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PubMed1985

Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin-hemoglobin cross-linking.

To further define the conditions for forming spectrin-hemoglobin cross-linking in human erythrocyte membranes and to examine its possible effects on membrane function, we incubated normal human erythrocytes for up to 3 h in concentrations of H2O2, varying from 45 to 180 microM, in an azide phosphate buffer, pH 7.4. The chemical changes observed indicated that methemoglobin formation occurred early and at a low concentration (45 microM). Morphologic changes characterized by increased echinocyte formation occurred in a dose-dependent fashion. In addition, decreased cell deformability commensurate with increased membrane rigidity was found. Finally, an increase in cell recognition as determined by monocyte phagocytosis and adherence in vitro, as well as decreased phosphatidylcholine accessibility to bee venom phospholipase A2, was found in H2O2-treated erythrocytes compared with controls. Both of these latter changes were closely correlated with the extent of spectrin-hemoglobin cross-linking. In addition to these protein-mediated interactions, lipid peroxidation also occurred after H2O2 exposure, as shown by generation of fluorescent amino propene derivatives. The addition of the antioxidant, butylated hydroxytoluene, decreased the fluorescent derivatives, but did not prevent the effects on membrane function. This suggests that lipid peroxidation, though present, was not necessary for the membrane changes found. In contrast, spectrin-hemoglobin aggregation and the alterations in membrane function were completely prevented by prior exposure of the erythrocytes to carbon monoxide.

Carbon Monoxide↗

PubMed1989

Sequence and exon-intron organization of the DNA encoding the alpha I domain of human spectrin. Application to the study of mutations causing hereditary elliptocytosis.

We have determined the exon-intron organization and the nucleotide sequence of the exons and their flanking intronic DNA in cloned genomic DNA that encodes the first 526 amino acids of the alpha I domain of the human red cell spectrin polypeptide chain. From the gene sequence we designed oligonucleotide primers to use in the polymerase chain reaction technique to amplify the appropriate exons in DNA from individuals with three variants of hereditary elliptocytosis characterized by the presence of abnormal alpha I spectrin peptides, 46-50 and 65-68 kD in size, in partial tryptic digests of spectrin. The alpha I/68-kD abnormality resulted from a duplication of leucine codon 148 in exon 4: TTG-CTG to TTG-TTG-CTG. The alpha I/50a defect was associated in different individuals with two separate single base changes in exon 6: CTG to CCG (leucine to proline) encoding residue 254, and TCC to CCC (serine to proline) encoding residue 255. In another individual with the alpha I/50a polypeptide defect, the nucleotide sequence encoding amino acid residues 221 through 264 was normal. The alpha I/50b abnormality resulted from a single base change of CAG (glutamine) to CCG (proline) encoding residue 465 in exon 11 in two unrelated individuals. In a third individual with alpha I/50b-kD hereditary elliptocytosis, the entire exon encoding residues 445 through 490 was normal. The relationship of the alpha I domain polypeptide structure to these mutations and the organization of the gene is discussed.

Amino Acid Sequence↗

PubMed1984

Molecular defect of spectrin in the family of a child with congenital hemolytic poikilocytic anemia.

We present the study of a black family in which the proband suffered from a severe neonatal hemolytic anemia with poikilocytosis. Both the parents, sister's, and brother's proband were clinically normal. The presence of poikilocytes in proband led to a search for a red cell membrane skeleton defect. Owing to recent improvements in the erythrocyte membrane knowledge, it is now possible to approach the diagnosis by means of biochemical evaluation of both parents, even if they are asymptomatic. So, the first time discovery of a spectrin self-association defect in both parents allowed us to suspect double inheritance of this abnormality in the proband. A complete morphological and biochemical evaluation of the family allowed us to propound the diagnosis of heterozygous type I hereditary elliptocytosis (HE) for both parents and the sister and the diagnosis of homozygous type I HE for the proband owing to the following reasons: slight ovalocytosis was present in both parents and the sister; cell deformability ektacytometric studies gave the same profiles of curve as those observed in patients with HE. Defective spectrin dimer self-association found in both parents was also observed in the sister and proband, associated with the same abnormal spectrin digest pattern, namely a decrease in the amount of a 80,000-dalton peptide and a corresponding increase in a 74,000-dalton peptide. However, clinical presentation of the proband was consistent either with hereditary pyropoikilocytosis or homozygous hereditary elliptocytosis; erythrocyte thermal sensitivity studies in the proband could not be conclusive because of the presence of transfused cells. Both these diagnoses are discussed in detail.(ABSTRACT TRUNCATED AT 250 WORDS)

Anemia, Hemolytic, Congenital↗

PubMed1995

Integrin and spectrin homologues, and cytoplasm-wall adhesion in tip growth.

Saprolegnia ferax contains an integrin homologue, identified by crossreactivity with antiserum to the consensus sequence of human/chick/Xenopus cytoplasmic domain beta 1-integrin, which is highly conserved. In non-reduced samples, this integrin was larger than the reported size range for beta 1-integrins, at 178 kDa. In reduced samples, there was a reducing agent-concentration-dependent conversion from 178 kDa to 120 kDa, well within the reported size range for beta 1-integrins in other organisms. The integrin antiserum stained plasma membrane-associated patches, which had a shallow tip-high gradient. This population was reduced and its distribution perturbed in hyphae whose growth rate was reduced by half with tetrapentyl ammonium chloride. The expected integrin function in cytoplasm-cell wall attachment was shown by differential resistance to plasmolysis-induced separation, which positively correlated with integrin abundance. However, when there was separation, remnants of cytoplasm stayed attached to the wall. These were enriched in actin and integrin. Saprolegnia also has a spectrin homologue identified by crossreactivity with an erythrocyte spectin antibody, which has a size (246 kDa) similar to other organisms. This spectrin had a superficially similar distribution to that of integrin, but it did not participate in cytoplasm-wall anchoring. These data suggest that Saprolegnia hyphae have a plasma membrane which is strengthened by spectrin, and cytoplasm which is attached to the cell wall by integrin.

Animals↗

PubMed1995

The membrane skeleton of acetylcholine receptor domains in rat myotubes contains antiparallel homodimers of beta-spectrin in filaments quantitatively resembling those of erythrocytes.

I used immunogold labeling and quick-freeze, deep-etch, rotary replication to characterize the membrane skeleton at regions with high concentrations of acetylcholine receptor domains in receptor clusters of cultured rat muscle cells. This membrane skeleton consists of a network of filaments closely applied to the cytoplasmic membrane surface. The filaments are specifically decorated by immunogold labeling with a monoclonal antibody, VIIF7, that recognizes an isoform of beta-spectrin colocalizing with acetylcholine receptors. The filaments are 32 +/- 11 nm in length and three to four filaments (average 3.1-3.3) join at each intersection to form the network. These parameters are nearly identical to those reported previously for the membrane skeleton of erythrocytes. Depending on the amount of platinum coating, filament diameters range from 9 to 11 nm in diameter, and are 1.4 nm larger on average than spectrin filaments of erythrocytes replicated at the same time. Filaments are decorated with gold particles close to one end, consistent with the location of the epitope recognized by the monoclonal antibody. Computer modeling shows that all filament intersections in the membrane skeletal network are equally capable of being labeled by the monoclonal antibody. This pattern of labeling is consistent with a network containing antiparallel homodimers of beta-spectrin.

Animals↗

PubMed2006

Spectrins and ankyrinB constitute a specialized paranodal cytoskeleton.

Paranodal junctions of myelinated nerve fibers are important for saltatory conduction and function as paracellular and membrane protein diffusion barriers flanking nodes of Ranvier. The formation of these specialized axoglial contacts depends on the presence of three cell adhesion molecules: neurofascin 155 on the glial membrane and a complex of Caspr and contactin on the axon. We isolated axonal and glial membranes highly enriched in these paranodal proteins and then used mass spectrometry to identify additional proteins associated with the paranodal axoglial junction. This strategy led to the identification of three novel components of the paranodal cytoskeleton: ankyrinB, alphaII spectrin, and betaII spectrin. Biochemical and immunohistochemical analyses revealed that these proteins associate with protein 4.1B in a macromolecular complex that is concentrated at central and peripheral paranodal junctions in the adult and during early myelination. Furthermore, we show that the paranodal localization of ankyrinB is disrupted in Caspr-null mice with aberrant paranodal junctions, demonstrating that paranodal neuron-glia interactions regulate the organization of the underlying cytoskeleton. In contrast, genetic disruption of the juxtaparanodal protein Caspr2 or the nodal cytoskeletal protein betaIV spectrin did not alter the paranodal cytoskeleton. Our results demonstrate that the paranodal junction contains specialized cytoskeletal components that may be important to stabilize axon-glia interactions and contribute to the membrane protein diffusion barrier found at paranodes.

Animals↗

PubMed2005

Extending a spectrin repeat unit. I: linear force-extension response.

Nonequilibrium molecular dynamics simulations were used to calculate the elastic properties of a spectrin repeat unit. A contiguous alpha-helical linker was constructed by employing periodic boundary conditions, allowing a novel scheme for evaluating the thermodynamic force as a function of extension. By measuring the force-extension response under small extensions, spectrin was observed to behave primarily as an elastic material with a spring constant of 1700 +/- 100 pN/nm. The implications of this spring constant, in terms of the properties of the spectrin tetramer, are also discussed.

Algorithms↗

PubMed1993

Crystal structure of the SH3 domain in human Fyn; comparison of the three-dimensional structures of SH3 domains in tyrosine kinases and spectrin.

The Src-homology 3 (SH3) region is a protein domain consisting of approximately 60 residues. It occurs in a large number of eukaryotic proteins involved in signal transduction, cell polarization and membrane--cytoskeleton interactions. The function is unknown, but it is probably involved in specific protein--protein interactions. Here we report the crystal structure of the SH3 domain of Fyn (a Src family tyrosine kinase) at 1.9 A resolution. The crystals have two SH3 molecules per asymmetric unit. These two Fyn SH3 domains are not related by a local twofold axis. The crystal structures of spectrin and Fyn SH3 domains as well as the solution structure of the Src SH3 domain show that these all have the same basic fold. A protein domain which has the same topology as SH3 is present in the prokaryotic regulatory enzyme BirA. The comparison between the crystal structures of Fyn and spectrin SH3 domains shows that a conserved surface patch, consisting mainly of aromatic residues, is flanked by two hairpin-like loops (residues 94-104 and 114-118 in Fyn). These loops are different in tyrosine kinase and spectrin SH3 domains. They could modulate the binding properties of the aromatic surface.

Bacterial Proteins↗

PubMedDate not supplied

Influence of the spectrin network on fusion of influenza virus with red blood cells.

We examined the influence of the physical state of the membrane skeleton on low pH fusion of influenza virus A/PR 8/34 with intact human red blood cells. Spectrin, the major component of the skeleton, is known to become denaturated at 50 degrees C. After heat treatment of erythrocytes at 50 degrees C we observed an enhanced kinetics of fusion monitored spectrofluorometrically by the octadecylrhodamine fluorescence dequenching assay, while the extent of fusion was not affected. The accelerated fusion of influenza virus after preincubation of red blood cells at 50 degrees C is not mediated by alterations of the lipid phase of the target. From ESR measurements using spin-labelled phospholipids we conclude that heat-induced alterations of the spectrin network did not affect either the phospholipid asymmetry or the fluidity of the exoplasmic and the cytoplasmic leaflets of the erythrocyte membrane. Moreover, as deduced from our previous investigations, the swelling behaviour of red blood cells could not be responsible for the observed effect. Possible mechanisms for the spectrin effect include a change in the ability of the target membrane to bend locally, and a change in the rate of formation and development of the fusion pore.

Erythrocyte Membrane↗

PubMed1999

Association study of a polymorphism of nonerythroid alpha-spectrin gene with schizophrenia.

The specificity of cytoarchitectural abnormalities in limbic structures of patients with schizophrenia and their contributions towards the etiology of schizophrenia remain unknown. We have recently reported an increased breakdown of nonerythroid alpha-spectrin (fodrin), a major component of neuronal cytoskeletal proteins, in schizophrenic left superior temporal cortices [Kitamura et al., 1998: Biol Psychiatry 43:254-262], suggesting that polymorphisms of the alpha-spectrin gene might contribute to the vulnerability to schizophrenia. We screened for genetic variations associated with schizophrenia through the C-terminus sequences of the human nonerythroid alpha-spectrin gene (SPTAN1) spanning two EF-hands and also tested a possible contribution of the polymorphism to the development of schizophrenia by an association study. We found a polymorphic region of an intron located in the second EF-hand of SPTAN1 gene. There was no significant difference between patients with schizophrenia and controls in allele frequencies or genotype distribution. There is evidence that the Psh BI SPTAN1 gene polymorphism does not play a major role in the genetic component of schizophrenia.

Adult↗

PubMed1999

Changes in passive electric parameters of human erythrocyte membrane during hyperthermia: role of spectrin phosphorylation.

In prefixed by 1 mmol/l OsO4 human erythrocytes, the discocyte shape was preserved upon heating to temperatures which include the denaturation temperature of the main peripheral protein spectrin. Nevertheless, the suspension of fixed cells displayed threshold decrease in its capacitance and resistance at the temperature range where spectrin denaturates. The same changes were established using intact cells and their resealed ghosts. For packed cells (ghosts), the capacitance and resistance decreased about 17% (31%) and 30% (19%). These data indicate a decrease in the beta dispersion of erythrocyte membrane associated, according to a previous study (Ivanov 1997), with the heat denaturation of spectrin at 49.5 degrees C. The amplitude of the 49.5 degrees C decrease in beta dispersion was reversibly reduced in intact erythrocytes and white ghosts following reversible decrease in the phosphorylation of their membrane proteins. It was fully eliminated in ghosts following their resealing with alkaline phosphatase (0.1 mg/ml) which dephosphorylated membrane proteins. These findings are discussed in relation to similar changes found in normal and tumour tissues and cells during hyperthermia.

Adenosine Triphosphate↗

PubMed2000

The presynaptic calcium channel is part of a transmembrane complex linking a synaptic laminin (alpha4beta2gamma1) with non-erythroid spectrin.

Nerve regeneration studies at the neuromuscular junction (NMJ) suggest that synaptic basal lamina components tell the returning axon where to locate neurotransmitter release machinery, including synaptic vesicle clusters and active zones. Good candidates for these components are the synaptic laminins (LNs) containing alpha4, alpha5, or beta2 chains. Results from a beta2 laminin knockout mouse have suggested a linkage of this extracellular laminin to cytosolic synaptic vesicle clusters. Here we report such a transmembrane link at the electric organ synapse, which is homologous to the NMJ. We immunopurified electric organ synaptosomes and found on their surface two laminins of 740 and 900 kDa. The 740 kDa laminin has a composition of alpha4beta2gamma1 (laminin-9). Immunostaining reveals that as in the NMJ, alpha4 and beta2 chains are concentrated at the electric organ synapse. Using detergent-solubilized synaptosomes, we immunoprecipitated a complex containing alpha4beta2gamma1 laminin, the voltage-gated calcium channel, and the cytoskeletal protein spectrin. Other presynaptic proteins such as 900 kDa laminin are not found in this complex. We hypothesize that alpha4beta2gamma1 laminin in the synaptic basal lamina attaches to calcium channel, which in turn is attached to cytosolic spectrin. Spectrin could then organize synaptic vesicle clusters by binding vesicle-associated proteins.

Animals↗

PubMed2001

New insights into red cell network structure, elasticity, and spectrin unfolding--a current review.

The red cell membrane's well-recognized ability to withstand the stresses of circulation clearly has its origins in various levels of spectrin-actin network structure. We highlight recently obtained insights into this sub-structure and also briefly explain the implications to membrane components that interact with the network. Novel insights into the resilience of this cytoskeleton are being provided by experiments that range from atomic force microscopy (AFM) tests of single, unfoldable spectrin chains to micropatterned photobleaching of a pipette-deformed network. Continued progress in atomic level structure determinations of non-erythroid spectrin and related repeats are further complemented by theoretical efforts--computational approaches most notably--that have begun to correlate molecular scale aspects of structure with micro-mechanical measures. All of this recent activity in the biophysics of red cell structure-function challenges and refines some of the most basic tenets in cell membrane response.

Actin Cytoskeleton↗

PubMed2001

Mechanoprotection of the plasma membrane in neurons and other non-erythroid cells by the spectrin-based membrane skeleton.

Though the cytomechanics of spectrin have been explored only for erythrocytes, it is thought that the spectrin skeleton acts universally to support the otherwise mechanically vulnerable cell surface bilayer. Evidence for this role is beginning to accumulate and is reviewed here. Compared to that for erythrocytes, cells whose simplicity facilitates biophysical approaches, the evidence is indirect. One way that membrane skeleton/bilayer interactions have been probed is via the behavior of mechanosusceptible ion channels - channel whose gating is perturbed by abnormally high bilayer tension. These initially unresponsive channels become progressively more mechanoresponsive as stretch and chemical reagents damage the membrane skeleton. The straightforward implication is that the intact membrane skeleton is mechanoprotective. In non-erythroid cells there is continual trafficking of bilayer to and from the plasma membrane. Some of the traffic involves spectrin-lined vacuolar membrane. Several lines of evidence suggest that when neurons elongate and remodel their neurites, membrane skeleton-based mechanoprotection allows the dynamic vacuoles and the plasma membrane to participate in mechanosensitive surface area expansion and retrieval.

Animals↗

PubMed1992

[Hereditary elliptocytosis caused by a spectrin deficiency (Sp alpha I/46). 1st patient described in Cuba].

We report biochemical studies of membrane proteins performed in a patient with hereditary elliptocytosis (HE). The presence of 90% of elliptocytes on wet smears of glutaraldehyde-fixed cells, the increased red cell thermal sensitivity, the normal erythrocyte membrane electrophoresis, the increased spectrin dimer in the 4 degrees C extract (35%) and the 46 Kd peptide present in electrophoresis after limited tryptic digestion of spectrin, allows us to classify this disorder as type I HE. The patient has common HE with compensated mild hemolysis. This is the first case of HE with alpha chain molecular variant of spectrin found in a Cuban population.

Blood Protein Electrophoresis↗

PubMedDate not supplied

[Contractile proteins and nonerythroid spectrin in the oogenesis of the clawed toad].

Distribution of contractile proteins, actin and myosin, and spectrin was studied in oogenesis of X. laevis. These proteins are present already at the previtellogenic stages, where they are diffusely distributed. During vitellogenesis actin and myosin are distributed in the animal region in a fibril-like way, while in the vegetal one they are concentrated around the yolk platelets. In the mature oocyte, distribution of actin and myosin again becomes diffuse. Spectrin forms in the vitellogenic oocyte a network all over the cytoplasm, while in the full-grown oocyte it is localized mostly in the subcortex of the animal region and disappears during oocyte maturation. All these proteins are present in the nuclei of oocytes. Changes in distribution of actin, myosin and spectrin during oocyte maturation are discussed with reference to the cortical contractility, spatial distribution of yolk platelets and regional sensitivity to cytochalasin B.

Actins↗

PubMed2004

Ankyrin is a target of spectrin's E2/E3 ubiquitin-conjugating/ligating activity.

Ubiquitin is a small protein of 8.6 kDa molecular weight. When polyubiquitin is attached to target proteins, they are tagged for destruction by cytoplasmic organelles called proteasomes. We now know that ubiquitination of target proteins also regulates functions as diverse as the sorting of proteins to different intracellular destinations, cell signaling, cell division, gene transcription, and protein-protein interactions. The ubiquitination of target proteins requires a cascade of enzymes: E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme and E3 ubiquitin ligating enzyme. Recently we have demonstrated that the red blood cell (RBC) membrane skeletal protein, spectrin, has E2/E3 enzymatic activities in its alpha-subunit, that can transfer ubiquitin to itself. We have now created a cell free assay using biotinylated ubiquitin that allows detection of target proteins by streptavidin peroxidase. This approach coupled with immunoprecipitation, purification and micro liquid chromatography coupled to tandem mass spectrometry has identified ankyrin as a target of spectrin's E2/E3 activity. Western blotting, with ubiquitin antibody, of purified ankyrin and its well characterized functional domains, has demonstrated that both the spectrin and band 3 binding domains are ubiquitinated in vivo.

Ankyrins↗

PubMed2004

NMR studies of calcium-binding to mutant alpha-spectrin EF-hands.

The co-operative calcium binding mechanism of the two C-terminal EF-hands of human alphaII-spectrin has been investigated by site-specific mutagenesis and multi-dimensional NMR spectroscopy. To analyse the calcium binding of each EF-hand independently, two mutant structures (E33A and D69S) of wild type alpha-spectrin were prepared. According to NMR analysis both E33A and D69S were properly folded. The unmutated EF-hand in these mutants remained nearly intact and active in calcium binding, whereas the mutated EF-hand lost its affinity for calcium completely. The apparent calcium binding affinity of the E33A mutant was much lower compared to the D39S mutant (approximately 2470 microM and approximately 240 microM, respectively). When the chemical shift perturbations were followed upon calcium titration, a positive correlation between the D69S mutant and the binding of the first calcium ion to the wild type was revealed. These observations showed that the first EF-hand in spectrin binds the first calcium ion and thereby triggers a conformational change that allows the second calcium ion to bind to the other EF-hand.

Amino Acid Sequence↗