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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↗
PubMed1997

Spectrin St Claude, a splicing mutation of the human alpha-spectrin gene associated with severe poikilocytic anemia.

An alpha-spectrin variant with increased susceptibility to tryptic digestion, alpha(II/47), was previously observed in a child with severe, recessively inherited, poikilocytic anemia. The molecular basis of this variant, spectrin St Claude, has now been identified as a splicing mutation of the alpha-spectrin gene due to a T --> G mutation in the 3' acceptor splice site of exon 20. This polypyrimidine tract mutation creates a new acceptor splice site, AT --> AG, and leads to the production of two novel mRNAs. One mRNA contains a 12 intronic nucleotide insertion upstream of exon 20. This insertion introduces a termination codon into the reading frame and is predicted to encode a truncated protein (108 kD) that lacks the nucleation site and thus cannot be assembled in the membrane. In the other mRNA, there is in-frame skipping of exon 20, predicting a truncated (277 kD) alpha-spectrin chain. The homozygous propositus has only truncated 277 kD alpha-spectrin chains in his erythrocyte membranes. His heterozygous parents are clinically and biochemically normal. This allele was identified in 3% of asymptomatic individuals from Benin, Africa.

Adult↗
PubMed1983

A monoclonal antibody against erythrocyte spectrin reacts with both alpha- and beta-subunits and detects spectrin-like molecules in non-erythroid cells.

A panel of nine monoclonal antibodies against the characteristic erythrocyte membrane protein spectrin has been isolated. One antibody reacts with both the 240 000 and 220 000 D alpha- and beta-subunits of spectrin after denaturation. The same antibody reacts with a 240 000 D protein present in various hemopoietic and other cell lines, as well as some smaller polypeptides, as established by western blotting and immunoautoradiography. These results indicate that the alpha- and beta-subunits of spectrin, a polypeptide of 240 000, and some smaller polypeptides present in non-erythroid cell types possess a considerable region of sequence homology, but it is not yet clear just how extensively the spectrin-like molecules and other polypeptides are related.

Amino Acid Sequence↗
PubMed1996

Epidemiological studies of spectrin mutations related to hereditary elliptocytosis and spectrin polymorphisms in Benin.

We studied an African population in Benin and discovered an unexpectedly high frequency (1.6%) of hereditary elliptocytosis (HE) among the 1447 subjects studied. In approximately two-thirds of HE individuals we identified molecular defects, primarily those in erythrocyte alpha-spectrin (dupL154, L260P and L207P mutations), as well as a novel mutation of erythrocyte beta-spectrin (beta-W2061R mutation). We also identified the genetic basis of a previously identified protein polymorphism of the alpha III domain of spectrin (R1331I mutation). The genetic background of HE in the African population was studied using a number of polymorphisms of the alpha-spectrin gene, including the alpha III domain polymorphism. These studies suggest that the HE mutations appear to have originated from separate genetic backgrounds in this population.

Benin↗
PubMed1995

Spectrin Anastasia (alpha I/78): a new spectrin variant (alpha 45 Arg-->Thr) with moderate elliptocytogenic potential.

We describe a white Italian kindred in which hereditary elliptocytosis (HE) is associated with abnormal level of alpha I/78 peptide in spectrin digest. Clinical phenotype varied among the family members ranging from asymptomatic to mild haemolytic HE. The original mutation responsible is a G-C substitution of the spectrin alpha-gene: alpha 45 Arg-->Thr (AGG-->ACG). The corresponding spectrin is designated spectrin Anastasia. Utilizing a secondary structure predictive method we suggest that this mutation has a poor capability to induce conformational changes of the tetramerization site and thus shows a moderate elliptocytogenic potential.

Adult↗
PubMed1982

Brain spectrin, a membrane-associated protein related in structure and function to erythrocyte spectrin.

An immunoreactive analogue of erythrocyte spectrin has been purified from brain membranes. This protein co-sediments with and cross-links actin filaments, associates with spectrin-binding sites on erythrocyte membranes, and has been visualized by rotary shadowing as an extended, flexible rod. The brain spectrin comprises 3% of the total membrane protein, and may have a major role in mediating linkage of actin to membranes.

Actins↗
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↗
PubMed1977

Release of spectrin-free vesicles from human erythrocytes during ATP depletion. I. Characterization of spectrin-free vesicles.

Human erythrocytes incubated without glucose at 37 degrees C (in vitro aging) release spectrin-free vesicles after 12 or more hours. The release of vesicles is dependent upon ATP depletion. If the endogenous level of ATP is maintained, vesicle release is completely inhibited up to 54 h. Vesicle release is independent of hemolysis because in vitro aged cells and cells that maintain their ATP levels lose identical amounts of hemoglobin up to 45 h. 93 percent of all membrane particles released constitute a uniform population of spheres with a diameter of 185 +/- 23nm. These vesicles are of slightly varying densities due to varying contents of hemoglobin. Vesicles contain half the amount of membrane protein that is found in intact membranes when referred to the content of phospholipids phosphorus. This is primarily due to the absence of spectrin. However, their content of protein component III, glycophorin, and cholesterol remains the same as in intact membranes. Thus, the major integral membrane proteins are present in vesicles in similar quantities were surface area as in cells except for the enzyme acetylcholinesterase that is enriched up to twofold. The phospholipids composition of these vesicles is representative of the intact membrane except that the amount of phosphatidic acid is 10-fold higher and the amount of phosphatidylethanolamine is slightly lower than in erythrocytes. These results suggest a selective release of membrane domains that lack peripheral membrane proteins and are enriched in acetylcholinesterase. This release of spectrin-free vesicles from cells aged in vitro could represent an acceleration of the physiological aging process.

Acetylcholinesterase↗
PubMed1993

Beta spectrin kissimmee: a spectrin variant associated with autosomal dominant hereditary spherocytosis and defective binding to protein 4.1.

We analyzed the DNA sequence of the cDNA encoding the NH2 terminal region of beta spectrin from members of a kindred with autosomal dominant hereditary spherocytosis associated with defective protein 4.1 binding. We found a point mutation at codon 202 within the 272 amino acid NH2-terminal region of beta spectrin. TGG was changed to CGG, resulting in the replacement of tryptophan by arginine. The base change eliminates a normally occurring PvuII restriction site and creates a new MspI site. This finding enabled rapid detection or exclusion of the mutation at the DNA level among the family members, including one member for whom this analysis was performed prenatally. The mutation was found only in the affected family members and occurred as a de novo mutation in the proband. It has not been found in 20 other kindreds. The recombinant peptide derived from the normal cDNA retains the capacity to sediment with protein 4.1 and F-actin. The mutant peptide spontaneously degrades. This variant represents both the first point mutation and the first beta spectrin mutation demonstrated in autosomal dominant hereditary spherocytosis. Furthermore, the mutation is located within a conserved sequence among spectrinlike proteins and may define an amino acid critical for protein 4.1 binding activity.

Adult↗
PubMed1994

A surface replica method: a useful tool for studies of the cytoskeletal network in red cell membranes of normal subjects and patients with a beta-spectrin mutant (spectrin Le Puy: beta 220/214).

Visualization of the components of the red cell membranes, and especially the structure of cytoskeletal proteins in situ, has become a requisite in studies of red cell membrane disorders. There has been a search for a consistent and dependable method for detecting these structures. In the present study, the surface replica method was used with transmission electron microscopy to examine the cytoskeletal network of the red cell ghosts of a normal control and patients with a beta-spectrin mutant (beta-spectrin Le Puy). The surface replica method is well-suited to observation of the cytoskeletal network of the membranes in a nearly native in situ condition. Immunogold labelling with anti-membrane protein antibodies is easily applicable to the identification of each component of the cytoskeletal proteins. The findings obtained under normal and pathological conditions using the surface replica method corresponded with those made by the quick-freeze, deep-etching method.

Cytoskeleton↗
PubMed1986

Spectrin, red cell shape and deformability. I. Membrane curvature in genetic spectrin deficiency.

Using novel microscopic techniques for observing individual cells in suspension, the shape and deformability of the erythrocytes of a spectrin-deficient strain of mouse were investigated in vitro (fresh and after fixation) and in vivo (intravital microscopy of the mesenteric capillaries). The animals were identical to those studied by Greenquist et al. [14]; however, in contrast to the descriptions by these authors, in the present study, spherical cells were seen only exceptionally. Instead, sphero-echinocytes, spherules with tethered microvesicles, myelin figures and occasional stomato-spherocytes were observed. This pleomorphy, also seen in scanning electron micrographs, can be explained by the fact that in the majority of these cells the membrane assumes an extremely positive outward curvature. After osmotic dehydration, all cells responded by developing progressive membrane protrusions with a positive curvature. Osmotic inflation led to reincorporation of the membrane tethers. After hypoosmolar lysis, the ghosts of the spectrum-deficient mouse cells also showed a pronounced tendency to assume shapes characterized by a progressively positive outward curvature. In vivo observation of the mouse erythrocytes using high magnification interference contrast optics confirmed this in vitro observation. Thus, it is concluded that spectrin deficiency does not primarily lead to spherocytosis: instead, the equilibrium shapes assumed are associated with membrane evaginations which are easily torn off from the main cell body by mechanical forces.

Animals↗
PubMed1987

Interaction of spectrin with phospholipids. Quenching of spectrin intrinsic fluorescence by phospholipid suspensions.

Phospholipid suspensions prepared of phosphatidylethanolamine, phosphatidylserine and their mixtures are able to influence the intrinsic protein fluorescence of spectrin. In the case of phosphatidylethanolamine suspension up to 75% of protein fluorescence can be quenched. The interaction of phospholipid aggregates with spectrin is modulated by pH and ionic strength. Phospholipids, particularly phosphatidylethanolamine display a 'stabilizing' effect against the changes of protein fluorescence induced by increasing ionic strength and by thermal denaturation.

Erythrocyte Membrane↗
PubMed1987

Structural analogy among mammalian spectrins and spectrin-like proteins revealed by molybdenum labeling.

Pentavalent complex of 99Mo with ascorbic acid binds in vitro to the plasma membranes of human, rabbit, rat and mouse red cell membranes and to bovine synaptic and rat intestinal brush border membranes. Red cell spectrins and spectrin-like proteins from non-erythroid cells were determined as the molybdenum-binding proteins in the membranes. Specificity of this binding among all membrane proteins suggests structural analogy in this group of proteins.

Animals↗
PubMed1998

Spectrin self-association site: characterization and study of beta-spectrin mutations associated with hereditary elliptocytosis.

Most of hereditary elliptocytosis (HE) cases are related to a spectrin dimer (SpD) self-association defect. The severity of haemolysis is correlated with the extent of the SpD self-association defect, which itself depends on the location of the mutation regarding the tetramerization site. This site is presumed to involve the first C helix of the alpha chain and the last two helices, A and B, of the beta chain to reconstitute a triple helical structure (A, B and C), as observed along spectrin. Using recombinant peptides, we demonstrated that the first C helix of the alpha chain and the last two helices of the beta chain alone are not sufficient to establish interactions, which only occurred when a complete triple-helical repeat was added to each partner. One adjacent repeat is necessary to stabilize the conformation of both N- and C-terminal structures directly involved in the interaction site and is sufficient to generate a binding affinity similar to that observed in the native molecule. Producing peptides carrying a betaHE mutation, we reproduced the tetramerization defect as observed in patients. Therefore, the betaW2024R and betaW2061R mutations, which replace the invariant tryptophan and a residue located in the hydrophobic core, respectively, affect alpha-beta interactions considerably. In contrast, the betaA2013V mutation, which modifies a residue located outside any presumed interacting regions, has a minor effect on the interaction.

Binding Sites↗
PubMed1985

Pig thyroid spectrin. A membrane-associated protein related in structure and function to brain spectrin.

Thyroid spectrin has been obtained pure from pig thyroid glands. This protein, composed of two non-identical polypeptide chains of 240 kDa and 235 kDa, appears to possess the same structural and immunological properties as well as the same calmodulin and actin-binding properties as brain spectrin. Through cross-linking of actin filaments it is a potent gelation factor for F-actin solutions. It represents one of the major protein of the cytoskeleton underlying the thyroid plasma membrane together with myosin, alpha-actinin and actin.

Actins↗
PubMed1990

Hereditary pyropoikilocytosis and elliptocytosis in a white French family with the spectrin alpha I/74 variant related to a CGT to CAT codon change (Arg to His) at position 22 of the spectrin alpha I domain.

We describe a white French family in which 12 subjects presented with hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP). Eight of these subjects were shown to be heterozygous for a spectrin (Sp) alpha I/74 variant, as demonstrated by analysis of partial tryptic digestion fragments of spectrin. This abnormal peptide pattern was associated with a decreased ability of Sp dimers to self-associate. In this kindred, in which four generations were available for study, the clinical expression varied from mild HE to HPP with an intermediate status of hemolytic HE. The severity of the disease appeared to be correlated both with the estimated amount of variant Sp (42% to 65%) and the excess of Sp dimers found in the membrane (30% to 51%, with a normal value of 3.7% +/- 1.6%). Reassociation studies using isolated Sp alpha and beta chains from an affected patient and an unaffected control subject showed that the Sp alpha I/74 Kd abnormal tryptic peptide resulted from a defect in the Sp alpha chain. Partial amino acid sequencing showed that the Sp alpha I/74 Kd peptide resulted from cleavage at lysine residue 42 of the Sp alpha I/80 Kd domain. Knowledge of the exon/intron organization of the human alpha Sp gene allowed us to amplify by the polymerase chain reaction the second exon of the alpha Sp gene in total cellular DNA of the HPP proposita. The amplified fragment was subcloned and sequenced. We found a G to A base substitution in the 22nd codon (CAT for CGT), which changes the normal arginine to a histidine. Hybridization of amplified DNAs with allele-specific oligonucleotides corresponding to the normal and mutant sequences confirmed the presence of the mutation in six other HE and HPP members of the family. The identification of this mutation at the DNA level confirmed the transmission of the same molecular defect in Sp through four generations but with different patterns of clinical expression.

Adult↗
PubMed1990

Structural and functional heterogeneity of alpha spectrin mutations involving the spectrin heterodimer self-association site: relationships to hematologic expression of homozygous hereditary elliptocytosis and hereditary pyropoikilocytosis.

Defects involving alpha spectrin (Sp) are found in patients with hereditary elliptocytosis and a related disorder, hereditary pyropoikilocytosis (HPP). We have previously found that the severity of hemolysis was related to the total spectrin content of the cells and the percentage of unassembled dimeric Sp (SpD) in the membranes, which, in turn, reflected the amount of mutant Sp in the cell. However, no data are available comparing differences in the function of various alpha Sp mutations to clinical severity. We now report studies of nine homozygotes or double heterozygotes for four alpha Sp mutations: alpha 1/74, alpha 1/46, alpha 1/65, and alpha 1/61, whose red blood cells (RBCs) contained only the mutant Sp and no normal Sp. Sp alpha 1/74, Sp alpha 1/46, and alpha 1/65 homozygotes differed strikingly in the severity of hemolysis that correlated with the severity of mutant Sp dysfunction, as reflected by the fraction of unassembled SpD in the membranes and the self-association of mutant Sp on inside-out vesicles. Homozygotes for Sp alpha 1/74 had a very severe hemolytic anemia and their SpD were virtually incapable of self-association, whereas SpD alpha 1/46 were not as severely affected. The Sp alpha 1/65 homozygotes had a relatively mild hemolytic anemia and their SpD showed the least impairment of function. Ultrastructural examination of membrane skeletons from subjects whose SpD self-association was severely impaired showed gross skeletal disruption and loss of hexagonal structure. In striking contrast, the homozygote for the mildly dysfunctional Sp alpha 1/65 had only a moderate disruption of the skeleton. Some of the homozygous or doubly heterozygous subjects also exhibited a partial deficiency of Sp that correlated with a RBC morphology characteristic of HPP, namely, marked microspherocytosis with virtual absence of elliptocytes. These data demonstrate striking differences in the function and structure of various alpha Sp mutants that underlie differences in clinical expression.

Adolescent↗
PubMed1979

Proteolytic fragmentation of spectrin:effect of removal of terminal phosphopeptides on spectrin binding to human erythrocyte membranes.

The involvement of phosphorylated regions of spectrin, from human erythrocytes, in membrane binding has been investigated. Spectrin is phosphorylated at several sites, all near a terminal of the band 2 polypeptide. This region can be removed from the bulk of the molecule by brief trypsinization. Membrane binding of the bulk of the protein is unaffected by brief proteolysis, whereas phosphofragments generated by this treatment exhibit no membrane binding.

Binding Sites↗