Download Effects of phosphatidylethanolamine glycation on lipid–protein

Biochem. J. (2008) 416, 145–152 (Printed in Great Britain)
145
doi:10.1042/BJ20080618
Effects of phosphatidylethanolamine glycation on lipid–protein interactions
and membrane protein thermal stability
Valeria LEVI*, Ana M. VILLAMIL GIRALDO†, Pablo R. CASTELLO†1 , Juan P. F. C. ROSSI† and F. Luis GONZÁLEZ FLECHA†2
*Departamentos de Fı́sica y Quı́mica Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria-1428 Buenos Aires, Argentina, and
†Instituto de Quı́mica y Fisicoquı́mica Biológicas, Facultad de Farmacia y Bioquı́mica, Universidad de Buenos Aires-CONICET, Junı́n 956-1113 Buenos Aires, Argentina
Non-enzymatic glycation of biomolecules has been implicated in
the pathophysiology of aging and diabetes. Among the potential
targets for glycation are biological membranes, characterized by
a complex organization of lipids and proteins interacting and
forming domains of different size and stability. In the present
study, we analyse the effects of glycation on the interactions
between membrane proteins and lipids. The phospholipid
affinity for the transmembrane surface of the PMCA (plasmamembrane Ca2+ -ATPase) was determined after incubating the
protein or the phospholipids with glucose. Results show that
the affinity between PMCA and the surrounding phospholipids
decreases significantly after phosphospholipid glycation, but
remains unmodified after glycation of the protein. Furthermore,
phosphatidylethanolamine glycation decreases by ∼ 30 % the
stability of PMCA against thermal denaturation, suggesting that
INTRODUCTION
The reaction of glucose with biological molecules has been known
since the early works of Maillard [1]. The first steps of this
reaction are reversible and begin with the nucleophilic addition of
a primary amino group of the biomolecule to the carbonyl group
of glucose, and the rearrangement of the Schiff base to form an
Amadori product. In later stages Amadori products undergo a
series of complex irreversible inter- and intra-molecular reactions
to produce a heterogeneous group of products called AGEs
(advanced glycation end-products) [2]. Both the early products
and the end-products of glycation affect the physicochemical, and
occasionally also the biological, properties of the target molecules
[3]. Several studies have demonstrated that proteins react in vivo
with glucose generating Amadori products and AGEs, which have
been implicated in the pathogenesis of diabetes and normal aging
[4].
In contrast with non-enzymatic glycation of proteins and
nucleic acids, the reaction of lipids with sugars has received little
attention. In 1993, Bucala et al. [5] demonstrated that aminophospholipids react with glucose to form fluorescent compounds.
Subsequent in vitro studies showed that the non-enzymatic
reaction of PE (phosphatidylethanolamine) and PS (phosphatidylserine) with glucose takes place through the same mechanism
described for proteins (Scheme 1) ([6,7] and references therein).
These authors observed that incubation of dioleoyl-PE with glucose forms Amadori-glycated PE and triggers lipid peroxidation.
Moreover, it was also verified that Amadori-PE levels were
glycated aminophospholipids induce a structural rearrangement
in the protein that makes it more sensitive to thermal unfolding.
We also verified that lipid glycation decreases the affinity of lipids
for two other membrane proteins, suggesting that this effect might
be common to membrane proteins. Extending these results to the
in vivo situation, we can hypothesize that, under hyperglycaemic
conditions, glycation of membrane lipids may cause a significant
change in the structure and stability of membrane proteins, which
may affect the normal functioning of membranes and therefore of
cells.
Key words: membrane protein, non-enzymatic glycation, phospholipid, plasma membrane Ca2+ -ATPase, protein–lipid interaction.
elevated almost 3-fold in diabetic patients [6], suggesting an
important role for in vivo lipid glycation in the pathogenesis of
atherosclerosis, diabetes and aging. Given the relevance of lipid
glycation to several pathologies, there is an increasing interest
in finding inhibitors of this process. Recently, Higuchi et al. [8]
have demonstrated that pyridoxal 5 -phosphate can prevent PE
glycation by forming adducts with PE. Moreover, these authors
found these complexes in human red blood cells, suggesting that
this compound may act as a lipid glycation inhibitor in vivo, and
demonstrated that supplementation of the diet of diabetic rats with
pyridoxal 5 -phosphate reduces the levels Amadori-PE.
For several years, biological membranes were considered to be
a homogeneous lipid matrix with membrane proteins immersed
within it [9]. A number of studies have later demonstrated that
membrane structure is more complex; their components can
form segregated domains of variable size and stability [10,11].
This inhomogeneous organization seems to be intimately related
to certain membrane functions [12]. Furthermore, the transmembrane domain of proteins establish hydrophobic interactions
with fatty acid chains of lipids, whereas the phospholipid
headgroups interact through electrostatic interactions with protein
residues located at the interface between the membrane and the
aqueous solution. Through these interactions, lipids and membrane proteins adapt structurally to each other minimizing the
free energy of the system [13,14].
It is well known that native membranes are composed of a
wide variety of lipids, some of these interact efficiently with the
transmembrane domain of membrane proteins, whereas others
Abbreviations used: AGE, advanced glycation end-product; C12 E10 , polyoxyethylene 10-laurylether; DMPC, dimyristoyl phosphatidylcholine;
DMPE, dimyristoyl phosphatidylethanolamine; HPPC, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PMCA, plasma-membrane Ca2+ -ATPase.
1
Present address: Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO, U.S.A.
2
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
146
V. Levi and others
chased from New England Nuclear. The fluorescent probe
HPPC [1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine] was obtained from Molecular Probes. All other
chemicals used in the present study were of analytical grade.
Recently drawn human blood was obtained from the Haematology
Section of the Hospital de Clı́nicas José de San Martı́n, Argentina
(Public Blood Transfusion Service). The donor provided informed
consent to the donation, and blood was taken and used according
to the law and Ministry of Health guidelines.
Purification of the Ca2+ pump from human erythrocytes
Scheme 1
DMPE glycation
Glucose reacts with the amino group of the phospholipid generating a Schiff base (I) which
further rearrange forming a stable Amadori product (II).
interact poorly [14]. Based on these observations, a model was
proposed in which transmembrane domains are surrounded by
a lipid monolayer that is enriched in high-affinity lipids and where
lipids of low affinity are preferentially excluded [15].
We have previously shown that the composition of the boundary
monolayer is intimately related to the stability of membrane
proteins [16]. This result suggests that alterations in the monolayer
composition are translated into modifications on the structure of
membrane proteins. In this context, a chemical modification
of either lipids or proteins, such as those occurring during nonenzymatic glycation, may affect the interaction between these
membrane components and consequently the membrane functionality. In the present study we assess this hypothesis using
the PMCA (plasma-membrane Ca2+ -ATPase) as a model protein
since its glycation produces a significant reduction in its biological
activity [17–19].
The erythrocyte PMCA is an integral membrane protein
consisting of a single polypeptide chain of M r 134 000 [20]
comprising a large intracellular domain, which includes catalytic
and regulatory sites, and short external loops connecting ten
transmembrane segments organized in three hydrophobic clusters
[21]. Under native conditions, PMCA reversibly dimerizes [22],
providing additional stability to PMCA structure [23].
In the present study we show that the affinity of PE for PMCA
significantly decreases after lipid glycation. This decrease in
affinity correlates with a significant loss of the stability of the
protein. We also found that the decrease of the lipid affinity to
the protein transmembrane surface is common to other membrane
proteins, in particular, the Na+ /K+ pump and erythrocyte band
3 suggesting that glycation of membrane lipids may cause a
significant change in the structure of membrane proteins, which
may affect the function of biological membranes and therefore of
cells.
EXPERIMENTAL
Calmodulin-depleted erythrocyte membranes were prepared as
described previously [19] using 15 mM Mops, 1 mM EGTA
and 0.1 mM PMSF (pH 7.4 at 4 ◦C) as a hypotonic solution.
PMCA was isolated by calmodulin-affinity chromatography [23].
Fractions exhibiting the highest activity were pooled together. The
phospholipid concentration was determined according to Chen
et al. [24]. Membrane protein concentrations were measured by
densitometric analysis after SDS/PAGE. Prior to use, the enzyme
(560 nM, specific Ca2+ -ATPase activity 11.5 μmol of Pi /mg per
min) was kept under liquid nitrogen in a medium containing:
300 mM KCl, 10 mM Mops/KOH (pH 7.4 at 4 ◦C), 1 mM MgCl2 ,
2 mM EDTA and 2 mM CaCl2 ([Ca2+ ]free = 70 μM), with the lipid
and detergent concentrations indicated in each case.
Preparation of Na+ /K+ -ATPase from pig kidney
Na+ /K+ -ATPase was partially purified from the pig kidney
outer medulla according to Jensen et al. [25] and was kindly
provided by Dr R.C. Rossi (Facultad de Farmacia y Bioquı́mica,
Universidad de Buenos Aires, Buenos Aires, Argentina). Fragmented membranes were washed with 130 mM NaCl, 20 mM
KCl, 1 mM DTT (dithiothreitol), 1 mM EGTA and 10 mM Tris/
HCl (pH 7.4) at 25 ◦C (buffer I). Proteins were solubilized by
adding 1 mg of C12 E10 per ml of the enzyme suspension (0.6 mg
of protein/ml), incubated for 10 min at 0 ◦C, and sedimented at
14 000 g for 10 min.
Isolation of the HCO3 − /Cl− exchanger band 3 from human
erythrocytes
Erythrocyte band 3 was isolated from red blood cells as described
previously [26] with some modifications. Erythrocyte membranes
were prepared as described for the purification of the Ca2+ pump.
The HCO3 − /Cl− exchanger band 3 was selectively solubilized by
adding C12 E10 to the membrane preparation, followed by 10 min
incubation at 25 ◦C and sedimentation at 14 000 g to eliminate the
non-solubilized material.
Measurement of ATPase activity
ATPase activity was measured at 37 ◦C as the initial velocity of
release of Pi from ATP, as described previously [23]. The incubation medium was: 120 mM KCl, 30 mM Mops/KOH (pH 7.4),
3.75 mM MgCl2 , 1 mM EGTA, 1.1 mM CaCl2 , 140 μM soya
bean phospholipids, 800 μM C12 E10 and 2 mM ATP. The protein
concentration was 7 nM. Ca2+ -dependent ATPase activity was
calculated as the difference between the ATPase activities in medium with and without Ca2+ . The concentration of Ca2+ (140 μM)
was determined using an Orion 9320 ion-selective Ca2+ electrode.
Reagents
DMPE (dimyristoyl PE), DMPC [dimyristoyl PC (phosphatidylcholine)], soya bean lipids and C12 E10 (polyoxyethylene 10-laurylether) were obtained from Sigma. D-[6-3 H]glucose was pur
c The Authors Journal compilation c 2008 Biochemical Society
Phospholipid and protein glycation
Glycation of phospholipids was performed as described
previously [27,28] with some modifications. Briefly, DMPC and
Glycation effects on lipid–protein interactions
147
DMPE (molar ratio 1:1) were dissolved in chloroform/methanol
[2:1; (v/v)] and the solvent was evaporated under nitrogen to
form a thin layer film. The lipids were suspended up to 14 mM in
100 mM glucose, 2 mM EDTA and 40 mM phosphate (pH 7.4
at 37 ◦C) and sonicated for 20 min. We included PC in the
mixture because this lipid is essential to obtain full active PMCA
preparations [29]. The resulting mixture was incubated in the dark
under nitrogen for up to 15 days at 37 ◦C.
PMCA glycation was performed as described [19], incubating,
at 37 ◦C, for various periods of time the purified enzyme
(20 μg/ml) supplemented with 280 μM soya bean lipids, 800 μM
C12 E10 and 10 mM glucose in the presence of 40 mM NaH2 PO4 /
Na2 HPO4 (pH 7.4) and 3 mM sodium azide, 1 μM pepstatin,
10 μM leupeptin, 1 μg/ml aprotinin and 0.1 mM PMSF to prevent
proteolysis of the enzyme and bacterial growth.
procedure described previously [32]. Briefly, the method is based
on the fact that fluorescence of most native proteins is dominated
by tryptophan fluorescence upon excitation at wavelengths
≈ 290 nm [33] and these residues in membrane proteins are
preferentially located in the membrane-aqueous phase interface
[34]. Thus trace quantities, approx. 1 molecule per micelle, of
a pyrene-labelled phospholipid (HPPC) can be used to monitor
the exchange among unlabelled amphiphiles on the boundary
monolayer covering the hydrophobic transmembrane surface of
the protein [16]. As a consequence of energy transfer between
tryptophan residues and the lipidic probe, the fluorescence
intensity of the protein (I d,a ) decreases according to (eqn 2):
Measurement of glucose incorporation into phospholipids
and protein
where Eapp is the apparent energy transfer efficiency, I d is the
intensity of the protein in the absence of the probe, X HPPC,mic and
X PL,mic are the mole fraction of HPPC and phospholipids in the
micelles, ξ is a constant parameter depending on the protein and
the probe, and β is the stoichiometric coefficient for the amphiphile exchange and was determined to be equal to 2 in the
experiments shown in the present study. The exchange constant
K ex,PL , defined as the ratio between the equilibrium constants of
adsorption of lipids and detergent to the hydrophobic transmembrane surface of the protein, gives a measure of the relative
affinity lipid/detergent for the protein.
Phospholipid and PMCA glycation was quantified following
the procedure described in the previous section but including
[6-3 H]glucose in the incubation medium. To measure glucose
incorporation into phospholipids, aliquots were taken at different
incubation times and lipids were isolated by partition in a mixture
composed of 16 vol. of chloroform/methanol [2:1; (v/v)] and
11 vol. of water. The phases were separated by centrifugation
(1200 g for 10 min at 10 ◦C), and the organic phase was transferred
to counting vials. To determine glucose incorporation into PMCA,
aliquots (200 μl) were taken at different times and mixed with a
solution of 100 mM glucose. After isotopic dilution, the enzyme
was precipitated with 7 % TCA (trichloroacetic acid) and filtered
through mixed cellulose ester membrane filters (0.20 μm pore
size). The filters were washed three times with 15 ml of an icecold solution of 10 mM glucose and 15 mM Mops-KOH (pH 7.4),
dried and transferred to counting vials. Radioactivity was
measured in a Wallac 1214 liquid-scintillation counter.
Measurement of phospholipid peroxidation
Lipid oxidation was tested by UV spectrophotometry registering
the absorbance ratio between 230 nm and 215 nm as described
previously [30,31]. According to Klein [31] this method allows
detection as low as 2 nmol of early-stage phospholipid oxidation
products.
Fluorescence measurements
Protein emission spectra were registered at 25 ◦C in a 3 mm ×
3 mm quartz cuvette using a SLM-AMINCO BOWMAN
Series 2 spectrofluorimeter with excitation at 290 nm. Both
excitation and emission bandwidths were set at 4 nm. Each spectrum was corrected for background emission.
The total fluorescence intensity of the membrane protein was
determined according to (eqn 1):
I (λi )λ
(1)
I=
I (λ)dλ ∼
=
λi
where I(λi ) represents the fluorescence intensity at λi .
Determination of lipid affinity for the transmembrane surface of
membrane proteins
The affinity of phospholipids for the hydrophobic transmembrane
surface of membrane proteins was determined according to a
E app = 1 −
ξ
Id,a
= X HPPC,mic
Id
K ex,PL · X PL,mic + (1 − X PL,mic )β
(2)
Thermal inactivation experiments
Thermal inactivation of PMCA was assayed incubating 10 μg of
protein at 44 ◦C as described previously [18]. The time course
of thermal inactivation is described by an exponential function
(eqn 3):
v = vo · e−kinact t
(3)
where v and vo are the remaining and the initial Ca2+ -ATPase
activities, t is the incubation time and kinact is the kinetic coefficient
for thermal inactivation.
Data analysis
Data shown in the present study are representative of at least
three independent experiments. Equations were fitted to the
experimental data by non-linear regression [35]. The dependent
variables were assumed to be homoscedastic and the independent variable was considered to have negligible error. Best-fitted
parameter values were expressed as the means +
− S.E.M. Joint
confidence regions for the regression parameters were calculated
as described by Box et al. [36]. Briefly, the sum of quadratic
residues S is calculated as follows (eqn 4):
(yi − f i )2
(4)
S=
i
where yi is the experimental value of the dependent variable and fi
is the value calculated using the equation to be fitted and a given
set of parameter values.
The joint confidence region is defined by the parameter values
satisfying (eqn 5):
p
(5)
S = Smin · 1 + F1−P ( p, n − p) ·
(n − p)
c The Authors Journal compilation c 2008 Biochemical Society
148
Figure 1
V. Levi and others
Time course of phospholipid and protein glycation
(A) Glycation of DMPE. Glucose incorporation to DMPE was monitored as described in the
Experimental section. The same procedure was followed with a control sample in which glucose
was replaced by mannitol and [6-3 H]glucose was added at the end of each incubation period.
The difference in radioactivity measured for the sample and the control was plotted as a function
of the incubation time. The data were modelled with an exponential function (continuous line)
−1
obtaining a kinetic constant k glu = 0.20 +
− 0.05 days . (B) Glycation of PMCA. Glucose incorporation to PMCA was measured as described in the Experimental section. Unspecific
incorporation was determined by incubating the enzyme with non-radioactive glucose followed
by the addition of [6-3 H]glucose at the end of the incubation, and was subtracted from all
of the determinations. The data were modelled with an exponential function (continuous line)
−1
obtaining a kinetic constant k glu = 0.04 +
− 0.01 min . Maximum glycation levels were ∼ 5 %
of the available amino groups for both DMPE and PMCA. These values were similar to those
reported previously [18,28].
where n is the number of data points, p is the number of parameters
in the equation, Smin is the value of S corresponding to the best-fitting parameter values and F1−P (p,n − p) is the inverse Fisher distribution with probability P, and p and n − p degrees of freedom.
RESULTS AND DISCUSSION
Time course of PMCA and PE glycation
Phospholipid and PMCA glycation were performed at 37 ◦C by
incubating the samples with glucose as described in the Experimental section above. To monitor the glycation process, trace
quantities of D-[6-3 H]glucose were included in the incubation
medium. Phospholipid glycation was evaluated after partition in
a chloroform/methanol/water two-phase system by measuring the
amount of D-[6-3 H]glucose in the organic phase. Figure 1(A)
shows that glucose incorporation to PE occurs in a time-scale
of days (t0.5 = 3.5 days). Two control experiments were run in
parallel. In the first experiment, we measured the concentration of
phospholipid in the aqueous phase and non-detectable amounts
c The Authors Journal compilation c 2008 Biochemical Society
were found. The second control was performed replacing DMPE
by DMPC. In contrast with PE, PC does not contain a primary
amino group susceptible to react with glucose and to form a Schiff
base. In this case, no time-dependent incorporation of glucose to
DMPC was detected (results not shown).
It is known that long incubations of phospholipids with glucose
can generate reactive oxygen species that trigger lipid oxidation
which can also contribute to modifications of proteins [7]. To avoid
glyco-oxidation we used a saturated PE (DMPE) and performed
the experiments in the dark under nitrogen, and a metal ion
chelator was added to the incubation medium to eliminate traces
of metals that could catalyse peroxidation [37].
We determined oxidation products of phospholipids after incubation with glucose and found no significant differences between
non-incubated and incubated samples, showing that no peroxidation occurs under the experimental conditions chosen.
Bucala et al. [5] showed that PE-linked AGEs present an
excitation maximum at 360 nm and an emission maximum at
440 nm. Thus we evaluated whether AGEs are formed during the
incubation by registering the excitation and emission fluorescence
spectra of the glucose-incubated samples. We did not detect
fluorescence upon excitation at 360 nm, indicating that no
AGEs were formed. Thus the covalent adduct formed under the
experimental conditions used in the present study could be an
Amadori product of PE as was described by Oak et al. [7].
In a previous study we have demonstrated that glucose reacts
covalently with PMCA, and glycation of one essential lysine
residue located near the catalytic site produces the enzyme inactivation [19]. We followed the time course of glucose incorporation
into PMCA finding that it followed an exponential function with
t0.5 = 17 min at 37 ◦C (Figure 1B) in agreement with our previous
results [18]. We verified that amino-phospholipids present in the
enzyme reconstitution medium were not significantly glycated at
this time scale (results not shown). Longer protein incubations
were not assayed because PMCA irreversibly denatures [16].
Effects of PE and PMCA glycation on lipid–protein interactions
The effects of glycation on the lipid–protein interactions were
explored using PMCA reconstituted in mixed micelles composed
of DMPC/DMPE and C12 E10 . Given that the phase state of dilute
aqueous lipid–detergent mixtures depends exclusively on the effective detergent mole fraction in the phase [38], we have selected
for all of the experiments X det > 0.4 so that only isotropic mixed
micelles were present in the systems. For these experiments,
the enzyme or the phospholipid mixture were incubated with
glucose as described below. As controls, similar experiments were
performed replacing glucose with mannitol. This non-carbonylic
analogue of glucose shares the hydrogen-bonding capability of
glucose but it cannot react with amino groups to form the Schiff
base.
We first tested the effects of PE glycation on lipid–protein
interactions. The purified enzyme was reconstituted in mixed
micelles containing glycated or control phospholipids (the mixture DMPC/DMPE) and supplemented with increasing quantities
of C12 E10 and HPPC. The fluorescence intensity was registered
once the steady state was reached (∼ 1 min). Figure 2 shows the
results obtained following this procedure. Eqn (2) was fitted to the
experimental data obtaining the K ex,PL values showed in Table 1.
This result indicates that lipid glycation significantly decreased
the affinity of lipids for the protein. The 98 % confidence
regions for the regression parameters corresponding to the experiment performed with control and glycated lipids did not overlap
(Figure 2E), indicating that the observed differences were
statistically significant.
Glycation effects on lipid–protein interactions
Figure 2
149
Effects of phospholipid glycation on the phospholipid-detergent exchange constant for the PMCA
The protein was purified and reconstituted in a medium containing 100 mM glucose, C12 E10 , and either 280 μM phospholipids incubated with mannitol (A and C) or 280 μM glycated phospholipids
(B and D). Samples were supplemented with C12 E10 up to the mole fractions within the range 0.40–0.70. PMCA fluorescence intensity (I d,a ) was measured as indicated in the Experimental section
after adding increasing quantities of HPPC and mixing for 1 min. The final HPPC concentration was always lower than 3.6 μM. I d values, corresponding to the intensity measured in the absence of
HPPC were corrected for the dilution caused by the addition of the probe; this correction was never higher than 7 % of the total intensity. E app was calculated as the relative decrease of fluorescence
intensity due to the presence of acceptor (eqn 2) and plotted as a function of the mole fraction of HPPC and phospholipids (A and B). The surface represented in each Figure represents the fit of eqn (2)
to the experimental data with K ex,PL values shown in Table 1. To better visualize these results, E app data corresponding to experiments with initial mole fraction of detergent of 0.4 and 0.7, were
projected over the E app − XHPPC plane (C and D). Continuous lines were obtained generating the same projection with eqn (2) and the best-fit parameter values corresponding to the global fitting.
98 % confidence regions for K ex,PL and ξ . (E) were constructed as described in the Experimental section. The point in the centre of each region represents the values of the parameters obtained by
fitting eqn (2) to the experimental data for glycated (䊊) and control (䊉) lipids. The shape of the confidence regions denotes the existence of positive correlation between the regression parameters.
Table 1 Phospholipid-detergent exchange constants on the transmembrane
surface of proteins
K ex,PL
Membrane protein
Phospholipids composing
the micelle
PMCA
PMCA
Na+ /K+ -ATPase
Erythrocyte band 3
DMPC/DMPE
Soya bean phospholipids
DMPC/DMPE
Soya bean phospholipids
Control
Glycated
phospholipid
1.8 +
− 0.2
9+
−1
1.7 +
− 0.3
10 +
−2
1.0 +
− 0.1
1.1 +
− 0.6
0.9 +
− 0.1
1.9 +
− 0.5
The effect of lipid glycation on lipid–protein interactions was
also assayed in a micellar system composed of C12 E10 and soya
bean lipids (Figure 3). This mixture includes approx. 21 % of
PE in its phospholipid composition [39]. We tested this system
because the enzyme is more stable in this micellar environment
due to a higher overall affinity for the protein of the lipids included in the soya bean mixture with respect to the DMPC/DMPE
system assayed previously [16]. Importantly, we did not detect
either oxidation products or AGEs in this sample as was also
observed for the DMPE/DMPC system. Table 1 shows K ex,PL
values determined after treating these lipids with glucose or
mannitol in the conditions previously described. Lipid glycation
also reduced significantly the affinity for the protein in the soya
bean lipids/C12 E10 micellar system. The high value of K ex,PL
obtained in the control experiment with micelles composed of
soya bean phospholipids has been previously reported and was
attributed to the presence of acidic phospholipids [16].
Next, we investigated the effects of protein glycation on
lipid–protein interactions. In this case the enzyme (control or
PMCA glycated as described above, incubating the enzyme with
glucose for 2 h) was reconstituted with non-glycated soya bean
phospholipids. The obtained K ex,PL values were 8.7 +
− 1 for PMCA
incubated with mannitol and 9.3 +
− 2 for glycated PMCA. The
differences between these values were not significant, indicating
that interactions between PMCA and lipids are not affected by
glycation of PMCA.
Effects of PE glycation on PMCA thermal stability
In the previous section we verified that glycation of lipids
produces a significant change in their affinity for PMCA. As was
mentioned above, the structure of membrane proteins depends
on the amphiphilic environment of their transmembrane domain
[13]. Therefore we explored whether glycation of lipids induces
a structural rearrangement of PMCA by following the kinetics
of PMCA thermal inactivation in the presence of glycated and
non-glycated lipids. Because the stability of a protein is inversely
related to the rate of its unfolding, the kinetic coefficient of thermal
inactivation (kinact ) gives a measure of its stability [16].
c The Authors Journal compilation c 2008 Biochemical Society
150
Figure 3
V. Levi and others
Effects of protein and phospholipid glycation on the soya bean phospholipid-detergent exchange constant for the PMCA
Purified PMCA was reconstituted in a medium containing 100 mM glucose, C12 E10 , and either 280 μM control (A) or glycated (B) soya bean phospholipids. Glycated PMCA was reconstituted in
a medium containing 100 mM glucose, 140 mM C12 E10 and 280 μM of non-glycated soya bean phospholipids (C). The enzyme preparations were supplemented with C12 E10 up to mole fractions
0.74, 0.76, 0.79, 0.82 and 0.92. The emission intensity (I d,a ) was measured after adding increasing quantities of HPPC as indicated in Figure 2. E app data corresponding to experiments with an initial
mole fraction of detergent of 0.74 and 0.92 were projected over the E app − XHPPC plane to better visualize the data. Continuous lines were obtained generating the same projection with eqn (2) and
the best-fit parameter values corresponding to the global fitting (Table 1). The 99 % confidence regions for these parameters (D) were constructed as described in the Experimental section.
Figure 4 shows that PMCA inactivated faster in the presence
of glycated lipids (t0.5 = 15 min) with respect to the control
(t0.5 = 19 min). The 99 % confidence regions for the regression
parameters shown in the inset to Figure 4 indicate that the 26 %
decrease in the stability of the enzyme is statistically significant.
Also, the values for the initial ATPase activity in the presence
of control and glycated lipids are not different, in accordance
with our previous findings [17–19]. This result suggests that the
changes in the phospholipid affinity due to glycation trigger
a structural rearrangement of the protein that makes it more
sensitive to thermal unfolding.
Effects of PE glycation on lipid–protein interactions in other
reconstituted membrane proteins
To assess whether reduction of lipid affinity due to glycation is
specific for PMCA or whether it is common to other membrane
proteins, we determined K ex,PL for the Na+ /K+ -ATPase and the
erythrocyte band 3 after glycating the phospholipids in which
the proteins are reconstituted.
Na+ /K+ -ATPase was solubilized in micelles composed of
C12 E10 and control or glycated samples of DMPC/DMPE.
Na+ /K+ -ATPase fluorescence intensity was determined as a function of HPPC and phospholipid mole fractions (Figure 5). Eqn (2)
was fitted to the experimental data and the K ex,PL values showed
c The Authors Journal compilation c 2008 Biochemical Society
Figure 4
2+
Effects of PE glycation on the stability of the PMCA
The Ca pump was purified as described in the Experimental section and supplemented up
to 800 μM C12 E10 and 280 μM of control (䊉) or glycated (䊊) DMPC/DMPE. Samples were
incubated at 45 ◦C for different periods of time, and Ca2+ -ATPase activity was measured and
plotted as a function of incubation time. Eqn (3) was fitted to the experimental data obtaining the
−1
−1
+
following best-fitting values for k inact : 2.14 +
− 0.03 h (control) and 2.70 − 0.06 h (glycated).
The inset shows 99 % confidence regions for these parameters constructed as described in the
Experimental section.
Glycation effects on lipid–protein interactions
151
Figure 5 Effect of phospholipid glycation on the phospholipid-detergent
exchange constant for the Na+ /K+ -ATPase
Figure 6 Effects of phospholipids glycation on the phospholipid-detergent
exchange constant for the HCO3 − /Cl− exchanger band 3
Na+ /K+ -ATPase was reconstituted in buffer I with 100 mM glucose, 140 mM C12 E10 and 280 μM
of control (A) or glycated (B) DMPC/DMPE. The enzyme preparations were supplemented with
C12 E10 up to mole fractions 0.30, 0.45, 0.53, 0.58 and 0.63. The emission intensity (I d,a )
was measured after adding increasing quantities of HPPC as indicated in Figure 2. E app data
corresponding to experiments with an initial mole fraction of detergent of 0.30 and 0.63 were
projected over the E app − XHPPC plane to help with the visualization of the data. Continuous lines
were obtained generating the same projection with eqn (2) and the best-fit parameter values
corresponding to the global fitting (Table 1).
Band 3 was extracted from erythrocyte membranes and supplemented with 100 mM glucose,
140 μM C12 E10 and 280 μM control (A) or glycated (B) soya bean lipids. The enzyme
preparations were supplemented with C12 E10 up to mole fractions 0.74, 0.76, 0.79, 0.82 and
0.90. The emission intensity (I d,a ) was measured after adding increasing quantities of HPPC
as indicated in Figure 2. E app data corresponding to experiments with an initial mole fraction
of detergent of 0.74 and 0.90 were projected over the E app − X HPPC plane to help with the
visualization of the data. Continuous lines were obtained generating the same projection with
eqn (2) and the best-fit parameter values corresponding to the global fitting (Table 1).
Concluding remarks
in Table 1 were obtained. This parameter decreased significantly
after glycation of the lipids, indicating a preferential adsorption
of PE over glucose-treated PE to the transmembrane surface of
the Na+ /K+ -ATPase.
The HCO3 − /Cl− exchanger band 3 is one of the more abundant
proteins in the erythrocyte membrane constituting approx. 25 %
of the total membrane proteins [40]. We selectively extracted band
3 from erythrocyte membranes by using increasing concentrations
of C12 E10 (See Supplementary Figure S1 at http://www.BiochemJ.
org/bj/416/bj4160145add.htm). This procedure provided a solubilized preparation of erythrocyte membrane proteins in which
band 3 represents more than 90 % of the total proteins. The
protein was reconstituted in micelles of C12 E10 and control or
glycated soya bean phospholipids. The affinity of phospholipids
to the hydrophobic transmembrane surface of band 3 was assayed
following the procedure described above (Figure 6), obtaining
the K ex,PL values shown in Table 1. A significant decrease on lipid
affinity for the transmembrane surface of band 3 after glycation
can be observed, as was also found for PMCA and Na+ /K+ ATPase.
In the present study, we have analysed the effect of non-enzymatic
glycation on the interactions between lipids and membrane
proteins. PMCA is an ideal membrane protein to study this process
since glycation of this protein is well described. We further
extended the analysis to other membrane proteins i.e. the Na+ /
K+ -ATPase and the erythrocyte band 3 which is one of the most
abundant proteins in erythrocyte membranes.
We first assayed the effects of PMCA glycation on phospholipid–protein interactions. PMCA has 80 lysine residues, all
of them outside the transmembrane regions thus constituting
potential targets for glycation [20]. Despite the fact that there
are no lysine residues in the transmembrane helices, glycation of
water-exposed lysine residues could produce a rearrangement
of the structure including changes in the lipid-exposed surface.
Our results suggest that this is not the case, since the exchange
constant K ex,PL is unaffected by glycation of the pump. On the other
hand, PE glycation produces a significant reduction in the phospholipid affinity for PMCA.
The decrease of affinity upon lipid glycation seems to be
common to several membrane proteins. All of the membrane
c The Authors Journal compilation c 2008 Biochemical Society
152
V. Levi and others
proteins assayed in the present study showed similar effects on
K ex,PL upon glycation of lipids.
We did not detect a short-term effect of lipid glycation on
PMCA function since it did not affect the ATPase activity of the
enzyme. However, we observed that the stability of the enzyme
significantly decreased upon glycation. This result agrees with
previous evidence demonstrating that the stability of membrane
proteins is intimately related to the composition of the lipid
environment of the transmembrane domain [16,41,42]
Taken together, the results of the present study show that
glycation of phospholipids decreases their affinity for the transmembrane surface of membrane proteins and probably diminishes
the stability of the folded structure of these proteins. Extending
these results to the in vivo situation, we can hypothesize that
under hyperglycaemic conditions (uncontrolled diabetes) or longterm exposure to glucose (aging), glycation of lipids of cellular
membranes may cause a significant change in the structure and
stability of membrane proteins which may affect the normal
functioning of the membranes and therefore of the cells.
This work was supported by grants from UBACYT (B110), CONICET (PIP 6168) and
ANPCyT (PICT 01741).
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doi:10.1042/BJ20080618
SUPPLEMENTARY ONLINE DATA
Effects of phosphatidylethanolamine glycation on lipid–protein
interactions and membrane protein thermal stability
Valeria LEVI*, Ana M. VILLAMIL GIRALDO†, Pablo R. CASTELLO†1 , Juan P. F. C. ROSSI† and F. Luis GONZÁLEZ FLECHA†2
*Departamentos de Fı́sica y Quı́mica Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria-1428 Buenos Aires, Argentina, and
†Instituto de Quı́mica y Fisicoquı́mica Biológicas, Facultad de Farmacia y Bioquı́mica, Universidad de Buenos Aires-CONICET, Junı́n 956-1113 Buenos Aires, Argentina
Figure S1 SDS/PAGE analysis of erythrocyte membrane proteins (lane 4)
and the solubilized material using C12 E10 at the following concentrations:
0.5 mg/ml (lane 1), 0.7 mg/ml (lane 2) and 1 mg/ml (lane 3)
Received 19 March 2008/29 May 2008; accepted 18 June 2008
Published as BJ Immediate Publication 18 June 2008, doi:10.1042/BJ20080618
1
2
Present address: Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO, U.S.A.
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society