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PubMed1978

Physical-chemical studies of spectrin.

In recent years considerable progress has been made in the understanding of the structure and function of the red blood cell membrane. The protein spectrin, of high molecular weight and propensity for self-association, appears to play a major role, in concert with actin, in maintaining the shape and integrity of the membrane. A study of the physical-chemical properties of spectrin, and its size, shape, self-association pattern, and its interaction with other components, leads to a plausible model for the way this protein performs its biological role. The evidence from the structure and interactions of spectrin suggests a structure which is relatively symmetrical yet highly expanded, and which allows extensive, two-dimensional network formation with actin. In these respects, the structure of spectrin is quite different from that of myosin, to which it has often been likened.

Chemical Phenomena↗
PubMed1979

Substructure of human erythrocyte spectrin.

The human erythrocyte structural protein spectrin and its subunits I, II were isolated in the presence of Na-dodecyl-sulfate by gel filtration and preparative gel electrophoresis. After removal of the detergent, spectrin alpha-helical content is comparable to spectrin isolated without detergent. Subunits I and II formed single bands in isoelectric focusing (pI = 5.6) and in Ornstein-Davis disc gel electrophoresis systems, indicating the individual subunits are homogenous in nature. The molecular weights of the subunits I and II, determined by Ferguson plot, are 237,500 and 238,600, respectively, which is in good agreement with values obtained by the standard SDS gel relative mobility method. Limited tryptic digestion of spectrin and two-dimensional peptide maps of the individual subunits cleaved by S-cyanylation reaction showed dissimilar patterns, suggesting differences in primary structure between the two subunits.

Amino Acids↗
PubMed1979

Erythrocyte spectrin alteration induced by low-density lipoprotein.

Addition of human plasma low-density lipoproteins (LDL) to intact human erythrocytes induces the erythrocytes to undergo morphologic transition from biconcave disks to echinocytes and spherocytes. The transformation is time-dependent. Two hours are required before echinocytes are detected by scanning electron microscopy. After two hours, LDL also decrease the phosphate content of spectrin by 40% relative to the control, suggesting that these lipoproteins modulate cell shape by influencing phosphorylation-dephosphorylation of a membrane-associated cytoskeletal protein. LDL do not induce depletion of intracellular adenosine triphosphate (ATP), nor do they inhibit cyclic adenosine monophosphate-independent protein kinases which phosphorylate spectrin. LDL stimulate membrane-bound phosphatases by a factor of two, thereby reducing the amount of phosphate covalently bound to membrane proteins. The observed effects are specific for LDL. High-density lipoproteins (HDL) do not stimulate dephosphorylation of spectrin or alter erythrocyte morphology. However, HDL protect the erythrocytes against LDL-induced alterations. These data suggest that the circulating lipoproteins have a role in maintaining erythrocyte morphology by regulating the extent of phosphorylation of spectrin.

Erythrocyte Membrane↗
PubMed1993

Band 4.1 enhances spectrin binding to phosphatidylserine vesicles.

Erythroid band 4.1 enhances the binding of erythroid spectrin to phosphatidylserine vesicles under conditions of ionic strength and at protein concentrations similar to those in the red cell. The extent of enhancement depends on the concentration of band 4.1; at 2 microM 4.1, spectrin binding increases approximately 10-fold (to 600 mumoles/mole lipid). The Kd is 0.5 microM, as measured by SDS-PAGE of protein-bound vesicles recovered by ficoll gradient centrifugation. The 4.1-enhanced binding of spectrin was also measured by a gel filtration assay. The electrostatic nature of the enhancement of spectrin binding is indicated by its dependence on the phosphatidylserine content of the vesicles.

Chromatography, Ion Exchange↗
PubMed1996

Fluorescence of spectrin-bound prodan.

A large blue-shift (delta lambda max = 87 nm) in the fluorescence emission maximum and 2-fold enhancement in the emission intensity of PRODAN in the presence of 1.5 microM dimeric spectrin from the same aqueous buffer was observed. Binding of PRODAN to spectrin was estimated from the concentration-dependent change in the ratio of two fluorescence intensities, I520nm for the unbound and I430nm corresponding to the spectrin-bound PRODAN. PRODAN binds to spectrin with a higher affinity (Kapp = 2 X 10(6)M-1) than those reported for BSA and tubulin (Kapp = 1 X 10(5)M-1 for BSA and 5 X 10(4)M-1 for tubulin). This is the first report of PRODAN binding at such a low protein concentration.

2-Naphthylamine↗
PubMed1999

Engagement of spectrin and actin in capping of FcgammaRII revealed by studies on permeabilized U937 cells.

Plasma membrane receptors can undergo translocation in the plane of plasma membrane after binding of polyvalent ligands. Ligand/receptor clusters, named patches, can collect into a polar cap, presumably due to their association with the submembrane actin-based cytoskeleton. We found that the assembly of Fcgamma receptor II caps in human monocytic U937 cells was accompanied by the accumulation of spectrin and actin in the cap region. Permeabilization of cells with streptolysin O rendered capping sensitive to inhibition by phalloidin, an actin filament stabilizing agent. A rabbit antibody directed against the chicken erythrocyte alpha-subunit of spectrin, an actin- and membrane-binding protein, also blocked the capping in a dose dependent manner. The inhibition reached approximately 50% after 20 minutes of cell treatment with the antibody. Anti-alpha-spectrin targeted specifically its submembrane antigen, in contrast to unspecific antibodies which remained dispersed in the cell interior and had no influence on the cap assembly. Our results indicate an active engagement of spectrin and actin filaments in the capping of Fcgamma receptor II.

Actins↗
PubMed1997

Solution structure of the spectrin repeat: a left-handed antiparallel triple-helical coiled-coil.

Cytoskeletal proteins belonging to the spectrin family have an elongated structure composed of repetitive units. The three-dimensional solution structure of the 16th repeat from chicken brain alpha-spectrin (R16) has been determined by NMR spectroscopy and distance geometry-simulated annealing calculations. We used a total of 1035 distance restraints, which included 719 NOE-based values obtained by applying the ambiguous restraints for iterative assignment (ARIA) method. In addition, we performed a direct refinement against 1H-chemical shifts. The final ensemble of 20 structures shows an average RMSD of 1.52 A from the mean for the backbone atoms, excluding loops and N and C termini. R16 is made up of three antiparallel alpha-helices separated by two loops, and folds into a left-handed coiled-coil. The basic unit of spectrin is an antiparallel heterodimer composed of two homologous chains, beta and alpha. These assemble a tetramer via a mechanism that relies on the completion of a single repeat by association of the partial repeats located at the C terminus of the beta-chain (two helices) and at the N terminus of the alpha-chain (one helix). This tetramer is the assemblage able to cross-link actin filaments. Model building by homology of the "tetramerization" repeat from human erythrocyte spectrin illuminates the possible role of point mutations which cause hemolytic anemias.

Amino Acid Sequence↗
PubMed2001

Isolation of spectrin subunits by reverse-phase high-performance liquid chromatography.

We present a one-step uncomplicated method of separation of spectrin subunits. The method is based on reverse-phase HPLC employing an analytical C4 column. Reverse-phase HPLC combines the steps of dissociation and separation of spectrin subunits. The method can be applied to different spectrin isoforms. It can be used for analytical purposes, as well as for small-scale (<0.4 mg) isolation of spectrin subunits.

Animals↗
PubMed1997

CS2-mediated cross-linking of erythrocyte spectrin and neurofilament protein: dose response and temporal relationship to the formation of axonal swellings.

Using model proteins, a mechanism for CS2-mediated covalent cross-linking of proteins has been demonstrated previously. The biologic importance of CS2-promoted protein cross-linking is apparent as a possible dosimeter of CS2 exposure and as a potential mechanism to account for the identical neuropathies produced by 2,5-hexanedione and CS2. The present investigation examines the utility of erythrocyte spectrin cross-linking as a biomarker of effect for inhalation exposure to CS2 and examines the ability of CS2 to cross-link neurofilament proteins, a potential neurotoxic target. Rats were exposed to CS2 via inhalation at control, 50-, 500-, and 800-ppm levels for 2, 4, 8, and 13 weeks and spectrin dimer formation was quantified using denaturing gel electrophoresis and densitometry. Neurofilament preparations were also obtained from spinal cords and examined for cross-linking using Western blotting methods. The results obtained for protein cross-linking were compared to morphologic changes in the cervical and lumbar spinal cord using light and electron microscopy. The spectrin dimer exhibited a cumulative dose response and was detectable at both the 50-ppm level employed that did not produce axonal swellings and prior to the development of axonal swellings for the 500- and 800-ppm levels used. Neurofilament protein cross-linking involved all three subunits and the temporal relationship of cross-linking was consistent with a contributing role in the development of axonal swellings. These results establish the sensitivity of spectrin cross-linking for evaluating inhalation exposures and extend the similarities observed for 2,5-hexanedione and CS2 in both clinical settings and in vitro models to their effects exerted on neurofilaments in the axon.

Administration, Inhalation↗
PubMed1990

Evidence that expression of Sp alpha I/65 hereditary elliptocytosis is compounded by a genetic factor that is linked to the homologous alpha-spectrin allele.

Many cases of hereditary elliptocytosis (HE) result from mutated spectrin alpha-chains. It has repeatedly been observed that the amount of a mutant alpha-chain is different in various affected individuals, resulting in clinical pictures of variable severity. The different levels are thought to result from different percentages of the alpha-spectrin allele in trans. Such percentages, in turn, could be under genetic control. We tested this hypothesis in a large Algerian family with Sp alpha I/65 HE. In an informative sibship, we found three persons with a distinctly high level of expression of the Sp alpha I/65 variant, suggesting the existence, in trans, of a low percentage alpha-allele. The alpha-spectrin gene haplotype associated with the latter was constantly - + -, based on the XbaI, PvuII, and MspI polymorphic sites. In contrast, a basal level of expression of the Sp alpha I/65 variant in the same sibship indicated, in trans, the existence of a normal percentage alpha-allele. The haplotype corresponding to this other alpha-allele was + - +. Study of another generation of the family showed, however, that the - + - haplotype could also be linked to a normal percentage alpha-allele. These results are consistent with the view that the expression level of alpha I/65 spectrin (and of other types of alpha-variants) is compounded by a genetic factor that is linked to the normal alpha-allele in trans. The low percentage allele itself remains silent in the simple heterozygous state.

Elliptocytosis, Hereditary↗
PubMed1987

Hereditary pyropoikilocytosis and elliptocytosis in a Caucasian family. Transmission of the same molecular defect in spectrin through three generations with different clinical expression.

Hereditary pyropoikilocytosis (HPP) is a severe hemolytic anemia characterized by a material instability of the red cell membrane leading to cell fragmentation. This fragility may be correlated with functional and structural defects of spectrin. Most HPP patients have been black. We now report three HPP patients from a Caucasian family, the proposita and her two maternal uncles. The proposita's mother and daughter presented mild type I hereditary elliptocytosis (HE), while the proposita's father was clinically and hematologically normal. Our studies revealed a defective ability of spectrin to self-associate, resulting in an excess of spectrin dimer in 4 degrees C extracts in the three HPP patients and to a similar extent in HE relatives. Limited tryptic digestion of spectrin showed a molecular variant in the alpha I domain as expressed by a decreased amount of 80,000-dalton peptide with a concomitant increase in the 74,000-dalton peptide. Investigations in the proposita's father revealed no abnormalities of the erythrocyte membrane. The co-transmission of HPP and HE phenotypes in the same lineage might suggest variability in the clinical expression of the same molecular defect and lead us to discuss the hypothesis of a double heterozygosity in HPP patients.

Adult↗
PubMed1995

Localization of immuno-analogues of erythrocyte protein 4.1 and spectrin in epidermis of psoriasis vulgaris.

The presence and localization of immuno-analogues of human erythrocyte protein 4.1 and spectrin were examined in the epidermis of psoriasis vulgaris. Immunoblot analysis with antibodies against human erythrocyte protein 4.1 revealed that psoriatic epidermis contains a 4.1-like protein of 80 kDa, and also minor immunoreactive polypeptides, including a 45-kDa polypeptide. The 45-kDa band was not detected in non-lesional epidermis. Lesional epidermis of psoriasis contains spectrin-like proteins of 240 kDa. Analysis with immunofluorescence microscopy revealed that 4.1-like proteins were detected mainly in the cytoplasm of the suprabasal cells in lesional epidermis and in the peripheral cytoplasm of the basal cells in non-lesional epidermis. On the other hand, spectrin-like proteins were localized to the peripheral cytoplasm of basal keratinocytes in both lesional and non-lesional psoriatic epidermis. The present results indicate that proteins related to protein 4.1 and spectrin are consistently detected within epidermal cells of psoriasis, a chronic skin disease characterized by epidermal hyperplasia; the expression and distribution of protein 4.1 in lesional epidermis of psoriasis differs from that in non-lesional epidermis. These membrane skeletal proteins may be of significance in the hyperproliferative epidermis of psoriasis.

Adult↗
PubMed1986

Localization of alpha-spectrin in chicken and monkey ventral horns by immunoelectron microscopy.

Localization of alpha-spectrin in chicken and monkey ventral horns has been studied by immunoperoxidase techniques at the electron microscopic level. For this purpose, an antiserum specific for chicken alpha-spectrin (240 kD subunit of spectrin) was prepared. The characteristics of the staining patterns of both chicken and monkey ventral horns were essentially identical. The reaction product for peroxidase was contained in the somata of large cells (presumably motor neurons), dendrites and axons. No specific staining was seen with either preimmune or blocked sera. The staining within the cell somata was primarily localized in cortical cytoplasm. Within dendrites and axons the immunocytochemical label was associated predominantly with the cortical cytoplasm and with microtubules. Staining was heavy over postsynaptic densities. Although presynaptic terminals showed weak staining as a whole, heavy staining was sometimes observed in areas adjacent to the presynaptic plasma membrane facing the postsynaptic density. These results indicate that spectrin distributes widely and functions in many biological activities in the nervous system.

Animals↗
PubMed1993

Combined ankyrin and spectrin deficiency in hereditary spherocytosis.

Hereditary spherocytosis is characterized by a reduced spectrin content of the erythrocytes. However, the underlying primary defect remains unclear in the majority of cases. Genetic studies have revealed a linkage to the gene for ankyrin in some families. By means of ELISA we measured the ankyrin, spectrin, and band-3 contents in erythrocytes of 45 patients with typical spherocytosis. They were classified as having mild or moderate spherocytosis, according to clinical severity. Sixteen patients with mild spherocytosis showed slight reductions of ankyrin and spectrin contents. In contrast, 29 patients with moderate spherocytosis exhibited a clear reduction of both ankyrin and spectrin to about 60% of normal. Band 3 and lipid phosphorus, as measures for membrane surface area, were only slightly reduced to 85%. Our results, together with the molecular genetic data indicating the linkage between spherocytosis and the gene for ankyrin, suggest an ankyrin defect or deficiency as the primary lesion in most cases of spherocytosis.

Adolescent↗
PubMed1987

Assignment of mouse beta-spectrin gene to chromosome 12.

The structural gene for the beta-subunit of the mouse erythrocyte spectrin, hereinafter designated as Sp-b, was assigned to the mouse chromosome 12. This assignment was made by Southern analysis of genomic DNA from mouse X Chinese hamster hybrid cells using cloned mouse erythrocyte beta-spectrin cDNA as a probe. In the PstI-digested genomic hamster cell DNA a single band of 2.0 kb was detected, whereas PstI-digested mouse DNA gave a band of 4.2 kb, when probed with the mouse erythroid beta-spectrin cDNA clone. This allowed us to analyze a panel of mouse X Chinese hamster somatic cell hybrids to map this gene to chromosome 12. Interestingly, this assignment is different from that observed for the alpha-subunit of spectrin, which has been mapped to chromosome 1 in mouse. These results serve as a basis for further genetic characterization of the mouse hemolytic anemias.

Animals↗
PubMed2006

Functional links between membrane transport and the spectrin cytoskeleton.

Membrane transporters precisely regulate which molecules cross the plasma membrane and when they can cross. In many cases it is also important to regulate where substances can cross the plasma membrane. Consequently, cells have evolved mechanisms to confine and stabilize membrane transport proteins within specific subdomains of the plasma membrane. A number of different transporters (including ion pumps, channels and exchangers) are known to physically associate with the spectrin cytoskeleton, a submembrane complex of spectrin and ankyrin. These proteins form a protein scaffold that assembles within discrete subdomains of the plasma membrane in polarized cells. Recent genetic studies in humans and model organisms have provided the opportunity to test the hypothesis that the spectrin cytoskeleton has a direct role in restricting transporters to specialized domains. Remarkably, genetic defects in spectrin and ankyrin can produce effects on cell physiology that are comparable to knockouts of the transporters themselves.

Amino Acid Transport System X-AG↗
PubMed1978

The incorporation of 32 P into spectrin aggregates following incubation of erythrocytes in 32 P-labelled inorganic phosphate.

32P was incorporated into spectrin by incubation of fresh erythrocytes with 32Pi and glucose. The dimer and tetramer aggregates revealed only covalently-bound incorporation of phosphorus, while a higher aggregate of spectrin revealed both covalent and non-covalent incorporation. The specific activity of the covalently-bound phosphorus in all oligomers was identical, suggesting that the state of association is independent of phosphorylation. The non-covalent incorporation was shown to be due to the association of ATP with this higher aggregate. The nucleotide appers not to be bound directly to spectrin but rather to component 5 (erythrocyte actin) which is also found to be associated with this highly aggregated spectrin structure.

Actins↗
PubMed1980

The effect of endogenous proteases on the spectrin binding proteins of human erythrocytes.

We have demonstrated that in human erythrocyte ghosts endogenous proteolytic activity is responsible for the digestion of the spectrin binding proteins (bands 2.1 to 2.6). The pH optimum, cofactor requirements and inhibitor sensitivity have been established. Our results indicate that proteolysis of bands 2.1 to 2.6 and the formation of 3', a fragment containing an active spectrin binding site, can occur through two enzymatic pathways: a cascade of consecutive proteolytic cleavages of the spectrin binding proteins inhibited by phenylmethylsulfonyl fluoride or a Ca2+-stimulated, phenylmethylsulfonyl fluoride-insensitive, EDTA-inhibited cleavage of band 2.1 to band 2.3, followed by digestion to band 3' by phenylmethylsulfonyl fluoride-inhibitable enzymes. These findings may provide the techniques necessary to prevent proteolysis of the spectrin binding proteins during purification and reconstitution experiments and provide insight into how they are formed in vivo.

Calcium↗