Is the linkage region of sphingolipids responsible for lipid raft formation?

Full text of DOI:10.1021/ja015715w

5124
J. Am. Chem. Soc. 2001, 123, 5124-5125
Chart 1
Is the Linkage Region of Sphingolipids Responsible
for Lipid Raft Formation?
Maki Uragami, Nobuya Tokutake, Xun Yan, and
Steven L. Regen*
Department of Chemistry and
Zettlemoyer Center for Surface Studies
Lehigh UniVersity, Bethlehem, PennsylVania 18015
ReceiVed February 23, 2001
ReVised Manuscript ReceiVed April 12, 2001
The hypothesis that sphingolipids (glycosphingolipids and
sphingomyelins) combine with cholesterol to form clusters in
biological membranes is of considerable current interest. In
particular, there is a growing body of evidence that suggests that
such clusters (commonly referred to as “lipid rafts”) are formed
in a “sea” of glycerolipids, and that these rafts may play a key
role in processes such as membrane trafficking and signal
transduction.1-10 If this hypothesis is correct, then understanding
those factors that are responsible for lipid raft formation is of
great importance.
In this paper, we report the synthesis of an exchangeable
sphingolipid dimer (1) and show that its monomer units mix,
nonideally, with those of a longer chain glycerolipid (2) in
cholesterol-rich fluid bilayers. We further show that such sphin-
golipid-glycerolipid mixing is closer to ideal than analogous
glycerolipid-glycerolipid mixing found with monomers of 2 and
3, i.e., heterolipid associations are faVored. Our principal results,
which are reported herein, provide the first quantitative insight
into how the linkage region of a sphingolipid and a glycerolipid
influences their mixing behavior in the physiologically relevant,
fluid bilayer state.
regionsthat portion of the lipid that connects the headgroup to
the hydrocarbon chains. Chart 1 highlights the major differences
that exist between the linkage region of glycerolipids and
sphingolipids. It should be noted that many but not all naturally
occurring sphingolipids also contain a trans double bond in this
region. In principle, the presence of amide and hydroxyl groups
could promote self-clustering and raft formation via intermolecular
hydrogen bonding.13,14 Such bonding should be significant,
especially if this region of the membrane were hydrophobic in
character, i.e., penetration of water beneath the headgroup was
minimal.
In the present study, we sought an exchangeable sphingolipid
that could be compared with an exchangeable glycerolipid having
the same chain length. Since we have previously shown that
monomers of 2 and 3 are nonideally miscible in cholesterol-rich
bilayers, homodimer 1 and the corresponding heterodimer, 4, were
viewed as attractive synthetic targets. In particular, by using
equilibrated bilayers made from 2 and 3 as a frame of reference,
insight into sphingolipid-glycerolipid mixing should be possible
by quantifying the relative mixing behavior of the monomers of
1 and 2. Specifically, a higher degree of nearest-neighbor
recognition for bilayers derived from 1 and 2 would indicate that
the linkage region favors segregation of the two different lipids;
a lower degree of recognition would reflect a preference for
sphingolipid-glycerolipid association.
Acylation of D-erythro-sphinganine (Avanti Polar Lipids) with
N-succinimidyl tetradecanoate afforded 5. Subsequent benzoyla-
tion and silylation of the primary and secondary hydroxyls,
respectively, to give 6, followed by debenzoylation, and introduc-
tion of a t-Boc-protected phosphoethanolamine moiety (via the
reaction sequence shown in Scheme 1) afforded 7. Deprotection
of the secondary hydroxyl group and the amino moiety, followed
by acylation of the latter with N-[O-1,2,3-benzotriazin-4(3H)one-
yl]-3-(2-pyridyldithio)propionate [BPDP] yielded 8.15 Finally,
reductive cleavage of the activated disulfide and reaction with
its precursor (8) afforded 1. Heterodimer, 4, was obtained by
reacting 8 with 1 equiv of the thiol monomer of 2.
(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.
To gain insight into the mixing properties of sphingolipids and
glycerolipids, we have begun to examine the miscibility of
exchangeable mimics by use of the nearest-neighbor recognition
(NNR) method.11 As described elsewhere, this chemical technique,
which probes nearest-neighbor interactions by measuring equi-
librium dimer distributions, provides quantitative insight into lipid
mixing. Thus, when equilibrium mixtures of dimers are found to
be statistical, such a finding establishes that the lipids are ideally
mixed. When homodimers are found to be in excess (i.e., NNR
is observed), and when this excess can be reduced or eliminated
by the presence of a nonexchangeable lipid that functions as a
mixing agent, nonideal mixing is indicated.12
(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) Davidson, S. K. M.; Regen, S. L. Chem. ReV. 1997, 97, 1269.
(12) (a) Vigmond, S. J.; Dewa, T.; Regen, S. L. J. Am. Chem. Soc. 1995,
117, 7838. (b) Dewa, T.; Miyake, Y.; Kezdy, F. J.; Regen, S. L. Langmuir
2000, 16, 3735.
(13) (a) Boggs, J. M. Biochim. Biophys. Acta 1987, 906, 353. (b) Lee, D.
C.; Miller, I. R.; Chapman, D. Biochim. Biophys. Acta 1986, 859, 266.
(14) Grainger, D. W.; Sunamoto, J.; Akiyoshi, K.; Goto, M.; Knutson, K.
Langmuir 1992, 8, 2479.
The purpose of the work described herein was to probe the
effects of one structural feature, which distinguishes all sphin-
golipids from glycerolipids, on lipid miscibility, i.e., the linkage
10.1021/ja015715w CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/03/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 21, 2001 5125
Scheme 1a
Figure 1. (A) High-sensitivity excess heat capacity profile of 1. (B)
Surface pressure-area isotherm for 1 and 3 over 1.0 M NaCl (used to
prevent solubilization) at 25 °C, compressed at 24 Å2/min/molecule.
Table 1. Equilibrium Heterodimer/Homodimer Ratiosa
exchangeable
monomersb
DPPC
(mol %)
heterodimerc
homodimer
entry
1
2
3
4
5
6
14/18
14/18
0
1.55 ( 0.02
(1.55 ( 0.08)12a
2.07 ( 0.07
50
(1.97 ( 0.06)12a
1.71 ( 0.01
14SL/18
14SL/18
0
50
2.02 ( 0.07
a All thiolate-disulfide interchange reactions were carried out at 60
°C to maintain a fluid phase. In each case, 29 mol % cholesterol plus
20 mol % of the corresponding thiol monomers were included in the
membrane. b Monomers of 1 (14SL) and 2 (18). c Convergent molar ratio
of heterodimer to 2; error values represent one standard deviation.
a Conditions: (a) N-Succinimidyl tetradecanoate, Et3N, CHCl3, 86%.
(b) Benzoyl chloride, DMPA, pyridine, 60%. (c) TIPSOTf, 2,6-lutidine,
CH2Cl2, 77%. (d) NaOCH3 (0.5 equiv), CH3OH, 79%. (e) PCl3, imidazole,
Et3N, toluene, 0 °C. (f) N-(tert-Butoxycarbonyl)ethanolamine, pivaloyl
chloride, pyridine. (g) I2, H2O, 62%. (h) HF/pyridine, CH3CN/CH2Cl2.
(i) TFA, CH2Cl2. (j) BPDP, Et3N, CHCl3, 56%. (k) DTT, CHCl3, 0 °C.
(l) 8, CHCl3.
A summary of our principal findings is shown in Table 1.
Miscibility measurements that were carried out with the ex-
changeable glycerolipids 2 and 3 in the presence, and in the
absence, of a mixing agent (i.e., 1,2-dipalmitoyl-sn-glycero-3-
phosphocholine, DPPC) gave results that were in excellent
agreement with those previously reported (entries 1-4).12a Here,
nonideal mixing is indicated by the elimination of the nearest-
neighbor recognition when DPPC is included in the membrane.
Analogous experiments that were carried out with 1 and 2 showed,
qualitatively, similar behavior, except that the degree of nearest-
neighbor recognition was lower in the absence of DPPC; i.e., the
dimer distribution was closer to the statistical value of 2.0,
reflecting random mixing (entries 5 and 6). Taken together, these
results indicate that the affinity of sphingolipids toward glycero-
lipids is greater than the affinity of sphingolipids toward
themselVes. This preference for heteroassociation corresponds to
the difference in free energy of mixing of ca. 130 cal/mol at 60
°C (calculated from the equilibrium constants for the two different
systems). The exact reason why heterolipid association is favored
is not clear at present.
Examination of a multilamellar dispersion of 1 in “borate
buffer” (10 mM borate, pH 7.4, 140 mM NaCl, 2 mM NaN3) via
high-sensitivity differential scanning calorimetry revealed a
melting temperature (Tm) of 25.5 °C and an enthalpy of 12.2 kcal/
mol (Figure 1A). This melting behavior is very similar to that
previously reported for the glycerophospholipid analogue, 3,
where the values of Tm and ∆H were 22.7 °C and 14.7 kcal/mol,
respectively; 2 melts at 54 °C.16,17 Monolayers of 1 that were
spread at the air/water interface showed a “lift-off” area of 1.94
nm2‚molecule-1 and a limiting area 1.65 ( 0.03 nm2‚molecule-1
.
The lift-off and limiting areas for 3 were 2.05 and 1.58 ( 0.03
nm2‚molecule-1, respectively. Thus, the melting behavior and
monolayer properties of 1 and 3 are similar in character.
On the basis of these model studies, we conclude that it is
highly unlikely that the difference in the linkage region of natural
sphingolipids and glycerolipids, by itself, provides a driving force
for lipid raft formation when both lipids are in the fluid phase.
That the self-association of these amide-bearing lipids is, in fact,
disfavored strongly suggests that the linkage region is “wet” and
that a hydrophilic microenvironment exists, which is not condu-
cive for intermolecular hydrogen bonding between neighboring
sphingolipids.
The extent to which other factors may contribute to raft
formation (e.g., the inclusion of transmembrane proteins, differ-
ences in headgroup structure and charge, etc.) remains to be
established. Systematic studies aimed at evaluating the influence
of such factors via the nearest-neighbor recognition method are
currently in progress.
Large unilamellar vesicles were formed from 1/1 molar
mixtures of homodimers (1/2 or 2/3) plus 29 mol % cholesterol
via reverse phase evaporation methods in borate buffer.18 To
promote monomer exchange via thiolate-disulfide interchange,
20 mol % of the corresponding thiol monomers were also included
in the membranes. Equilibrium was reached in all cases within
10 h. To confirm that true equilibrium values were obtained,
product mixtures were also generated from vesicles that were
made from the corresponding heterodimer. Specific experimental
procedures that were used for synthesizing the lipids, forming
vesicles, initiating the thiolate-disulfide interchange reaction, and
analyzing dimer distributions by HPLC were similar to those
previously described.12,16
(15) Janout, V.; Lanier, M.; Regen, S. L. Tetrahedron Lett. 1999, 40, 1107.
(16) Krisovitch, S. M.; Regen, S. L. J. Am. Chem. Soc. 1992, 114, 9828.
(17) Mixtures of C14 and C18 phospholipids tend to have intermediate
melting temperatures.11
(18) Cholesterol concentrations of ca. 29 mol % in closely related
membranes place them in the liquid ordered/liquid disordered coexistence
region, which is thought to be biologically relevant: (a) Almeida, P. F. F.;
Vaz, W. L.; Thompson, T. E. Biochemistry 1992, 31, 6739. (b) Sankaram,
M. B.; Thompson, T. E. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 8686.
Acknowledgment. We are grateful to the National Institutes of Health
(PHS Grant GM56149) for support of this research
Supporting Information Available: Procedures for the synthesis of
1, 4, 5, 6, 7, and 8 (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
JA015715W