Lateral organization in Acholeplasma laidlawii lipid bilayermodels containing endogenous pyrene probes Patrik Storm1, Lu Li2, Paavo Kinnunen3,4and A˚ke Wieslander1 1 Department of Bioche
Trang 1Lateral organization in Acholeplasma laidlawii lipid bilayer
models containing endogenous pyrene probes
Patrik Storm1, Lu Li2, Paavo Kinnunen3,4and A˚ke Wieslander1
1
Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden;2Wallenberg Laboratory for
Cardiovascular Research, Go¨teborg University, Sweden;3Department of Medical Chemistry, Institute of Biomedicine,
Helsinki University, Finland;4Memphys – Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark
In membranes of the small prokaryote Acholeplasma
laid-lawiibilayer- and nonbilayer-prone glycolipids are major
species, similar to chloroplast membranes Enzymes of the
glucolipid pathway keep certain important packing
proper-ties of the bilayer in vivo, visualized especially as a monolayer
curvature stress (spontaneous curvature) Two key enzymes
depend in a cooperative fashion on substantial amounts of
the endogenous anionic lipid phosphatidylglycerol (PG)
for activity The lateral organization of five unsaturated
A laidlawiilipids was analyzed in liposome model bilayers
with the use of endogenously produced pyrene-lipid probes,
and extensive experimental designs Of all lipids analyzed,
PG especially promoted interactions with the precursor
diacylglycerol (DAG), as revealed from pyrene excimer ratio
(Ie/Im) responses Significant interactions were also recorded
within the major nonbilayer-prone monoglucosylDAG
(MGlcDAG) lipids The anionic precursor phosphatidic acid (PA) was without effects Hence, a heterogeneous lateral lipid organization was present in these liquid-crystalline bilayers The MGlcDAG synthase when binding at the PG bilayer interface, decreased acyl chain ordering (increase of membrane free volume) according to a bis-pyrene-lipid probe, but the enzyme did not influence the bulk lateral lipid organization as recorded from DAG or PG probes It is concluded that the concentration of the substrate DAG by
PG is beneficial for the MGlcDAG synthase, but that binding in a proper orientation/conformation seems most important for activity
Keywords: Acholeplasma; chemometrics; lipid heterogeneity; pyrene
Acholeplasma laidlawii A-EF22 is a simple cell-wall-less
prokaryotic parasite Its membrane lipid composition is
metabolically adjusted in response to environmental and
lipid-supply conditions Due to this, A laidlawii has been
used as a model system to study plasma membrane
properties and howthese are maintained by the lipid
synthesizing enzymes Membrane lipids are synthesized in
two competing pathways, both using phosphatidic acid (PA)
as a precursor, with one branch resulting in glucolipids and the other in phosphatidylglycerol (PG) as shown in the diagram below
At least five enzymes constitute the glucolipid pathway Phosphatidic acid phosphatase (PAP) makes diacylglycerol (DAG) from PA 1,2-diacylglycerol-3-glucosyltransferase
Correspondence to A˚ke Wieslander, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
Fax: + 46 8 15 36 79, Tel.: + 46 8 16 24 63, E-mail: ake@dbb.su.se
Abbreviations: bis-PyrPC, glycero-3-phosphatidylcholine; bis-PyrPG, 1,2-bis-[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phospho-rac-glycerol; CL, cardiolipin; 1,2-DOG, 1,2-dioleoylglycerol; DGlcDAG, 1,2-diacyl-3-O-[a- D
-glucopyranosyl-(1fi2)-O-a-D -glucopyranosyl]-sn-glycerol; MADGlcDAG, 1,2-diacyl-3-O-[a- D -glucopyranosyl-(1fi2)-O-(6-O-acyl-a- D -glucopyranosyl)]-sn-glycerol; MAMGlcDAG, 1,2-diacyl-3-O-[6-O-acyl(a- D -glucopyranosyl)]-sn-glycerol; MGlcDAG, 1,2-diacyl-3-O-(a- D -glucopyranosyl)-sn-glycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PD, pyrenedecanoic acid; PG, phosphatidylglycerol; PyrDAG, 1-palmioyl-2-pyrenedecanoyl-glycerol; PyrPA, 1-palmioyl-2-pyrenedecanoyl phosphatidic acid; PyrPG, 1-palmioyl-2-pyrenedecanoyl-phosphatidylglycerol.
Enzymes: 1,2-diacylglycerol-3-glucosyltransferase (MGlcDAG synthase; EC 2.4.1.157); 1,2-diacylglycerol-3-a-glucose (1fi2)-a-glucosyl transferase (DGlcDAG synthase; EC 2.4.1.208).
(Received 30 November 2002, revised 22 January 2003, accepted 19 February 2003)
Trang 2(MGlcDAG synthase) (EC 2.4.1.157; I above), makes
monoglucosyl diacylglycerol (MGlcDAG) from DAG plus
UDP-Glc 1,2-diacylglycerol-3-a-glucose (1fi2)-a-glucosyl
transferase (DGlcDAG synthase) (EC 2.4.1.208), II above,
makes diglucosyl diacylglycerol (DGlcDAG) from
MGlc-DAG and UDP-Glc Under certain circumstances, when
MGlcDAG turns bilayer-prone by saturated chains, the
more acylated and more nonbilayer-prone minor
glu-colipids, i.e MAMGlcDAG and MADGlcDAG, are
synthesized [1] Likewise, substantial amounts (20–30 mol/
100 mol) of the normally minor precursor 1,2-DAG may
accumulate in membranes with many saturated acyl chains [2]
It has been shown that the lipid composition is regulated
to maintain certain properties: (a) a balance between bilayer
and nonbilayer lipids (e.g MGlcDAG/DGlcDAG) yielding
phase equilibria close to a bilayer/nonbilayer transition; (b)
a certain surface charge density through the ratio between
the glucolipids (MGlcDAG, DGlcDAG and more acylated
variants) and the charged lipids (PG and the
phosphoryl-ated glucolipids) In vivo it has been shown that the ratio
between DGlcDAG and PG is nearly constant [3] The
regulation of these properties is sensed and performed by
the lipid synthesizing enzymes Each enzyme acts on a lipid
substrate with a specific headgroup, but are also sensitive to
the type of acyl chains and lipid composition in the
membrane [2]
MGlcDAG synthase (I) is activated by approximately
20 mol/100 mol PG or 10 mol/100 mol cardiolipin (CL),
but is not critically dependent on the nature of the
phosphate moiety and can be activated by other negatively
charged lipids, however, not as efficiently [4–7] The
activation by CL indicates no specificity for the PG
headgroup, but that the negatively charged phosphate is
important for the enzyme
DGlcDAG synthase (II) is activated by PG or CL in the
same way as MGlcDAG synthase, but also by other
phosphate-containing species such as certain metabolites
and dsDNA [8] However, PG is the strongest activator
among the naturally occurring lipids (strain A-EF22 does
not make CL) As for the MGlcDAG synthase, this process
is cooperative with respect to PG amounts and has a fairly
high Hill coefficient (4–6 for MGlcDAG synthase and 3–7
for DGlcDAG synthase) [4] Substrate fractions of
MGlc-DAG up to five mol/100 mol raise the activity, which then
levels out, most likely due to saturation of the active site [8]
More DGlcDAG is made from MGlcDAG in membranes
with more unsaturated or longer acyl chains, increased
temperature or increased amount of cholesterol The shift in
this lipid ratio stems from the more pronounced nonlamellar
tendency of the membrane, compensated by making more
DGlcDAG (from the nonbilayer prone MGlcDAG) This
curvature sensitivity implies a sensing mechanism of
mem-brane perturbation of nonbilayer-prone lipids Analogous
sensing features have been proposed for
CTP:phosphocho-line cytidylyltransferase [9–11] or protein kinase C [12]
Could lipids adopting a heterogeneous lateral distribution
have a bearing on the activity of the A laidlawii
glucosyl-transferases, in that substrates or surface charges become
locally concentrated? Mammalian plasma membranes show
transverse and lateral asymmetry In the outer leaflet, rafts
can form by the tight packing of saturated
glycosphingo-lipids and cholesterol in a L phase, possible to isolate
[13–16] The biological function seems to be enrichment of certain proteins, e.g doubly acylated or GPI anchored in the rafts [17–21] involved in signaling and transport over the membrane In the inner leaflet, lateral heterogeneity can form with phosphatidylserine and diacylglycerol, activating protein kinase C [22] In this respect DAG is special in the interspacing, dehydration and altering conformation of lipid headgroups, as well as conferring a nonbilayer propensity for the membrane [23]
The reason for lateral heterogeneity is preferential interaction between headgroups (Coulombic forces, hydro-gen bonding, divalent cations, hydration level) or acyl chains (London forces) of certain lipids [24,25] It is known that acyl hydrocarbon chain mismatch can cause lateral segregation, either by the length or the degree of saturation [5,26–30] Indeed, stability of rafts demands a critical mismatch, as POPE (1-palmitoyl-2-oleoyl-sn-glycerophos-phatidylethanolamine) but not PDPE (1-palmitoyl-2-docosahexanoyl-sn-glycerophosphatidylethanolamine) mix with raft lipids [31] In vivo lipid mixtures from Micrococcus luteus and A laidlawii, both containing gly-colipids and PG, reveal interactions between the individual lipids in monolayer experiments [32,33] Analogous features are also recorded for plant galactolipids The importance of the glycolipids for these properties are highlighted by the lower lateral diffusion for A laidlawii in vivo glucolipids compared to the E coli phospholipids [34]
To investigate whether lateral heterogeneity exists in the fluid glucolipid-rich membrane of A laidlawii A-EF22 as a function of headgroup composition, liposomes were made where composition of five different lipids (major lipids in the membrane of A laidlawii A-EF22), all with di-18:1c acyl chains, was varied according to a chemometrical experi-mental design Pyrene-derivatives of the same lipids, inclu-ding endogenous major glucolipids synthesized by
A laidlawii, were used as fluorescent probes A potential influence on the MGlcDAG synthase, the first regulating enzyme in the glucolipid pathway, was also investigated
Materials and methods
Lipids and probes MGlcDAG and DGlcDAG were prepared from A laidla-wii cells grown in a lipid-depleted medium supplemented with oleic acid [35] 1,2-dioleoylglycerol (1,2-DOG) was pur-chased from Larodan (Malmo¨, Sweden) Phosphatidylgly-cerol (PG) was purchased from Avanti polar Lipids (USA) Pyrenedecanoic (PD) acid, 1-palmitoyl-2-pyrenedecanoyl-phosphatidylglycerol (PyrPG) and 1,2-bis-[10-(pyren-1-yl)] decanoyl-sn-glycero-3-phosphatidylcholine (bis-PyrPC) was purchased from Molecular Probes Inc (Oregon, USA) 1-palmioyl-2-pyrenedecanoyl-glycerol (PyrDAG) and 1,2-bis-[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phospho-rac-glycerol (bis-PyrPG) and 1-palmioyl-2-pyrenedecanoyl phosphatidic acid (PyrPA) were from KKV Bioware (Espos, Finland)
Organism and growth conditions
A laidlawiistrain A-EF22 was grown at 30C in a lipid-depleted tryptose/bovine serum albumin medium [36] The
Trang 3fatty acids, oleic (18:1c) and palmitic (16:0) were
supple-mented from sterile ethanol stock solutions and
pyrene-decanoyl (PD) acid was supplemented from sterile
dimethyl sulfoxide stock solution Total concentration of
fatty acids was 150 lMin the growth medium Fatty acids
were radiolabeled with 10 lCiÆL)1 [14C]palmitic and
100 lCiÆL)1[3H]oleic acid (Amersham Pharmacia Biotech,
Uppsala, Sweden), respectively, after four consecutive
inoculations 2-hydroxy-propyl-b-cyclodextrin (10 mM)
was used in the medium as a carrier for the PD acid
[37] Cell growth was monitored by absorbance and by
phase contrast light microscopy Contamination by any
other bacteria was analyzed on standard bacteriological
agar plates
Extraction and analysis of lipids
Cells were harvested by centrifugation, washed twice in
buffer, and frozen at )80 C Membrane lipids were
extracted from the cell pellets using chloroform/methanol
(2 : 1, v/v)
One-dimensional thin layer chromatography (TLC) was
used to separate and characterize the different lipids in the
membrane The TLC plates coated with silica gel 60
(Merck, Darmstadt, Germany) were developed in
chloro-form/methanol/water (80 : 25 : 4, v/v/v) [14C]-labeled
lipids were visualized with electronic autoradiography
(Packard Instant Imager) Excised gel lipid spots were
digested in Soluene-350 (Packed) for 30 min at 37C and
quantified by double-channel liquid scintillation counting
To purify the pyrenyl lipids, the TLC plate was developed
first in chloroform/methanol/water (80 : 25 : 4, v/v/v) and
then in chloroform/methanol/ammonia (91 : 35 : 10, v/v/
v) Compared with a one-dimensionally developed TLC
plate of extracted lipids from medium 18:1c/PD
120 lM: 30 lM, the spots of pyrenyl lipids could be
separated better and become more concentrated in two
dimensions Excised gel spots of pure pyrenyl glucolipids
(MGlcDAG and DGlcDAG) were extracted by
chloro-form/methanol (2 : 1, v/v), and typical fluorescence
spec-tra of mono-pyrenyl and bis-pyrenyl glucolipids visualized
(Fig 2B,C)
Incorporation of PD and synthesis of pyrenyl
glucolipidsin vivo
No growth of A laidlawii could be observed with only 16:0
or PD, separately or together (Table 1) The presence of
18:1c fatty acid was very important for both the growth of
A laidlawiiand the incorporation of PD into pyrenyl lipids However, the yield of pyrenyl lipids was quite low (less than 10% on the basis of added fatty acids) compared to the nonpyrenyl lipids (30%)40%), but incorporation into MGlcDAG and DGlcDAG was fairly similar
The yield of nonpyrenyl MGlcDAG from A laidlawii strain A-EF22 was much lower than that of nonpyrenyl DGlcDAG when PD in growth media; revealed by quantitation of nonpyrenyl lipids from excised gel spots (data not shown)
In the same membrane 18:1c fatty acid preferred to incorporate PD acid to produce mono-pyrenyl glucolipid rather than nonpyrenyl glucolipid, whereas 16:0 dominated
in the latter (data not shown) The Ie/Im ratio from the fluorescence spectra increased for the extracted lipid mixture from cells when increasing the PD ratio in the medium, showing that more bis-pyrenyl lipids were synthesized at higher PD acid to fatty acid ratios (data not shown) The yield of synthesized pyrenyl glucolipids was determined from standard fluorescence intensity curves, obtained from synthetic PyrDAG and bis-PyrPG
Enzymes and assays Mixed lipid micelles were made by swelling dry lipid to a final concentration of 10 mM(1 mMsubstrate) in a buffer of
110 mMTris pH 8, 22 mMChaps, 22 mMMg2+ Purified MGlcDAG synthase (50 lL) or DGlcDAG synthase was incubated with 40 lL lipid micelles at 4C for 30 min The enzyme reaction was started by adding 10 lL of 10 mM (0.5 CiÆmol)1) UDP-[14C]glucose The reactions were ter-minated by the addition of 375 lL methanol/chloroform (2 : 1, v/v) Synthesized MGlcDAG or DGlcDAG was extracted according to a modified Bligh and Dyer method [38] and separated from other lipids by TLC The 14 C-labeled glucolipid products were quantified using electronic autoradiography (Packard Instant Imager) Homogeneous MGlcDAG synthase for liposome binding was purified from detergent-solubilized A laidlawii cells by three column chromatography methods, including ion exchange, gel filtration and hydroxyapatite chromatography [39] Experimental design
Chemometrics is howto design an experimental series in order to extract the maximum information from the minimum number of experiments [40] Chosen factors are varied simultaneously in a randomized run order to reduce
or eliminate unknown or uncontrolled influence on data
Table 1 Incorporation of PD and yield of A laidlawii pyrenyl lipids.
% Incorporation
of PD into lipids
Trang 4The response(s) Y is then fitted to the variables by a
mathematical model, e.g Y¼ m + Xb + e; w ere X is the
model terms/variables, b is the coefficient of effect and e is
the residuals
We have used the MODDE 3.0 package (Umetri AB,
Umea˚, Sweden) Here, variables were changed from low to
high and the response was plotted and analyzed in the
computer to give a measure of effects Variables in this case
are the amounts of different lipid headgroups (as all acyl
chains are 18:1c) and the amount of MGlcDAG synthase
Responses are excimer formation of pyrene-labeled
phos-pholipid or anisotropy of diphenylhexatriene (DPH)
DGlcDAG, considered the matrix lipid, was set as a filler
and a full factorial design was chosen In the simple case
of three variables (dimensions, factors), a full factorial
design is a cube in the experimental space, where data points
are in the corners and center of the cube (Fig 1), resulting
in a linear interaction model In a couple of cases the
investigation was expanded to a response surface model,
i.e composite face-centered (CCF) design, where the
design-cube also has data points on the face of the sides, making
quadratic models possible to obtain For the investigation
of chain ordering for pure lipids a mixture (D-optimal)
design was chosen, where matrix lipid DGlcDAG is not set
as a filler
Partial least squares (PLS) was used to fit the model
PLS finds the relationship between a matrix Y (response
variables) and a matrix X (model terms) Measure of model
fit is R2¼ 1) (RSS/YSS), where RSS is residual sum of
squares and YSS is the response sum of squares Internal
validation (crossvalidation or prediction ability) is measured
by the Q2value, i.e Q2¼ 1) (PRESS/YSS), where PRESS
is the predicted residual sum of squares Rules of thumb are
that R2should be at least 0.8 and Q2above 0.3 for linear
models and even closer to one for quadratic models R2and
Q2are the overall parameters for model accuracy, which
encompass analysis of variance (ANOVA), lack of fit, normal
distribution of residuals If these parameters are not
satisfactory, then outliers, wrong metric, inhomogeneous
data, range of the factors, etc., need to be investigated The
fitted model can then be presented as a response surface
(Fig 1B), a curve or a table
Furthermore, an important aspect of experimental design
is that interaction effects can be detected; this would not be possible if only one variable at a time was changed Inter-action means that the response of a variable is dependent on the level of another variable in a nonadditive fashion Preparation of large unilamellar vesicles
For each sample 0.25 lmol of total lipid was mixed to the desired composition according to the experimental design, where DOPG was varied 0–40 mol/100 mol, DOPA 0–10 mol/100 mol, MGlcDAG 0–30 mol/100 mol, DOG 0–10 mol/100 mol, and DGlcDAG was used as the (bal-ance) matrix The content of fluorescence probe was constant at 1 mol/100 mol for mono-pyrenyl lipids, and 0.5 mol/100 mol for bis-pyrenyl lipids or DPH The mixture was then dried under a nitrogen flow and then under reduced pressure (vacuum) overnight The resulting lipid film was hydrated with intermittent vortexing during 45 min
in filtered and deoxygenated 10 mM Hepes pH 8.0 with
5 mM MgCl2, and then extruded with a LiposoFast Basic extruder (Avestin Inc., Canada) 19 times through two stacked polycarbonate filters (Millipore; pore diameter
100 nm) This yields large unilamellar vesicles (LUV) with
an average diameter of nearly 100 nm [41] The quality of vesicles for all data points was verified with dithionite quenching of an NBD-probe, showing that all vesicles were LUVs, as only the outer leaflet is quenched and roughly 50% of the signal remained after quenching (data not shown)
Fluorescence and absorbance methods Absorbance measurements were performed with a Beckman
DU 70 spectrophotometer
Fluorescence measurements with labeled vesicles were carried out so that 50 lL of prepared liposomes were added
to 1950 lL buffer in an optical 1· 1 cm fluorescence cuvette, and fluorescence measured with a Spex Fluoro-Max-2 fluorometer with magnetic stirrer and temperature control (28C) Samples with pyrene probes, were excited at
344 nm and emission spectra collected between 360 and
500 nm Slits had a bandwidth of 1 nm for excitation and
4 nm for emission (step width 1 nm, integration time 0.5 s) Four scans were sampled, averaged, and subtracted by a blank consisting of the buffer, in order to obtain the fluorescence curve Vesicles without a probe do not particularly affect the spectra, as verified in a control design showing only noise that was virtually the same as the blank; therefore no such reference was used in any of the runs Ie/Im (excimer ratio) was calculated as the ratio between excimer emission at 480 nm (Ie), when two pyrenes are
in close proximity ( 3.5 A˚), and monomer emission at
398 nm (Im) Enzyme (MGlcDAG synthase) was incubated with 50 lL liposomes (protein : lipid 1 : 700–1 : 70) on ice for 30 min prior to measurement at room temperature in a
1· 0.2 cm quartz fluorescence cuvette using a Perkin-Elmer LB50 spectrofluorimeter
DPH anisotropy [42,43] was analyzed using a Spex Fluorolog 12 fluorometer (Department of Biophysical Chemistry, Umea˚ University), where bandwidths were 3.6 nm for excitation and 7.2 nm for emission Sample
Fig 1 Experimental design (A) Full factorial design cube with
cen-terpoint Variables, e.g lipids, are changed from lowto high amounts.
(B) Example of a response surface plot showing response variation
when varying two variables The purpose of the design is to extract
maximum information from a minimum number of experiments.
Trang 5solution was equilibrated for five minutes in the cuvette
holder (no magnetic stirrer) to reach a temperature of 28C
prior to measurement Absorption at the excitation
wave-length was less than 0.09, thus a minimal reabsorption
Anisotropy r¼ (II) I^)/(II+ 2I^) for each datapoint was
calculated and averaged in connection to the measurement
by a computer program
Results
Enzymes recognize pyrene derivatives
For analysis of potential interactions between the various
A laidlawiimembrane lipids we chose fluorescence
spectro-scopy, with pyrene-labeled lipids containing one normal and
one pyrene-labeled chain as proximity probes, as the studied
phenomena may be transient and not possible to isolate,
and too small (<300 nm) to be detected with microscopy
Pyrene-decanoyl chains locate in the membrane
hydro-phobic core and are virtually nonperturbing at a fraction of
1 mol/100 mol or less, partitioning preferentially in the fluid
phase [44–49] To investigate chain ordering, i.e membrane
free volume (Vf), a bis-pyrenyl lipid, with a pyrene on both
acyl chains, was used [47,50]
Native A laidlawii pyrene-labeled glucolipids (not
com-mercially available) were produced in vivo, and tested as
lipid enzyme substrates in vitro to monitor the impact of the
pyrenyl chain moiety on headgroup organization
Pyrenyl-decanoic acid (PD) was used for the incorporation of the
pyrene group into the lipids 16:0 and 18:1c fatty acid were
chosen for their approximately similar chain length to PD
Different compositions of growth medium fatty acids were
used to optimize the incorporation of PD acid, with or
without 16:0 and 18:1c, into the glucolipids (Table 1) Cell
growth and size of cells were checked by routine light
microscopy PD or 16:0 could not support growth, alone or
in combination A laidlawii cells became much bigger and
less aggregated, and the density of the culture became
low er, w hen the ratio of PD in fatty acids w as increased
One-dimensional thin layer chromatography developed in
chloroform/methanol/water (80 : 25 : 4, v/v/v) was used to
characterize the lipids extracted from the cells (Fig 2) The
Rfvalues of different lipids on a TLC plate were compared
according to the standard samples characterized by NMR
[1,51] Fluorescent spots (under UV light) are marked by
rings in Fig 2A Combined with the data from radiolabel
analysis, it is obvious that without a pyrene group in the
hydrocarbon chain of the lipid, there was no fluorescence
With one PD acyl chain and the other chain 16:0 or 18:1c,
as in mono-pyrenyl lipids, both fluorescence and isotope
signals were detected (data not shown) With two pyrenyl
chains, only fluorescence but no isotope signal could be
detected from the spot of the bis-pyrene lipid on the TLC
plate (Fig 2A) Note that mono-pyrenyl lipid migrated a
little further than the nonpyrenyl lipid, and bis-pyrenyl
lipid migrated even further, as expected from the larger
hydrocarbon regions of the pyrenyl-containing lipids
(Fig 2A)
Purified MGlcDAG synthase and partially purified
DGlcDAG synthase were used to study the potential
disturbance of the polar headgroup organization by the
purified pyrenyl-labeled glucolipids in vitro (Fig 3) The
enzymatic products, pyrenyl-MGlcDAG or pyrenyl-DGlc-DAG, from the in vitro enzyme reactions, were extracted from TLC plates Similar yields were obtained for pyrenyl-glucolipid and nonpyrenyl-pyrenyl-glucolipid products, from both the MGlcDAG synthase and DGlcDAG synthase reactions (Fig 3) Furthermore, the shape of the fluorescence spectra
of the product depends on which type of pyrenyl lipid was used as substrate; mono- and bis-pyrenyl lipid substrate produced mono- or bis-pyrenyl glucolipid products, respectively (data not shown) Thus, these enzymes do not discriminate between substrates with a pyrene moiety in the acyl chain Similar features have been observed for enzymes
Fig 2 In vivo synthesis of pyrenyl lipids (A) A laidlawii 14C/ 3 H-labeled glucolipids and pyrenyl-glucolipids after TLC separation Extracted lipids applied on TLC plates were developed in chloroform/ methanol/ammonia (91 : 35 : 10, v/v/v) The growth medium fatty acid composition (ratio 16:0/18:1c/PD) was from left to right:
120 : 30 : 0; 90 : 30 : 30; 30 : 30 : 90; 0 : 60 : 90; 0 : 30 : 120 and
0 : 10 : 140 Encircled spots represent the fluorescent mono-pyrenylMGlcDAG (lower) and bis-mono-pyrenylMGlcDAG (upper), respectively The fluorescence spectra of purified mono-pyrenyl (B) and bis-pyrenyl glucolipids (C) produced in vivo The samples (0.1 m M
lipid) were excited at 344 nm in chloroform/methanol (2 : 1, v/v) Synthetic mono-pyrenylDAG (B), and bis-pyrenylPG (C), were used
as references.
Fig 3 Pyrenyl lipids as enzyme substrates in vitro Synthesis of DGlcDAG from MGlcDAG and UDP-[14C]glucose by purified DGlcDAG synthase The contents of pyrenyl glucolipids were less than 1% (mol/mol) (s) [ 14 C]DGlcDAG produced from di-18:1c-MGlcDAG; (m) pyrenyl DGlcDAG produced from mono-pyrenyl MGlcDAG; and (d) bis-mono-pyrenyl DGlcDAG produced from bis-pyrenyl MGlcDAG, respectively, by the DGlcDAG synthase.
Trang 6acting on phosphatidylinositol lipids [52] This seems logical
in that the MGlcDAG synthase is attached to the
membrane interface [7] and does not recognize the acyl
chain region close to the bilayer center, where the pyrene
moiety is
Lipid organization as seen with pyrene derivatives
Potential interactions between the A laidlawii membrane
lipids were analyzed in liposome bilayer models containing
various pyrene-labeled probes of synthetic and in vivo
origin Excimer formation (Materials and Methods) for
lipids with one pyrene-acyl chain is an intermolecular event,
depending on the collision rate, and Ie/Im hence monitor
lateral mobility and concentration of these molecules A
series of full factorial designs were made, where the
compositions (87 conditions in total) were varied to cover
the limits occurring in vivo for the five important lipids of the
glucolipid pathway in the membrane of A laidlawii (PG,
PA, DAG, MGlcDAG and DGlcDAG) In in vitro bilayer
(liposome) models, the lipids adopted a heterogeneous
organization, as was seen with excimer formation of the
pyrene-labeled probes for each lipid type Table 2 lists lipid
composition and the statistically significant changes in Ie/Im
(the excimer ratio) for different pyrene derivatives as a
function of different headgroups, going from a lowmole
content to a high mole content for each lipid, and where all
acyl chains are 18:1c (dioleoyl) In two cases this
investiga-tion was expanded to a composite face-centered (CCF)
design
The distribution of DAG, as monitored by Ie/Im of the
PyrDAG probe (Table 2) was affected significantly by PG
and DAG (increased Ie/Im), but none of the other lipids,
according to a CCF design Interestingly, the model also
revealed a dependence (interaction) between the variables
PG and DAG (data not shown), i.e increasing both PG and
DAG does not increase the Ie/Im additively In accordance
with this model a one-variable titration, where DOPG was
varied 0–40 mol/100 mol (at constant 5 mol/100 mol
DOPA, 5 mol/100 mol DOG and DGlcDAG as balance),
showed a 1.5-fold increase in Ie/Im (0.080–0.124) for the
PyrDAG probe between 0 and 25 mol/100 mol PG, and
then a decrease to 0.105 between 25 and 40 mol/100 mol
PG (Fig 4) We therefore conclude that DAG has a
heterogeneous distribution, strongly promoted by
increas-ing amounts of PG It is interestincreas-ing to note that increasincreas-ing
the amount of CL (0–20 mol/100 mol) in a DGlcDAG
matrix with 5 mol/100 mol DOG and 1 mol/100 mol PyrDAG, did not produce any change in the Ie/Im (data not shown)
For the PyrPG probe Ie/Im increased 1.35-fold (0.051– 0.069) between 0 and 40% PG (Table 2) It has been noted before that decreasing amounts of DGlcDAG (the balance here) upon increasing PG, increased the collision rate between pyrenes [5] All other lipids had an insignificant effect on excimer formation As a comparison, increasing
PG amounts in a matrix with DGlcDAG did not change the
Fig 4 Glucosyltransferases and lipid organization (A) Response of pyrene-derivatives of activator PG and substrate DOG lipids, and order-sensing bis-PyrPC, upon increase in the DOPG content (B) Normalized enzyme activities (adapted from Dahlqvist et al [3]) of the MGlcDAG and DGlcDAG synthases.
Table 2 Lateral interactions between A laidlawii lipids in liposome bilayers Changes in Ie/Im of pyrene probes upon variation in lipid amounts according to a factorial design or, in the case of PyrDAG and PyrPA probes, to a composite face-centered design (Materials and methods) The balance (matrix) in the various lipid mixtures was always DGlcDAG Only statistically significant changes are shown NT, not tested R 2 and Q 2 are measures of model fit and vary between 0 and 1 (Materials and methods).
Probe
MGlcDAG
0–30 mol/100 mol
DAG 0–10 mol/100 mol
PG 0–40 mol/100 mol
PA 0–10 mol/100 mol
Replicate error (Ie/Im) R 2 /Q 2
PyrDAG – 0.078–0.087 (11.5%) 0.078–0.110 (41%) – ± 0.0017 0.97/0.72
PyrPA 0.058–0.062 (7%) 0.058–0.064 (10%) – – ± 0.0027 0.90/0.51
PyrPC 0.064–0.068 (6%) 0.064–0.065 (1.5%) 0.064–0.073 (14%) NT ± 0.0029 0.94/0.42
Trang 7excimer ratio for pyrene-labeled DGlcDAG, Ie/Im w as
always approximately 0.06 As the PyrPG signal increased
during these conditions (Table 2, Fig 4), this may indicate
affinity between like molecules for these two lipids
The Ie/Im for pyrene probes of MGlcDAG and PA were
not changed when increasing the amount of the enzyme
activator lipid PG (Table 2), indicating no interactions
However, increasing the amounts of normal MGlcDAG
gave an increase in Ie/Im for PyrMGlcDAG probe
(Table 2), supporting an interaction between the
MGlc-DAG molecules (note that decreasing DGlcMGlc-DAG amounts
was the balance) Also, headgroup interaction for
MGlc-DAG lipids is indicated in a monolayer study, showing a
much more compact fluid state than the equivalent
phosphatidylcholine (PC) lipid [53] For PA, precursor to
both the glucolipid and phospholipid pathways, an increase
in Ie/Im for PyrPA probe was also observed when
increasing the amount of DAG or MGlcDAG (Table 2)
in a CCF design, supporting a heterogeneous distribution,
however, the effect was small Note that there are only a few
mol percent of this lipid naturally occurring in the
membrane A small effect was also seen with PyrPC as a
probe (Table 2), probably reflecting exclusion from the
domains formed
Bilayer chain ordering
A starting point is the response at a homogenous
distribu-tion of probe in the membrane, as is the case for
1 mol/100 mol PyrPC in a matrix of DOPC [46] This gave
an Ie/Im of 0.07 in PC-matrix and 0.06 in
DGlcDAG-matrix (data not shown) The difference may reflect ease
of diffusion Diffusion is also indirectly related to chain
ordering in the membrane This property decreased with
increasing content of DGlcDAG and increased with
increasing content of DOPG or DOG when measured
with a bis-PyrPC probe For bis-pyrenyl lipid probes the
Ie/Im reflects an intramolecular event, where an increase
corresponds to increased chain-chain contacts (collision
rates) The Ie/Im was 1.4 at 40 mol/100 mol DOPG,
and 1.05 at 0 mol/100 mol DOPG (Table 3), indicating
a decreased Vf by increased PG (or increased Vf by
DGlcDAG) A complete inverse of this property was
indicated w hen measured w ith DPH (Table 3) Steady state
anisotropy r for DPH was between 0.10 at high DOPG (low
DGlcDAG) content and 0.14 at lowDOPG content (high DGlcDAG) DOG had the same effect as DOPG, although the smaller fraction in the membrane made its effect less pronounced This has been observed before [26] and was addressed to chain splaying motions of the bis-PyrPC The location of pyrene has nevertheless been determined to be located close to the bilayer center MGlcDAG, shown to increase order [54], is organized laterally in this five-lipid model system in a way that gives no significant contribution
to the ordering of the membrane bilayer, according to the experimental design model
Enzyme binding, lateral organization and chain order The MGlcDAG synthase (purified without detergent [55]), when binding to the lipid bilayers at initial protein : lipid-ratios of 1 : 700, 1 : 350 or 1 : 70 (mol/mol), did not affect the Ie/Im of PyrDAG or PyrPG (data not shown) Thus, the substrate lipid DAG and the activator lipid PG were not concentrated on a large scale by the enzyme Yet, binding and activity with liposomes, as here, was strongly correlated with increasing amounts of anionic lipid ([55] and Li et al submitted), with a Hill coefficient of 4–6, which is the potential number of PG molecules associated to the enzyme However, the number of lipid molecules bound under an enzyme makes it very unlikely that these be two probe molecules (1 mol/100 mol concentration), even at the highest enzyme-lipid ratio Binding was practically irrever-sible and more enzymes bound to liposomes as a function of mol/100 mol anionic lipid (promoted by nonbilayer lipid), revealed by surface plasmon resonance experiments using Biacore (Li et al submitted) Furthermore, the Ie/Im from a bis-PyrPC probe was reduced by the addition of enzyme,
as seen in a design (R2¼ 0.93, Q2¼ 0.41) with LUVs composed of DOPG (0–40%), CL (0–20%), DAG (0– 10%), bis-PyrPC (0.1%) and DGlcDOG as balance, and
1 : 700–1 : 70 (mol/mol) enzyme : lipid (Fig 5) PG and DAG increase the order, as seen in Table 3, as do CL There was also synergism between PG and the enzyme, and antagonism between DAG and enzyme, with respect to chain-ordering effects (Fig 5) Hence, interfacial binding of fairly large amounts of the MGlcDAG enzyme reduced chain order (increased Vf) but did not detectably change the lateral distribution of the A laidlawii A-EF22 polar lipid species
Table 3 Lipid composition and chain ordering Excimer formation (Ie/Im) and anisotropy (r) were monitored by bis-PyrPC and DPH, respectively.
A mixture design was made (Materials and methods) where DOPG 0–40 mol/100 mol, DODAG 0–10 mol/100 mol, DODGlcDAG 40–90 mol/
100 mol (40–99 mol/100 mol with DPH as a probe), DOMGlcDAG 0–30 mol/100 mol, DOPA 0–10 mol/100 mol Only lipids with a significant effect are shown, with variables from the modeled data The model is linear with R2¼ 0.95 and Q 2
¼ 0.91 for bis-PyrPC as the probe, and
R 2
¼ 0.89 and Q 2
¼ 0.79 for DPH as the probe.
Trang 8Synthesis of pyrenyl glucolipid
The existence of 18:1c fatty acid in the medium is very
important for both cell growth and the incorporation of PD
into glucolipids (Materials and Methods) A higher ratio of
PD in the growth medium produces more bis-pyrenyl
glucolipids, as A laidlawii must choose PD as the side
chain, as there are not enough of the other fatty acids in the
medium However, the cell cannot grow well with a high PD
ratio In vivo it might be difficult to incorporate PD acid into
the precursor PA as the occupied volume and
hydropho-bicity are larger than for 18:1 or 16:0 fatty acids, resulting in
lowyields of pyrenyl lipids In our experiment, less than
10% of PD could be incorporated into lipids Media with a
30 : 120 ratio of 18:1c to PD is appropriate to obtain a
reasonable yield of mono-pyrenyl and bis-pyrenyl
gluco-lipids In vitro, using PyrDAG, purified mono-pyrenyl
MGlcDAG or bis-pyrenyl together with extra large
amounts of nonpyrenyl lipids as substrates, the product
yields were approximately the same for these two glucolipid
synthases (Fig 2A) This suggests that the enzymes do not
discriminate between pyrenyl lipid and nonpyrenyl lipid in a
micelle system, which makes pyrenyl-glucolipid probes
possible to use in the study of metabolic stages of glucolipids
and biophysical properties of membranes This is also
the first time mono- and bis-pyrenyl glucolipids from
A laidlawiihave been synthesized and purified
Lateral organization ofA laidlawii lipids
The metabolism of glucolipids in A laidlawii depends on
several factors such as growth temperature, presence of
foreign molecules, and unsaturation and length of the
fatty acids [2,3,56] Not all lipids adopt a homogeneous
distribution in liquid-crystalline liposome model membranes
with A laidlawii lipids having 18:1c acyl chains, as indicated
from the present investigation The most prominent effect
was for DAG when increasing the molar fraction of PG
There was a good agreement between results from the
factorial design models (Table 2) and a one-variable
titra-tion showing (at most) a 1.5-fold increase in the excimer
ratio (Ie/Im) (Fig 4) Similar increase in Ie/Im was observed when ceramide (DAG analogue) was enzymatically split from sphingomyelin and forming microdomains [57,58], or the patching of DOPG in liquid-crystalline PC due to a hydrophobic (chain length) mismatch [5] This is most probably not due to an increased diffusion in the mem-brane Given an excited state lifetime of 100 ns for pyrene and diffusion coefficient of lipids approximately 5· 10)8
cm2Æs)1, the pyrene derivative move at most two lipid diameters and should not form excited dimers unless already being close [59] This is supported by PyrPC showing a small change in Ie/Im (Table 2) and by the ordering of lipids seen with bis-PyrPC (Table 3) Indeed, total lipid extracts from A laidlawii grow n at tw o different growth temperatures, thereby containing different lipid compositions, showa fairly similar lateral diffusion coeffi-cient [34] An interesting observation is that anisotropy of DPH is decreasing when Ie/Im of bis-PyrPC is increasing (Table 3) However, Ie/Im increases when cholesterol is included in the alloys with sugar lipids (data not shown), giving reason to believe that bis-PyrPC gives a good indication of the fluidity (free volume) in the membrane No further investigation was made into this phenomenon, but hypothesizing that it may have to do with the matrix of sugar lipids with 18:1c acyl chains, as a report by Kaiser and London [60] states that DPH is located close to bilayer center in DOPC bilayers
Thus, DAG is segregated in what seems like micro-domains by increasing the amount of PG in the membrane, possibly due to favorable hydrogen bonding between DAG and PG This is not due to charge entirely, as PA had
no appreciable effect on the PyrDAG-probe response The decrease in Ie/Im after 25 mol/100 mol PG (Fig 4) can be that a microdomain formed preferably by the DAG lipid is diluted DAG is a special lipid, as pointed out in a reviewby Goni and Alonso [23] Constituting only a small fraction of membranes, it can act as an intracellular second messenger
or metabolic intermediate and is involved in enzyme modulation, membrane fusion and membrane physical properties For membrane physical properties, unsaturated DAG imposes no phase separation at lowmolar fractions but does increase the chain order in an unsaturated phosphatidylcholine membrane This ordering was also
Fig 5 Enzyme binding and chain ordering Plot showing the effects of varying amount of MGlcDAG synthase (MGS) and three different lipids in LUVs with DGlcDAG as balance, going from low to high amount, and 0.1 mol/100 mol bis-PyrPC as probe (computed (R2¼ 0.93, Q 2
¼ 0.41) in
MODDE 3.0) Range: CL 0–20 mol/100 mol, DOG 0–15 mol/100 mol, DOPG 0–40 mol/100 mol and MGS/Lipid 1 : 700–1 : 70 (mol/mol) Interaction effects between pairs of variables are also plotted, where positive value means synergism and negative value antagonism The effect in Ie/Im is the sum of the response difference between high and low variable levels divided by two Effects for which error bars encompass zero are insignificant.
Trang 9observed in the system investigated here (Table 3 and
Fig 5) DAG also increases the spacing between
phospho-lipid (or glucophospho-lipid) headgroups, with its hydroxyl proton
participating in hydrogen bonding This is part of the reason
why ceramide assemble laterally when enzymatically
released from sphingomyelin [58,61] Hydrogen bonding
in the lipid interface plays a role in the interaction between
sugar headgroups, where a subtle difference as between
galactose and glucose may be important [62] For other lipid
species, modulation by divalent cations (in our case Mg2+)
coordinating negatively charged lipids and decreasing the
headgroup repulsion, is also important PG, which has a
flexible glycerol moiety in the headgroup, is shown to take
part in hydrogen bonding [63,64] and it has been noticed
that PG and dimannosyl-DAG is heterogeneously
distri-buted, due to interactions in the headgroup region [65]
Furthermore, lipids in total lipid mixtures from A laidlawii
have a smaller interfacial area than any of the individual
lipids [33,65], due to interactions in the headgroup region
causing a lateral condensation An interaction between
MGlcDAG or DGlcDAG species was revealed here from
the responses of the corresponding pyrene probes (Table 2
and Results above)
The patching of substrate lipid DAG with activator PG
(Fig 4), in combination with an increased charge density
creating an electrostatic interaction between the membrane
and the enzyme (L Li, unpublished observation), contribute
to the explanation of the anionic lipid preference for the
MGlcDAG synthase An increase in acyl chain order also
precedes enzyme activity (bis-PyrPC signal in Fig 4) For
the DGlcDAG synthase the patching of its substrate
MGlcDAG when the amount increases (Table 2) seems
not biologically relevant as PG is the only naturally
occurring activator, and that 3 mol/100 mol MGlcDAG
saturates the DGlcDAG synthase [8] However, the order
increase probably contributes to the activity (Fig 4) With
respect to the precursor PA, the PyrPA showan
addi-tively increased Ie/Im by DAG and MGlcDAG (Table 2),
but the biological function is less clear as the PA
phospha-tase is not regulated by changes in lipid composition [66]
In conclusion, this is the first time that up to five lipids
have been varied simultaneously in vitro and that a
fluorescent glucolipid probe has been synthesized in vivo
We find that DAG does not mix ideally but forms
microdomains, possibly in weak interaction with PG As
PG is the strongest activator in vivo for the two
glucolipid-synthesizing enzymes, this phenomenon has a biological
relevance in concentrating DAG, the substrate for
MGlc-DAG synthase, which is rate-limiting in glucolipid synthesis
Purified MGlcDAG synthase, free of lipids and detergent
[55], did not affect the organization (Ie/Im) for the tested
PyrDAG and PyrPG probes, but affects order in the
membrane Activity therefore seems to depend more on the
ability to bind to the membrane in a proper orientation/
conformation as Fig 5 suggests, and due to the fact that
CL, a strong activator, did not affect the Ie/Im for
PyrDAG
Acknowledgements
This work was supported by the Swedish Natural Science Research
Council, and the K & A Wallenberg foundation.
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