Effect of spectrin on structure properties of lipid bilayers formed from mixtures of phospholipids. Fluorescence and microcalorimetric studies.

Effect of spectrin from human erythrocytes on structure properties of lipid bilayers formed from a mixture of phosphatidylethanolamine/phosphatidylserine (PE/PS) and/or phosphatidylethanolamine/phosphatidylcholine (PE/PC) was studied with the use of fluorescence and microcalorimetric methods. Spectrin did not affect the order parameter of lipids in PE/PS vesicles. However, spectrin binding to liposomes did influence temperature, half-width and enthalpy of phase transitions in mixtures of dimyristoylphosphatidylethanolamine (DMPE) and dimyristoylphosphatidylcholine (DMPC), and this effect was dependent on DMPE to DMPC weight ratio. A change in miscibility of the components in the presence of spectrin was observed and it might be due to spectrin-PE interactions.     

Characteristics of spectrin-induced leakage of extruded, phosphatidylserine vesicles

At neutral pH spectrin induces modest leakage of trapped calcein from reverse-phase or extruded, but not sonicated, vesicles composed of phosphatidylserine, but not phosphatidylcholine. The extent of leakage from extruded vesicles is not or is only slightly affected by magnesium ions at a physiological concentration or calcium ions at a greater than physiological concentration, respectively. In addition to accounting for several previously discrepant observations on the lytic effects of spectrin, these findings indicate that some proteins like spectrin may destabilize vesicles with low curvature more readily than vesicles of high curvature, in contrast to certain amphiphilic peptides. 60% less leakage is induced from phosphatidylserine vesicles by heat-denatured than by native spectrin. In contrast, both trypsin- and subtilisin-treated spectrins, if sufficiently digested, induce several-fold more leakage than undigested spectrin. Since spectrin prepared either by 1 M Tris dissociation of Triton-extracted cytoskeletons or by low ionic strength extraction of ghosts released the same amounts of calcein from vesicles of various compositions, these effects are unlikely to reflect artifacts of spectrin preparation. Furthermore, spectrin is unlikely to promote leakage in vivo, since vesicles composed of phosphatidylserine, cholesterol and/or phosphatidylethanolamine, which constitute the lipid composition of the inner monolayer of the red cell membrane, did not leak on addition of spectrin, whereas vesicles composed of phosphatidylserine and phosphatidylcholine, did leak in the presence of spectrin.     

Chlorpromazine associates with phosphatidylserines to cause an increase in the lipid's own interfacial molecular area--role of the fatty acyl composition

Partition coefficients of the drug chlorpromazine were determined for five different molecular species of diacylglycerophosphatidylserine in a monolayer kept at constant surface pressure (20 mN/m). Two models of adsorption of chlorpromazine in phosphatidylserine monolayers were compared. The first model correlated the amount of inserted drug molecules with the induced increase in area. The second model introduced the effect of drug adsorption on the lipid's own area by comparing the effect of increasing temperature on the lipid's own interfacial area. From the second model, the extrapolated work of insertion of one drug molecule per lipid molecule in a monolayer kept at 20 mN/m was correlated to the partition of the drug in liposomes. The work of insertion of chlorpromazine was insignificant in the unsaturated dioleoylphosphatidylserine and was maximum in the saturated distearoylphosphatidylserine monolayers. The presence of one double bond in the acyl chains dramatically reduces the work of insertion of chlorpromazine between lipid molecules and also reduces the effect chlorpromazine induces on the lipids own interfacial area in monolayers.     

Interaction of anilinonaphtyl labeled spectrin with fatty acids and phospholipids: a fluorescence study

Anilinonaphtyl labeled spectrin exhibits a fluorescence emission spectrum characteristic of a highly hydrophobic environment. Quenching of the fluorescence intensity by nitroxide analogs of fatty acids of affinity 10(4) M-1 reveals that the sites of interaction of fatty acids lie very close to the anilinonaphtyl groups. Similar experiments performed with a nitroxide analog of phosphatidylserine yield a 30% quenching of fluorescence while the same phosphatidylcholine analog has essentially no effect. The changes in the fluorescence emission spectrum exhibited in the presence of sonicated phosphatidylserine vesicles further outline the specificity of interaction towards phosphatidylserine, with one spectrin binding site per about 750 exposed phospholipids. Moreover, they suggest a penetration of the anilinonaphtyl group into the lipid bilayer.     

Weak interaction of spectrin with phosphatidylcholine-phosphatidylserine multilayers: a 2H and 31P NMR study

Spectrin from human erythrocytes binds to bilayer dispersions of both DMPC and DMPS:DMPC (1:1, w/w). However, no effect of bound spectrin on the conformation of the lipid head groups, as measured from the deuterium quadrupolar splittings of DMPC or DMPS specifically deuterated in the polar head groups, was detected in 1:1 mixtures of the two lipids containing either deuterated DMPC or DMPS. Neither the phase transition of the DMPS:DMPC mixtures, nor the spin-lattice relaxation time (T1) of the deuterated DMPS head group, was affected by spectrin. These results argue against any strong interaction of spectrin with phosphatidylserine and rule out the possibility that spectrin is responsible for the maintenance of PS in the inner monolayer of the erythrocyte membrane during the whole life-span of this cell.     

Calcium-mediated DNA adsorption to yeast cells and kinetics of cell transformation by electroporation.

The interfacial elastic packing interactions of different galactosylceramides (GalCers), sphingomyelins (SMs), and phosphatidylcholines (PC) were compared by determining their elastic area compressibility moduli (Cs-1) as a function of lateral packing pressure (pi) in a Langmuir-type film balance. To assess the relative contributions of the lipid headgroups as well as those of the ceramide and diacylglycerol hydrocarbon regions, we synthesized various GalCer and SM species with identical, homogeneous acyl residues and compared their behavior to that of PCs possessing similar hydrocarbon structures. For PCs, this meant that the sn-1 acyl chain was long and saturated (e.g., palmitate) and the sn-2 chain composition was varied to match that of GalCer or SM. When at equivalent pi and in either the chain-disordered (liquid-expanded) or chain-ordered (liquid-condensed) state, GalCer films were less elastic than either SM or PC films. When lipid headgroups were identical (SM and PC), Cs-1 values (at equivalent pi) for chain-disordered SMs, but not chain-ordered SMs, were 25-30% higher than those of PCs. Typical values for fluid phase (liquid-expanded) GalCer at 30 mN/m and 24 degrees C were 158 (+/- 7) mN/m, whereas those of SM were 135 (+/- 7) mN/m and those of PC were 123 (+/- 2) mN/m. Pressure-induced transitions to chain-ordered states (liquid-condensed) resulted in significant increases (two- to fourfold) in the "in-plane" compressibility for all three lipid types. Typical Cs-1 values for chain-ordered GalCers at 30 mN/m and 24 degrees C were between 610 and 650 mN/m, whereas those of SM and of PC were very similar and were between 265 and 300 mN/m. Under fluid phase conditions, the pi-Cs-1 behavior for each lipid type was insensitive to whether the acyl chain was saturated or monounsaturated. Measurement of the Cs-1 values also provided an effective way to evaluate the two-dimensional phase transition region of SMs, GalCers, and PCs. Modest heterogeneity in the acyl composition led to transitional broadening. Our findings provide useful information regarding the in-plane elasticity of lipids that are difficult to investigate by alternative methods, i.e., micropipette aspiration technique. The results also provide insight into the stability of sphingolipid-enriched, membrane microdomains that are thought to play a role in the sorting and trafficking of proteins containing glycosylphosphatidylinositol anchors with cells.     

The interfacial properties of ApoA-I and an amphipathic alpha-helix consensus peptide of exchangeable apolipoproteins at the triolein/water interface

Apolipoprotein A-I (apoA-I) is the major protein in high density lipoprotein (HDL). During lipid metabolism, apoA-I moves among HDL and triacylglycerol-rich lipoproteins. The main structure and the major lipid binding motif of apoA-I is the amphipathic alpha-helix. To understand how apoA-I behaves at hydrophobic lipoprotein interfaces, the interfacial properties of apoA-I and an amphipathic alpha-helical consensus sequence peptide (CSP) were studied at the triolein/water (TO/W) interface. CSP ((PLAEELRARLRAQLEELRERLG)2-NH2) contains two 22-residue tandem repeat sequences that form amphipathic alpha-helices modeling the central part of apoA-I. ApoA-I or CSP added into the aqueous phase surrounding a triolein drop lowered the interfacial tension (gamma) of TO/W in a concentration- and time-dependent fashion. The gamma(TO/W) was lowered approximately 16 millinewtons (mN)/m by apoA-I at 1.4 x 10(-6) m and approximately 15 mN/m by CSP at 2.6 x 10(-6) m. At equilibrium gamma, both apoA-I and CSP desorbed from the interface when compressed and readsorbed when expanded. The maximum surface pressure CSP could withstand without being ejected (PiMAX) was 16 mN/m. The PiMAX) of apoA-I was only 14.8 mN/m, but re-adsorption kinetics suggested that only part of the apoA-I desorbed at Pi between 14.8 and 19 mN/m. However, above approximately 19 mN/m (PiOFF) the entire apoA-I molecule desorbed into the water. ApoA-I was more flexible at the TO/W interface than CSP and showed more elasticity at oscillation periods 4-128 s even at high compression, whereas CSP was elastic only at faster periods (4 and 8 s) and moderate compression. Flexibility and surface pressure-mediated desorption and re-adsorption of apoA-I probably provides lipoprotein stability during metabolic-remodeling reactions in plasma.     

Interaction of a nonspecific wheat lipid transfer protein with phospholipid monolayers imaged by fluorescence microscopy and studied by infrared spectroscopy.

The interaction of a nonspecific wheat lipid transfer protein (LTP) with phospholipids has been studied using the monolayer technique as a simplified model of biological membranes. The molecular organization of the LTP-phospholipid monolayer has been determined by using polarized attenuated total internal reflectance infrared spectroscopy, and detailed information on the microstructure of the mixed films has been investigated by using epifluorescence microscopy. The results show that the incorporation of wheat LTP within the lipid monolayers is surface-pressure dependent. When LTP is injected into the subphase under a dipalmytoylphosphatidylglycerol monolayer at low surface pressure (< 20 mN/m), insertion of the protein within the lipid monolayer leads to an expansion of dipalmytoylphosphatidylglycerol surface area. This incorporation leads to a decrease in the conformational order of the lipid acyl chains and results in an increase in the size of the solid lipid domains, suggesting that LTP penetrates both expanded and solid domains. By contrast, when the protein is injected under the lipid at high surface pressure (> or = 20 mN/m) the presence of LTP leads neither to an increase of molecular area nor to a change of the lipid order, even though some protein molecules are bound to the surface of the monolayer, which leads to an increase of the exposure of the lipid ester groups to the aqueous environment. On the other hand, the conformation of LTP, as well as the orientation of alpha-helices, is surface-pressure dependent. At low surface pressure, the alpha-helices inserted into the monolayers are rather parallel to the monolayer plane. In contrast, at high surface pressure, the alpha-helices bound to the surface of the monolayers are neither parallel nor perpendicular to the interface but in an oblique orientation.     

Aromatic residues in the C terminus of apolipoprotein C-III mediate lipid binding and LPL inhibition

Plasma apoC-III levels correlate with triglyceride (TG) levels and are a strong predictor of CVD outcomes. ApoC-III elevates TG in part by inhibiting LPL. ApoC-III likely inhibits LPL by competing for lipid binding. To probe this, we used oil-drop tensiometry to characterize binding of six apoC-III variants to lipid/water interfaces. This technique monitors the dependence of lipid binding on surface pressure, which increases during TG hydrolysis by LPL. ApoC-III adsorption increased surface pressure by upward of 18 mN/m at phospholipid/TG/water interfaces. ApoC-III was retained to high pressures at these interfaces, desorbing at 21–25 mN/m. Point mutants, which substituted alanine for aromatic residues, impaired the lipid binding of apoC-III. Adsorption and retention pressures decreased by 1–6 mN/m in point mutants, with the magnitude determined by the location of alanine substitutions. Trp42 was most critical to mediating lipid binding. These results strongly correlate with our previous results, linking apoC-III point mutants to increased LPL binding and activity at lipid surfaces. We propose that aromatic residues in the C-terminal half of apoC-III mediate binding to TG-rich lipoproteins. Increased apoC-III expression in the hypertriglyceridemic state allows apoC-III to accumulate on lipoproteins and inhibit LPL by preventing binding and/or access to substrate.     

Phosphatidylserine binding sites in erythroid spectrin: location and implications for membrane stability

The erythrocyte membrane is a composite structure consisting of a lipid bilayer tethered to the spectrin-based membrane skeleton. Two complexes of spectrin with other proteins are known to participate in the attachment. Spectrin has also been shown to interact with phosphatidylserine (PS), a component of the lipid bilayer, which is confined to its inner leaflet. That there may be multiple sites of interaction with PS in the spectrin sequence has been inferred, but they have not hitherto been identified. Here we have explored the interaction of PS-containing liposomes with native alpha- and beta-spectrin chains and with recombinant spectrin fragments encompassing the entire sequences of both chains. We show that both alpha-spectrin and beta-spectrin bind PS and that sites of high affinity are located within 8 of the 38 triple-helical structural repeats which make up the bulk of both chains; these are alpha8, alpha9-10, beta2, beta3, beta4, beta12, beta13, and beta14, and PS affinity was also found in the nonhomologous N-terminal domain of the beta-chain. No other fragments of either chain showed appreciable binding. Binding of spectrin and its constituent chains to mixed liposomes of PS and phosphatidylcholine (PC) depended on the proportion of PS. Binding of spectrin dimers to PS liposomes was inhibited by single repeats containing PS binding sites. It is noteworthy that the PS binding sites in beta-spectrin are grouped in close proximity to the sites of attachment both of ankyrin and of 4.1R, the proteins engaged in attachment of spectrin to the membrane. We conjecture that direct interaction of spectrin with PS in the membrane may modulate its interactions with the proteins and that (considering also the known affinity of 4.1R for PS) the formation of PS-rich lipid domains, which have been observed in the red cell membrane, may be a result.     

The effect of hydrostatic pressure on S-layer-supported lipid membranes

We report on the behavior of unsupported and surface layer (S-layer)-supported lipid membranes at the application of a uniform hydrostatic pressure. At a hydrostatic pressure gradient higher than 6 N/m(2), unsupported lipid membranes, independent from which side pressurized and S-layer-supported lipid membranes pressurized from the lipid-faced side revealed a pronounced increase in capacitance. A maximal hydrostatic pressure gradient of 11.0 N/m(2) resulted in an almost doubling of the capacitance of the (composite) membranes. S-layer-supported lipid membranes showed a hysteresis in the capacitance versus pressure plot, indicating that this composite structure required a certain time to reorient when the pressure gradient acting from the lipid-faced side was balanced. By contrast, the S-layer-supported lipid membrane pressurized from the protein-faced side revealed only a minute increase in capacitance (C/C(0,max)=1.17+/-0.05), reflecting only minor pressure-induced area expansion. In addition, no hysteresis could be observed, indicating that no rearrangement of the composite membrane occurred. The maximal induced tension was with 4.3+/-0.2 mN/m, significantly higher than that of unsupported (2.5+/-0.3 mN/m) and S-layer-supported lipid membranes pressurized from the lipid-faced side (2.6+/-0.1 mN/m).     

Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane

After treatment of intact human erythrocytes with SH-oxidizing agents (e.g. tetrathionate and diamide) phospholipase A₂ cleaves approx. 30% of the phosphatidylserine and 50% of the phosphatidylethanolamine without causing hemolysis (Haest, C.W.M. and Deuticke, B. (1976) Biochim. Biophys. Acta 436, 353–365). These phospholipids are scarcely hydrolysed in fresh erythrocytes and are assumed to be located in the inner lipid layer of the membrane (Verkleij, A.J., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and van Deenen, L.L.M. (1973) Biochim. Biophys. Acta 323, 178–193). The enhancement of the phospholipid cleavage is now shown to be accompanied by a 50% decrease of the membrane SH-groups and a cross-linking of spectrin, located at the inner surface of the membrane, to oligomers of < 10⁶ dalton.Blocking approx. 10% of the membrane SH groups with N-ethylmaleimide suppresses both the polymerization of spectrin and the enhancement of the phospholipid cleavage. N-Ethylmaleimide, under these conditions, reacts with three SH groups per molecule of spectrin, 0.7 SH groups per major intrinsic 100 000 dalton protein (band 3) and 1.1 SH groups per molecule of an extrinsic protein of 72 000 daltons (band 4.2). Blocking studies with iodoacetamide demonstrate that the SH groups of the 100 000-dalton protein are not involved in the effects of the SH-oxidizing agents.It is suggested that a release of constraints imposed by spectrin enables phosphatidylserine and phosphatidylethanolamine to move from the inner to the outer lipid layer of the erythrocyte membrane and that spectrin, in the native erythrocyte, stabilizes the orientation of these phospholipids to the inner surface of the membrane.     

Formation of three-dimensional protein-lipid aggregates in monolayer films induced by surfactant protein B

This study focuses on the structural organization of surfactant protein B (SP-B) containing lipid monolayers. The artificial system is composed of the saturated phospholipids dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) in a molar ratio of 4:1 with 0.2 mol% SP-B. The different "squeeze-out" structures of SP-B were visualized by scanning probe microscopy and compared with structures formed by SP-C. Particularly, the morphology and material properties of mixed monolayers containing 0.2 mol% SP-B in a wide pressure range of 10 to 54 mN/m were investigated revealing that filamentous domain boundaries occur at intermediate surface pressure (15-30 mN/m), while disc-like protrusions prevail at elevated pressure (50-54 mN/m). In contrast, SP-C containing lipid monolayers exhibit large flat protrusions composed of stacked bilayers in the plateau region (app. 52 mN/m) of the pressure-area isotherm. By using different scanning probe techniques (lateral force microscopy, force modulation, phase imaging) it was shown that SP-B is dissolved in the liquid expanded rather than in the liquid condensed phase of the monolayer. Although artificial, the investigation of this system contributes to further understanding of the function of lung surfactant in the alveolus.     

Elasticity of the human red blood cell skeleton

We have measured by optical tweezers micromanipulations the area expansion and the shear moduli of spectrin skeletons freshly extracted from human red blood cells, in different controlled salinity conditions. At medium osmolarity (150 mOsm/kg), we measure KC=9.7+/-3.4 microN/m, muC=5.7+/-2.3 microN/m, KC/muC=2.1+/-0.7. When decreasing the osmolarity, both KC and muC decrease, while KC/muC is nearly constant and equal to about 2. This result is consistent with the predictions made when modeling the spectrin skeleton by a two-dimensional triangular lattice of springs. From the measured elastic moduli we estimate the persistence length of a spectrin filament: xi approximately 2.5 nm at 150 mOsm/kg.     

Pulmonary surfactant protein A interacts with gel-like regions in monolayers of pulmonary surfactant lipid extract

Epifluorescence microscopy was used to investigate the interaction of pulmonary surfactant protein A (SP-A) with spread monolayers of porcine surfactant lipid extract (PSLE) containing 1 mol % fluorescent probe (NBD-PC) spread on a saline subphase (145 mM NaCl, 5 mM Tris-HCl, pH 6.9) containing 0, 0.13, or 0.16 microg/ml SP-A and 0, 1.64, or 5 mM CaCl(2). In the absence of SP-A, no differences were noted in PSLE monolayers in the absence or presence of Ca(2+). Circular probe-excluded (dark) domains were observed against a fluorescent background at low surface pressures (pi approximately 5 mN/m) and the domains grew in size with increasing pi. Above 25 mN/m, the domain size decreased with increasing pi. The amount of observable dark phase was maximal at 18% of the total film area at pi approximately 25 mN/m, then decreased to approximately 3% at pi approximately 40 mN/m. The addition of 0.16 microg/ml SP-A with 0 or 1.64 mM Ca(2+) in the subphase caused an aggregation of dark domains into a loose network, and the total amount of dark phase was increased to approximately 25% between pi of 10-28 mN/m. Monolayer features in the presence of 5 mM Ca(2+) and SP-A were not substantially different from those spread in the absence of SP-A, likely due to a self-association and aggregation of SP-A in the presence of higher concentrations of Ca(2+). PSLE films were spread on a subphase containing 0.16 microg/ml SP-A with covalently bound Texas Red (TR-SP-A). In the absence of Ca(2+), TR-SP-A associated with the reorganized dark phase (as seen with the lipid probe). The presence of 5 mM Ca(2+) resulted in an appearance of TR-SP-A in the fluid phase and of aggregates at the fluid/gel phase boundaries of the monolayers. This study suggests that SP-A associates with PSLE monolayers, particularly with condensed or solid phase lipid, and results in some reorganization of rigid phase lipid in surfactant monolayers.     

Conditions of two-dimensional crystallization of cholera toxin B-subunit on lipid films containing ganglioside GM1

A systematic study of the lipid-layer two-dimensional crystallization technique has been carried out on the system composed of cholera toxin B-subunit and monosialoganglioside GM1, by electron microscopy, image analysis, and lipid film surface pressure measurements. Concentrations of protein and lipid components required for two-dimensional crystallization of toxin-GM1 complexes have been determined. Crystals were only obtained in the presence of mixed lipid films, composed of GM1 and of unsaturated lipids, such as dioleoylphosphatidylcholine or dioleoylphosphatidylethanolamine, in agreement with a previous report [D. S. Ludwig et al., (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8585–8588]. Crystals were obtained with cholera toxin B-subunit concentration as low as 5 μg/ml, as well as in the presence of protein contaminants. They were obtained over a wide range of concentrations of both GM1 and unsaturated lipids. The minimal lipid amount needed for crystallization corresponded to a lipid monolayer at, or near, the maximal spreading pressure (50 mN/m). The use of an excess of lipid resulted in a stabilization of lipid monolayers and in a higher reproducibility or crystallization experiments.     

The mycocidal, membrane-active complex of Cryptococcus humicola is a new type of cellobiose lipid with detergent features.

The chemical composition of the mycocidal complex (formerly known as microcin) secreted by Cryptococcus humicola was investigated by chemical, mass spectrometric and nuclear magnetic resonance methods. The results indicate that the mycocidal complex is composed of glycolipids with a highly acetylated (up to five acetyl groups) cellobiose backbone [beta-D-Glcp-(1'-->4)-beta-D-Glcp] linked to the omega-hydroxyl group of alpha,omega-dihydroxy palmitate [16:0-alpha,omega-di-OH] with an unsubstituted carboxyl group. The acyl chain forming aglycon can be replaced by [18:0-(alpha,omega-di-OH)], [18:0-(alpha,omega-1,omega-tri-OH)], and [18:0-(alpha,omega-2,omega-tri-OH)]. The complex has a comparatively high surface activity; 0.5 mg/ml of it reduced the surface tension of 0.1 M NaHCO(3) from 71 mN/m to 37 mN/m and interfacial tension against n-hexadecane from 39 mN/m to 10 mN/m. The critical micelle concentration of the complex at pH 4.0, determined by the fluorometric method with N-phenyl-1-naphthylamine as fluorescent probe and by the De Nouy ring method, was 2 x 10(-5) M (taking the average molecular mass of the complex to be 750); it did not depend on the presence of 100 mM KCl and was an order of magnitude higher at pH 7.0. By fluorescence resonance energy transfer spectroscopy with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine as energy donor and N-(rhodamine B sulfonyl)-phosphatidylethanolamine as energy acceptor the complex was shown to intercalate into the liposomal lipid matrix. Primary lesions caused by the complex in planar lipid bilayers were revealed as short-living current fluctuations of a broad spectrum of amplitudes. The mycocidal effect of the complex is suggested to be associated with its detergent-like properties.     

Specific anchoring modes of two distinct dystrophin rod sub-domains interacting in phospholipid Langmuir films studied by atomic force microscopy and PM-IRRAS

Dystrophin rod repeats 1-3 sub-domain binds to acidic phosphatidylserine in a small vesicle binding assay, while the repeats 20-24 sub-domain does not. In the present work, we studied the adsorption behaviour of both sub-domains at the air/liquid interface and at the air/lipid interface in a Langmuir trough in order to highlight differences in interfacial properties. The adsorption behaviour of the two proteins at the air/liquid interface shows that they display surface activity while maintaining their alpha-helical secondary structure as shown by PM-IRRAS. Strikingly, R20-24 needs to be highly hydrated even at the interface, while this is not the case for R1-3, indicating that the surface activity is dramatically higher for R1-3 than R20-24. Surface-pressure measurements, atomic force microscopy and PM-IRRAS are used in a Langmuir experiment with DOPC-DOPS monolayers at two different surface pressures, 20 mN/m and 30 mN/m. At the lower surface pressure, the proteins are adsorbed at the lipid film interface while maintaining its alpha-helical structure. After an increase of the surface pressure, R1-3 subsequently produces a stable film, while R20-24 induces a reorganization of the lipid film with a subsequent decrease of the surface pressure close to the initial value. AFM and PM-IRRAS show that R1-3 is present in high amounts at the interface, being arranged in clusters representing 3.3% of the surface at low pressure. By contrast, R20-24 is present at the interface in small amounts bound only by a few electrostatic residues to the lipid film while the major part of the molecule remains floating in the sub-phase. Then for R1-3, the electrostatic interaction between the proteins and the film is enhanced by hydrophobic interactions. At higher surface pressure, the number of protein clusters increases and becomes closer in both cases implying the electrostatic character of the binding. These results indicate that even if the repeats exhibit large structural similarities, their interfacial properties are highly contrasted by their differential anchor mode in the membrane. Our work provides strong support for distinct physiological roles for the spectrin-like repeats and may partly explain the effects of therapeutic replacement of dystrophin deficiency by minidystrophins.     

ATP-dependent Mechanism Protects Spectrin against Glycation in Human Erythrocytes

Human erythrocytes are continuously exposed to glucose, which reacts with the amino terminus of the β-chain of hemoglobin (Hb) to form glycated Hb, HbA1c, levels of which increase with the age of the circulating cell. In contrast to extensive insights into glycation of hemoglobin, little is known about glycation of erythrocyte membrane proteins. In the present study, we explored the conditions under which glucose and ribose can glycate spectrin, both on the intact membrane and in solution and the functional consequences of spectrin glycation. Although purified spectrin could be readily glycated, membrane-associated spectrin could be glycated only after ATP depletion and consequent translocation of phosphatidylserine (PS) from the inner to the outer lipid monolayer. Glycation of membrane-associated spectrin led to a marked decrease in membrane deformability. We further observed that only PS-binding spectrin repeats are glycated. We infer that the absence of glycation in situ is the consequence of the interaction of the target lysine and arginine residues with PS and thus is inaccessible for glycation. The reduced membrane deformability after glycation in the absence of ATP is likely the result of the inability of the glycated spectrin repeats to undergo the obligatory unfolding as a consequence of interhelix cross-links. We thus postulate that through the use of an ATP-driven phospholipid translocase (flippase), erythrocytes have evolved a protective mechanism against spectrin glycation and thus maintain their optimal membrane function during their long circulatory life span.