Laurens van Deenen Lecture.
52nd ICBL Warsaw
Lipidomics: making sense of the data lode.
Peter J. Quinn
King’s College London
This lecture honours the legacy of a man who was a giant in the field of lipid research. As a
graduate student in the mid 1960’s I was greatly inspired by the research on the
physicochemical properties of membrane lipids and the role of enzymes responsible for their
metabolic turnover emanating from Utrecht at this time. Later on in my career I got to know
Laurens and developed a respect for not only his scientific achievements but for the man
himself. He impressed me by his enthusiasm for the subject and the way this infected
countless young scientists at the early stages of their careers. His influence spread beyond
placing The Netherlands at the forefront of membrane science at the time but had a lasting
effect on subsequent generations of lipid scientists throughout the world.
To provide you with a flavour of the challenges he faced as he approached the end of the first
decade of his post-doctoral studies I quote from a review he published in Progress in the
Chemistry of Fats and Other Lipids published in 1966. “It needs no argument that an exact
determination of the composition of a natural phospholipid mixture is no sinecure. Progress
in this difficult field was possible by the development of chromatographic techniques
including columns with silica or aluminium oxide, paper impregnated with silica or otherwise
pre-treated, thin-layer chromatography and application of various hydrolysis techniques
combined with chromatographic resolution of the split products” And he goes on, “The
overwhelming variations in chemical structure of the paraffinic acid connected with
biogenetic, dietary and other factors challenge the investigator attempting to establish
relations between the composition and function of phospholipids in membranes.” Many of
you working in the field today would not recognize the tools of the trade of that era not
forgetting high-voltage paper ionophoresis which I used in those days to separate watersoluble phospholipase products of brain lipids in the discovery of a pathway of lecithin
turnover mediated by a novel enzyme EC 3.1.4.38.
van Deenen recognised early on that using these techniques one could appreciate the diversity
of lipid molecular species present in the membranes of different cells and tissues. Moreover,
there was also an established literature of studies on the physical properties of polar lipids
exploiting infrared spectroscopy and calorimetry based on synthetic lipid analogues.
However, he reasoned that it was necessary to marry the particular lipid with its physical
property to understand how the functions of phospholipids in membranes were performed.
These principles were to guide his endeavours throughout the remainder of his distinguished
career.
1
An example that particularly impressed me was the way he approached a question that
bedevilled the experiment of another group of Dutchmen, Gorter and Grendel, who 50 years
earlier related the area occupied by lipid extracts of erythrocytes when spread at the air-water
interface to the area of membrane from which they were extracted and came to the conclusion
that the membrane consisted of a bilayer of lipid. Crucial to this experiment was the density
of lipids in the monolayer. The figure chosen was the density at a surface pressure of 2
mN.m-1. van Deenen and his erstwhile graduate student, Rudy Demel, adopted an entirely
novel approach to address this question by examining the surface pressure of substrate
monolayers that could be hydrolysed by phospholipases from a range of sources and
compared this with their action on cell membranes. Enzymes from a variety of venoms and
toxins haemolysed red cells and were able to attack substrate monolayers at surface pressures
greater than 40 mN.m-1. Phospholipases from endogenous and plant sources, however, could
neither haemolyse red cells nor monolayers at surface pressures greater than about 31
mN.m-1. The conclusion was unambiguous; membrane lipids were packed at a density giving
a surface pressure in the region of 32 mN.m-1.
The techniques of analysis of tissue lipids in use today are unimagined from that in the
1950’s. Improvements began with the introduction of gas-liquid chromatography followed
by liquid-liquid chromatography for separation of lipids from complex mixtures. These
advances were accompanied by the use of increasingly sensitive methods of detection of the
separated components from flame ionization through to mass spectrometry. Subsequent
advances in mass spectrometry have been achieved largely through improvements in creating
lipid ions that could be separated on the basis of their mass to charge ratio in the
spectrometer. Such ionization methods include electron impact, fast atom bombardment and
chemical ionization. These methods are problematic because they generate a very large
number of fragment ions which, in turn, leads to difficulties in characterising the individual
lipids in complex mixtures. To some extent this problem was handled by pre-separation of
the components of the mixture by chromatography prior to ionization but, in this mode, the
mobile phase of the chromatographic system had to be dealt with if unwanted handling of the
sample was to be avoided.
The methods of choice for analysis of tissue extracts nowadays are the, so called, soft
ionization methods of matrix-assisted laser desorption and electrospray ionization. These
methods, referred to as shotgun lipidomics when applied to tissue lipid extracts, generate
molecular ions without extensive fragmentation of the molecules and hence identification and
quantitation of the lipids in a mixture is greatly simplified. This significant advance in
technology was recognised by award of the Nobel Prize in Chemistry in part to Fen and
Tanaka in 2002 for their development of soft desorption ionization methods for mass
spectrometric analysis of biological macromolecules. Their procedures generate quasimolecular ions in yields and polarities which can be modulated by the acidic or basic
properties of the analyte. This development in conjunction with more sophisticated sample
handling methods, data reduction and analysis now means that the entire lipidome can be
revealed rapidly in samples of ever diminishing size.
2
These powerful techniques have been exploited in imaginative ways to characterise the
lipidome. They include amongst others the diagnosis of lipidoses and disease states, the
development and monitoring of drug therapies and imaging of lipid distribution and
interactions in living tissues. A trawl through PubMed using key words “mass spectrometry
and lipid” for publications during the past year yielded a figure of more than 1600. Rather
than attempt a comprehensive survey of the different scientific approaches I will just mention
two that appeared most interesting to me. The first is in the field of cancer surgery. Laser
desorption ionization-mass spectrometry has been developed for imaging of tissues by Zoltan
Takats and his colleagues in Budapest. The mass ions generated largely originate from
phospholipids and spectral analysis can distinguish one tissue type from another as well as
healthy from diseased tissue. A surgical CO2 laser has been combined with a mass
spectrometer to produce a sufficiently high ion current to allow a principle component
analysis of the spectral data in real time. Thus the surgeon is guided during the operation by
mass spectral analysis of the phospholipids that provide a fingerprint to distinguish healthy
from malignant tissue even though this may not be obvious to the unaided eye.
The second and related method I wish to mention is the use of mass spectrometry to provide a
two, and sometime a three, dimensional image of the lipidome within tissues even down to
subcellular resolutions. A variety of different methods of generating lipid ions have been
employed but matrix-assisted laser desorption ionization is presently used most commonly in
lipid imaging. This method, however, lacks spacial resolution appropriate for interrogation
of the lipidome on a subcellular scale. Such a resolution can nevertheless be achieved by
secondary ion generating methods combined with time of flight mass spectrometry. The, so
called, MALDI-TOF method is now sufficiently versatile to be of general use. The method
involves preparation of frozen sections and the application of a suitable matrix to the surface
of the specimen. Spacial resolution is greatly influenced by the size of the matrix crystals
and migration of the analyte during matrix application. Ways to optimize the conditions are
to use nano sized particles of iron oxide for phospholipid distribution, silver nanoparticles for
fatty acid analysis and gold nanoparticles for glycosphingolipids. With reduction of laser
beam diameter to 10 μm or so and tissue translation distances of 1μm combined with the use
of highly focused ion beam clusters like Buckminsterfullerene, a subcellular resolution of the
complete lipidome is rapidly moving into the realms of reality.
Given this abundance of data how do we make sense of this in terms of what are lipids doing
and how do they perform their functions? My particular interest here is the structural role of
membrane lipids. It has been argued that biophysics has yet to delve into the detailed
molecular aspects of membrane lipid function and that we have become stuck at describing
lipid assemblies by averaging techniques only suitable for characterising lipid phase
behaviour. I want to plead a case for an advance of our understanding of lipid bilayers
merely as structures comprised of phases of differing fluidity. It seems to me we should be
exploiting advances in technology to examine the structure of lipid bilayers at least at the
molecular level. The primary structural tools for this endeavour are scattering methods with
wavelengths capable of providing information at atomic resolution and these are X-ray and
neutron scattering.
3
To give an example of how the technology has changed in the case of X-ray diffraction I
show two images of powder diffraction patterns in Fig. 1. The first was recorded using a
conventional rotating anode to generate nickel-filtered CuKα X-rays, a camera equipped with
toroidal optics and photographic film to record the scattering intensity pattern accumulating
over several hours (Fig. 1A & B). This is the most recent published diffraction pattern by
Graham Shipley, the acknowledged expert in this field, and is of a dispersion of a pure
molecular species of sphingomyelin with a stearoyl N-acyl chain. The other is a powder
pattern of an aqueous dispersion of bovine brain sphingomyelin recorded on an image plate
exposed for 30 sec to an X-ray beam of wavelength 0.12 nm at beamline BL40B2 of the
Spring8 synchrotron radiation source in Japan (Fig. 1C &D). The two main advantages that
are achieved by synchrotron X-ray methods are the orders of magnitude greater flux of
tuneable radiation and secondly, the greater angular resolution that can be achieved by
increasing the distance between the sample and the detector.
Fig.1. X-ray powder
diffraction using conventional
(A & B) and synchrotron Xradiation (C & D).
How then are these images interpreted? The concentric rings represent conditions where
Bragg’s Law is satisfied and the dimensions of the diffracting units oriented 360o in real
space are inversely related to the X-ray wavelength. The rings closest to the direct beam that
hits the beam-stop in the centre of the pattern gives information about the mesophase
structure and the scattering at wider angles tells us about the manner of packing of the
hydrocarbon chains. In the example given, the concentric rings represent the orders of
reflection differing by exactly one wavelength and, because they are evenly spaced, they
indicate a bilayer structure. About one in 10,000 X-rays incident on the sample are scattered
by interaction with the electrons surrounding the constituent atoms. It is possible to use the
pattern of scattering intensity of the different orders of reflection to calculate the density of
electrons through the repeating unit cells. The credit for proposing the current model of the
unit cell goes to my former colleague at King’s College, Maurice Wilkins, the co-recipient of
the 1962 Nobel Prize in Physiology and Medicine for the discovery of the structure of DNA.
The model is illustrated in Fig. 2 which shows the relative electron density calculated for
multilayers of egg lecithin hydrated with 21 or 14 % water and the molecular interpretation
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above. He reasoned that the distance separating the regions of greatest electron density that
were responsive to changes in hydration was where the water was located. The regions of
high electron density were the phosphate groups of the phospholipids and the lowest electron
density occurs along the central plane of the bilayer where the terminal methyl groups of the
hydrocarbon chains
Fig. 2. Fit of molecular
arrangement of phospholipids to
relative electron density profiles
through the unit cell of multilayer
structures.
reside. Thus the repeating unit is a centrosymmetric bilayer of lipid and a layer of water.
The model has remained unchallenged for the past 40 years and is the benchmark upon which
nearly all biophysical investigations of membranes are interpreted.
When I began my experiments to investigate the structure of membrane lipids at synchrotron
sources more than 25 years ago I was satisfied that the basic principles of bilayer structure
and phase behaviour had been well grounded using conventional scattering methods and
chemically-defined molecular species of lipids. I resolved, therefore, to ask questions about
lipid structures that were more demanding and relevant to cell membranes and this required
examination of complex lipid mixtures of the type extracted from biological tissues. I began
with dispersions of total polar lipid extracts of cell membranes such as chloroplast thylakoid
membranes but met with no success. The reason was evident from freeze-fracture electron
microscopy and from the known physical properties of the lipids present in the extract. All
biological membranes contain a proportion of polar lipids that when dispersed in aqueous
media under physiological conditions form non-bilayer structures generally of the hexagonalII type. These lipids have specific functions such as introducing instability to the bilayer
during membrane fusion. They are prevented from forming non-bilayer structures in
membranes because of their interactions with other membrane components but are
unconstrained when these components are removed from the extract.
What remained for examination were the interactions that take place between the different
membrane lipids in a bilayer configuration. I first looked at a mixture that had been
thoroughly scrutinized by many investigators using a range of biophysical methods and that
was phosphatidylcholine and sphingomyelin. These two phospholipids are the major
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phospholipids of the outer monolayer of the mammalian plasma membrane. The molecular
species of these two lipids differ principally in the temperature at which they undergo a
transition from a gel to a fluid structure and this is partly due to the intermolecular hydrogen
bonds that can form between sphingolipids causing them to be stable at higher temperatures.
The accepted belief was that gel-fluid phase separation occurs up to the temperature when the
sphingomyelin is converted to a fluid structure at about 40oC, above which the two
phospholipids were completely miscible in all proportions. This is not the case. The
evidence is seen in Fig. 3 which shows a sequence of X-ray scattering intensity patterns of
Fig. 3. A sequence of X-ray
scattering intensity profiles of
second-order peaks recorded from
an equimolar mixture of eggsphingomyelin and eggphosphatidylcholine during a
heating scan from 20o to 50oC.
the second-order Bragg peak recorded from an aqueous dispersion of an equimolar mixture of
egg-phosphatidylcholine and egg sphingomyelin during a heating scan from 20o to 50oC. The
scattering band can be resolved into separate peaks assigned to bilayers of
phosphatidylcholine in fluid structure throughout the temperature scan and peaks assigned to
sphingomyelin in gel structure at temperatures below 40oC and a fluid structure at higher
temperatures. The transition from gel to fluid structure could be confirmed by deconvolution
of a sharp gel peak from the scattering in the wide-angle region at temperatures below 40oC.
The assignments of the bilayer structures were also confirmed by relating the scattering
intensities of individual peaks in mixtures of the two lipids in different proportions.
Thus it can be seen that the X-ray method allows the identification of the phospholipid from
the particular dimensions of the bilayer repeat structure, the configuration of the acyl chain
packing and the relative number of electrons or mass of the phospholipid in the mixture. It is
important to point out that because the diffracting units of the two phospholipids retain the
same identity they have in dispersions of the pure phospholipid it means that they must
remain in register in the mixture. That is to say phosphatidylcholine is only coupled with
phosphatidylcholine and likewise sphingomyelin is only coupled with sphingomyelin in their
respective unit cells otherwise hybrid unit cells would appear. This tells us that line tension
between the two leaflets of the respective bilayers is lower than that between the interfacial
6
edges of adjacent coupled unit cells. These differences in interfacial energy prevent mixing
of the two phospholipids. Line tension and intermolecular hydrogen bonds between
sphingomyelin molecules at the lipid-water interface ensures that the two phospholipids are
completely immiscible in both gel and fluid structures.
I next turned my attention to one of the most exhaustively tested lipid mixtures and that is the
interaction between phospholipids and cholesterol. The condensing effect of cholesterol has
been known since the 1920’s and a preferential interaction between cholesterol and more
saturated molecular species of phospholipids is well documented in both monolayers and
bilayers. Thus in ternary mixtures of phosphatidylcholine, sphingomyelin and cholesterol the
accepted view was that cholesterol partitions between the phosphatidylcholine and
sphingomyelin with an approximately 3-fold preference for the sphingomyelin but in either
case interaction with cholesterol produces a liquid-ordered phase. As its name implies this
phase has properties intermediate between a gel and a fluid phase. I used the same mixture
shown in the previous diagram, which is an equimolar mixture of egg phosphatidylcholine
and egg sphingomyelin, and examined the effect of adding increasing amounts of cholesterol.
Fig. 4. A. X-ray scattering intensity profiles of the
first two orders of lamellar repeat structures formed by
equimolar mixtures of egg-sphingomyelin and eggphosphatidylcholine cantaining indicated amounts of
cholesterol. B. Relative scattering intensities of the
peaks assigned as sphingomyelin as a function of mass
fraction of cholesterol in the mixture.
Fig. 4A shows the first and second orders of reflection from mixtures containing increasing
proportions of cholesterol recorded at 37oC. The peak assigned as sphingomyelin is
increasing relative to the phosphatidylcholine as the proportion of cholesterol in the mixture
increases. An analysis of the scattering intensities of the peaks is shown in Fig. 4B. In the
absence of cholesterol the relative scattering intensity from the peak assigned to
sphingomyelin is proportional to its mass in the equimolar mixture. Knowing the mass of all
the components in the different mixtures it is possible to calculate the relative scattering
7
intensity due to the sphingomyelin plus the cholesterol and this is indicated in the diagram.
Adding 5 mole% cholesterol causes some sphingomyelin to partition into the
phosphatidylcholine presumably because the cholesterol disrupts the intermolecular hydrogen
bonding network that preserves the coupled bilayer structure of the sphingomyelin. With
increasing proportions of cholesterol sphingomyelin molecules are recruited back from the
phosphatidylcholine until a point is reached when a maximum amount of cholesterol interacts
with the sphingomyelin. Beyond this the excess cholesterol partitions into the
phosphatidylcholine. The inflection point occurs when virtually all the cholesterol is
complexed with sphingomyelin and the complex coexists with bilayers of pure
phosphatidylcholine.
The stoichiometry of the complex can be obtained from the mass fraction of cholesterol at the
inflection point and this corresponds to 1.7 molecules of sphingomyelin per cholesterol. To
put this stoichiometry into a molecular context it means that each cholesterol molecule must
be in contact with at least 3.5 hydrocarbon chains. Cholesterol-cholesterol contacts can be
discounted because these could easily be detected in the X-ray scattering patterns and, in any
event, are energetically unfavourable. This means that each hydrocarbon chain is shared
between two cholesterol molecules. Fig. 5A shows such an array. It turns out that
Fig. 5A. An array of
cholesterol (C) and
sphingomyelin
hydrocarbon chains
(1:1.7). B. GUV with
sphingolipid-cholesterol
raft.
cholesterol is somewhat of a goldilocks molecule in that it’s cross sectional area fits precisely
into an array of seven hydrocarbon chains and its length and amphiphilic balance positions it
at just the right depth with respect to the lipid-water interface. This is the reason why the
function of cholesterol cannot be substituted by even closely related sterols in mammalian
membranes. One of the features of the structure is that intermolecular hydrogen bond
interactions between the sphingomyelin molecules appears to be a crucial factor in stabilizing
the structure. The ordered assembly of the lipid components creates a quasicrystalline matrix
into which signalling molecules may be interpolated in a very specific manner. This would
include lipid-anchored proteins. The matrix may also be modified by incorporation of
sphingolipids of the type involved in ceramide signalling which, in turn, provides either
docking facilities or a structure that activates other signalling proteins. Just to illustrate what
a complex between sphingolipids and cholesterol looks like a giant unilamellar vesicle
comprised of a mixture of glycerophospholipids, sphingolipids and cholesterol is shown in
8
Fig. 5B in which a seven-lobed domain of liquid-ordered structure is present in a fluid bilayer
of phospholipid.
Next I want to show you the importance of molecular species of lipids in the structure of
bilayers. It is becoming increasingly apparent that molecular species of sphingolipids with
N-acyl chains of 22-26 carbons in length have a special role in signalling and related
functions performed by membrane rafts. These asymmetric lipids behave very differently
from molecular species with N-acyl chain lengths of 16-18 carbons in which the chains are
symmetrical with respect to the hydrocarbon substituent of the sphingosine base. This
behaviour is illustrated in Fig. 6 which is an X-ray diffraction analysis of an aqueous
dispersion of bovine brain sphingomyelin performed at 50oC. At this temperature the
hydrocarbon chains are in a fluid state. The extract is comprised of a mass proportion of
symmetric to asymmetric molecular species of sphingomyelin of 1:3. Panel A is a
synchrotron X-ray scattering intensity pattern in the small-angle region from an aqueous
dispersion of bovine brain sphingomyelin at 50oC recorded on an image plate set 1 m from
the sample. The Bragg reflections are the first two orders of the lamellar repeat structure and
Fig. 6. Synchrotron X-ray diffraction
analysis of bovine brain
sphingomyelin. A. Image plate showing
first two orders of a lamellar repeat
structure. B. Scattering intensity
profiles of the image in A. C.
Deconvolution of the first-order
reflection by the fit of two Voigt
functions showing two bilayer
structures. D & E. Relative electron
density distributions through the unit
cells of Peak 1 and 2, respectively,
using the first 4 orders of reflection.
it can be seen that the distribution of scattering intensities within each reflection, shown in
Panel B, are not symmetric. Looking in detail at the first-order lamellar reflection we can see
in Panel C that this reflection can be fitted by two Voigt functions with confidence limits,
R2>0.99. Panels D & E show how this type of data for the first four orders of reflection can
be used to calculate the relative electron density distributions through the respective unit
cells. It is not necessary to assign the molecular species composition to each of the two
bilayers to be able to say that the lipid compositions must be different. Most conspicuously
9
the unit cell of the bilayer with the greater lamellar repeat spacing has a thinner bilayer and a
thicker water channel.
Can we learn something of the composition of the two bilayers that are resolved from the
complex mixture of bovine brain sphingomyelin extract by comparing them with the
properties of pure molecular species of symmetric and asymmetric sphingomyelin? Fig. 7
shows an X-ray diffraction analysis of multibilayer structures formed by C-24 and C-16
molecular species of sphingomyelin again recorded at 50oC when both lipids are in a fluid
state. The asymmetric sphingomyelin was recorded on an image plate as was that shown in
Fig. 7. Synchrotron X-ray
diffraction analysis of pure
N-acyl molecular species of
sphingomyelin. A. Image
plate of C-24 sphingomyelin.
B & D. scattering intensity
profiles of C-24 and C-16
sphingomyelins. C & E.
Relative electron density
profiles of C-24 and C-16
sphingomyelins.
Fig. 6 (Panel A) and the symmetric sphingomyelin was recorded using a multiwire quadrant
detector, hence no image is available. Both the scattering intensity profiles of the C-24 and
C-16 sphingomyelins (Fig. 6B & D) can be fitted to single symmetric peaks with lamellar dspacings of 7.21 and 6.66 nm, respectively. The relative electron densities through the unit
cells of the individual bilayers are similar with water channels of almost the same dimension
but with bilayer thicknesses that differ according to the extent of the respective hydrocarbon
moieties of the molecules. When comparing the relative electron density profiles of the two
pure lipids with that of the two bilayers deconvolved from the biological extract in Fig. 6 the
most obvious difference is the bilayer structure of greatest lamellar repeat spacing. This
bilayer has a thickness more akin to that formed by the symmetric molecular species of
sphingomyelin but the water channel is much thicker.
This is convincing evidence that the bilayer of greater repeat spacing is comprised of a
combination of symmetric and asymmetric molecular species that confers a particular
physicochemical property on the complex that is not shared by the pure molecular species.
Moreover, because the structure formed by this entity gives rise to a single Bragg peak the
particular combination of molecular species of sphingomyelin that comprises the bilayer
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remain in register across the bilayer even in the fluid state and coexist with structures with
features that assign them as symmetric molecular species of sphingomyelin.
The biological implications of this observation are quite profound. It means that the free
energy of bilayers of pure asymmetric molecular species of sphingomyelin in the fluid state is
greater than a bilayer formed by a combination of asymmetric and symmetric molecular
species of sphingomyelin. We might say this is obvious from the fact that asymmetric
molecular species of glycosphingolipids that are found abundantly in membrane rafts are not
phase separated into ordered, as opposed to liquid-ordered structures, despite having solidfluid phase transition temperatures greater than 60oC. What then is known about the
interactions between symmetric and asymmetric molecular species of membrane lipids?
We have already seen that symmetric molecular species of sphingomyelin and
phosphatidylcholine are immiscible even in the fluid state but what of mixtures of
asymmetric sphingolipids with glycerophospholipids? To address this question I firstly
examined mixtures of dipalmitoylphosphatidylcholine and glucosylceramide extracted from
the spleen of a patient with Gaucher’s disease. I chose this mixture for two reasons; the
glycosphingolipid is comprised almost entirely of molecular species with N-acyl fatty acids
of 22-26 carbon atoms in length and secondly the phase diagram for this system had been
published. An example of the type of data obtained in the study is illustrated in Fig. 8. This
Fig. 8. Synchrotron X-ray scattering
intensity profiles in the region of the firstorder lamellar repeat recorded from a mixed
aqueous dispersion of 40 moles of
glucosylceramide per 100 moles of
dipalmitoylphosphatidylcholine during a
heating and subsequent cooling scan
between 25o and 85oC
shows synchrotron X-ray scattering intensity profiles of the first-order reflections recorded
from an aqueous dispersion of dipalmitoylphosphatidylcholine containing 40 moles of
glucosylceramide per 100 moles of phospholipid during a heating and cooling scan between
25o and 85oC. The Bragg peak can be deconvolved into two coexisting bilayer structures at
temperatures up to about 75oC when the glucosylceramide is transformed from a solid to a
fluid state and only a single bilayer structure is observed. There is a significant decrease in
lamellar repeat spacing associated with the transition of the pure phospholipid from a gel to a
fluid state at 41oC. Note that there is considerable hysteresis in relaxation of the mixture to
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an equilibrated state at 25oC. The bilayer structure designated Peak 2 in Fig. 8 is assigned on
the basis of lamellar repeat spacings and gel-fluid phase transition temperature as
dipalmitoylphosphatidylcholine. Calculations were performed to assign the molecular
species composition of the glucosylceramide-rich structure, designated Peak 1, and this was
found to consist of an equimolar proportion of the phospholipid and glycosphingolipid at
temperatures both above and below the phase transition temperature of the phospholipid. The
structure can be classed as a complex because it has a lamellar repeat spacing that differs
from either of the parent lipids and, because it dissociates at temperatures below the melting
temperature of the glycosphingolipid, it cannot be classed as a compound.
I have performed similar studies of mixtures of asymmetric glucosylceramide with symmetric
molecular species of different phospholipids and found that complexes are formed only under
specific conditions. With symmetric egg-sphingomyelin, for example, complexes are formed
Table 1. Mol PE per mol GlcCer in the
GlcCer-rich bilayer structure in binary mixtures
of the two lipids at T<5oTm and T>5oTm.
PE
Tm
(oC)
mol PE/ GlcCer
<Tm
>Tm
DLPE
43
0
0
DMPE
49
0
0.3
DPPE
64
0
0
DEPE
38
1.4
2.2
POPE
26
1.6
2.0
OPPE
36
1.4
2.1
SOPE
30
1.1
1.6
POPE-C12GlcCer
26
1.6
1.8
with a molar stoichiometry of 1:1 at temperatures below the gel to fluid transition
temperature of the phospholipid at 40oC but increase to 2:1; phospholipid:glycosphingolipid
at higher temperatures. A similar story is seen with phosphatidylethanolamines. A summary
of these findings is presented in Table 1. This shows that unsaturated molecular species of
phosphatidylethanolamine are immiscible with asymmetric glucosylceramide at any
temperature between 20o and 90oC. Complexes, however, are formed with unsaturated
molecular species irrespective of whether these bonds are in a cis or trans configuration or
are present on fatty acids esterified to the sn-1 or sn-2 position of the glycerol. Interestingly,
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complexes are also formed between glucosylceramide which has an N-acyl substituent of
only 12 carbons but so far a systematic study of these complexes has not been performed.
I will conclude this lecture with some perspective of how we might rationalize the functions
of the immense diversity of polar lipid molecular species that are revealed by current
lipidomic investigations. Firstly, there is a clear distinction between membrane lipids that
form bilayer structures under physiological conditions and those that do not. The role of nonbilayer forming lipids appears to be to package intrinsic membrane proteins into functional
complexes and to seal the irregular lipid-protein interface to the passage of solutes. The
thermotropic transition between bilayer and non-bilayer configuration of these lipids
proscribes the limits of stability of a functional membrane. Amongst the bilayer forming
lipids that I have been concerned with on this occasion there is a clear distinction between
membrane lipids which have hydrocarbon chains of similar length, which I have referred to
as symmetric lipids, and those with hydrocarbon chains of unequal length. It turns out that
the asymmetric lipids are mainly sphingolipids comprised of a sphingosine base of
comparable length to the fatty acyl residues of the glycerophospholipids and an N-acyl linked
fatty acid of 22-26 carbon atoms in length. I have presented evidence that some classes of
symmetric lipids are immiscible even in the fluid state whereas many mixtures of asymmetric
and symmetric lipids form complexes of defined stoichiometry.
There has been much discussion of the assembly of bilayer lipids into, so called, membrane
rafts. Indeed the current definition of rafts embodies the notion of domain size. As methods
for defining membrane domains improve with technological developments their size is
shrinking to the point where the concept of nanodomains consisting of ever fewer numbers of
lipids is now considered. From the results of the studies I have described it requires no leap
of imagination to conceptualize signalling complexes comprised of asymmetric sphingolipids
and particular lipid-anchored proteins. Several conditions must be satisfied for this model to
be plausible. Firstly, there must be specific interactions between asymmetric sphingolipids
and lipid-anchored proteins comprising the signalling complex. At this stage one may only
speculate that the carbohydrate moiety of the glycosphingolipid may play some part in this
process but at least it is a subject worthy of further investigation. Secondly, the complexes
comprising receptors in the exoplasmic side of the membrane must be in register with
complexes responsible for transducing the signal located in the cytoplasmic side of the
structure. Physical contact between the signalling elements is a crucial step in signal
transduction and the idea that this takes place through the properties of a lipid phase is
difficult to conceptualize.
The success with which marriages between receptors and effectors may be consummated and
annulled, for signals must be terminated as well as initiated, is built into the properties of
biological membranes. Considerable energy is thus consumed in uncoupling membrane
lipids by pumping specific lipids across the structure to ensure that the distribution of lipids is
asymmetric. Few asymmetric sphingolipids are located on the cytoplasmic side of the
membrane, glucosylceramide being the notable exception, and these will readily register with
asymmetric complexes in the exoplasmic side. The forces responsible for this coupling have
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yet to be explored but they have been convincingly demonstrated in the results described in
this lecture. Finally, biochemical turnover of the asymmetric glycosphingolipids orchestrated
from within the cytoplasm provides a means of strict control over the signalling process.
I believe that the abundant fruits of lipidomic investigations will only be fully exploited by
dedicated efforts to explain the diverse structural roles they play in the living cell. We need
to move beyond the description of lipid assemblies merely in terms of their thermotropic
phase behaviour and begin to look in molecular detail at the structures that are formed in
complex mixtures. The development of more sophisticated biophysical technologies will
doubtless lead us into these uncharted waters.
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