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Sugar-Based Lipid Headgroups: How Sticky Are They? Michihiro Sugahara, Maki Uragami, Nobuya Tokutake, Xun Yan, and Steven L. Regen* Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015 Received December 10, 2001 This paper reports the synthesis of a disulfide-based exchangeable glycophospholipid and establishes that this lipid mixes, ideally, with a shorter-chain, phospholipid analogue in cholesterol-rich fluid bilayers. These findings indicate that associative interactions between carbohydrate headgroups are unlikely to provide a significant driving force for the clustering of glycolipids in biological membranes.
Introduction The hypothesis that sphingolipids (glycosphingolipids and sphingomyelins) combine with cholesterol to form transient clusters (“lipid rafts”) in biological membranes and that such structures sequester other lipid-based components (e.g., glycosylphosphatidylinositol-anchored proteins), is a subject of considerable current interest.1-10 Despite the significant role that lipid rafts are presumed to play in cellular function, the forces that drive their formation, and even their existence, remain debatable. One intriguing proposal that has been made is that interactions between carbohydrate headgroups of neighboring glycolipids provide a driving force for raft formation.1a There is, in fact, some experimental data in the literature that gives credence to this notion. In particular, hydrogen bonding between sugars of certain glycosylated bile acids has been implicated as a driving force for micelle formation.11 However, classic studies by Klotz et al. suggest that associative interactions between sugar headgroups of lipids are unlikely to be significant in fluid bilayers.12 In this paper, we report the synthesis of an exchangeable glycophospholipid and show that it mixes, ideally, with a shorter-chain phospholipid analogue in fluid bilayers. Specifically, we show that the monomer units of 1 and 2 become randomly distributed within liposomal membranes containing 0, 29, and 40 mol % cholesterol, when they are free to undergo exchange. These findings indicate that associative interactions between these carbohydrate headgroups do not provide a significant driving force for the clustering. Results and Discussion To probe the self-association behavior of a sugar-based lipid headgroup (i.e., its “stickiness”), we synthesized exchangeable dimers 1, 2, and 3, and examined the mixing (1) (a) Simons, K.; Ikonen, E. Nature 1997, 387, 569. (b) Simons, K.; Ikonen, E. Science 2000, 290, 1721. (2) Chunbo, Y.; Johnston, L. J. Biophys J. 2000, 79, 2768. (3) Kasahara, K.; Watanabe, K.; Takeuchi, K.; Kaneko, H.; Oohira, A.; Yamamoto, T.; Sanai, Y. J. Biol. Chem. 2000, 275, 34701. (4) Field, K. A.; Apgar, J. R.; Hong-Geller, E.; Siraganian, R. P.; Baird, B.; Holowka, D. Mol. Biol. Cell 2000, 11, 3661. (5) Dhanvantari, S.; Loh, Y. P. J. Biol. Chem. 2000, 275, 29887. (6) Czech, M. P. Nature 2000, 407, 147. (7) Wang, T. Y.; Silvius, J. R. Biophys. J. 2000, 79, 1478. (8) Heino, S.; Lusa, S.; Somerharju, P.; Ehnholm, C.; Olkkonen, V. M.; Ikonen, E. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8375. (9) Nelson, K. L.; Buckley, J. T. J. Biol. Chem. 2000, 275, 19839. (10) Radhakrishnan, A.; Anderson, T. G., McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 12422. (11) Venkatesan, P.; Cheng, Y.; Kahne, D. J. Am. Chem. Soc. 1994, 116, 6955. (12) Klotz, I. M.; Franzen, J. S. J. Am. Chem. Soc. 1962, 84, 3461.
behavior of their monomer units by use of the nearestneighbor recognition (NNR) method.13 For this purpose, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-Nlactose (Avanti) was hydrogenated and then acylated with N-[O-1,2,3-benzotriazin-4(3H)one-yl]-3-(2-pyridyldithio) propionate (BPDP), to give 4 (Scheme 1).14 Subsequent reduction with tris(2-carboxyethyl)phosphine) (TCEP), followed by reaction of the resulting thiol monomer with 4, afforded 1. An alternate reaction of this same thiol monomer with an activated form of the thiol monomer of 2 afforded 3. The synthesis of 2, as well as its activated precursor, has previously been described.15 Examination of multilamellar dispersions of 1 and 3 in borate buffer (140 mM NaCl, 10 mM boric acid, 2 mM NaN3, pH 7.4) by high-sensitivity differential scanning calorimetry revealed gel to liquid-crystalline phase transition temperatures (Tm) of 50.6 and 33.6 °C, respectively; the Tm value for 2 (22.7 °C) has previously been reported.15 Comparison of the surface pressure-area isotherm of 1, with an analogue that does not contain the lactose moiety (i.e., 5), reveals similar behavior except that the latter packs more tightly; i.e., the limiting areas for 1 and 5 are (13) Davidson, S.; Regen, S. L. Chem. Rev. 1997, 97, 1269. (14) Janout, V.; Lanier, M.; Regen, S. L. Tetrahedron Lett. 1999, 40, 1107. (15) Krisovitch, S. M.; Regen, S. L. J. Am. Chem. Soc. 1992, 114, 9828.
10.1021/la015722y CCC: $22.00 © 2002 American Chemical Society Published on Web 01/23/2002
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Langmuir, Vol. 18, No. 4, 2002
Letters
Scheme 1
Figure 1. Surface pressure-area isotherms for 1 and 5 over 0.14 M NaCl at 25 °C, compressed at 24 Å2 min-1 molecule-1. The isotherm showing the larger area/molecule is that of 1. Table 1. Mixing Behavior of Exchangeable Lipids
154 ( 2.4 and 112 ( 4 Å2/molecule, respectively (Figure 1). In essence, the nearest-neighbor recognition method detects and measures the thermodynamic tendency of two lipids to become nearest neighbors in the bilayer state.13 Thus, two exchangeable lipids of interest are converted into dimers and then allowed to undergo monomer interchange via thiolate-disulfide displacement. The equilibrium dimer distribution is then analyzed as formal, noncovalent bonds between pairs of adjacent lipids. When equilibrium mixtures are found to be statistical, such a finding establishes that the monomers are ideally mixed within the membrane. When there is a thermodynamic preference for forming homodimers or heterodimers, nonideal mixing is indicated; the deviation from a statistical mixture being a quantitative measure of this nonideality. Large unilamellar vesicles were formed from an equimolar mixture of 1 and 2, plus varying mole percentages of cholesterol, using reverse phase evaporation methods. Prior to monomer exchange, each dispersion was heated to 60 °C in order to place the membrane in the physiologically relevant fluid phase. Experimental protocols that were used to form vesicles, to promote monomer interchange after partial reduction (
