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Sphingolipids (Galactosylceramide and Sulfatide) in Lamellar-Hexagonal Phospholipid Phase Transitions and in Membrane Fusion† Asier Sa´ez-Cirio´n,‡ Gorka Basa´n˜ez,‡ Gerardo Fidelio,§ Fe´lix M. Gon˜i,‡ Bruno Maggio,§ and Alicia Alonso*,‡ Unidad de Biofı´sica (CSIC-UPV/EHU) and Departamento de Bioquı´mica, Universidad del Paı´s Vasco, Aptdo. 644, 48080 Bilbao, Spain, and Departamento de Quı´mica Biolo´ gica-CIQUIBIC, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´ rdoba, 5000 Co´ rdoba, Argentina Received April 10, 2000. In Final Form: June 26, 2000 The effects of galactosylceramide (cerebroside) and sulfogalactosylceramide (sulfatide) from bovine brain on the lamellar-to-inverted hexagonal phase transition of dielaidoylphosphatidylethanolamine are examined using differential scanning calorimetry. When mixed with dielaidoylphosphatidylethanolamine, cerebroside increases the transition temperature (ca. 0.2 °C/mol % added cerebroside) and increases the transition ∆H. Sulfatide increases the transition temperature by ca. 0.4 °C/mol % added sulfatide and decreases ∆H. Both lipids are seen to hinder the formation of the nonlamellar phase, although sulfatide is more effective in this respect. When incorporated into vesicles formed by phosphatidylcholine/phosphatidylethanolamine/ cholesterol (2:1:1 mole ratio), which are a good substrate for phospholipase C and undergo fusion as a consequence of the enzyme activity (Nieva et al. Biochemistry 1989, 28, 7364), cerebroside at all concentrations and sulfatide at >5 mol % inhibit enzyme activity and vesicle fusion. Cerebroside inhibition of fusion is due not only to a reduced enzyme activity but also to the impaired formation of nonlamellar phases. Sulfatide at low concentrations (e.g., 1 mol %) enhances phospholipase C activity and vesicle fusion, probably because its net negative charge causes hyperpolarization of the interface, which is known to activate phospholipase C. Under these conditions, its enzyme-activating effect predominates over its bilayer-stabilizing properties. Thus, sulfatide at low concentrations is an exception to the rule that amphiphiles hindering the lamellar-hexagonal transition inhibit both phospholipase C activity and membrane fusion.
Introduction The equilibrium organization of lipids in cell membranes resembles the structure of the lamellar phase in aqueous dispersions of phospholipids, e.g., phosphatidylcholines. However, it is increasingly recognized that, under nonequilibrium conditions, formation of transient nonlamellar structures would explain a number of cell membrane phenomena, including membrane fusion, budding, and some instances of macromolecular transport. These nonlamellar structures would have geometries comparable to those of the inverted hexagonal and inverted cubic phases that have been observed in pure lipid-water dispersions.1,2 Therefore, research is being devoted to a variety of systems based on natural lipids that may undergo lamellar to nonlamellar (inverted hexagonal or cubic) phase transitions.3-6 Studies from this laboratory have addressed the implication of nonbilayer intermediates in the phenomenon * Corresponding author. Fax: +34-94-464-8500. E-mail:
[email protected]. † Part of the Special Issue “Colloid Science Matured, Four Colloid Scientists Turn 60 at the Millennium”. ‡ Universidad del Paı´s Vasco. § Universidad Nacional de Co ´ rdoba. (1) Nieva, J. L.; Alonso, A.; Basa´n˜ez, G.; Gon˜i, F. M.; Gulik, A.; Vargas, R.; and Luzzati, V. FEBS Lett. 1995, 368, 143. (2) Siegel, D. P. Biophys. J. 1999, 76, 291. (3) Siegel, D. P.; Epand, R. M.Biophys. J. 1997, 73, 3089. (4) Jime´nez-Monreal, A. M.; Aranda, F. J.; Micol, V.; Sa´nchez-Pin˜era, P.; de Godos, A.; Go´mez- Ferna´ndez, J. C. Biochemistry 1999, 38, 7747. (5) Siegel, D. P.; Banschbach, J.; Alford, D.; Ellens, H.; Lis, L. J.; Quinn, P. J.; Yeagle, P. L.; Bentz, J. Biochemistry 1989, 28, 3703. (6) Veiga, M. P.; Arrondo, J. L.; Gon˜i, F. M.; Alonso, A. Biophys. J., 1999, 76, 342.
of vesicle membrane fusion induced by phospholipase C.1,7,8 This enzyme catalyzes the hydrolysis of phospholipids in a bilayer, giving rise to diacylglycerol and to a watersoluble phosphoryl compound (e.g., phosphorylcholine). In turn, diacylglycerol facilitates vesicle aggregation and formation of nonlamellar structures that are instrumental in generating fusion pores.1,7 The transient intermediate, or “stalk” as it has been called,9 is a semitoroidal structure with a “negative” curvature, i.e., curved in the sense opposite to that of the outer monolayer of a cell membrane.10 Thus lipids that facilitate negative curvature in a hemibilayer, e.g., phosphatidylethanolamine or free polyunsaturated fatty acids, are known to facilitate phospholipase C-induced fusion, and the opposite is true of those that have a propensity to form positively curved hemibilayers, e.g., lysophosphatidylcholine.7,11,12 The complex glycosphingolipids known as gangliosides13 modify in a very interesting way membrane fusion induced by phospholipase C. On the basis of two independent effects, namely, inhibition of phospholipase C activity14,15 (7) Gon˜i, F. M.; Basa´n˜ez, G.; Ruiz-Argu¨ello, M. B.; Alonso, A. Faraday Discuss. Chem. Soc. 1998, 111, 55. (8) Basa´n˜ez, G.; Fidelio, G. D.; Gon˜i, F. M.; Maggio, B.; Alonso, A. Biochemistry 1996, 35, 7506. (9) Chernomordik, L.; Kozlov, M. M.; Zimmerberg, J. J. Membr. Biol. 1995, 146, 1. (10) Helfrich, W. Z. Naturforsch. 1973, C28, 693. (11) Basa´n˜ez, G.; Gon˜i, F. M.; Alonso, A. Biochemistry 1998, 37, 3901. (12) Chernomordik, L. V.; Frolov, V. A.; Leikina, E.; Bronk, P.; Zimmerberg, J. J. Cell Biol., 1998. 140, 1369. (13) Maggio, B. Prog. Biophys. Mol. Biol. 1994, 62, 55. (14) Perillo, M. A.; Guidotti, A.; Costa, E.; Yu, R. K.; Maggio, B. Mol. Membr. Biol. 1994, 11, 119. (15) Daniele, J. J.; Maggio, B.; Bianco, I. D. Gon˜i, F. M.; Alonso, A.; Fidelio, G. D. Eur. J. Biochem. 1996, 239, 105.
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and stabilization of lamellar versus nonlamellar phases8 (the latter due to the tendency to form positively curved surfaces), gangliosides can largely inhibit the enzymeinduced fusion at concentrations as low as 1 mol % in a bilayer.8 In the present paper, we explore the effects on lipid phase behavior and on vesicle fusion of two sphingolipids whose polar headgroups are far less complex than those of gangliosides, namely, galactosylceramide (cerebroside) and its sulfate ester (sulfatide). Both are naturally found in cell membranes, particularly in the myelin membrane, where they are believed to contribute to structural stability. Like gangliosides, albeit at higher concentrations, these lipids have the tendency to oppose inverted hexagonal phase formation, and they both, under certain conditions, inhibit membrane fusion. However, the presence of a net negative charge in sulfatide provides the molecule with some properties that make it clearly different from cerebroside, so that it acts, e.g., as an activator of phospholipase C at low concentrations. Materials and Methods Phospholipase C (EC 3.1.4.1) from Bacillus cereus was supplied by Boehringer-Mannheim. Glycosphingolipids were purified from bovine brain as described previously.16 Egg phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were obtained from Lipid Products (South Nutfield, England), cholesterol (Ch) was purchased from Sigma (St. Louis, MO), dielaidoylphosphatidylethanolamine (DEPE) was supplied by Avanti Polar Lipids (Birmingham, AL), and 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) and p-xylylenebis(pyridinium bromide) (DPX) were purchased from Molecular Probes (Eugene, OR). Large unilamellar vesicles (LUVs) were prepared at room temperature by extrusion7,8 using Nuclepore filters of 0.1 µm pore diameter. The lipid composition of these liposomes was PC/ PE/Ch (2:1:1 mole ratio). When required, the appropriate amounts of glycosphingolipids or phosphatidylglycerol (PG) were added. These amounts are indicated as additional mole percentages. LUVs were prepared in a mixture of 10 mM Hepes, 200 mM NaCl, and 10 mM CaCl2, pH 7.0. All experiments were performed at 37 °C. The total lipid concentration was 0.3 mM and the enzyme was used at 1.6 units/mL, corresponding approximately to the optimum ratio of 10 enzyme molecules/vesicle.7 Phospholipase C activity was assayed by determining the amounts of phosphorus in samples of the aqueous phase of an extraction mixture (chloroform/methanol, 2:1) after addition of aliquots from the reaction mixtures at different times. Phosphorus was assayed according to Bartlett.17 Because of the 1:1 phosphorus:diacylglycerol stoichiometry in the substrates of phospholipase C, the enzyme activity could be equally expressed as hydrolyzed phospholipid, released phosphate, or released diacylglycerol. Liposome aggregation was estimated as an increase in light scattering, measured in an Perkin-Elmer LS50B spectrofluorometer with both monochromators set at 520 nm. Fusion was detected as mixing of aqueous contents using the ANTS/ DPX fluorescent probe system described by Ellens et al.18 The reader is directed to our previous publications8,11,19 for additional details of the spectroscopic methods. DEPE dispersions for differential scanning calorimetry were prepared by rehydrating lipid films from chloroform under high vacuum. The final DEPE concentration was 7 mM. Again, the appropriate amounts of glycosphingolipids or PG were added and are indicated as additional mole percentages. The buffer consisted of 20 mM Pipes, 150 mM NaCl, and 1 mM EDTA, pH 7.4. The samples were dispersed at 45 °C with shaking and left to equilibrate for 1 h at this temperature. Both lipid suspensions and buffer were degassed before being loaded into the sample or reference cell of an MC-2 high-sensitivity scanning calorimeter (16) Maggio, B.; Cumar, F. A.; Caputto, R. Biochem. J. 1978. 171, 559. (17) Bartlett, G. D. R. J. Biol. Chem. 1959, 234, 466. (18) Ellens, H.; Bentz, J.; Szoka, F. C. Biochemistry 1986, 25, 285. (19) Ruiz-Argu¨ello, M. B.; Gon˜i, F. M.; Alonso, A. Biochemistry 1998, 37, 11621.
Figure 1. Influence of galactosylceramide (cerebroside) on the lamellar-to-inverted hexagonal phase transition of dielaidoylphosphatidylethanolamine (DEPE). (A) Differential scanning calorimetry thermograms of the L-H transitions of pure DEPE and of DEPE/cerebroside mixtures. The cerebroside concentration is given for each thermogram in mole percent. (B) Midpoint L-H transition temperatures Tm (b) and transition enthalpies ∆H (O) corresponding to the thermograms in panel A. (Microcal, Northampton, MA). Three heating scans were recorded for each sample. After the first scan, succesive heating scans of the same sample gave always superimposable thermograms.
Results Lamellar-Hexagonal Phase Transitions. Dielaidoylphosphatidylethanolamine exhibits a cooperative lamellar-to-inverted hexagonal (L-H) thermotropic transition at ca. 66 °C and neutral pH. This transition can be conveniently monitored by differential scanning calorimetry.6 Figure 1A shows representative thermograms of the L-H transitions of pure DEPE and mixtures containing DEPE + cerebroside. The latter causes dose-dependent shifts of the transitions to higher temperatures, indicating that its presence in this concentration range tends to stabilize the lamellar phase of DEPE. The transition temperatures (Tm) and enthalpies (∆H) corresponding to the above thermograms are plotted in Figure 1B. The L-H transition enthalpy of DEPE increases with 10 mol % cerebroside, to remain unchanged at higher sphingolipid concentrations. A similar series of studies with the sulfated derivative of galactosylceramide, or sulfatide (Figure 2), show a partially different effect of this negatively charged lipid on the L-H transition of DEPE. In common with cerebroside, sulfatide shifts the transition to higher temperatures, thus stabilizing the lamellar phase, but it is more effective than cerebroside in doing so, ∆Tm being ca. 0.45 °C/mol % sulfatide vs 0.21 °C/mol % cerebroside. An important difference is that sulfatide at 10 mol % considerably decreases the transition enthalpy. The combined effect of increasing Tm and decreasing ∆H
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Figure 3. Influences of cerebroside (O) and sulfatide (0) on phospholipase C activity. The substrates consisted of large unilamellar vesicles of PC/PE/Ch (2:1:1) containing various percentages of sphingolipid. The total lipid concentration was constant at 0.3 mM; the enzyme concentration was 0.16 U/mL. Enzyme activity was measured as an increase in liposomal suspension turbidity:20 the values on the y axis correspond to maximum slopes of “turbidity vs time” plots as shown in Figures 4B, 5B, and 6B. y-Axis units are % change in turbidity/s.
Figure 2. Influence of sulfogalactosylceramide (sulfatide) on the lamellar-to-inverted hexagonal phase transition of DEPE. (A) Differential scanning calorimetry thermograms of the L-H transitions of pure DEPE and of DEPE/sulfatide mixtures. The sulfatide concentration is given for each thermogram in mole percent. A thermogram for a DEPE sample containing 10 mol % phosphatidylglycerol is also included. (B) Midpoint L-H transition temperatures Tm (b) and transition enthalpies ∆H (O) corresponding to the sulfatide thermograms in panel A.
resembles the effects of the more complex negatively charged sphingolipids, the gangliosides, although the latter completely abolish the transition enthalpy at mole ratios below 5%.8 A further characteristic of sulfatide is that, at 1 mol %, it has a clear effect in decreasing ∆H, which appears to remain constant up to 5 mol % and to decrease again at higher sulfatide concentrations (Figure 2B). This nonlinear character of sulfatide effects is further examined below. Figure 2 also includes data for a DEPE/phosphatidylglycerol mixture at 10 mol % PG. This negatively charged glycerophospholipid was included in our study for comparison purposes. The effects of 10 mol % PG and 10 mol % sulfatide on the L-H transition of DEPE are rather similar, perhaps underlining the importance of the negative charge and/or hydration sphere of the lipid headgroup. Phospholipase C Activity. Phospholipase C catalyzes the hydrolysis of phospholipids into diacylglycerol and a water-soluble phosphoryl derivative (e.g., phosphorylcholine or phosphorylethanolamine). The PC/PE/Ch mixture (2:1:1 mole ratio) is a particularly good substrate for phospholipase C.7 Phospholipase C action on liposomes is closely paralleled by vesicle aggregation; thus, enzyme activity can be conveniently monitored through changes in liposome suspension turbidity.19,20 Enzyme activities on the above lipid mixture in the form of large unilamellar liposomes, containing in addition various proportions of cerebroside or sulfatide, are shown in Figure 3. Cerebroside (20) Basa´n˜ez, G.; Nieva, J. L.; Gon˜i, F. M.; Alonso, A. Biochemistry 1996, 35, 15183.
causes a small, gradual decrease in enzyme activity, while sulfatide, in agreement with the calorimetric observations, has a nonlinear effect. It enhances phospholipase activity at concentrations up to 1 mol %, but its effect is reversed at higher concentrations, becoming clearly inhibitory by 10 mol %. The importance of the net negative charge of sulfatide in phospholipase C inhibition was investigated by performing a similar experiment, in which 10 mol % of the negatively charged phosphatidylglycerol was used instead of the sulfatide. As shown in Figure 4, both negatively charged lipids inhibit enzyme activity and vesicle aggregation in almost exactly the same way. These results, together with those in Figure 2, point to a correlation among negative charge, stabilization of the lamellar versus inverted hexagonal phase, and inhibition of phospholipase C. The inhibitory effect of 10 mol % sulfatide on phospholipase C activity contrasts with our previous report15 stating that sulfatide is ineffective in this respect. However, the substrate in our previous study was pure phosphatidylcholine, and phospholipase C activity is 1 order of magnitude lower with pure PC than with the mixture used in the present paper.7,20 It is thus understandable that any inhibitory effect of sulfatide would be more easily detected when absolute activity values are larger. Vesicle-Vesicle Fusion. Phospholipase C activity on large unilamellar vesicles composed of PC/PE/Ch (2:2:1 mole ratio) leads not only to vesicle aggregation but also to intervesicular mixing of lipids and of aqueous contents, i.e., to liposome fusion.17 Thus the effects of cerebroside and of sulfatide on vesicle fusion (represented here as mixing of vesicular aqueous contents) were examined next. Figure 5 summarizes the effects of cerebroside on phospholipase C-dependent diacylglycerol production (Figure 5A), vesicle aggregation (Figure 5B), and vesicle fusion (Figure 5C) as functions of time. A comparison of the cerebroside effects on enzyme activity and on liposome fusion (Figure 5A,C) reveals an interesting difference. The main cerebroside effect on phospholipase activity is a decrease in rate (slope), while the effect on fusion includes an increase in lag time. This is particularly clear when the plots corresponding to 10% and 20% cerebroside are compared (Figure 5C). The
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Figure 4. Comparative study of the effects of sulfatide (SUL) and phosphatidylglycerol (PG) on phospholipase C activity. The substrate consisted of LUVs containing PC/PE/Ch (2:1:1) ( 10% SUL or PG. Lipid and enzyme concentrations were as for Figure 3. Upper panel: enzyme activity measured as production of diacylglycerol. Lower panel: enzyme activity measured as the increase in vesicle suspension turbidity, i.e., vesicle aggregation.
increased fusion lag times are probably due to the fact that fusion depends on the formation of a nonlamellar intermediate to connect the two juxtaposed vesicles. In turn, formation of the nonlamellar “stalk” is highly dependent on lipid composition and requires in our system a certain proportion of diacylglycerol.1 generated by the enzyme. Cerebroside is perhaps exerting here the same dual effect that has been reported elsewhere for gangliosides,8 namely, inhibition of phospholipase activity and hindrance of formation of nonlamellar structures (Figure 1). On one hand, higher proportions of diacylglycerol are required order to overcome the latter effect. On the other, diacylglycerol is being produced at a lower rate by the cerebroside-inhibited enzyme. The combined result is that a longer time is required for the fusion pore(s) to open (Figure 5C). Once this occurs, exchange of vesicular aqueous contents proceeds without inhibition, virtually as in the absence of cerebroside. The effect of sulfatide on membrane fusion is shown in Figure 6. In this case, the slopes of enzyme activity, vesicle aggregation, and fusion change almost in parallel, with fusion lagging slightly behind aggregation. The presence of 1 mol % sulfatide, which was seen to enhance enzyme activity (Figure 3), also increases the rate of vesicle fusion. Thus, unlike cerebrosides, sulfatides appear to influence fusion mainly through modulation of phospholipase C activity, their bilayer-stabilizing properties being in this case less important for the final outcome of the process. Discussion Sphingolipids and Lipid Phases. Natural cerebroside and sulfatide contain mainly long, saturated fatty acids, and they are believed to contribute to the stability of the membranes in which they reside.12 Probably because
Figure 5. Effect of cerebroside on phospholipase C-induced fusion of large unilamellar vesicles. Vesicles contained PC/PE/ Ch (2:1:1) ( varying proportions of cerebroside, as indicated for each curve. Lipid and enzyme concentrations were as for Figure 3. (A) Phospholipid hydrolysis. (B) Vesicle suspension turbidity (liposome aggregation). (C) Intervesicular mixing of aqueous contents (liposome fusion).
of their peculiar fatty acid composition, aqueous dispersions of these lipids are found to form thermally metastable phases, as well as highly stable crystalline ones.13,21-24 Sulfatides give rise to interdigitated lipid bilayers.25 In mixtures with dipalmitoylphosphatidylcholine (DPPC), a lipid that exhibits in the pure state a narrow gel-fluid phase transition at 41 °C, cerebroside does not have a major effect on the transition, as detected by differential scanning calorimetry at concentrations below 20 mol %, but a shift toward higher temperatures is seen with increasing sphingolipid concentrations.26 When mixed with the same phospholipid, sulfatide has a similar effect.26-28 In addition, sulfatide, but not cerebroside, (21) Curatolo, W.; Jungalwala, F. B. Biochemistry 1985, 24, 6608. (22) Boggs, J. M.; Koshy, K. M.; and Rangaraj, G. Biochim. Biophys. Acta. 1988, 938, 361. (23) Ali, S.; Smaby, J. M.; Brown, R. E. Biochemistry 1993, 32, 11696. (24) Maggio, B.; Monferran, C. G.; Montich, G. G.; Bianco, I. D. In New Trends in Ganglioside Research. Neurochemical and Neurodegenerative Aspects; Ledeen, R. W., Hogan, E. L., Tettamanti, G., Yates, A. J., Yu, R. K., Eds.; Liviana Press: Padova, Italy, 1988; p 105. (25) Boggs, J.; Koshy, K. M.; Rangaraj, G. Biochim. Biophys. Acta. 1988, 938, 373. (26) Maggio, B.; Ariga, T.; Sturtevant, J. M.; Yu, R. K. Biochim. Biophys. Acta 1985, 818, 1.
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Figure 6. Effect of sulfatide on phospholipase C-induced fusion of large unilamellar vesicles. Vesicles contained PC/PE/Ch (2: 1:1) ( varying proportions of sulfatide, as indicated for each curve. Lipid and enzyme concentrations were as for Figure 3. (A) Phospholipid hydrolysis. (B) Vesicle suspension turbidity (liposome aggregation). (C) Intervesicular mixing of aqueous contents (liposome fusion).
increases the interfacial micropolarity of phosphatidylcholine bilayers, with a clear effect being already seen at 10 mol % sphingolipid.24,26 In agreement with these studies, sulfatide was shown to increase hydration in the headgroup region of PE vesicles.29 Probably because of hydration, sulfatide stabilizes phosphatidylcholine liposomes against spontaneous aggregation.27 Infrared spectroscopy has shown that interdigitation of long-chain sulfatide fatty acyl chains occurs also in mixed bilayers with phosphatidylcholine.30 Studies on the effects of sphingolipids on lamellar-tononlamellar phase transitions in lipid/water systems are scarce. Perillo et al.31 observed that the complex gangliosides GD1a and GM1 completely inhibited formation of the inverted hexagonal phase in PE at concentrations above 2-3 mol %. This was attributed to the geometry (27) Pedersen, T. B.; Frokjaer, S.; Mouritsen, O.; Jørgensen, K. J. Liposome Res. 1999, 9, 261. (28) Ruocco, M. J.; Shipley, G. G. Biochim. Biophys. Acta 1986, 859, 246. (29) Wu, X.; Li, Q. T. Biochim. Biophys. Acta 1999, 1416, 285. (30) Nabet, A.; Boggs, J. M.; Pe´zolet, M. Biochemistry 1996, 35, 6674. (31) Perillo, M. A.; Scarsdale, N. J.; Yu, R. K.; Maggio, B. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 10019.
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(conical shape10,32) of the gangliosides, which would oppose the curvature required for inverted hexagonal phases to form.33 The results of the present study (Figures 1 and 2) show that cerebroside and sulfatide have effects qualitatively similar to those of gangliosides on L-H transitions, increasing the transition temperatures and, at least in the case of sulfatide, decreasing ∆H. However, this lamellar-stabilizing effect is only seen at bilayer concentrations of sphingolipid 1 order of magnitude higher than those of gangliosides.31 This correlates well with the geometrical considerations, since the headgroup of cerebroside or sulfatide is certainly smaller than those of gangliosides; thus the conical shape of the molecules studied in this paper is not as marked as that of gangliosides. Sulfatide appears to be more effective than cerebroside at inhibiting the L-H transition (Figures 1 and 2), perhaps because its net negative charge and the corresponding large hydration sphere, discussed above, act against the formation of the inverted hexagonal phase geometry.33,34 Charge and hydration are also two features that sulfatide shares with the more complex gangliosides, but not with cerebroside. This may be at the origin of the different effects of cerebroside and sulfatide on the L-H transition of DEPE. Cerebroside increases the transition enthalpy, i.e., increases the energetic requirements of the transition, because its molecular geometry stabilizes the lamellar phase. Sulfatide, however, because of its large hydration sphere, makes the transition inaccessible to a certain population of DEPE molecules, and the transition enthalpy decreases concomitantly. Sphingolipids and Phospholipase C Activity. In general, sphingolipids tend to decrease phospholipase C activity when the substrate is present in either monolayer14,35 or bilayer form.8,15 Gangliosides are particularly effective in this respect. They do not affect the process of enzyme adsorption to the monolayer/bilayer surface but decrease the maximum rate of catalysis of the enzyme already adsorbed and the availability of the substrate in a suitable form for enzyme catalysis to take place.15,35 In our hands (Figures 3 and 5) cerebroside does inhibit phospholipase C, but again at concentrations 1 or 2 orders of magnitude larger than those at which gangliosides exert comparable inhibitory effects.8 The strengths of the different inhibitory effects of various gangliosides on phospholipase C and phospholipase A2 activities have been correlated to their capacities to modify (depolarize) the local electrostatic fields at the lipid-water interfaces, depolarization increasing in proportion to the complexity of the ganglioside polar headgroups.14,15,36 The small polar moiety of cerebroside, in comparison to those of the gangliosides also, explains its reduced potency as a phospholipase C inhibitor. Sulfatide is unique among the commonly found sphingolipids in that, under most conditions, it stimulates phospholipase C activity.14,15,35 The basis of this behavior may be that, in contrast to gangliosides, sulfatide generates a local hyperpolarized field at its surface.14,36 In addition, sulfatide-containing bilayers, especially in the presence of calcium ions, have considerably more inhomogeneties, or lateral defects, than those containing gangliosides or cerebrosides.13,26,37 These lateral defects (32) Israelachvili, J. N.; Marcelja, S.; Horn, R. G. Q. Rev. Biophys. 1980, 13, 121. (33) Maggio, B.; Albert, J.; Yu, R. K. Biochim. Biophys. Acta 1988, 945, 145. (34) Castresana, J.; Nieva, J. L.; Rivas, E.; Alonso, A. Biochem J. 1992, 282, 467. (35) Bianco, I. D.; Fidelio, G. D.; Maggio, B. Biochim. Biophys. Acta 1990, 1026, 179. (36) Maggio, B. J. Lipid Res. 1999, 40, 930.
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are known to facilitate enzyme activity, at least in the case of phospholipase A2.38 Our results show that sulfatide does activate phospholipase C (Figures 3 and 6), but only at mole fractions lower than 0.05. Higher sulfatide concentrations in the bilayer cause a decrease in enzyme activity. The reason for this dual behavior may reside in two different properties of sulfatide, namely, its electrical (hyperpolarizing) and geometrical (positive-curvature-inducing) characteristics, the former prevailing at low sulfatide concentrations and the latter, at high concentrations. As explained above, creation of a local hyperpolarized field tends to increase phospholipase activity.14,36 Why the tendency to form positively curved monolayers should inhibit the enzyme is not immediately obvious, but the fact is that phospholipase C, acting on PC/PE/Ch bilayers, is inhibited by a variety of conically shaped lipids and stimulated by lipids having inverted-cone shapes.11 Several authors39,40 have postulated that, even in purely lamellar phases, the presence of inverted-cone lipids would give rise to interfacial tension and incipient topographical distortion due to their propensity to adopt an inverted phase. Certain membrane-related enzymes would sense these inhomogeneities and have their activities modulated thereby. We have proposed7 that phospholipase C from B. cereus is one such enzyme, and the data in this paper are in general agreement with the prediction (with the exception of sulfatide at low concentrations, where the electrical effect predominates). The inhibitory properties of sulfatide were not observed in previous studies.14,15,35 However, studies in monolayers14,35 are unlikely to detect any tendencies toward nonlamellar phase formation and our previous data on the effect of sulfatide in vesicles15 were obtained with pure phosphatidylcholine as a substrate. PC, unlike the mixtures used in this and other (see ref 7 for a review) studies, forms very stable lamellae, and it is conceivable that, in this case, bilayer-destabilizing tendencies would not be easily manifested. Sphingolipids and Membrane Fusion. Several anionic and neutral glycosphingolipids, including gangliosides, cerebrosides, and sulfatides, are very effective modulators of membrane fusion induced chemically in cells and bilayers.41,42 In general, glycosphingolipids inhibit fusion of bilayer vesicles induced by either Ca2+ or myelin basic protein43-45 to extents that are related to the (37) Maggio, B.; Sturtevant, J. M.; Yu, R. K. Biochim. Biophys. Acta 1987, 901, 173. (38) Burack, W. R.; Biltonen, R. L. Chem. Phys. Lipids. 1994, 73, 209. (39) De Boeck, H.; and Zidovetzki, R. Biochemistry, 1989, 28, 7439. (40) Kinnunen, P. K. J. Chem. Phys. Lipids, 1996, 81, 151. (41) Maggio, B.; Cumar, F. A.; Caputto, R. FEBS Lett. 1978, 90, 149. (42) Maggio, M.; Cumar, F. A.; Caputto, R. Biochim. Biophys. Acta 1981, 650, 69.
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complexities of their oligosaccharide chains and to the presence or absence of negative charges. Vesicle fusion induced by phospholipase C is totally abolished at 1-3 mol % ganglioside. This inhibition was found to have two components, namely, inhibition of enzyme activity and stabilization of lamellar phases by gangliosides,15 resulting in inhibition of the putative nonlamellar fusion intermediates. Various sphingolipids have also been independently assessed as fusion suppressors.46,47 Similar to the case for the inhibition of myelin basic protein-induced vesicle fusion, cerebrosides (Figure 5) reproduce the effect of gangliosides, only at higher concentrations, as expected from the results discussed above. The extended fusion lag time in the 20% cerebroside sample (Figure 5C) is consistent with the idea that formation of the fusion pores is being hindered beyond mere enzyme inhibition (Figure 5A), the latter being directly responsible for the (smaller) change in vesicle aggregation (Figure 5B). The data for 1 mol % sulfatide (Figure 6) reflect a good parallelism in increased phospholipid hydrolysis, vesicle aggregation, and fusion with respect to the control mixture, suggesting that, for this system, enzyme activity is the limiting step. For a 10 mol % sulfatide concentration, however, the amount of diacylglycerol generated after 60 s would be more than enough to produce extensive fusion in the absence of sulfatide (compare 0% and 10% curves in Figure 6). The observed low rates and extent of fusion in the presence of 10% sulfatide at these late stages of the process reflect the bilayer-stabilizing properties that sulfatide, in common with most other glycosphingolipids, has at this concentration. Concluding Remarks. In a number of studies, summarized in ref 7, we have assayed the effects of a large variety of lipids on phospholipase C-induced fusions of phospholipid vesicles. We found that, without exception, those lipids that facilitate L-H transitions facilitate fusion and enhance enzyme activity. Conversely, lipids that stabilize lamellar phases inhibit fusion as well as phospholipase activity. Our present contribution presents the case of cerebroside, whose behavior confirms the general rule, and the more interesting case of sulfatide, which by virtue of its electric charge departs from the general behavior at low (
