogenous calmodulin or resealed around purified calmodulin exhibit norepinephrine- and Ca+2-stimulated phosphorylation only in the presence of [gamma-32P]-ATP. Ghosts resealed with or without calmodulin in the presence of unlabelled ATP show no net gain or loss of 32P in membrane proteins when exposed to norepinephrine or calcium stimulation. These observations suggest that calcium and norepinephrine stimulation of membrane protein phosphorylation is mediated by calmodulin-dependent spectrin kinase activity, rather than by increased turnover by spectrin ATPase or by inhibition of phosphospectrin phosphatase.

Adenosine Triphosphate↗
PubMedDate not supplied

ESR investigation of the structural transition of spectrin in ghosts and in solution.

The effect of temperature on the human erythrocyte ghosts and on isolated spectrin, both labelled with a protein spin label (2, 2, 6, 6 tetramethylpiperidin - 1 - oxyl - 4 - maleimide), was studied by the electron spin resonance spin label method. The results show that the spectra associated with the membrane undergo significant changes with temperature and a well-defined transition is detected at 52 degrees C. The observed transition is removed from the membrane by the extraction of spectrin and in the supernatant the transition is unchanged from that exhibited by the native ghosts. The results show that it is possible to observe the heat dependent transition of the spectrin in whole ghosts avoiding the extraction procedure.

Electron Spin Resonance Spectroscopy↗
PubMedDate not supplied

[Pyruvate and glucose transport through the erythrocyte membranes and the role of spectrin in these processes].

Effect of antibody to peripheral protein spectrin and antibody to integral protein of band 3 on kinetic parameters of pyruvate and glucose transport in the pink erythrocyte ghosts has been studied. It is shown that spectrin structure reorganization induced by the antibody to this protein has different effect on pyruvate and glucose transport parameters. Band 3 protein modification with the help of the antibody to this protein changes pyruvate transport parameters, while glucose transport is not changed. The data obtained show that facilitated diffusion of glucose and anions in the erythrocyte membrane is carried out by different carriers, the action of these carriers essentially depending on the structure state of spectrin.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1983

Spectrin oligomers of the red cell membrane extracts in hereditary spherocytosis.

Spectrin oligomers were extracted from the erythrocyte membrane at 37 degrees C with the low ionic strength buffer. The spectrin extractability of HS membranes was enhanced compared with controls in some cases. The major spectrin component in the extract was dimer which was, apart from a tetramer, purified by gel filtration on the Sepharose 4B column. No changes of dimer-tetramer interconversion in the extract from HS membranes were observed. Similarly, the protein composition of the membrane extracts from two types of red cells remained unchanged.

Erythrocyte Membrane↗
PubMed1983

Increased sensitivity of isolated alpha subunits of normal human hemoglobin to oxidative damage and crosslinkage with spectrin.

After peroxidation, alpha and beta subunits of normal human hemoglobin demonstrated a significant differential reactivity in their ability to form methemoglobin subunits and irreducible crosslinkages with spectrin. The alpha subunits formed crosslinks with spectrin in the absence of exogenous hydrogen peroxide, whereas both the beta subunit and the intact hemoglobin molecule required a minimum of 40 and 4 microM peroxide, respectively, in order to form these crosslinks. Changes in the amount of methemoglobin occurred at much higher concentrations of hydrogen peroxide for the beta hemoglobin subunit (100 microM H2O2) than for the alpha subunit (0.1 microM H2O2). A possible explanation for the existing reactivity between each of the two hemoglobin subunits and spectrin is considered and discussed. In our notation, alpha subunit = alpha SH and the beta subunit = beta SH.

Cross-Linking Reagents↗
PubMed1980

Presence of spectrin tetramer on the erythrocyte membrane.

In order to examine the subunit structure of membrane-associated spectrin, human erythrocyte membranes were cross-linked with a variety of photosensitive heterobifunctional reagents by flash photolysis millisecond cross-linking (Kiehm, D.J., and Ji, T.H. (1977) J. Biol. Chem. 252, 8524-8531). Cross-linking of ghosts produced a series of new bands with apparent molecular weights of approximately 420,000, 710,000, and 910,000, as well as a band at the top of the gels. Evidence is presented that these three bands represent spectrin dimer, trimer, and tetramer, respectively. We propose that spectrin tetramer exists on the membrane as the predominant structural unit and that dimer is not a repeating structural unit.

Cross-Linking Reagents↗
PubMed1995

Evolutionarily conserved alternative pre-mRNA splicing regulates structure and function of the spectrin-actin binding domain of erythroid protein 4.1.

A developmental alternative splicing switch, involving exon 16 of protein 4.1 pre-mRNA, occurs during mammalian erythropoiesis. By controlling expression of a 21-amino acid peptide required for high-affinity interaction of protein 4.1 with spectrin and actin, this switch helps to regulate erythrocyte membrane mechanical stability. Here we show that key aspects of protein 4.1 structure and function are conserved in nucleated erythroid cells of the amphibian Xenopus laevis. Analysis of protein 4.1 cDNA sequences cloned from Xenopus erythrocytes and oocytes showed that tissue-specific alternative splicing of exon 16 also occurs in frogs. Importantly, functional studies with recombinant Xenopus erythroid 4.1 demonstrated specific binding to and mechanical stabilization of 4.1-deficient human erythrocyte membranes. Phylogenetic sequence comparison showed two evolutionarily conserved peptides that represent candidate spectrin-actin binding sites. Finally, in situ hybridization of early embryos showed high expression of 4.1 mRNA in ventral blood islands and in developing brain structures. These results demonstrate that regulated expression of structurally and functionally distinct protein 4.1 isoforms, mediated by tissue-specific alternative splicing, has been highly evolutionarily conserved. Moreover, both nucleated amphibian erythrocytes and their enucleated mammalian counterparts express 4.1 isoforms functionally competent for spectrin-actin binding.

Actins↗