Erythrocyte Cytoskeletal Proteins Research Articles

Interaction of the DNA-binding antitumor antibiotics, chromomycin and mithramycin with erythroid spectrin.

The aureolic acid group of antitumor antibiotics, chromomycin A3 and mithramycin, are well established as transcription inhibitors, which bind reversibly to DNA at and above physiological pH, in the presence of divalent metal ions such as Mg2+. As part of our broad objective to elucidate their intracellular mode of action, other than association with DNA, we studied their interactions with the erythrocyte cytoskeletal protein, spectrin, in the absence and presence of magnesium. Different spectroscopic studies, such as absorbance, fluorescence and CD, have shown that both free chromomycin and mithramycin and their Mg2+ complexes bind to spectrin with an affinity higher than that reported for DNA. The affinity constants for the association of chromomycin and mithramycin (or their Mg2+ complexes) with spectrin are comparable with those for the association of spectrin with other cytoskeletal proteins, for example F-actin, ankyrin, protein 4.1, etc. The nature of the binding of the two antibiotics to spectrin is different. The mode of binding of the antibiotics with spectrin also changes in the presence of Mg2+. The interaction leads to a change in the tertiary structure of the protein. The relevance of the results to our understanding of the mode of action of the antibiotics is discussed.

Evidence for age-related deamidation reactions in erythrocyte p55 and glycophorins C and D: implications for signal transduction involving tumour-suppressor proteins in higher eukaryotes

The methylation of erythrocyte cytoskeletal proteins by methyltransferases is a strategy for the repair of deamidated asparagine residues. Evidence from extant data shows that the junction point proteins of the erythrocyte cytoskeleton, band 3, ankyrin, glycophorins C/D, band 4.1, calmodulin, and p55 are all methylated in situ. It is suggested that sequence-dependent deamidation of asparagine residues in p55, 4.1 and glycophorins C/D could affect the interactions of these junction point proteins in an age-dependent manner. Comparison of the asparagine content of 4.1 binding regions of p55 analogues dig and hdlg acts as a caveat to the extrapolation of findings made in erythrocytes to other eukaryotic cells, and has important implications for transmembrane signalling pathways and age-dependent changes in the membrane-binding characteristics of tumour-suppressor proteins in higher cell systems.

Cytoskeletal protein binding kinetics at planar phospholipid membranes

It has been hypothesized that nonspecific reversible binding of cytoskeletal proteins to lipids in cells may guide their binding to integral membrane anchor proteins. In a model system, we measured desorption rates k(off) (off-rates) of the erythrocyte cytoskeletal proteins spectrin and protein 4.1 labeled with carboxyfluorescein (CF), at two different compositions of planar phospholipid membranes (supported on glass), using the total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) technique. The lipid membranes consisted of either pure phosphatidylcholine (PC) or a 3:1 mixture of PC with phosphatidylserine (PS). In general, the off-rates were not single exponentials and were fit to a combination of fast, slow, and irreversible fractions, reported both separately and as a weighted average. By a variation of TIR/FRAP, we also measured equilibrium affinities (the ratio of surface-bound to bulk protein concentration) and thereby calculated on-rates, k(on). The average off-rate of CF-4.1 from PC/PS (approximately 0.008/s) is much slower than that from pure PC (approximately 1.7/s). Despite the consequent increase in equilibrium affinity at PC/PS, the on-rate at PC/PS is also substantially decreased (by a factor of 40) relative to that at pure PC. The simultaneous presence of (unlabeled) spectrin tends to substantially decrease the on-rate (and the affinity) of CF-4.1 at both membrane types. Similar experiments for CF-spectrin alone showed much less sensitivity to membrane type and generally faster off-rates than those exhibited by CF-4.1. However, when mixed with (unlabeled) 4.1, both the on-rate and off-rate of CF-spectrin decreased drastically at PC/PS (but not PC), leading to a somewhat increased affinity. Clearly, changes in affinity often involve countervailing changes in both on-rates and off-rates. In many of these studies, the effect of varying ionic strength and bulk concentrations was examined; it appears that the binding is an electrostatic attraction and is far from saturation at the concentrations employed. These results and the techniques implemented carry general implications for understanding the functional role of nonspecific protein binding to cellular lipid membranes.

Spontaneous recovery of fluorescence by photobleached surface-adsorbed proteins.

Fluorescence photobleaching of a carboxyfluorescein-labeled protein (erythrocyte cytoskeletal protein 4.1) immobilized on bare glass is found to be spontaneously reversible, provided that the sample is deoxygenated. After a short (hundredths of seconds) photobleaching laser flash, the subsequent fluorescence excited by a dim probe beam partly recovers on a long (tenths of second) time scale, even in the absence of chemical exchange or diffusion processes. Neither the fraction of the fluorescence that bleaches reversibly nor its recovery rate is a strong function of fluorophore surface concentration. At a fixed surface concentration, the reversibly photobleached fraction and its recovery rate decreases with increasing duration or intensity of the bleaching flash. On the other hand, nondeoxygenated air-equilibrated samples exhibit almost total irreversible bleaching on this time scale. Quantitative fluorescence microscopy experiments occasionally require deoxygenation to avoid photochemical crosslinking or photobleaching or to enhance the triplet state population. The observation presented here indicate that fluorescence recovery after photobleaching (FRAP) experiments performed under deoxygenated conditions for measuring diffusion or chemical kinetics should be interpreted with caution: fluorescence recoveries may be due to intrinsic photochemical processes rather than fluorophore mobility. The recovery effect appears too slow to be ascribed simply to a relaxation of a triplet state; other possible explanations are offered.

Hemolysis in primary lipoprotein lipase deficiency

A slight to moderate hemolysis is often present in plasma from patients with primary lipoprotein lipase (LPL) deficiency. To determine the nature of this hemolysis, we measured erythrocyte hypo-osmotic fragility, plasma free hemoglobin, and phospholipid composition in 26 patients with primary LPL deficiency and 21 unrelated controls. In some patients, these investigations were completed by erythrocyte cytoskeletal protein determinations and abdominal echography. Osmotic fragility was similar between control subjects and patients. However, there was a significantly increased concentration of plasma free hemoglobin in primary LPL deficiency (0.282 ± 0.331 v 0.048 ± 0.038 g/L in controls, P < .005). In LPL-deficient patients, an increase of plasma lysophosphatidylcholine concentration (12.6% ± 5.8% v 6.4% ± 1.9% in controls, P < .0001) was also found. The protein composition of the erythrocyte membrane skeleton was abnormal in some LPL-deficient patients and splenomegaly was present in 12, but these abnormalities did not correlate with plasma free hemoglobin levels. Bilirubin and haptoglobin levels were also within physiologic ranges in these patients, suggesting that the observed hemolysis did not result from hypersplenism. It appears likely that the accumulation of lysophosphatidylcholine was due to an impairment in the reverse metabolic pathway converting lysophosphatidylcholine back to phosphatidylcholine. Collectively, these data, along with a positive correlation between plasma free hemoglobin and lysophosphatidylcholine levels ( r = .58, P = .0001), suggest that the hemolysis observed in primary LPL deficiency is mediated to some extent by the abnormally elevated concentration of lysophosphatidylcholine.

Effect of lipopolysaccharide on the physical conformation of the erythrocyte cytoskeletal proteins

Red blood cell deformability is important for effective circulation in the capillaries. It is known that red cell deformability is significantly reduced during septic shock. Surface to volume ratio, physical effects of the cytoskeletal proteins and the fluidity of lipid bilayer are some of the important intrinsic factors that regulate this mechanical function. Alterations in the physical conformation of cytoskeletal proteins in septic conditions could significantly alter their function. In this study, erythrocytes in whole blood were treated with lipopolysaccharide, the outer covering of Gram-negative bacteria released during Gram-negative sepsis. Electron paramagnetic resonance spectroscopy in conjunction with a protein-specific maleimide nitroxide spin label covalently bound to cytoskeletal proteins was used to investigate the resulting changes occurring in the physical state of cytoskeletal proteins in isolated membranes. Treatment of red blood cells with a lipopolysaccharide concentration as low as 40 μg/mL of blood solution for 90 minutes showed a significant decrease in the relevant EPR parameter (p0̌.01) of the spin label bound to subsequently isolated membranes, suggestive of a decreased segmental motion of the spin label and an increase in cytoskeletal protein-protein interactions. These results suggest a marked conformational alteration in the cytoskeletal proteins induced by the lipopolysaccharide and may explain, in part, the marked reduction in red blood cell deformability during septic shock. Bacterial lipopolysaccharide does not exert most of its effects on the host directly, but rather elicits the production of host factors that leads to complex septic shock. Leukocytes, endothelial tissue and many other cells release these mediators. Leukocytes are thought to be a particularly important source of such mediators, including cytokines (tumor necrosis factor, interleukins, etc.), oxygen free radicals, proteases, and hydrolyses. In order to characterize the possible mechanism by which the lipopolysaccharide acts on the physical state of the erythrocyte cytoskeleton, erythrocytes void of leukocytes and plasma were treated with lipopolysaccharide. The relevant EPR parameter showed no significant change over the control value. These results indicate that the leukocytes and their factors are responsible for the rearrangements seen in the cytoskeletal proteins of the erythrocyte membrane.

Interaction of erythrocyte cytoskeletal proteins with band 3: rotational diffusion measurements

Interaction of erythrocyte cytoskeletal proteins with band 3: rotational diffusion measurements

Assignment of the gene for β-spectrin (SPTB) to chromosome 14q23→q24.2 by in situ hybridization

Type I hereditary spherocytosis results from a molecular defect in the beta-polypeptide of the erythrocyte cytoskeletal protein spectrin. Using a cDNA probe, we had previously assigned the gene for human erythrocyte beta-spectrin (SPTB) to chromosome 14 based upon analysis of its segregation in panels of human x rodent somatic cell hybrids (Winkelmann et al., 1988). Here we report the regional localization of this gene by in situ hybridization to 14q23----q24.2.

CDNA cloning and sequencing of the protein-tyrosine kinase substrate, ezrin, reveals homology to band 4.1.

Ezrin is a component of the microvilli of intestinal epithelial cells and serves as a major cytoplasmic substrate for certain protein-tyrosine kinases. We have cloned and sequenced a human ezrin cDNA and report here the entire protein sequence derived from the nucleotide sequence of the cDNA as well as from partial direct protein sequencing. The deduced protein sequence indicates that ezrin is a highly charged protein with an overall pI of 6.1 and a calculated molecular mass of 69,000. The cDNA clone was used to survey the distribution of the ezrin transcript, and the 3.2 kb ezrin mRNA was found to be expressed in the same tissues that are known to express the protein and at the same relative levels. Highest expression was found in intestine, kidney and lung. The cDNA clone hybridized to DNAs from widely divergent organisms indicating that its sequence is highly conserved throughout evolution. The amino acid sequence of ezrin revealed a high degree of similarity within its N-terminal domain to the erythrocyte cytoskeletal protein, band 4.1 and secondary structure predictions indicate that a second region of ezrin contains a long alpha-helix, a feature also common to band 4.1. The structural similarity of ezrin to band 4.1 suggests a mechanism for the observed localization to the membrane, and a role for ezrin in modulating the association of the cortical cytoskeleton with the plasma membrane.

Amelin and synapsin I are 4.1 related spectrin binding proteins in brain

How do synaptic vesicles move towards the presynaptic plasma membrane, fuse with that membrane, and release their contents during synaptic transmission? The answers to these questions at the molecular level are just beginning to be understood. Synapsin I is a neuron specific phosphoprotein that is associated with the cytoplasmic surface of synaptic vesicles. During synaptic transmission, the translocation of the synaptic vesicles to the presynaptic membrane of the neuron is thought to be mediated through changes in the phosphorylation state of synapsin I. It has been suggested that synapsin I is a spectrin binding protein related to the erythrocyte cytoskeletal protein 4.1, which binds to the terminal ends of the erythrocyte spectrin tetramer. The interaction of synapsin I (through brain spectrin) with the neuronal cytoskeleton may be essential for regulating the movement of synaptic vesicles towards the presynaptic plasma membrane. In addition, we have identified another protein in brain that is immunologically and structurally more closely related to erythrocyte 4.1 than is synapsin I. This protein, termed amelin, is localized in the cell body and dendrites of the neuron, whereas synapsin I is found exclusively in the synaptic terminals, suggesting that there is a family of erythrocyte 4.1 related proteins present in brain with distinct subcellular distribution and functions.

Determination and analysis of the primary structure of the nerve terminal specific phosphoprotein, synapsin I.

A rat brain cDNA clone containing an open reading frame encoding the neuron-specific protein synapsin I has been sequenced. The sequence predicts a protein of 691 amino acids with a mol. wt of 73 kd. This is in excellent agreement with the size of rat brain synapsin Ib measured by SDS--polyacrylamide gel electrophoresis. Inspection of the predicted primary structure has revealed the probable sites for synapsin I phosphorylation by the cAMP-dependent and Ca2+/calmodulin-dependent protein kinases. All of the biochemically observed intermediates of synapsin I digestion by collagenase can be verified by inspection of the sequence, and the collagenase-resistant fragment has been defined as the amino-terminal 439 amino acids of the molecule. Predictions of sequence secondary structure and hydrophobicity suggest that a central domain of approximately 270 amino acids may exist as a folded, globular core. The carboxyl-terminal domain of the protein (the region sensitive to collagenase digestion) contains sites for Ca2+/calmodulin-dependent protein kinase phosphorylation. These sites are flanked by three regions of repeating amino acid sequence that are proposed to be the synaptic vesicle-binding domain of synapsin I. This region also shares homology with the actin-binding proteins profilin and villin. The characteristics of the synapsin I sequence do not support extensive homology with the erythrocyte cytoskeletal protein 4.1.

Mechanisms of cytoskeletal regulation: modulation of aortic endothelial cell protein band 4.1 by the extracellular matrix.

The bovine aortic endothelial cell (BAEC) cytoskeleton is a complex structure modulated by many stimuli including release from contact inhibition and various components of the extracellular matrix (ECM). Transduction of information from the ECM to the cell nucleus proceeds via several complex pathways including the cytoskeleton. We have demonstrated the presence of an immunoreactive isoform of the human erythrocyte cytoskeletal protein band 4.1 (4.1) in BAEC. BAEC 4.1 is similar in molecular weight to the erythroid protein by immunoblot analyses and produces a similar pattern of cysteine specific cleavage products consistent with a cluster of cysteine residues previously described in the erythroid molecule. We have also examined the effects of defined ECM proteins on the distributions of cultured BAEC 4.1 and actin filaments (AF) at confluency and following release from contact inhibition. The distribution of 4.1 in BAEC on a plasma fibronectin substrate is complex, having partial codistribution with cytoplasmic AF and a unique perinuclear staining. In contrast, on a collagen type I/III substrate, 4.1 is localized, in part, to peripheral areas of cell-cell contact distinct from the dense peripheral band staining of AF. During migration on this substrate, 4.1 had a filamentous distribution having partial codistribution with AF. Indirect immunofluorescence staining of cross-sections of bovine calf aortae revealed a cortical staining pattern in the aortic endothelial cells with staining noted on the luminal and basolateral aspects of the cells. These data suggest that, in endothelial cells, protein 4.1 is a cortical membrane protein which may function to link actin filaments to other skeletal proteins such as spectrin. These findings also suggest an active role for protein 4.1 in cytoskeletal reorganization events which can occur in response to external stimuli, such as the extracellular matrix or contact with other cells.

Spectrin phosphorylation in senescent rat erythrocytes

The rates of phosphorylation and dephosphorylation of the erythrocyte cytoskeletal protein, spectrin, were analyzed in young and old rat erythrocytes. Endogenous membrane protein kinase activity was measured in age-seperated rat erythrocytes, and was found to decrease as a function of cell age. Membranes prepared from young and old erythrocytes contained comparable levels of protein phosphatase activity. Spectrin phosphatase activity was readily observed in erythrocyte membranes. Partially purified spectrin kinase and spectrin were prepared from membranes obtained from young and old erythrocytes, and the phosphorylation of the spectrin fractions was measured with the isolated kinases. The kinases prepared from young or old cells phosphorylated spectrin from young cells to the same extent. When spectrin from old cells was used as the substrate, it was phosphorylated ten-fold less extensively by the spectrin kinase prepared from old cells than by the spectrin kinase from young cells. This finding indicated that the decrased phosphorylation of spectrin observed in membranes prepared from age-separated red cells was due to a structural alteration in the spectrin. A structural basis for the decreased phosphorylation of spectrin in older erythrocytes was sought. Treatment of erythrocyte membranes with malonyldialdehyde, a product of lipid peroxidation which accumulates in erythrocyte membranes during senescence, adversely affected spectrin phosphorylation. The results presented here indicate that intramolecular derivatization of spectrin was sufficient to impair its function as a substrate for protein kinase.