Biochemistry 1986, 25, 1321-1330
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Ricinus communis Agglutinin-Mediated Agglutination and Fusion of Glycolipid-Containing Phospholipid Vesicles: Effect of Carbohydrate Head Group Size, Calcium Ions, and Spermine? Dick Hoekstra* Laboratory of Physiological Chemistry, University of Groningen, Bloemsingel 10, 971 2 K Z Groningen, The Netherlands Nejat Duzguneg Cancer Research Institute and Department of Pharmaceutical Chemistry, University of California, San Francisco, California 941 43 Received August 14, 1985
ABSTRACT: The glycolipids galactosylcerebroside (GalCer), lactosylceramide (LacCer), and trihexosylceramide
(Gb,) were inserted into phospholipid vesicles, consisting of phosphatidylethanolamine and phosphatidic acid. T h e extent to which their carbohydrate head groups protruded beyond the vesicle surface and their interference with membrane approach were examined by determining vesicle susceptibility toward type I Ricinus communis agglutinin (RCA,) induced agglutination and Ca2+- and spermine-induced aggregation and fusion either in the presence or in the absence of the lectin. The initial agglutination rates increased in the order GalCer Gb3 > LacCer (Figures 3 and 8; Hoekstra et al., 1985a). Interestingly, subthreshold concentrations of spermine did not facilitate RCAl-induced agglutination, nor was spermine-induced fusion facilitated by RCA,, indicating that its
EFFECT OF GLYCOLIPIDS ON MEMBRANE FUSION
mode of interaction with the bilayer differs from that of Ca2+. These differences between Ca2+ and spermine were also reflected by their distinctly different effect on membrane stability, as Ca2+induced extensive leakage of vesicle contents, in contrast to spermine (Figure 10B). Taken together, these results can be explained in terms of the different capacity of spermine and Ca2+to (locally) dehydrate (apposed) bilayers, a necessary condition for triggering membrane fusion (Wilschut et al., 1980; Hoekstra, 1982a; Wilschut & Hoekstra, 1984; Diizgiineg, 1985). This different dehydrating capacity can be correlated to the enhanced lectin-induced vesicle agglutination in the presence of Ca2+,as opposed to spermine. A picture thus emerges that involves a (partial) dehydration of the bilayer upon binding of Ca2+ to PA, which in turn induces a reorientation of the strongly hydrophilic carbohydrate head group by forcing it to tilt toward a more aqueous environment, Le., by increasing the distance of the terminal sugar residue relative to the bilayer/water interface. As a result, fusion will be interrupted when the glycolipid contains at least three carbohydrate residues, Le., Gb3 and globoside Gb4. Because of its relatively short carbohydrate chain length (Grant & Peters, 1984), the lactosyl head group will, even in its extended form, still provide a close intermembrane contact and fusion will be sustained. The same mechanism also explains the enhanced lectin agglutinability. Effect of Temperature on RCAl-Glycolipid Interaction. At temperatures above 25 OC, an RCA,-mediated enhancement of Ca2+-induced fusion became apparent also for GalCer vesicles. The direct involvement of an RCA,/galactosyl moiety interaction was revealed by abolishment of the lectin-mediated effect by the haptenic sugar lactose. This result suggests that the galactosyl moiety of the cerebroside was insufficiently exposed at room temperature, while with increasing temperature a modification was induced, involving either the glycolipid structure per se or a temperature-induced alteration in the lipid matrix, thus affecting GalCer/RCA, interaction in an indirect manner. Since the phase transition of the glycolipid is centered around 60 "C (Maggio et al., 1981), the latter possibility seems more likely, particularly in light of the observation that a similar temperature-dependent effect was seen on both LacCer and Gb3 vesicles (Figure 4). Up to 25 "C, the fusion rates for GalCer vesicles, either in the presence or in the absence of RCA,, were essentially the same (Figure 6), while these rates increased proportionally for Gb3 vesicles. However, in the case of LacCer vesicles, the initial fusion rate in the presence of RCA, increased faster than in its absence, indicating an enhanced glycolipid/receptor interaction with increasing temperature. Differences in lectin/receptor affinity as a result of temperature-dependent modifications of the lectin can be excluded since such an effect should have been also reflected by an enhanced interaction with Gb3vesicles. Rather, it would appear that the relative enhancement of LacCer vesicle fusion in the presence of RCA, can be attributed to physical alterations in the vesicle bilayer. In line with the above notion on Ca2+-facilitatedagglutination, such alterations will be most sensitive to the carbohydrate head group (disaccharide vs. trisaccharide), which is relatively close to the vicinity of the phospholipid head group region. Presumably, above 25 "C, these bilayer modifications become even more pronounced as the initial fusion rates in the presence of RCA, relative to the rates in the absence of the lectin became also enhanced for Gb3 and GalCer vesicles. This temperaturedependent effect may he attributed to the transformation of egg PE from a lamellar to a hexagonal phase, which takes place at a temperature around 28 OC (Cullis & De Kruijff,
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1979). As a result, lipid phase separation (Papahadjopoulos, 1978; Hoekstra, 1982b) may subsequently increase the local density of glycolipid receptors in the lateral plane of the bilayer, which in turn will increase lectin agglutinability (Surolia et al., 1975; Curatolo et al., 1978; Goodwin et al., 1982; Grant & Peters, 1984). CONCLUSIONS The present results, in conjunction with those previously reported on globoside Gb4 containing vesicles (Hoekstra et al., 1985a), indicate that close approach of adjacent bilayers can be regulated by the size of the glycolipid carbohydrate head group. The effective protrusion can be further regulated by CaZ+,when the glycolipids are inserted into negatively charged bilayers. This effect is indirectly accomplished by Ca2+binding to the supporting matrix, which causes the head group to adopt a more perpendicular orientation relative to the lateral plane of the bilayer; the results indicate this to be related to the ability of Ca2+to dehydrate the bilayer. As a consequence, the distance between the terminal sugar receptor and the plane of the bilayer will increase, while within the agglutinated vesicle complex the interbilayer distance increases, which causes the arrest of membrane fusion. The latter phenomenon becomes apparent for glycolipids containing at least three carbohydrate residues such as trihexosylceramide (this study) and globoside Gb4 (Hoekstra et al., 1985a). Micromolar amounts of Ca2+sufficed to induce fusion after preagglutination of the vesicles with the lectin, or when in addition, a subthreshold (30 pM) concentration of spermine was used. In the latter case, i.e., under conditions that may bear physiological relevance, very high fusion rates were observed. Spermine has been suggested to function as an intracellular modulator of membrane fusion occurring during exocytotic events or membrane biosynthesis (Schuber et al., 1983). The routing of such and other intracellular vesicles to their specific sites of action would presumably require a specific recognition system that could involve an interaction between carbohydrate structures and specific membrane localized lectins. That certain glycolipids show a particular tendency to regulate intermembrane distances and thus membrane fusion is rather intriguing and merits further investigation. ACKNOWLEDGMENT We gratefully acknowledge Rinske Kuperus for preparing the manuscript. Registry No. Ca, 7440-70-2;GalCer, 85305-88-0; Gb3,71965-57-6; LacCer, 4682-48-8;spermine, 71-44-3. REFERENCES Barondes, S . H. (198 1) Annu. Rev. Biochem. 50, 207-23 1. Bentz, J., Diizgiineg, N., & Nir, S . (1983) Biochemistry 22, 3 3 20-3 330. Cullis, P. R., & De Kruijff, B. (1979) Biochim. Biophys. Acta 559, 399-420. Curatolo, W., Yau, A. O., Small, D., & Sears, B. (1978) Biochemistry 17, 5740-5744. Diizgiineg, N. (1975) Biosystems 6, 209-216. Diizgiineg, N. (1985) Subcell. Biochem. 11, 195-286. Diizgiineg, N., Wilschut, J., Hong, K., Fraley, R., Perry, C., Friend, D. S., James, T. L., & Papahadjopoulos, D. (1983) Biochim. Biophys. Acta 732, 289-299. Diizgiineg, N., Hoekstra, D., Hong, K., & Papahadjopoulos, D. (1984) FEBS Lett. 173, 80-84. Ekerdt, R., & Papahadjopoulos, D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2273-2277.
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Goldstein, I. J., & Hayes, C. E. (1978) Adu. Carbohydr. Chem. Biochem. 35, 127-340. Goodwin, G. C., Hammond, K., Lyle, I. G., & Jones, M. N. (1982) Biochim. Biophys. Acta 689, 80-88. Grant, C. W. M., & Peters, M. W. (1984) Biochim. Biophys. Acta 779, 403-422. Hampton, R. Y., Holz, R. W., & Goldstein, I. J. (1980) J . Bioi. Chem. 255, 6766-6771. Hoekstra, D. (1 982a) Biochemistry 21, 2833-2840. Hoekstra, D. (1982b) Biochemistry 21, 1055-1061. Hoekstra, D. (1982~)Biochim. Biophys. Acta 692, 171-175. Hoekstra, D., Diizgiines, N., & Wilschut, J. (1985a) Biochemistry 24, 565-572. Hoekstra, D., Wilschut, J., & Scherphof, G. (1985b) Eur. J. Biochem. 146, 131-140. Kabat, E. A. (1978) J. Supramol. Struct. 8, 79-88. Kelly, R. B., Deutsch, J. W., Carlson, S . S., & Wagner, J. A. (1979) Annu. Rev. Neurosci. 2, 399-446. Maggio, B., Cumar, F. A., & Caputto, R. (1981) Biochim. Biophys. Acta 650, 69-87. Nicolson, G. L., Blaustein, J., & Etzler, M. E. (1974) Biochemistry 13, 196-204. Olden, K., Parent, J. B., & White, S. L. (1982) Biochim. Biophys. Acta 650, 209-232.
Papahadjopoulos, D. (1978) Cell Surf. Rev. 5 , 765-790. Rauvala, H. (1983) Trends Biochem. Sci. (Pers. Ed.) 8, 323-325. Rendi, R., Vatter, A. E., & Gordon, J. A. ( 1 979) Biochim. Biophys. Acta 550, 318-327. Schuber, F., Hong, K., Duzgiineg, N., & Papahadjoulos, D. (1983) Biochemistry 22, 61 34-6140. Shechter, Y., Chang, K., Jacobs, S., & Cuatrecasas, P. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2720-2724. Slama, J. S., & Rando, R. R. (1980) Biochemistry 19, 4595-4600. Sundler, R. (1982) FEBS Left. 141, 11-13. Sundler, R., & Wijkander, J. (1 983) Biochim. Biophys. Acta 730, 391-394. Surolia, A., Bachhawat, B. K., & Podder, S. K. (1 975) Nature (London) 257, 802-804. Utsumi, H., Suzuki, T., Inoue, K., & Nojima, S. (1984) J . Biochem. ( T o k y o ) 96, 97-105. White, J., Kielian, M., & Helenius, A. (1983) Q. Rec. Biophys. 16, 151-195. Wilschut, J., & Hoekstra, D. (1984) Trends Biochem. Sci. (Pers. E d . ) 9, 479-483. Wilschut, J., Duzgiineg, N., Fraley, R., & Papahadjopoulos, D. (1980) Biochemistry 19, 6011-6021.
X-ray Diffraction Evidence for Fully Interdigitated Bilayers of 1-Stearoyllysophosphatidylcholinet S. W. Hui* Biophysics Department, Roswell Park Memorial Institute, Buffalo, New York 14263 Ching-hsien Huang* Department of Biochemistry, University of Virginia School of Medicine, Charlottesville, Virginia 22908 Received July 17, 1985
X-ray diffraction experiments have been performed on 1-stearoyllysophosphatidylcholineor C ( 18):C(O)PC as a function of hydration a t temperatures below the order/disorder transition ( T , = 26.2 "C). At these temperatures, hydrated C ( 18):C(O)PC forms lamellae. The bilayer thickness, as determined by the saturation hydration method and electron-density profile, is 35-36 A, and the average area per C(18):C(O)PC molecule a t the lipid/water interface is 45.5 A2. The packing geometry of C(lS):C(O)PC in the lamella is proposed to adopt a fully interdigitated model in which the long C ( 18) acyl chain extends across the entire hydrocarbon width of the bilayer. Thus far, three different types of interdigitated bilayers are known for phosphatidylcholines. These various types of chain interdigitation are discussed in terms of the chain length difference between the sn-1 and sn-2 acyl chains. ABSTRACT:
I t is generally recognized that in biological membranes most phospholipids self-assemble into two-dimensional arrays of the noninterdigitated lamellar type; that is, each phospholipid molecule is oriented with its polar head group toward the membrane surface and its chain methyl groups toward the geometric center of the hydrophobic core in the membrane interior. Recently, several spectroscopic studies have been directed to the investigation of aqueous dispersions of mixed-chain phospholipids including sphingomyelin, whose two 'Supported, in part, by National Institutes of Health Grants GM28120 and GM-17452.
0006-2960/86/0425-1330$01 S O / O
hydrocarbons chains are inequivalent in terms of carbon number (Huang et al., 1984; Hui et al., 1984; Levin et al., 1985; McIntosh et ai., 1984). Results of these studies indicate that in the gel state the packing mode of pure mixed-chain phospholipids in the two-dimensional array of lamellae is that of a highly ordered interdigitated bilayer, which is a marked deviation from the generally recognized noninterdigitated bilayer form of symmetric phospholipids. It is now established that at temperatures below the main order/disorder phase transition, mixed-chain phospholipid molecules can be accommodated in at least three different types of interdigitated packing modes, depending on the chain-length difference
0 1986 American Chemical Society
