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Proteinoligomerizationinducedbyoleicacidat the
solid–liquid interface–equinelysozyme cytotoxic
complexes
Kristina Wilhelm
1
, Adas Darinskas
2
, Wim Noppe
3
, Elke Duchardt
1,4
, K. Hun Mok
5
,
Vladana Vukojevic
´
6
,Ju
¨
rgen Schleucher
1
and Ludmilla A. Morozova-Roche
1
1 Department of Medical Biochemistry and Biophysics, Umea
˚
University, Sweden
2 Institute of Immunology, Vilnius University, Lithuania
3 Interdisciplinary Research Center, Campus Kortrijk, Leuven University, Belgium
4 Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt, Germany
5 Trinity College, School of Biochemistry and Immunology, University of Dublin, Ireland
6 Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden
Introduction
The process of protein self-assembly has become the
focus of much current research as a broad manifestation
and consequence of protein instability. The propensity
of protein molecules to aggregate markedly increases if
they are destabilized or partially unfolded [1–3]. The
increased exposure of hydrophobic surfaces in partially
Keywords
amyloid; HAMLET; lysozyme; oleic acid;
oligomers
Correspondence
L. A. Morozova-Roche, Department of
Medical Biochemistry and Biophysics, Umea
˚
University, 901 87 Umea
˚
, Sweden
Fax: +46 90 786 9795
Tel: +46 90 786 5283
E-mail: ludmilla.morozova-roche@
medchem.umu.se
(Received 30 March 2009, revised 6 May
2009, accepted 21 May 2009)
doi:10.1111/j.1742-4658.2009.07107.x
Protein oligomeric complexes have emerged as a major target of current
research because of their key role in aggregation processes in living systems
and in vitro. Hydrophobic and charged surfaces may favour the self-assembly
process by recruiting proteins and modifying their interactions. We found
that equinelysozyme assembles into multimeric complexes with oleic acid
(ELOA) atthesolid–liquidinterface within an ion-exchange chromatography
column preconditioned with oleic acid. The properties of ELOA were charac-
terized using NMR, spectroscopic methods and atomic force microscopy,
and showed similarity with both amyloid oligomers and thecomplexes with
oleic acid and its structural homologous protein a-lactalbumin, known as
humana-lactalbumin made lethal for tumour cells (HAMLET). As deter-
mined by NMR diffusion measurements, ELOA may consist of 4–30 lyso-
zyme molecules. Each lysozyme molecule is able to bind 11–48 oleic acids in
various preparations. Equinelysozyme acquired a partially unfolded confor-
mation in ELOA, as evident from its ability to bind hydrophobic dye
8-anilinonaphthalene-1-sulfonate. CD and NMR spectra. Similar to amyloid
oligomers, ELOA also interacts with thioflavin-T dye, shows a spherical mor-
phology, assembles into ring-shaped structures, as monitored by atomic force
microscopy, and exerts a toxic effect in cells. Studies of well-populated
ELOA shed light on the nature of the amyloid oligomers and HAMLET
complexes, suggesting that they constitute one large family of cytotoxic
proteinaceous species. The hydrophobic surfaces can be used profitably to
produce complexes with very distinct properties compared to their precursor
proteins.
Abbreviations
AFM, atomic force microscopy; ANS, 8-anilinonaphthalene -1-sulfonate; CLSM, confocal laser scanning microscopy; ELOA, complex of
equine lysozyme with oleic acid; FCS, fluorescence correlation spectroscopy; HAMLET, human a-lactalbumin made lethal to tumour cells;
PFG, pulse field gradient; ThT, thioflavin-T.
FEBS Journal 276 (2009) 3975–3989 ª 2009 The Authors Journal compilation ª 2009 FEBS 3975
unfolded states leads to spontaneous protein aggrega-
tion. Protein destabilization can be achieved using mild
denaturing conditions, such as acidic or basic pH, heat-
ing, chemical denaturants and ligands, as well as at
solid–liquid interfaces [4–7]. Among self-assembled pro-
tein complexes, oligomers have attracted special atten-
tion because of their involvement in amyloid formation
and their distinct properties, which often differ from
those of their precursor monomers. Specifically, during
amyloid formation, oligomers may serve as nuclei for
further aggregation [8–10]. It has also been suggested
that they can fulfil the role of major cytotoxic agents
compared with more inert amyloid fibrils [11–15].
Because of the transient nature of oligomeric species,
which tend to associate into larger aggregates or split
into monomers, it is difficult to produce their stable
fractions [16–20]. A number of attempts have been made
to stabilize the oligomers of amyloidogenic polypeptides
using fatty acids and surfactants [21–25]. In our
research, we have produced stable oligomeric complexes
of equinelysozyme with oleicacid (ELOA), which we
subsequently studied in detail with regard to their struc-
tural and cytotoxic properties.
Complexes of human a-lactalbumin with oleic acid
were first described in the 1990s by Svanborg and
coworkers, and named human a-lactalbumin made
lethal to tumour cells (HAMLET) [26,27]. HAMLET
was produced in vitro in an affinity column loaded
with oleic acid, and it was also shown that HAMLET
is present naturally in the casein fraction of human
milk [26]. Recently, HAMLET has been formed at
higher temperatures of 50 and 60 °C, which facilitated
the dispersal of oleicacid and structural changes in the
protein [28]. Complexes of bovine a-lactalbumin with
oleic acid have also been produced using column chro-
matography and were designated as bovine a-lactalbu-
min made lethal for tumor cells (BAMLET) [29,30].
Because of their unique antitumor activity, the struc-
ture and function of HAMLET and BAMLET have
been studied extensively, however, the nature of the
conformational changes occurring in the proteins upon
their complex formation and the mechanisms of cyto-
toxicity of thecomplexes are still debated [26,34].
It has been shown that in both complexes human
and bovine a-lactalbumins are partially unfolded or
misfolded even under physiological conditions, and
this may be crucial for the cytotoxicity of their com-
plexes [30,32]. A complex of bovine a-lactalbumin with
polyamines has also been produced and denoted as
LAMPA [35]; the partially unfolded state of a-lactal-
bumin within this complex was distinct from all other
states of monomeric a-lactalbumin characterized to
date. The same authors have shown that monomeric
a-lactalbumin in the absence of fatty acids can bind to
histone H3, which is the primary target of HAMLET
[36], but free a-lactalbumin has not been found to have
antitumor activity. Recently, it has been also shown
that oleicacid can inhibit the amyloid fibril formation
of bovine a-lactalbumin, acting atthe initial stages of
oligomerization and fibrillation [37].
Equine lysozyme was selected as the subject of our
studies because it is the closest structural homologue
of a-lactalbumin. Equinelysozyme has been used
extensively as a model in protein folding and amyloid
studies over the last two decades [4–6,16,38–44], and
this has enabled us to reveal a wealth of information
on the mechanisms underlying these processes. By con-
trast to conventional non-calcium-binding c-type lyso-
zymes and similar to a-lactalbumins, equine lysozyme
is a calcium-binding protein [38]; however, it still dis-
plays an enzymatic activity that is characteristic of
lysozymes. As a consequence, it possesses a combina-
tion of the structural and folding properties of both
superfamilies of structurally homologous proteins –
lysozymes and a-lactalbumins. Equinelysozyme is char-
acterized by significantly lower stability and cooper-
ativity than non-calcium-binding lysozymes [4,5,39,
40,44]. It forms a range of partially folded states under
equilibrium destabilizing conditions similar to a-lactal-
bumins [4,5], and also populates kinetic folding inter-
mediates during the refolding reaction similar to c-type
lysozymes [45,46]. Its equilibrium and kinetic interme-
diates as characterized by similar structural properties.
Specifically, equinelysozyme possesses a very stable
core, which retains its native-like conformation even in
the molten globule state [5,6] and which is rapidly
folded and persists in kinetic intermediates [45,46].
Equine lysozyme also forms oligomeric and fibrillar
amyloid assemblies under acidic conditions, where its
partially folded state is populated [42,47]. Its amyloid
oligomers, ranging from tetramers to ecosinomers, dis-
play an amyloid gain-on function, such as apoptotic
activity [42,48]. These oligomers are populated in
very small quantities of only a few percent and tend
to convert rapidly to amyloid protofilaments. There-
fore, the study of stable and well-populated
oligomers of equinelysozyme with oleic acid, which
share HAMLET-like and amyloid properties, may
shed light on both phenomena. The application of a
solid–liquid interface, facilitating protein self-assembly
and protein–oleic acid interactions, proved to be an
efficient approach to produce such complexes and to
model their interactions, which may occur at the
hydrophobic and charged surfaces in both biological
systems and in vitro during the storage of proteina-
ceous materials.
Protein oligomerizationinducedbyoleicacid K. Wilhelm et al.
3976 FEBS Journal 276 (2009) 3975–3989 ª 2009 The Authors Journal compilation ª 2009 FEBS
Results
ELOA complex formation
ELOA complexes were formed using an anion-
exchange column preconditioned with oleic acid, as
described in Materials and methods. ELOA was eluted
as a strong peak at $ 1 m NaCl using a NaCl gradient
of 0–1.5 m (Fig. 1). In the absence of oleic acid, equine
lysozyme was eluted as a narrow peak at a lower NaCl
concentration of 0.67 m. Atthe front of the ELOA
elution profile there is a small peak, possibly corre-
sponding to equinelysozyme according to its position
in the salt gradient; this was not analysed further.
CD spectroscopy of ELOA
The far- and near-UV CD spectra of ELOA and equine
lysozyme in 10 mm Tris buffer (pH 9.0) are presented in
Fig. 2. The near-UV CD spectrum of ELOA (Fig. 2A)
at 25 °C is much less structured than that of the native
state equine lysozyme, i.e. the minima at 305 and
291 nm, and the maximum at 294 nm are no longer
present, and the magnitude of the ellipticity is dimin-
ished (Fig. 2A). Thermal unfolding of equine lysozyme
at pH 9.0 (Fig. 2C) closely resembles theprotein unfold-
ing transition observed previously at pH 4.5, leading to
formation of the partially folded state of a molten glob-
ule type at 57 °C [39]. The near-UV CD spectrum of the
equine lysozyme molten globule at 57 °C is character-
ized by pronounced peaks atthe same wavelengths as in
the native state (Fig. 2A), in accord with results
described previously [4,6,39]. By contrast, the overall
amplitude of the near-UV CD spectrum of ELOA at
57 °C is significantly reduced compared with signals
recorded at 25 °C, and resembles the spectrum of ther-
mally unfolded equinelysozymeat 91 °C (Fig. 2A).
The thermal unfolding transition of ELOA was
monitored by changes in ellipticity at 222 nm in the
far-UV CD region (Fig. 2C). It was manifested in an
overall decrease of the CD signal and occurred over
a very board range of temperatures starting at
$ 35 °C and proceeding up to 91 °C. In equine lyso-
zyme alone, two unfolding transitions were observed
over the same thermal range, with the first transition
taking place between $ 35 and 57 °C, leading to an
increase in the amplitude of the CD signal, and the
second occurring between 57 and 91 °C, resulting in
an overall decrease in CD ellipticity.
ELOA spectra in the far-UV CD region recorded at
both 25 and 57 °C do not display the minimum at
230 nm typical of the native state equine lysozyme
spectrum at 25 °C, but exhibit the same shape as the
spectrum for theequinelysozyme molten globule at
57 °C (Fig. 2B). At 91 °C, both ELOA and equine
lysozyme are characterized bythe same residual ellip-
ticity typical of the thermally unfolded state (Fig. 2B).
The near- and far-UV CD spectra of ELOA incu-
bated at 37 °C for 24 h did not exhibit any changes,
indicating that ELOA remained stable and did not
undergo any structural changes under these conditions
(data not shown). The CD spectra of equine lysozyme
did not reveal any changes when theprotein was coin-
cubated with a 50 fold excess of oleicacid in solution
for 2 h at 20 °C (Fig. 2D). This indicates the impor-
tance of the column environment for ELOA formation.
Binding of fluorescent dyes to ELOA
ELOA binds hydrophobic dye 8-anilinonaphthalene-1-
sulfonate (ANS), which leads to an $ 10-fold increase
in dye fluorescence compared with the free dye in solu-
tion (Fig. 3A). A shorter wavelength shift of the spec-
trum maximum from 515 to 495 nm was also
observed, indicating that ANS is present in the bound
form in a more hydrophobic environment. These
results suggest that the ELOA complex is characterized
by exposed hydrophobic surfaces.
ELOA also binds thioflavin-T (ThT) dye, which is
known for its ability to bind specifically to amyloid
species. In the presence of ELOA, the fluorescence of
ThT increases by approximately sixfold compared with
the free dye in solution (Fig. 3B). This indicates that
ELOA possesses the tinctorial property of amyloids.
UV absorbance at 280 nm
Conductivity (mS·cm
–1
)
0:00 1:00 2:00
200
150
100
50.0
0.00
250
2.00
1.50
1.00
0.50
0.00
Time (h : min)
Fig. 1. ELOA production by anion-exchange chromatography.
Elution profile of ELOA (bold line) produced in the anion-exchange
column preconditioned with oleicacid and the control peak of
equine lysozyme (fine dotted line) eluted from the column without
oleic acid preconditioning. Elution profiles were measured by UV
absorbance at 280 nm (left y axis). The NaCl gradient correspond-
ing to the conductivity of the eluent in mSÆcm
)1
(right y axis) is
shown by a solid line.
K. Wilhelm et al. Proteinoligomerizationinducedbyoleic acid
FEBS Journal 276 (2009) 3975–3989 ª 2009 The Authors Journal compilation ª 2009 FEBS 3977
Atomic force microscopy
ELOA was analysed using atomic force microscopy
(AFM) and the images are presented in Fig. 4. ELOA
is characterized by a spherical morphology reflected in
spherical-cup specs of 10–30 A
˚
height as measured in
AFM cross-sections (Fig. 4A). In samples deposited on
mica preincubated with 10 mm NaCl, we observed
ring-shaped assemblies of spherical species, with a
height of $ 10 A
˚
measured along the circumference
(Fig. 4B–D) and a diameter of $ 30 nm between the
highest points of the circumference. Because NaCl bal-
ances negative charges on the mica surface and facili-
tates the adhesion of ELOA, which is also negatively
charged at pH 9.0, this may stabilize the ring assem-
blies of the ELOA oligomers.
1D and NOESY
1
H NMR spectra
The 1D
1
H NMR spectrum of ELOA at pH 9.0
exhibits very broad aromatic resonances at 8–
6 p.p.m. (Fig. 5C,D) and a complete absence of
resolved methyl peaks in the low-field region of 2.5–
0.5 p.p.m. (data not shown). By contrast, the 1D
1
H NMR spectra of equine lysozyme, either eluted
from the column without oleicacid preconditioning
(Fig. 5A) or freshly dissolved in D
2
O, are character-
ized by well-dispersed resonances in both the aro-
matic and aliphatic regions, closely resembling the
spectra reported previously and assigned to the
native equinelysozymeat pH 4.5 [6].
The positions of resolved resonances of oleicacid in
ELOA were compared with those of free oleicacid in
solution (Fig. 5D). All are consistently shifted up-field:
the peak for free oleicacidat 5.4 p.p.m. is positioned
at 5.24 p.p.m. in the ELOA complex, the 2.1 p.p.m.
peak is at 1.9 p.p.m., the 1.3 p.p.m. peak is at
0
10
20
30
40
50
410 460 510 560
460 480 500 520
Wavelength (nm)
ANS fluorescence
0
2
4
6
8
10
12
Wavelength (nm)
THT fluorescence
A
B
Fig. 3. Interaction of ELOA with fluorescent dyes. (A) Interaction of
ELOA with ANS. The fluorescence spectrum of dye bound to ELOA
is shown by a solid line of the free dye in solution is shown by a
dashed line. (B) Interaction of ELOA with ThT. The fluorescence
spectrum of dye bound to ELOA is shown by a solid line and the
free dye in solution is shown by a dashed line.
260 270 280 290 300 310 320
260 270 280 290 300 310 320
200 210 220 230 240 250
–100
–80
–60
–40
–20
0
20
40
60
80
100
(deg cm
2
·dmol
–1
)
]
[
(deg cm
2
·dmol
–1
)
][
10
–3
(deg cm
2
·dmol
–1
)
]
[
Wavelength (nm)
Wavelength (nm) Wavelength (nm)
–80
–60
–40
–20
0
20
40
60
80
0 102030405060708090100
–8
–7
–6
–5
–4
–3
–2
Temperature (°C)
][
222 nm
10
–3
(deg cm
2·
dmol
–1
)
–12
–8
–10
–6
–4
–2
0
A
B
C
D
Fig. 2. CD spectra of ELOA and equine
lysozyme. (A) Near-UV and (B) far-UV CD
spectra of ELOA at 25 °C (–—), 57 °C( )
and 91 °C(-
‘
-), and equinelysozyme at
25 °C(–Æ – Æ), 57 °C(ÆÆÆ) and 91 °C(-ÆÆ-),
respectively. (C) Thermal unfolding of ELOA
(
) and equinelysozyme (s) monitored by
recording ellipticity at 222 nm. (D) Near-UV
CD spectra of ELOA (—–) and equine lyso-
zyme directly after the addition of a 50-fold
access of oleicacid (- ÆÆ -) and after 2 h incu-
bation with oleicacid (- Æ - Æ -).
Protein oligomerizationinducedbyoleicacid K. Wilhelm et al.
3978 FEBS Journal 276 (2009) 3975–3989 ª 2009 The Authors Journal compilation ª 2009 FEBS
1.2 p.p.m. and the 0.9 p.p.m. peak is at 0.8 p.p.m.,
respectively. This indicates that oleicacid exists in a
different environment within the ELOA complex than
its free form. The spectrum of ELOA was also
recorded in 10 mm NaCl ⁄ P
i
at pH 7.4 (data not
shown), and closely resembled the spectrum shown in
Fig. 5C. The amount of bound oleicacid in ELOA
was determined by comparing the peak area of oleic
acid in its bound form at 5.24 p.p.m. (2 olefinic pro-
tons) with the peak area corresponding to aromatic
proton resonances of lysozyme in the 1D
1
H NMR
spectrum of ELOA. In approximately 20 consecutive
preparations, the ratio of oleicacid to equine lysozyme
in ELOA varied from 11 to 48 depending on the spe-
cific chromatographic conditions during the complex
formation. In general, repetitive saturation of the col-
umn with oleicacid resulted in the formation of
ELOA with a higher oleicacid content.
The 2D
1
H NOESY spectrum of ELOA at pH 9.0 is
shown in Fig. 6 and similar results were obtained at
pH 7.0 (data not shown). The spectrum arising from
the proteinaceous part is characterized by very broad
resonances and we present it at a high contour level to
demonstrate the resonances from oleicacid molecules
integrated into the complex structure. Indeed, the posi-
tive NOE cross-peaks between oleicacid signals at 5.2,
1.8 and 1.1 p.p.m. (Fig. 6A) indicate that oleicacid is
not present in its free form, but within a large mole-
cular complex. Positive NOE cross-peaks were also
observed between oleicacid proton resonances and the
aromatic residue resonances of equinelysozyme in the
region of 6.5–7.5 p.p.m., as shown in Fig. 6B. This
indicates intermolecular binding between lysozyme and
oleic acid and that aromatic residues of equine lyso-
zyme are involved in oleicacid binding.
Pulsed field gradient diffusion measurements
The diffusion coefficients of ELOA, native monomeric
equine lysozyme and molten globular equine lysozyme
at pH 2.0 were determined using pulse field gradient
16
12
8
4
0
0 200 400
Vector length (Å)
Height (Å)
600 800
D
A
B
C
Fig. 4. AFM imaging of ELOA. (A) ELOA on a mica surface is
shown as round particles. Scale bar = 200 nm. (B) Ring-shaped
assemblies of ELOA. Scale bar = 100 nm. (C) Individual ring-shaped
assembly. Scale bar = 25 nm. (D) Height profile of ELOA ring
shown in the AFM cross-section; the arrows in (C) and (D) indicate
the position of the cross-section.
p.p.m.
8765
3210
p.p.m.
8 7 68 7 6 8 7 6
ELOA
Oleic acid
A
D
CB
Fig. 5. 1D
1
H NMR spectra of ELOA and equine lysozyme. Aro-
matic regions of 1D
1
H NMR spectra of (A) native equine lysozyme
in 10 m
M Tris, pH 9.0, 25 °C, (B) equinelysozyme molten globule
in 10 m
M glycine, pH 2.0, 25 °C, (C) ELOA in 10 mM Tris, pH 9.0,
25 °C. (D) 1D
1
H NMR spectrum of ELOA (upper) and free oleic
acid (lower), the left-hand panel has been scaled up for demonstra-
tion purposes.
K. Wilhelm et al. Proteinoligomerizationinducedbyoleic acid
FEBS Journal 276 (2009) 3975–3989 ª 2009 The Authors Journal compilation ª 2009 FEBS 3979
(PFG) diffusion measurements (Fig. 7). Diffusion coef-
ficients were calculated by analysing diffusion decays
(a representative example is shown in Fig. 7A) accord-
ing to Eqn (1). Because equinelysozyme is present in a
molten globule state within ELOA, the diffusion coeffi-
cient of the molten globule was used as a reference
when calculating the molecular volumes and masses of
ELOA complexes, according to Eqn (2). The diffusion
coefficient of the native state of equinelysozyme was
1.18 times larger than the corresponding value for the
molten globule, indicating an $ 18% larger hydro-
dynamic radius and an $ 60% larger molecular
volume for the molten globule state.
The diffusion coefficients for the ELOA complexes
were determined by following separately the strong
signals of the aromatic residues of equine lysozyme
and theoleicacid protons at 1.15 p.p.m. The diffu-
sion coefficients determined by following the proton
resonances of aromatic residues of lysozyme molecules
were slightly smaller than those derived from monitor-
ing the signals of theoleicacid protons, indicating
that the ELOA preparations contain a small amount
of free oleic acid, estimated to be < 10%. The diffu-
sion coefficients for the ELOA complexes were 0.28–
0.56 times that of the molten globule state of equine
lysozyme (Fig. 7B). Using these values and taking into
account the amount of oleicacid bound to each pro-
tein molecule, the number of equinelysozyme mole-
cules in the ELOA complexes was estimated to be
4–9 in most cases and 30 molecules in one particular
preparation.
Trypan blue cell-viability assay
The effect of ELOA, oleic acid, equinelysozyme and
the mixture of equinelysozyme with oleicacid on cell
viability was examined using a Trypan blue staining
assay. A mouse embryonic liver cell culture (Fig. 8A)
and mouse embryonic fibroblasts (Doc. S1) were used
for this purpose. ELOA was added at a concentration
of 1.8–12.4 lm. The concentrations of equine lysozyme
5.5
0.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
5.0
4.5
0.0
4.0
3.0
2.0
1.0
5.0
0.0
Water Buffer
4.0
3.0
2.0
1.0
5.0
6.5
7.0
7.5
p.p.m.
p.p.m.
HC=CH
RCH
3
RC(=0)CH
2
(CH
2
)
n
CH
2
CH=CHCH
2
CH
3
(CH
2
)
7
CH=CH(CH
2
)
7
COOH
Oleic acid:
0.0
¨
A
B
Fig. 6. 2D
1
H NOESY spectrum of ELOA. (A) Assignment of oleic
acid signals in 1D
1
H NMR-spectrum of ELOA (upper) and 2D
1
H
NOESY spectrum of ELOA (lower), showing mostly cross-peaks of
oleic acidatthe chosen contour level. (B) Intermolecular cross-
peaks between the proton resonances of oleicacid and the aro-
matic residues of equine lysozyme.
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1 : 11 1 : 16 1 : 24 1 : 30 1 : 43
Relative diffusion coefficient
Molten globule
Native
–1.2
–0.8
–0.4
0.0
0 4000 8000
G
2
ln (I/I
0
)
A
B
Fig. 7. PFG diffusion NMR measurements of ELOA and equine
lysozyme. (A) Representative integral decays ln(I ⁄ I
0
) as a function
of gradient strength G
2
of folded equinelysozyme (s), equine lyso-
zyme molten globule (
) and ELOA (h) (corresponds to an equine
lysozyme ⁄ oleicacid ratio of 1 : 11). (B) Relative diffusion coeffi-
cients of the ELOA complexes with different ratios of equine lyso-
zyme to oleicacid molecules shown above the stripped bars. The
diffusion coefficients of equinelysozyme in the native (white bar)
and molten globule (grey bar) states were used as controls.
Protein oligomerizationinducedbyoleicacid K. Wilhelm et al.
3980 FEBS Journal 276 (2009) 3975–3989 ª 2009 The Authors Journal compilation ª 2009 FEBS
and oleicacid used were equivalent to their content in
the ELOA complex. The cells were incubated with
the corresponding compounds for 1.5, 5 and 24 h. The
viability of mouse embryonic liver cells decreased
significantly within 1.5 h of incubation at all ELOA
concentrations used; in the presence of 1.8–8.9 lm
ELOA it decreased by $ 20%, at a higher ELOA con-
tent of 12.4 lm it decreased by $ 40%. Cell viability
decreased by $ 70% upon the addition of 8.9 lm
ELOA after 5 h and by $ 80% after 24 h of incuba-
tion. The survival of cells treated with 12.4 lm ELOA
did not exceed $ 20% after either 5 or 24 h of incuba-
tion. Even at its highest concentration, equine lyso-
zyme alone did not affect the viability of mouse
embryonic liver cells (data not shown). The reduction
in cell viability inducedby 85–596 lm oleicacid was
within $ 10% (Fig. 8B); the same effect was observed
when cells were added to a mixture of oleicacid within
the same concentration range and equinelysozyme at
its highest concentration (data not shown).
Mouse embryonic fibroblast culture was also treated
with ELOA and the results of the cell viability assessed
by Trypan blue staining assay are presented in Fig. S1.
Cell viability decreased by $ 90% in the presence of
8.9 lm ELOA after 1.5–24 h of incubation, whereas
85–596 lm oleicacid reduced cell viability by $ 10%
Equine lysozymeOleicacid ELOA
Oleic acid added (µM)
Control
85
255 426 596
Cell viability (%) Cell viability (%)
0
20
40
60
80
100
120
0
20
40
60
80
100
120
Control
1.8
5.3 8.9 12.4
Complex added (µM)
**** ****** ****** ******
C
A
B
Fig. 8. Effect of ELOA on cell viability. Via-
bility of mouse embryonic liver cell culture
coincubated with (A) ELOA and (B) oleic
acid. Untreated cells were used as a control
and their viability was set at 100% (black
bars). The viability of cells coincubated with
ELOA or oleicacid for 1.5 h is shown by
grey bars, the viability of cells coincubated
for 5 h is shown by white bars and the via-
bility of cells coincubated for 24 h is shown
by striped bars. *P < 0.05, **P < 0.01. (C)
Acridine orange and ethidium bromide stain-
ing of murine embryonic liver cells treated
with ELOA and its components. Alive cells
treated with 12.4 l
M equinelysozyme (left)
and 596 l
M oleicacid (central) for 5 h are
stained with acridine orange, showing a
green fluorescence. Cells exposed to
12.4 l
M ELOA for 5 h (right) show both acri-
dine orange (green) and ethidium bromiden
(orange) staining, indicating cell death. Scale
bar = 100 lm.
11 min 58 min 59 min 60 min
10 µm
Fig. 9. Imaging ELOA interactions with live cells. Time-dependent accumulation of ELOA labelled with Alexa Fluor (shown in bright green) in
the vicinity of live PC12 cells up to 58 min of coincubation. At 59 min, the cell wall was ruptured, allowing ELOA to stream in and fill the cell
interior (60 min).
K. Wilhelm et al. Proteinoligomerizationinducedbyoleic acid
FEBS Journal 276 (2009) 3975–3989 ª 2009 The Authors Journal compilation ª 2009 FEBS 3981
after 1.5 h and by $ 30% after 24 h of incubation.
Equine lysozyme alone did not induce cellular toxicity
and a mixture of equinelysozyme and oleicacid at
their highest concentrations within the range examined
here produced the same effect as oleicacid alone (data
not shown).
The ELOA complexes with different protein to oleic
acid ratios were used in the cytotoxicity experi-
ments, including ratios of 1 : 20, 1 : 40 and 1 : 48.
Their cytotoxicity depended on the concentration of the
proteinaceous component, determined by measuring
absorbance spectra. This indicates that the proteina-
ceous component, but not oleic acid, is a critical factor
in defining the cytotoxicity of ELOA complexes. Fur-
ther studies are needed to provide more detail on the
structure–function relationship of ELOA complexes.
Acridine orange
⁄
ethidium bromide staining
Mouse embryonic liver cells treated with ELOA
(12.4 lm), equinelysozyme (12.4 lm) and oleic acid
(596 lm) for 1.5, 5 and 24 h were subjected to acridine
orange and ethidium bromide staining. Representative
images of the stained cells after 5 h of treatment are
given in Fig. 8C. Acridine orange permeates all cells
leading to green fluorescence. In the presence of equine
lysozyme and oleic acid, live cells appeared green in
$ 90% of cases. Ethidium bromide is taken up by
cells if their cytoplasmic membrane integrity is lost.
Ethidium bromide interacts with DNA in apoptotic
cells, giving an orange fluorescence; ethidium bromide
fluorescence usually predominates over acridine orange
uptake. Orange ⁄ green staining was seen in $ 80% of
all cells treated with ELOA (Fig. 8C), indicating
apoptotic type cell death [49].
Imaging of ELOA interactions with live cells
In order to observe interactions between ELOA and
live cells, the complex was fluorescently labelled with
the amine-reactive dye Alexa Fluor 488 and live PC12
cells were subsequently incubated with fluorescently
labelled ELOA. A concentration of fluorescently
labelled ELOA of 850 nm was determined in bulk
medium, using quantitative imaging by confocal laser
scanning microscopy (CLSM) [50] and fluorescence
correlation spectroscopy (FCS), techniques that enable
nondestructive observation of molecular interactions in
live cells with single-molecule sensitivity. The time
course of ELOA interactions with live cells was studied
using time-lapsed CLSM (Fig. 9). We observed that
ELOA accumulated continuously in the vicinity of the
cell membrane over a period of 58 min, reaching a
10-fold higher local concentration than the bulk con-
centration in solution. During this time, cells were able
to ‘resist’ ELOA and significant uptake of the complex
was not detected. At a pivotal time point of coincuba-
tion (59 min), cell membranes ruptured in a coopera-
tive manner and ELOA streamed into the cells, filling
the whole cellular interior almost instantaneously
(60 min). Such effect was not observed for equine lyso-
zyme alone (data not shown), which did not disrupt
+
+
+
+
+
+
+
+
Sepharose matrix
Sepharose matrix
Sepharose matrix
A
D
BC
Sepharose matrix
Fig. 10. Schematic representation of the
ELOA formation atthesolid–liquid interface
within column chromatography. (A) The
Sepharose matrix is positively charged
under our experimental conditions. (B) Bind-
ing of oleicacid to the matrix precedes
ELOA formation. (C) Folded equine lyso-
zyme molecules added to the column are
shown in space-filling and ribbon-diagram
representations. The exposed hydrophilic
residues are denoted in purple and the bur-
ied hydrophobic residues in grey. (D) During
interaction with thesolid–liquidinterface in
the column, the hydrophobic residues (grey)
become exposed in the molten globule
state of equinelysozyme and its molecules
assemble with each other and with oleic
acids to form ELOA (encircled sche-
matically).
Protein oligomerizationinducedbyoleicacid K. Wilhelm et al.
3982 FEBS Journal 276 (2009) 3975–3989 ª 2009 The Authors Journal compilation ª 2009 FEBS
cellular membranes and did not cause cell damage over
6 h of observation.
Discussion
We demonstrated that the self-assembly of equine lyso-
zyme into stable oligomers can be induced in an
anion-exchange chromatography column precondi-
tioned with oleic acid, as outlined in Fig. 10. It is
important to note that coincubation of a 50 fold excess
of oleicacid with equinelysozyme in solution did not
lead to ELOA formation, as evident from the near-UV
CD measurements (Fig. 2C). Oleicacid molecules
bound to the ion-exchange matrix constitute an
extended surface, facilitating both charged and hydro-
phobic interactions with equinelysozyme molecules.
Such a surface may effectively model the cell lipid
membranes able to induce protein–ligand interactions,
which would not otherwise occur in solution. Indeed,
in solution, oleic acid, like many other small aliphatic
molecules, would be present as a micelle. Concomi-
tantly, thesolid–liquidinterface may induce partial
unfolding of equinelysozyme and exposure of the
hydrophobic surfaces buried in the native state; this
may also be critical for ELOA complex formation. It
is important to note that extensive studies have
recently been conducted to characterize the conforma-
tional changes occurring atthe solid hydrophobic
interfaces in hen egg white lysozyme, which is a struc-
tural homologue to equinelysozyme [44]. A suggested
model of conformational change included conversion
of the initial a-helical structures into random coil ⁄ turn
and subsequently into b sheet [51–53]. Such structural
changes are a key event in oligomeric and fibrillar
amyloid assembly. Equinelysozyme is significantly less
cooperative than hen egg white lysozyme [5,6,46] and
is more prone to structural rearrangement and aggre-
gation. Therefore, under our experimental conditions,
it readily assembled into well-defined ELOA com-
plexes, preserved as a stable fraction in solution for
up to a week. It is worth noting that complexes of
hen egg white lysozyme with oleicacid were also
produced under the same conditions, but they were
significantly less populated and easily lost oleic acid
(data not shown). Complexes of human a-lactalbumin
with oleic acid, HAMLET, were also produced using
column chromatography [32]. Remarkably, a multi-
meric active complex of a-lactalbumin with oleic acid
was isolated and purified from the casein fraction of
human milk [26,27] and denoted as multimeric a-lact-
albumin (MAL), which indicates that the solid–liquid
interfaces of the chromatography column may mimic
in vivo conditions.
Equine lysozyme within the ELOA complex is pres-
ent in a partially unfolded state, as evident from the
near- and far-UV CD spectra (Fig. 2A,D), ANS bind-
ing (Fig. 3A) and the decreased dispersion seen in the
1D
1
H NMR spectrum (Fig. 5C). The near-UV CD
spectrum of ELOA exhibits lower ellipticity values and
largely overlapping peaks compared with the native
and even molten globule states of equine lysozyme
(Fig. 2A) [6,39]. This indicates that theprotein tertiary
structure within ELOA may be even more disordered
than in its molten globule state. Examination of the
1D
1
H NMR spectrum of ELOA clearly shows up-field
shifts of the resonance of oleicacid incorporated
within the complex compared with the resonances of
free oleic acid, demonstrating that oleicacid molecules
are an integral part of ELOA. They interact directly
with the aromatic residues of lysozyme, as demon-
strated bythe presence of cross-peaks between the pro-
tons of aromatic residues and oleicacid observed in
the
1
H NOESY spectrum of ELOA (Fig. 6B).
The number of protein and oleicacid molecules
varies within ELOA complexes produced in different
preparations. We have shown that 11–48 oleic acids
can bind to each equinelysozyme molecule, depending
on the specific chromatographic conditions during
complex formation. The number of equine lysozyme
molecules in ELOA can also vary from 4 to 30, as
determined by PFG diffusion measurements. Previ-
ously, we observed the formation of oligomers of
equine lysozyme under amyloid-inducing conditions at
acidic pH, which also ranged from tetramers to ecosi-
nomers and larger [42], however, they never constituted
more than a few percent of the total amount of mono-
meric equinelysozyme in solution. This is in contrast
to ELOA, which constitutes the majority of molecular
species in the samples. In this respect, ELOA resembles
the HAMLET-type complex of a-lactalbumin with
oleic acid extracted from the casein fraction of human
milk, which is also oligomeric in nature [26,31].
ELOA complexes display properties similar to those
of equinelysozyme amyloid oligomers, for example,
ThT binding and their morphological appearance as
shown by AFM. In a similar way to equine lysozyme
oligomers, ELOA also forms ring-shaped assemblies
(Fig. 4). By contrast to equinelysozyme and a-lactal-
bumin amyloid oligomers, which are populated
on-pathway to amyloid fibrils [47,54], the ELOA com-
plex did not produce polymeric structures upon pro-
longed incubation in our experiments. This suggests
that oleicacid stabilizes the oligomeric complex, pre-
venting its further conversion and assembly into larger
polymers. Some other surfactants and compounds such
as SDS and fatty acids were also applied to Ab pep-
K. Wilhelm et al. Proteinoligomerizationinducedbyoleic acid
FEBS Journal 276 (2009) 3975–3989 ª 2009 The Authors Journal compilation ª 2009 FEBS 3983
tide, a-synuclein and other amyloidogenic proteins to
stabilize their oligomers as opposed to fibrils [55].
Although prefibrillar proteinaceous structures encom-
pass a wide variety of species, studies of kinetically
trapped ELOA complexes can shed light on the struc-
tural and functional properties of pre-fibrillar species
and their role in ‘on-’ and ‘off’-pathway’ amyloid
assembly.
It is interesting to note that the thermal unfolding
transition of ELOA occurs over a very wide tempera-
ture range and broadly coincides with two unfolding
transitions of equinelysozyme alone under the same
conditions. However, two transitions were not noticed
in ELOA and we did not observed an increase in
ellipticity signals during ELOA unfolding, which is a
distinguishing feature of the first transition in equine
lysozyme [39,46]. This indicates that the confor-
mational changes in ELOA and equine lysozyme
alone may have different structural origina. Similarly,
HAMLET was slightly less stable than human a-lactal-
bumin in the presence of calcium towards thermal
denaturation and exhibited the same stability as
human a-lactalbumin towards urea denaturation [56].
This indicates that oleicacid has a similar effect on the
structural stability of both complexes.
We have shown that ELOA is cytotoxic towards dif-
ferent cell types, including mouse embryonic liver cell
culture, mouse embryonic fibroblast culture, a neuro-
blastoma cell line (SH-SY5Y) and a rat phreochromcy-
toma (PC12) cell line. Combined staining with acridine
orange and ethidium bromide indicated that ELOA
induces apoptotic-type cell death. In order to gain fur-
ther insight into the mechanisms underlying cellular tox-
icity, we studied the interactions of ELOA with live cells
by using single molecular techniques such as CLSM and
FCS (Fig. 9). Our results showed that ELOA initially
accumulated actively in the vicinity of the cell mem-
brane, implying that the cell membrane is a primary
target for ELOA toxic activity. We presume that inter-
actions of ELOA with the cell membrane trigger apop-
totic stimuli, proceeding from the plasma membrane to
the cell interior without ELOA internalization per se
and consequently trigger cell death. ELOA internaliza-
tion occurred after the cell membrane rupture.
It is important to note that equinelysozyme oligo-
mers are also cytotoxic, inducing apoptosis in similar
cell types [42]. HAMLET complexes have been shown
to cause cell death in cancer and immature cells, but
not in healthy differentiated cells [30,57]. Thus, a range
of various protein oligomeric complexes can induce
cytotoxicity, even though their structural properties
differ from each other, and this requires further
detailed investigation [7,11,58]. In all these complexes,
including ELOA, cytotoxicity is a newly gained prop-
erty, acquired as a result of their self-assembly and, in
the case of ELOA, also because of the interaction with
oleic acid. Oleicacid itself can induce some cytotoxic
effects [59–62], but its cytotoxicity is significantly lower
than that of proteinaceous complexes (Figs 8 and S1).
These results emphasize the role of protein self-assem-
bly in producing thecytotoxic effect. To date, exten-
sive information has been gathered on the mechanisms
behind the cytotoxicity of HAMLET and amyloid
oligomers, however, there is no clear consensus.
Because equinelysozyme can form both ELOA com-
plexes and amyloid oligomers, in-depth studies of their
molecular properties and induced cytotoxicity would
provide a clearer insight into both these phenomena
and any link between them.
In conclusion, using hydrophobic surfaces in column
chromatography, we produced highly populated
ELOA complexes, composed of partially unfolded pro-
tein molecules and oleic acid. These complexes have
some common structural and cytotoxic features with
amyloid oligomers of equinelysozyme and with HAM-
LET. These complexes are stable and therefore amena-
ble to structural characterization at atomic resolution,
whereas the amyloid oligomers are often transient in
nature and not populated in significant proportions.
By producing ELOA, we have shown that other pro-
teins besides human and bovine a-lactalbumins can
form such structures, which widens the scope of the
HAMLET-type phenomenon. Proteins provide an
unlimited source of varying properties and functions,
among them protein complexes, which if well-charac-
terized, can be used profitably in various therapeutic
and biotechnological applications with the potential to
target specifically undesirable cells.
Materials and methods
Materials
Equine lysozyme was purified from horse milk, as described
previously [63]. Oleicacid and all chemicals were purchased
from Sigma (Stockholm, Sweden), unless stated otherwise.
The protein concentration was determined by absorbance
measurements on a NanoDrop spectrophotometer (Nano-
Drop Technologies, Wilmington, DE, USA) at 280 nm
using an extinction coefficient of E
1%
= 23.5.
Production of ELOA by anion-exchange
chromatography
ELOA was produced using 1 or 5 mL DEAE FF Sepharose
columns (Amersham Biosciences, Piscataway, NJ, USA) con-
Protein oligomerizationinducedbyoleicacid K. Wilhelm et al.
3984 FEBS Journal 276 (2009) 3975–3989 ª 2009 The Authors Journal compilation ª 2009 FEBS
[...]... Journal 276 (2009) 397 5–3 989 ª 2009 The Authors Journal compilation ª 2009 FEBS K Wilhelm et al Proteinoligomerizationinducedbyoleicacid References 1 Uversky VN (2003) Protein folding revisited A polypeptide chain atthe folding–misfolding–nonfolding cross-roads: which way to go? Cell Mol Life Sci 60, 185 2–1 871 2 Dobson CM (2003) Protein folding and misfolding Nature 426, 88 4–8 90 3 Morozova-Roche... Mechanisms involved in Jurkat cell death inducedbyoleic and linoleic acids Clin Nutr 25, 100 4–1 014 Proteinoligomerizationinducedbyoleicacid 61 De Gottardi A, Vinciguerra M, Sgroi A, Moukil M, Ravier-Dall’Antonia F, Pazienza V, Pugnale P, Foti M & Hadengue A (2007) Microarray analyses and molecular profiling of steatosis induction in immortalized human hepatocytes Lab Invest 87, 79 2–8 06 62 Gill CI, Boyd... lyophilized ELOA in 500 lL D2O, 10 mm Tris or 10 mm NaCl ⁄ Pi pH at 9.0 or 7.2 to yield a protein concentration of $ 50 lm The molar ratio between oleicacid and lysozyme in the complex was determined by comparing the peak areas of oleicacid olefinic proton resonances with thelysozyme aromatic signals 2D 1H NOESY spectra were recorded at 25 °C, using a mixing time of 150 ms, 8 scans and 272 increments... 3985 Proteinoligomerizationinducedbyoleicacid K Wilhelm et al nol prior to the addition to culture media The effect on cells of equivalent concentrations of ethanol was examined and shown to not affect the cell viability PC12 cells, pheochromocytoma cells derived from rat adrenal medulla, were obtained from the American Type Culture Collection (ATCC) The cells were cultured in collagen-coated... Neurobiol Dis 30, 21 2–2 20 Nagarajan S, Ramalingam K, Neelakanta Reddy P, Cereghetti DM, Padma Malar EJ & Rajadas J (2008) Lipid -induced conformational transition of the amyloid core fragment Abeta(28-35) and its A30G and A30I mutants FEBS J 275, 241 5–2 427 FEBS Journal 276 (2009) 397 5–3 989 ª 2009 The Authors Journal compilation ª 2009 FEBS 3987 Proteinoligomerizationinducedbyoleicacid K Wilhelm et... & Privalov PL (1995) The unfolding thermodynamics of c-type lysozymes: a calorimetric study of the heat denaturation of equinelysozyme J Mol Biol 252, 44 7–4 59 41 Chowdhury FA, Fairman R, Bi Y, Rigotti DJ & Raleigh DP (2004) Protein dissection experiments reveal key differences in the equilibrium folding of alpha-lactalbumin and the calcium binding lysozymes Biochemistry 43, 996 1–9 967 42 Malisauskas... humidified atmosphere at 37 °C Cells were plated at a density of 104 cellsÆwell in 96-well plates, cell viability was assayed after 1.5, 5 and 24 h of coincubation with ELOA and respective controls ELOA was diluted in serum-free culture medium to the required concentrations and then added to the cells Oleicacid was diluted in etha- FEBS Journal 276 (2009) 397 5–3 989 ª 2009 The Authors Journal compilation... respectively Assuming that the proteinaceous particles are spherical and neglecting density changes, the mass of thecomplexes was determined using Eqn (2): MELOA =MEL ¼ ðDEL =DELOA Þ3 ð2Þ; where MELOA and MEL are molecular masses, and DELOA and DEL diffusion coefficients of the ELOA complex and monomeric equinelysozyme in the molten globule state used as a reference The number of equinelysozyme molecules... (2005) Does thecytotoxic effect of transient amyloid oligomers from common equinelysozyme in vitro imply innate amyloid toxicity? J Biol Chem 280, 626 9– 6275 43 Permyakov SE, Khokhlova TI, Nazipova AA, Zhadan AP, Morozova-Roche LA & Permyakov EA (2006) Calcium-binding and temperature induced transitions in equine lysozyme: new insights from the pCa-temperature ‘phase diagrams’ Proteins 65, 98 4–9 98 44... using the 488 nm line of the Ar ⁄ ArKr laser Quantitative measurements were achieved by quantitative APD imaging [50] performed on an integrated FCS/CSLM instrument Statistical analysis All cell viability experiments were performed in triplicate The experimental results were analysed by Student’s paired t-test and are shown as mean ± SEM The level of statistical significance was set at P < 0.05 for the . Protein oligomerization induced by oleic acid at the solid–liquid interface – equine lysozyme cytotoxic complexes Kristina Wilhelm 1 , Adas Darinskas 2 ,. display the minimum at 230 nm typical of the native state equine lysozyme spectrum at 25 °C, but exhibit the same shape as the spectrum for the equine lysozyme molten globule at 57 °C (Fig. 2B). At. cm 2 ·dmol –1 ) ] [ (deg cm 2 ·dmol –1 ) ][ 10 –3 (deg cm 2 ·dmol –1 ) ] [ Wavelength (nm) Wavelength (nm) Wavelength (nm) –8 0 –6 0 –4 0 –2 0 0 20 40 60 80 0 102030405060708090100 –8 –7 –6 –5 –4 –3 –2 Temperature