Phosphatidylserine Large Unilamellar Vesicles Research Articles

Annexin A4 binding to anionic phospholipid vesicles modulated by pH and calcium

Annexin A4 belongs to a class of Ca(2+)-binding proteins for which different functions in the cell have proposed, e.g. involvement in exocytosis and in the coagulation process. All these functions are related to the ability of the annexins to bind to acidic phospholipids. In this study the interaction of annexin A4 with large unilamellar vesicles (LUV) prepared from phosphatidylserine (PS) or from phosphatidic acid (PA) is investigated at neutral and acidic pH. Annexin A4 strongly binds to either lipid at acidic pH, whereas at neutral pH only weak binding to PA and no binding to PS occurs. Addition of 40 microM Ca(2+) leads to a strong binding to the lipids also at neutral pH. This is caused by the different electric charge of the protein below and above its isoelectric point. Binding of annexin A4 induces dehydration of the vesicle surface. The strength of the effects is much greater at pH 4 than at pH 7.4. At pH 7.4 annexin A4 reduces the Ca(2+)-threshold concentration necessary to induce fusion of PA LUV. The Ca(2+) induced fusion of PS LUV is not affected by annexin A4 at pH 7.4. At pH 4 annexin A4 induces fusion of either vesicles without Ca(2+). Despite the low binding extents at neutral pH annexin A4 induces a Ca(2+) independent leakage of PS- or PA-LUV. The leakage extent is increased at acidic pH. From the data two suggestions are made: (1) At pH 4 annexin A4 (at least partially) penetrates into the bilayer in contrast to the preferred location at the vesicle surface at neutral pH. The conformation of annexin A4 seems to be different at the two conditions. (2) At neutral pH, Annexin A4 seems to be able to bind two PA vesicles simultaneously; however, only one PS vesicle at the same time. This behavior might be related to a recently described double Ca(2+) binding site, which appears to be uniquely suited for PS.

Annexin V interaction with phosphatidylserine-containing vesicles at low and neutral pH.

Annexin V belongs to a class of Ca2+-binding proteins for which different functions in the cell are discussed, e.g., involvement in exocytosis, inhibition of protein kinase C, and calcium channel activity in cartilage matrix vesicles. All these functions are related to the ability of the annexins to bind to acidic phospholipids. In this study, the interaction of annexin V with large unilamellar vesicles (LUV) prepared from phosphatidylserine (PS) at low pH was compared to that at neutral pH. Annexin V strongly binds to PS LUV at low pH, whereas at neutral pH 20-100 microM Ca2+ are required to induce binding. This is caused by the different electric charge of the protein. The binding ability of the PS LUV for annexin V is higher at low pH. Binding of annexin V induces dehydration of the vesicle surface and a decrease of the lateral diffusion within the bilayer. While this dehydration is due to vesicle contact at pH 4, at pH 7.4 it is due to surface covering by annexin V. Annexin V promotes the phospholipid intermixing between LUVs at pH 5 and below but inhibits it at pH 7.4. A substitution of up to 80% of the PS by the uncharged phosphatidylcholine does not impair the extent of phospholipid intermixing at pH 4. The high binding capacity of PS LUV, the disappearance of the inhibiting action, and a calculated increase of the annexin V hydrophobicity make it likely that annexin V is able to penetrate into the membrane at low pH. At neutral pH, annexin V molecules act as steric barriers, preventing close apposition of two vesicles. At pH 5, annexin V lowers the threshold concentration of the Ca2+-induced phospholipid intermixing. Such a promotion is well-known for annexin VII (synexin). The effect may be related to the isoelectric points of the two annexins which have been reported as 4.8 (annexin V) and 7.0 (annexin VII), respectively.

Poly(ethylene glycol)--lipid conjugates regulate the calcium-induced fusion of liposomes composed of phosphatidylethanolamine and phosphatidylserine.

The effect of poly(ethylene glycol)--lipid (PEG--lipid) conjugates on liposomal fusion was investigated. Incorporation of PEG--lipids into large unilamellar vesicles (LUVs) composed of equimolar phosphatidylethanolamine (PE) and phosphatidylserine (PS) inhibited calcium-induced fusion. The degree of inhibition increased with increasing molar ratio of the PEG conjugate and with increasing size of the PEG moiety. Inhibition appeared to result from the steric barrier on the surface of the liposomes which opposed apposition of bilayers and interbilayer contact. In the presence of a large excess of neutral acceptor liposomes, however, fusogenic activity was restored. The rate of fusion under these conditions depended on the initial molar ratio of the PEG conjugate in the PE:PS vesicles and the length and degree of saturation of the acyl chains which composed the lipid anchor. These results are consistent with spontaneous transfer of the PEG--lipid from PE:PS LUVs to the neutral lipid sink reducing the steric barrier and allowing fusion of the PE:PS LUVs. The primary determinant of the rate of fusion was the rate of transfer of the PEG--lipid, indicating that liposomal fusion could be programmed by incorporation of appropriate PEG--lipid conjugates. Interestingly, increasing the size of the PEG group did not appear to affect the rate of fusion. The implications of these results with respect to the design of fusogenic liposomal drug delivery systems are discussed.

Diacylglycerol and hexadecane increase divalent cation-induced lipid mixing rates between phosphatidylserine large unilamellar vesicles

Bovine brain phosphatidylserine (BBPS) vesicles were prepared with traces of dioleoylglycerol (18:1, 18:1 DAG) or hexadecane (HD) to determine the influence of changes in headgroup or acyl chain packing on divalent cation-induced lipid mixing rates. A stopped-flow apparatus was used to combine vesicles with 3 mM Ca2+ or Ba2+. Aggregation was monitored by light scattering and lipid mixing by lipid probe dilution. Neither 3-6 mol% 18:1, 18:1 DAG nor up to 10 mol % HD significantly altered the BBPS chain melting temperature, vesicle diameter, or vesicle aggregation rates. Lipid mixing rates doubled by adding either 3 mol % 18:1, 18:1 DAG or 6 mol % HD to BBPS with no change in the Ca2+ concentration threshold. The Arrhenius slopes of the lipid mixing rates for control, 3 mol % 18:1, 18:1 DAG, and 6 mol % HD vesicles were identical. 2H-nuclear magnetic resonance spectra of perdeuterated dipalmitoylglycerol and HD in BBPS in the absence and presence of Ca2+ and Ba2+ showed that the solutes occupied different time-averaged positions in the bilayer under each condition. These data suggest that: 1) the enhanced lipid mixing rate is related to the volume of the added alkyl chains; 2) 18:1, 18:1 DAG and HD may alter the activation entropy or the attempt frequency at one or more steps in the lipid mixing process; 3) 18:1, 18:1 DAG and HD are likely to act at a different spatial or temporal point than the divalent cation; and 4) it is unlikely that the effect of these solutes on lipid mixing is due to their equilibrium time-averaged positions in the bilayer. Others have shown that apolar lipids accelerate fusion in nonbilayer phase-forming systems, but BBPS does not form these phases under these conditions. Therefore, we propose that the effect of very small amounts of apolar substances may be very general, e.g., stabilizing the hydrophobic interstices associated with a variety of proposed intermediate structures.

Function of saposin C in the reconstitution of glucosylceramidase by phosphatidylserine liposomes

The function of saposin C (Sap C), a glucosylceramidase activator protein, in the enzyme stimulation by phosphatidylserine (PS) liposomes has been investigated. Using gel filtration experiments evidence was obtained for Sap C binding to PS large unilamellar vesicles (LUV) but not to glucosylceramidase. PS LUV, which by themselves are unable to tightly bind and stimulate the enzyme, acquire the capacity to also bind the enzyme after interaction with Sap C, making it express its full activity. Our results indicate that the primary step in the Sap C mode of action resides in its association with PS membranes; in turn, this association promotes the interaction between the membranes and glucosylceramidase.

Studies on glucosylceramidase binding to phosphatidylserine liposomes: the role of bilayer curvature

The influence of phosphatidylserine (PS) liposome size on their capacity to activate and bind purified glucosylceramidase was investigated. Gel filtration and flotation experiments showed that large unilamellar vesicles (LUV) of either pure PS or PS in admixture with phosphatidylcholine (PC) are unable to tightly bind purified glucosylceramidase, and thus, to fully stimulate its activity. By contrast, small unilamellar vesicles (SUV) of PS adsorb glucosylceramidase on their surface, the smaller vesicles within the SUV preparation being the most effective. Reconstitution of glucosylceramidase can either be favoured or inhibited by factors affecting the bilayer curvature of PS liposomes. An increase of PS vesicle size induced by a fusogenic agent such as poly(ethylene glycol) (PEG), decreased enzyme binding and activity. On the contrary, the reduction of PS LUV size by sonication increased their stimulating ability. Enzyme association with PS SUV is reversible. In fact, glucosylceramidase bound to PS SUV was released from the lipid surface when the SUV were transformed into larger vesicles by PEG; dissociation from the vesicles resulted in a dramatic decrease of enzyme activity. Although PS LUV are unable to reconstitute glucosylceramidase, their association with oleic acid (OA) promotes the interaction with glucosylceramidase. This phenomenon is best explained in terms of OA-induced surface defects of PS LUV, with consequent exposure of the more hydrophobic part of the membrane and hence the improved binding of hydrophobic region/s of glucosylceramidase. Our data indicate that the physical organization of the PS-containing liposomes is of critical importance for glucosylceramidase reconstitution. The observation that physical changes of the lipid surface can markedly affect the enzyme activity offers a new approach to the study of glucosylceramidase regulation.

Interaction of erythrocyte protein 4.1 with phospholipids. A monolayer and liposome study

We have studied the interaction of purified human erythrocyte protein 4.1 with phospholipid membranes by monitoring both the increase in surface pressure of monolayers at the air/water interface and the change in permeability in liposomes to fluorescent molecules, in the presence of protein 4.1. Protein 4.1 penetrated into monolayers of brain phosphatidylserine (PS) and egg phosphatidylcholine (PC), even above surface pressures of 30 mN/m. Protein 4.1 increased the permeability of negatively charged PS, but not PC, liposomes, measured as the increase in fluorescence when encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and p- xylenebispyridinium bromide (DPX) or carboxyfluorescein were released into the medium. The interaction of protein 4.1 with PS large unilamellar vesicles (LUV) was increased as the pH and the ionic strength were lowered, and decreased as the Ca 2+ or Mg 2+ concentrations and ionic strength were raised. In order to study the relevance of these measurements to the erythrocyte, we prepared LUV of synthetic lipid mixtures characteristic of both the inner and the outer membrane leaflets. Protein 4.1 increased the permeability of inner, but not outer, leaflet LUV at both pH 6.0 and 7.4. These observations suggest that negatively charged phospholipid domains around the protein 4.1 high-affinity protein-binding site(s) may contribute to the anchoring of protein 4.1 to the cytoplasmic surface of the red cell membrane.

Lipid mixing during membrane aggregation and fusion: why fusion assays disagree.

The kinetics of lipid mixing during membrane aggregation and fusion was monitored by two assays employing resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) and N-(lissamine Rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE). For the "probe mixing" assay, NBD-PE and Rh-PE were incorporated into separate populations of phospholipid vesicles. For the "probe dilution" assay, both probes were incorporated into one population of vesicles, and the assay monitored the dilution of the molecules into the membrane of unlabeled vesicles. The former assay was found to be very sensitive to aggregation, even when the internal aqueous contents of the vesicles did not intermix. Examples of this case were large unilamellar vesicles (LUV) composed of phosphatidylserine (PS) in the presence of Mg2+ and small unilamellar vesicles (SUV) composed of phosphatidylserine in the presence of high concentrations of Na+. No lipid mixing was detected in these cases by the probe dilution assay. Under conditions where membrane fusion (defined as the intermixing of aqueous contents with concomitant membrane mixing) was observed, such as LUV (PS) in the presence of Ca2+, the rate of probe mixing was faster than that of probe dilution, which in turn was faster than the rate of contents mixing. Two assays monitoring the intermixing of aqueous contents were also compared. The Tb/dipicolinic acid assay reported slower fusion rates than the 1-aminonaphthalene-3,6,8-trisulfonic acid/N,N'-p-xylylene-bis(pyridinium bromide) assay for PS LUV undergoing fusion in the presence of Ca2+. These observations point to the importance of utilizing contents mixing assays in conjunction with lipid mixing assays to obtain the rates of membrane destabilization and fusion.(ABSTRACT TRUNCATED AT 250 WORDS)