Protein oligomerization induced by oleic acid at the solid-liquid interface - equine lysozyme cytotoxic complexes

Protein oligomerization induced by oleic acid at thesolid–liquid interface – equine lysozyme cytotoxiccomplexesKristina Wilhelm1, Adas Darinskas2, Wim Noppe3, Elke Duchardt1,4, K. Hun Mok5,Vladana Vukojevic6, Jurgen Schleucher1 and Ludmilla A. Morozova-Roche1

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 equine lysozyme assembles into multimeric complexes with oleic acid

(ELOA) at the solid–liquid interface 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 the complexes 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. Equine lysozyme 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 equine lysozyme with oleic acid (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 oleic acid 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 the complexes 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 oleic acid can inhibit the amyloid fibril formation

of bovine a-lactalbumin, acting at the 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. Equine lysozyme 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. Equine lysozyme 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, equine lysozyme 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 equine lysozyme 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 oligomerization induced by oleic acid 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. At the front of the ELOA

elution profile there is a small peak, possibly corre-

sponding to equine lysozyme 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 the protein 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 at the 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 equine lysozyme at 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 the equine lysozyme molten globule at

57 �C (Fig. 2B). At 91 �C, both ELOA and equine

lysozyme are characterized by the 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 the protein was coin-

cubated with a 50 fold excess of oleic acid 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

abs

orba

nce

at 2

80 n

m

Con

duct

ivity

(m

S·c

m–1

)

0:00 1:00 2:00

200

150

100

50.0

0.00

2502.00

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 oleic acid 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. Protein oligomerization induced by oleic acid


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 1H NMR spectra

The 1D 1H 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 1D1H NMR spectra of equine lysozyme, either eluted

from the column without oleic acid preconditioning

(Fig. 5A) or freshly dissolved in D2O, 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 equine lysozyme at pH 4.5 [6].

The positions of resolved resonances of oleic acid in

ELOA were compared with those of free oleic acid in

solution (Fig. 5D). All are consistently shifted up-field:

the peak for free oleic acid at 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)

AN

S fl

uore

scen

ce

02468

1012

Wavelength (nm)

TH

T fl

uore

scen

ce

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 320200 210 220 230 240 250

–100–80–60–40–20

020406080

100(d

eg c

m2 ·

dmol

–1)

][

(deg

cm

2 ·dm

ol–1

)]

[10–3

(de

g cm

2 ·dm

ol–1

)]

[

Wavelength (nm)

Wavelength (nm) Wavelength (nm)

–80

–60

–40

–20

0

20

40

60

80

0 10 20 30 40 50 60 70 80 90 100–8

–7

–6

–5

–4

–3

–2

Temperature (°C)

][

222

nm10

–3 (d

eg c

m2·

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 equine lysozyme at

25 �C (– Æ – Æ), 57 �C (ÆÆÆ) and 91 �C (-ÆÆ-),

respectively. (C) Thermal unfolding of ELOA

( ) and equine lysozyme (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 oleic acid (- ÆÆ -) and after 2 h incu-

bation with oleic acid (- Æ - Æ -).



1.2 p.p.m. and the 0.9 p.p.m. peak is at 0.8 p.p.m.,

respectively. This indicates that oleic acid exists in a

different environment within the ELOA complex than

its free form. The spectrum of ELOA was also

recorded in 10 mm NaCl ⁄Pi at pH 7.4 (data not

shown), and closely resembled the spectrum shown in

Fig. 5C. The amount of bound oleic acid 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 1H NMR

spectrum of ELOA. In approximately 20 consecutive

preparations, the ratio of oleic acid 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 oleic acid resulted in the formation of

ELOA with a higher oleic acid content.

The 2D 1H 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 oleic acid molecules

integrated into the complex structure. Indeed, the posi-

tive NOE cross-peaks between oleic acid signals at 5.2,

1.8 and 1.1 p.p.m. (Fig. 6A) indicate that oleic acid is

not present in its free form, but within a large mole-

cular complex. Positive NOE cross-peaks were also

observed between oleic acid proton resonances and the

aromatic residue resonances of equine lysozyme 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 oleic acid 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

00 200 400

Vector length (Å)

Hei

ght (

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.

8 7 6 5 3 2 1 0p.p.m.

8 7 68 7 6 8 7 6

ELOA

Oleic acid

A

D

CB

Fig. 5. 1D 1H NMR spectra of ELOA and equine lysozyme. Aro-

matic regions of 1D 1H NMR spectra of (A) native equine lysozyme

in 10 mM Tris, pH 9.0, 25 �C, (B) equine lysozyme molten globule

in 10 mM glycine, pH 2.0, 25 �C, (C) ELOA in 10 mM Tris, pH 9.0,

25 �C. (D) 1D 1H NMR spectrum of ELOA (upper) and free oleic

acid (lower), the left-hand panel has been scaled up for demonstra-

tion purposes.



(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 equine lysozyme 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 equine lysozyme 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 the oleic acid 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 the oleic acid 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 oleic acid bound to each pro-

tein molecule, the number of equine lysozyme 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, equine lysozyme and

the mixture of equine lysozyme with oleic acid 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 1.05.0 0.0

Water Buffer

4.0 3.0 2.0 1.05.0

6.5

7.0

7.5

p.p.m.

p.p.m.

HC=CH

RCH3RC(=0)CH2

(CH2)n

CH2CH=CHCH2

CH3(CH2)7CH=CH(CH2)7COOHOleic acid:

0.0

¨

A

B

Fig. 6. 2D 1H NOESY spectrum of ELOA. (A) Assignment of oleic

acid signals in 1D 1H NMR-spectrum of ELOA (upper) and 2D 1H

NOESY spectrum of ELOA (lower), showing mostly cross-peaks of

oleic acid at the chosen contour level. (B) Intermolecular cross-

peaks between the proton resonances of oleic acid 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

Rel

ativ

e di

ffusi

on c

oeffi

cien

t

Mol

ten

glob

ule

Nat

ive

–1.2

–0.8

–0.4

0.00 4000 8000

G2

ln (

I/I0)

A

B

Fig. 7. PFG diffusion NMR measurements of ELOA and equine

lysozyme. (A) Representative integral decays ln(I ⁄ I0) as a function

of gradient strength G2 of folded equine lysozyme (s), equine lyso-

zyme molten globule ( ) and ELOA (h) (corresponds to an equine

lysozyme ⁄ oleic acid ratio of 1 : 11). (B) Relative diffusion coeffi-

cients of the ELOA complexes with different ratios of equine lyso-

zyme to oleic acid molecules shown above the stripped bars. The

diffusion coefficients of equine lysozyme in the native (white bar)

and molten globule (grey bar) states were used as controls.



and oleic acid 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 induced by 85–596 lm oleic acid was

within � 10% (Fig. 8B); the same effect was observed

when cells were added to a mixture of oleic acid within

the same concentration range and equine lysozyme 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 oleic acid reduced cell viability by � 10%

Equine lysozyme Oleic acid ELOA

Oleic acid added (µM)

Control 85 255 426 596C

ell v

iabi

lity

(%)

Cel

l via

bilit

y (%

)

0

20

40

60

80

100

120

0

20

40

60

80

100

120

Control 1.8 5.3 8.9 12.4Complex 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 oleic acid 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 lM equine lysozyme (left)

and 596 lM oleic acid (central) for 5 h are

stained with acridine orange, showing a

green fluorescence. Cells exposed to

12.4 lM 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).



after 1.5 h and by � 30% after 24 h of incubation.

Equine lysozyme alone did not induce cellular toxicity

and a mixture of equine lysozyme and oleic acid at

their highest concentrations within the range examined

here produced the same effect as oleic acid 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), equine lysozyme (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

+

+

+

+

+

+

+

+

Sepharo

se m

atr

ix

Sepharo

se m

atr

ix

Sepharo

se m

atr

ix

A

D

B CS

epharo

se m

atr

ix

Fig. 10. Schematic representation of the

ELOA formation at the solid–liquid interface

within column chromatography. (A) The

Sepharose matrix is positively charged

under our experimental conditions. (B) Bind-

ing of oleic acid 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 the solid–liquid interface in

the column, the hydrophobic residues (grey)

become exposed in the molten globule

state of equine lysozyme and its molecules

assemble with each other and with oleic

acids to form ELOA (encircled sche-

matically).



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 oleic acid with equine lysozyme in solution did not

lead to ELOA formation, as evident from the near-UV

CD measurements (Fig. 2C). Oleic acid molecules

bound to the ion-exchange matrix constitute an

extended surface, facilitating both charged and hydro-

phobic interactions with equine lysozyme 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, the solid–liquid interface may induce partial

unfolding of equine lysozyme 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 at the solid hydrophobic

interfaces in hen egg white lysozyme, which is a struc-

tural homologue to equine lysozyme [44]. A suggested

model of conformational change included conversion

of the initial a-helical structures into random coil ⁄ turnand subsequently into b sheet [51–53]. Such structural

changes are a key event in oligomeric and fibrillar

amyloid assembly. Equine lysozyme 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 oleic acid 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 1H 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 the protein tertiary

structure within ELOA may be even more disordered

than in its molten globule state. Examination of the

1D 1H NMR spectrum of ELOA clearly shows up-field

shifts of the resonance of oleic acid incorporated

within the complex compared with the resonances of

free oleic acid, demonstrating that oleic acid molecules

are an integral part of ELOA. They interact directly

with the aromatic residues of lysozyme, as demon-

strated by the presence of cross-peaks between the pro-

tons of aromatic residues and oleic acid observed in

the 1H NOESY spectrum of ELOA (Fig. 6B).

The number of protein and oleic acid molecules

varies within ELOA complexes produced in different

preparations. We have shown that 11–48 oleic acids

can bind to each equine lysozyme 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 equine lysozyme 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 equine lysozyme 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 equine lysozyme 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 oleic acid 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-



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 equine lysozyme 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 oleic acid 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 equine lysozyme 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. Oleic acid 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 the cytotoxic 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 equine lysozyme 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 equine lysozyme 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]. Oleic acid 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 E1% = 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-



nected to a Bio-Rad chromatographic system (BioLogic

Wokstation, Bio-Rad, Hercules, CA, USA) and conditioned

with oleic acid. Fifty microlitres of 99.5% oleic acid were dis-

solved in 50 lL of 99.5% ethanol and sonicated in a Trans-

sonic 310 sonicator (Elma, Singen, Germany) for 15 min.

Then, 700 lL of 10 mm Tris ⁄HCl buffer, pH 9.0 were added

and the final solution was sonicated again for 15 min. The

resulting mixture was loaded onto the column and dispersed

through the DEAE Sepharose matrix, using a linear NaCl

gradient of 0–1.5 m in 10 mm Tris ⁄HCl buffer, pH 9.0. The

column was washed with a 10-bed volume of 10 mm

Tris ⁄HCl buffer, pH 9.0. Equine lysozyme in 10 mm

Tris ⁄HCl, pH 9.0, was loaded onto the column and the

ELOA complex was eluted by a linear NaCl gradient of

0–1.5 m. ELOA was dialysed against a 3 · 200-fold volume

excess of 10 mm ammonium acetate, containing 60 lm fatty

acid free bovine serum albumin, pH 9.0, for a minimum of

2 h each time (Slide-A-Lyzer, membrane cut-off 3 kDa;

Pierce, Rockford, IL, USA) and lyophilized.

Spectroscopic measurements

CD spectra were recorded in a Jasco J-810 spectropolarime-

ter (Jasco, Tokyo, Japan) equipped with a Jasco CDF-426L

thermostat, using 0.1- and 0.5-cm path length cuvettes. At

least three scans were averaged for each spectrum.

Fluorescence measurements were performed on a Jasco

spectrofluorometer FP-6500 (Jasco). The ThT-binding amy-

loid assay was carried out using a modification of the

method described by LeVine [64]. ThT fluorescence was

recorded using excitation at 440 nm, emission between 450

and 550 nm and setting the excitation and emission slits at

5 nm. The fluorescence of the hydrophobic dye ANS was

recorded using excitation at 350 nm and emission between

410 and 600 nm, with the excitation and emission slits set

at 3 nm.

AFM measurements

AFM measurements were performed on a Pico Plus micro-

scope (Agilent, Santa Clara, CA, USA) in tapping mode,

using a 100 nm scanner with acoustically driven cantilevers.

TESP model cantilevers with etched silicon probes of diame-

ter £ 10 nm (Veeco, Plainview, NY, USA) operated at

frequencies of 170–190 or 320–370 kHz. The scanning reso-

lution was 256 · 256 pixels. Scanning was performed in

trace and retrace to avoid scan artefacts. Images were flat-

tened and plane adjusted. Samples were diluted in Milli-Q

water to a final concentration of 20–100 lgÆmL)1, placed on

mica, left for up to 5 min, rinsed three times with Milli-Q

water and air-dried at room temperature overnight. Freshly

cleaved mica (GoodFellow, Devon, PA, USA) or mica

preincubated with 10 mm NaCl for 10 min was used. The

dimensions of ELOA species were measured in cross-section

in AFM height images using pico plus software (Agilent).

NMR spectroscopy

1D 1H NMR spectra were recorded using a Bruker

600 MHz spectrometer equipped with a 1H, 13C, 15N cryo

probe. NMR samples were prepared by dissolving

� 0.5 mg 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 oleic

acid and lysozyme in the complex was determined by

comparing the peak areas of oleic acid olefinic proton

resonances with the lysozyme aromatic signals. 2D 1H

NOESY spectra were recorded at 25 �C, using a mixing

time of 150 ms, 8 scans and 272 increments (experimental

time � 1.5 h).

PFG diffusion measurements were performed using the

bipolar pulse-pair diffusion experiment [65] and analysed as

described previously [66]. In the diffusion experiments, the

signal intensity was attenuated as a function of gradient

strength and, if other factors were constant, signal intensity

(I) relative to that in the absence of gradients (I0) was given

by Eqn (1):

I=I0 ¼ exp�ððcHÞ2d2ðD� d=3� s=2ÞDT G2Þ ð1Þ;

where G, d, D and s correspond to amplitude, duration,

time between PFGs, and recovery time after PFGs, respec-

tively; ch is 1H gyromagnetic ratio and Dt is translational

diffusion coefficient, respectively. Assuming that the pro-

teinaceous particles are spherical and neglecting density

changes, the mass of the complexes was determined using

Eqn (2):

MELOA=MEL ¼ ðDEL=DELOAÞ3 ð2Þ;

where MELOA and MEL are molecular masses, and DELOA

and DEL diffusion coefficients of the ELOA complex and

monomeric equine lysozyme in the molten globule state

used as a reference. The number of equine lysozyme mole-

cules in ELOA was calculated by dividing the molecular

mass of the complex (MELOA) by the molecular mass of

equine lysozyme coordinated with oleic acids, according to

the stochiometry determined from 1D 1H NMR spectrum.

Cell cultures

Mouse embryonic liver cell culture, mouse embryonic fibro-

blasts and human neuroblastoma SH-SY5Y cell line were

cultured in a Dulbecco’s modified Eagle’s medium supple-

mented with 10% (v ⁄ v) fetal bovine serum and antibiotics

in a 5% CO2 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. Oleic acid was diluted in etha-



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 col-

lagen-coated flasks in RPMI 1640 medium supplemented

with 5% fetal bovine serum, 10% heat-inactivated horse

serum, 100 UÆmL)1 penicillin and 100 lgÆmL)1 streptomy-

cin (all from Invitrogen, Stockholm, Sweden), and main-

tained in a 5% CO2 humidified atmosphere at 37 �C. Themedium was replaced every 2–3 days. For CLSM experi-

ments, cells were plated in eight-well chambered coverslips

(Nalge Nunc International, Rochester, NY, USA) and

grown in phenol-red-free RPMI medium supplemented

with 10% horse serum, 5% fetal bovine serum, penicillin

(100 unitsÆmL)1) and streptomycin (100 lgÆmL)1). Average

cell density at plating was � 1 · 105 cellsÆcm)2 in 300 lLmedium. The cells were observed for 2–3 days after

plating.

Trypan blue assay

The viability of mouse embryonic liver cells and mouse

embryonic fibroblasts (Supporting information) was mea-

sured by using a Trypan blue exclusion assay. Cells were

harvested from the plates after 5 min treatment with 0.25%

trypsin and 0.02% EDTA solution containing phenol red

(Biological Industries, Kibbutz beit Haemek, Israel). Cells

were maintained in culture media containing 10% fetal

bovine serum (Biological Industries), then sedimented by

centrifugation for 10 min at 400 g and resuspended in

100 lL NaCl ⁄Pi solution (without Ca and Mg). Cells were

then stained by adding 100 lL 0.4% Trypan blue solution

for 5 min and counted under a light microscope using a

Neubauer counting chamber. The viable cells were

unstained, whereas the dead cells displayed a blue colour;

the counts were evaluated using standard statistical analysis

techniques.

Acridine orange/ethidium bromide staining

The dyes acridine orange and ethidium bromide were used

to discriminate between live and dead cells on the basis of

their membrane integrity. Acridine orange and ethidium

bromide (100 lgÆmL)1) were mixed in a ratio of 1 : 1.

After harvesting, cells were centrifuged for 10 min at

400 g, and the cell pellet resuspended in 25 lL NaCl ⁄Pi.

Then 1 lL of the dye mixture was added and cells were

examined immediately under a Leica fluorescent micro-

scope (Leica, Wetzlar, Germany) equipped with a green

excitation filter block G-2E ⁄C. Cells were counted in four

randomly selected areas each containing � 100 cells to

quantify cell viability.

Fluorescent labelling of ELOA

ELOA was labelled with Alexa Fluor 488 dye, using the

protein labelling protocol provided by the producer

(Invitrogen). Excess free dye was removed using a protein

desalting column PD10 (GE Healthcare, USA).

FCS/CLSM measurements

FCS ⁄CLSM measurements were performed on a uniquely

modified LSM 510 instrument (Carl Zeiss, Jena, Germany),

equipped with an inverted microscope for transmitted light

and epifluorescence (Axiovert 200 m); a VIS-laser module

comprising the Ar ⁄ArKr (458, 477, 488 and 514 nm), HeNe

543 nm and HeNe 633 nm lasers and the scanning module

LSM 510 mETA. The instrument was modified to enable

avalanche photodiode imaging using silicon avalanche pho-

todiodes (SPCM-AQR-1X; Perkin–Elmer, Fremont, CA,

USA). Images were recorded without averaging, using a

scanning speed of h = 25.6 lsÆpixel)1 and 512 · 512 pixel

resolution. The C-Apochromat 40· ⁄ 1.2 W UV-VIS-IR

objective was used in all measurements. Alexa Fluor was

excited using the 488 nm line of the Ar ⁄ArKr laser. Quanti-

tative 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 statisti-

cal significance was set at P < 0.05 for the ELOA-treated

cells versus the cells treated with oleic acid. *, P < 0.05;

**, P < 0.01.

Molecular graphics

Images of equine lysozyme [67] were produced using

molmol graphic program.

Acknowledgements

We thank Catharina Svanborg for drawing our atten-

tion to the very promising HAMLET field, Christo-

pher Aisenbrey and Sohyun Kim for valuable research

assistance and Nils Elfving for supplying equine milk

from his horse farm in Arnasvall, Sweden. This

research was supported by grants from the Swedish

Research Council, the Wallenberg and the Kempe

foundations, The Swedish Brain Foundation, the Bio-

technology program, and Insamlingstiftelsen, Umea,

Sweden.



References

1 Uversky VN (2003) Protein folding revisited. A poly-

peptide chain at the folding–misfolding–nonfolding

cross-roads: which way to go?. Cell Mol Life Sci 60,

1852–1871.

2 Dobson CM (2003) Protein folding and misfolding.

Nature 426, 884–890.

3 Morozova-Roche LA & Malisauskas M (2007) A false

paradise - mixed blessings in the protein universe: the

amyloid as a new challenge in drug development. Curr

Med Chem 14, 1221–1230.

4 Van Dael H, Haezebrouck P, Morozova L, Arico-

Muendel C & Dobson CM (1993) Partially folded states

of equine lysozyme. Structural characterization and

significance for protein folding. Biochemistry 32, 11886–

11894.

5 Morozova LA, Haynie DT, Arico-Muendel C, Van

Dael H & Dobson CM (1995) Structural basis of the

stability of a lysozyme molten globule. Nat Struct Biol

2, 871–875.

6 Morozova-Roche LA, Arico-Muendel CC, Haynie DT,

Emelyanenko VI, Van Dael H & Dobson CM (1997)

Structural characterisation and comparison of the

native and A-states of equine lysozyme. J Mol Biol 268,

903–921.

7 Stefani M (2007) Generic cell dysfunction in neurode-

generative disorders: role of surfaces in early protein

misfolding, aggregation, and aggregate cytotoxicity.

Neuroscientist 13, 519–531.

8 Vestergaard B, Groenning M, Roessle M, Kastrup JS,

van de Weert M, Flink JM, Frokjaer S, Gajhede M &

Svergun DI (2007) A helical structural nucleus is the

primary elongating unit of insulin amyloid fibrils. PLoS

Biol 5, e134.

9 Morozova-Roche LA, Zurdo J, Spencer A, Noppe W,

Receveur V, Archer DB, Joniau M & Dobson CM

(2000) Amyloid fibril formation and seeding by wild-

type human lysozyme and its disease-related mutational

variants. J Struct Biol 130, 339–351.

10 Harper JD & Lansbury PT Jr (1997) Models of amyloid

seeding in Alzheimer’s disease and scrapie: mechanistic

truths and physiological consequences of the time-

dependent solubility of amyloid proteins. Annu Rev Bio-

chem 66, 385–407.

11 Gharibyan AL, Zamotin V, Yanamandra K, Moskaleva

OS, Margulis BA, Kostanyan IA & Morozova-Roche

LA (2007) Lysozyme amyloid oligomers and fibrils

induce cellular death via different apoptotic ⁄ necroticpathways. J Mol Biol 365, 1337–1349.

12 Haataja L, Gurlo T, Huang CJ & Butler PC (2008) Islet

amyloid in type 2 diabetes, and the toxic oligomer

hypothesis. Endocrin Rev 29, 303–316.

13 Vieira MN, Forny-Germano L, Saraiva LM, Sebollela

A, Martinez AM, Houzel JC, De Felice FG &

Ferreira ST (2007) Soluble oligomers from a non-

disease related protein mimic Abeta-induced tau hyper-

phosphorylation and neurodegeneration. J Neurochem

103, 736–748.

14 Kayed R, Head E, Thompson JL, McIntire TM, Milton

SC, Cotman CW & Glabe CG (2003) Common struc-

ture of soluble amyloid oligomers implies common

mechanism of pathogenesis. Science 300, 486–489.

15 Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli

L, Zurdo J, Taddei N, Ramponi G, Dobson CM &

Stefani M (2002) Inherent toxicity of aggregates implies

a common mechanism for protein misfolding diseases.

Nature 416, 507–511.

16 Morozova-Roche LA, Zamotin V, Malisauskas M,

Ohman A, Chertkova R, Lavrikova MA, Kostanyan

IA, Dolgikh DA & Kirpichnikov MP (2004) Fibrillation

of carrier protein albebetin and its biologically active

constructs. Multiple oligomeric intermediates and

pathways. Biochemistry 43, 9610–9619.

17 Kirkitadze MD, Condron MM & Teplow DB (2001)

Identification and characterization of key kinetic inter-

mediates in amyloid beta-protein fibrillogenesis. J Mol

Biol 312, 1103–1119.

18 Zamotin V, Gharibyan A, Gibanova NV, Lavrikova

MA, Dolgikh DA, Kirpichnikov MP, Kostanyan IA &

Morozova-Roche LA (2006) Cytotoxicity of albebetin

oligomers depends on cross-beta-sheet formation. FEBS

Lett 580, 2451–2457.

19 Harper JD, Wong SS, Lieber CM & Lansbury PT

(1997) Observation of metastable Abeta amyloid

protofibrils by atomic force microscopy. Chem Biol 4,

119–125.

20 Canet D, Sunde M, Last AM, Miranker A, Spencer A,

Robinson CV & Dobson CM (1999) Mechanistic stud-

ies of the folding of human lysozyme and the origin of

amyloidogenic behavior in its disease-related variants.

Biochemistry 38, 6419–6427.

21 Sliwinski-Korell A, Engelhardt H, Kampka M & Lutz

F (1999) Oligomerization and structural changes of the

pore-forming Pseudomonas aeruginosa cytotoxin. Eur J

Biochem 265, 221–230.

22 Sharon R, Bar-Joseph I, Frosch MP, Walsh DM, Ham-

ilton JA & Selkoe DJ (2003) The formation of highly

soluble oligomers of alpha-synuclein is regulated by

fatty acids and enhanced in Parkinson’s disease. Neuron

37, 583–595.

23 Gellermann GP, Byrnes H, Striebinger A, Ullrich K,

Mueller R, Hillen H & Barghorn S (2008) A beta-

globulomers are formed independently of the fibril

pathway. Neurobiol Dis 30, 212–220.

24 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, 2415–2427.



25 Otzen DE, Nesgaard LW, Andersen KK, Hansen JH,

Christiansen G, Doe H & Sehgal P (2008) Aggregation

of S6 in a quasi-native state by sub-micellar SDS. Bio-

chim Biophys Acta 1784, 400–414.

26 Hakansson A, Zhivotovsky B, Orrenius S, Sabharwal H

& Svanborg C (1995) Apoptosis induced by a human

milk protein. Proc Natl Acad Sci USA 92, 8064–8068.

27 Hakansson A, Andreasson J, Zhivotovsky B, Karpman

D, Orrenius S & Svanborg C (1999) Multimeric alpha-

lactalbumin from human milk induces apoptosis

through a direct effect on cell nuclei. Exp Cell Res 246,

451–460.

28 Kamijima T, Ohmura A, Sato T, Akimoto K, Itabashi

M, Mizuguchi M, Kamiya M, Kikukawa T, Aizawa T,

Takahashi M et al. (2008) Heat-treatment method for

producing fatty acid-bound alpha-lactalbumin that

induces tumor cell death. Biochem Biophys Res Commun

376, 211–214.

29 Svensson M, Fast J, Mossberg AK, Duringer C,

Gustafsson L, Hallgren O, Brooks CL, Berliner L,

Linse S & Svanborg C (2003) Alpha-lactalbumin

unfolding is not sufficient to cause apoptosis, but is

required for the conversion to HAMLET (human

alpha-lactalbumin made lethal to tumor cells). Protein

Sci 12, 2794–2804.

30 Mok KH, Pettersson J, Orrenius S & Svanborg C

(2007) HAMLET, protein folding, and tumor cell

death. Biochem Biophys Res Commun 354, 1–7.

31 Svensson M, Sabharwal H, Hakansson A, Mossberg

AK, Lipniunas P, Leffler H, Svanborg C & Linse S

(1999) Molecular characterization of alpha-lactalbumin

folding variants that induce apoptosis in tumor cells.

J Biol Chem 274, 6388–6396.

32 Svensson M, Hakansson A, Mossberg AK, Linse S &

Svanborg C (2000) Conversion of alpha-lactalbumin to

a protein inducing apoptosis. Proc Natl Acad Sci USA

97, 4221–4226.

33 Svensson M, Duringer C, Hallgren O, Mossberg AK,

Hakansson A, Linse S & Svanborg C (2002) Hamlet - a

complex from human milk that induces apoptosis in

tumor cells but spares healthy cells. Adv Exp Med Biol

503, 125–132.

34 Svensson M, Mossberg AK, Pettersson J, Linse S &

Svanborg C (2003) Lipids as cofactors in protein fold-

ing: stereo-specific lipid–protein interactions are

required to form HAMLET (human alpha-lactalbumin

made lethal to tumor cells). Protein Sci 12, 2805–2814.

35 Permyakov SE, Pershikova IV, Zhadan AP, Goers J,

Bakunts AG, Uversky VN, Berliner LJ & Permyakov

EA (2005) Conversion of human alpha-lactalbumin to

an apo-like state in the complexes with basic poly-

amino acids: toward understanding of the molecular

mechanism of antitumor action of HAMLET. J Prote-

ome Res 4, 564–569.

36 Duringer C, Hamiche A, Gustafsson L, Kimura H &

Svanborg C (2003) HAMLET interacts with histones

and chromatin in tumor cell nuclei. J Biol Chem 278,

42131–42135.

37 Yang F Jr, Zhang M, Zhou BR, Chen J & Liang Y

(2006) Oleic acid inhibits amyloid formation of the

intermediate of alpha-lactalbumin at moderately acidic

pH. J Mol Biol 362, 821–834.

38 Stuart DI, Acharya KR, Walker NP, Smith SG, Lewis

M & Phillips DC (1986) Alpha-lactalbumin possesses a

novel calcium binding loop. Nature 324, 84–87.

39 Morozova L, Haezebrouck P & Van Cauwelaert F

(1991) Stability of equine lysozyme I. Thermal unfold-

ing behaviour. Biophys Chem 41, 185–191.

40 Griko YV, Freire E, Privalov G, van Dael H & Priva-

lov PL (1995) The unfolding thermodynamics of c-type

lysozymes: a calorimetric study of the heat denaturation

of equine lysozyme. J Mol Biol 252, 447–459.

41 Chowdhury FA, Fairman R, Bi Y, Rigotti DJ &

Raleigh DP (2004) Protein dissection experiments reveal

key differences in the equilibrium folding of alpha-lact-

albumin and the calcium binding lysozymes. Biochemis-

try 43, 9961–9967.

42 Malisauskas M, Ostman J, Darinskas A, Zamotin V,

Liutkevicius E, Lundgren E & Morozova-Roche LA

(2005) Does the cytotoxic effect of transient amyloid

oligomers from common equine lysozyme in vitro

imply innate amyloid toxicity? J Biol Chem 280, 6269–

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-tempera-

ture ‘phase diagrams’. Proteins 65, 984–998.

44 Morozova-Roche LA (2007) Equine lysozyme: the

molecular basis of folding, self-assembly and innate

amyloid toxicity. FEBS Lett 581, 2587–2592.

45 Mizuguchi M, Arai M, Ke Y, Nitta K & Kuwajima K

(1998) Equilibrium and kinetics of the folding of equine

lysozyme studied by circular dichroism spectroscopy.

J Mol Biol 283, 265–277.

46 Morozova-Roche LA, Jones JA, Noppe W & Dobson

CM (1999) Independent nucleation and heterogeneous

assembly of structure during folding of equine lyso-

zyme. J Mol Biol 289, 1055–1073.

47 Malisauskas M, Zamotin V, Jass J, Noppe W, Dobson

CM & Morozova-Roche LA (2003) Amyloid protofila-

ments from the calcium-binding protein equine lyso-

zyme: formation of ring and linear structures depends

on pH and metal ion concentration. J Mol Biol 330,

879–890.

48 Malisauskas M, Darinskas A, Zamotin VV, Gharibyan

A, Kostanyan IA & Morozova-Roche LA (2006) Inter-

mediate amyloid oligomers of lysozyme: is their cyto-



toxicity a particular case or general rule for amyloid?

Biochemistry (Mosc) 71, 505–512.

49 Renvoize C, Biola A, Pallardy M & Breard J (1998)

Apoptosis: identification of dying cells. Cell Biol Toxicol

14, 111–120.

50 Vukojevic V, Heidkamp M, Ming Y, Johansson B,

Terenius L & Rigler R (2008) Quantitative single-mole-

cule imaging by confocal laser scanning microscopy.

Proc Natl Acad Sci USA 105, 18176–18181.

51 Ishiguro R, Yokoyama Y, Maeda H, Shimamura A,

Kameyama K & Hiramatsu K (2005) Modes of confor-

mational changes of proteins adsorbed on a planar

hydrophobic polymer surface reflecting their adsorption

behaviors. J Colloid Interface Sci 290, 91–101.

52 Sethuraman A & Belfort G (2005) Protein structural

perturbation and aggregation on homogeneous surfaces.

Biophys J 88, 1322–1333.

53 Sethuraman A, Vedantham G, Imoto T, Przybycien T

& Belfort G (2004) Protein unfolding at interfaces: slow

dynamics of alpha-helix to beta-sheet transition. Pro-

teins 56, 669–678.

54 Goers J, Permyakov SE, Permyakov EA, Uversky VN

& Fink AL (2002) Conformational prerequisites for

alpha-lactalbumin fibrillation. Biochemistry 41, 12546–

12551.

55 Wahlstrom A, Hugonin L, Peralvarez-Marin A, Jarvet

J & Graslund A (2008) Secondary structure conversions

of Alzheimer’s Abeta(1-40) peptide induced by mem-

brane-mimicking detergents. FEBS J 275, 5117–5128.

56 Fast J, Mossberg AK, Svanborg C & Linse S (2005)

Stability of HAMLET - a kinetically trapped alpha-

lactalbumin oleic acid complex. Protein Sci 14, 329–

340.

57 Pettersson-Kastberg J, Aits S, Gustafsson L, Mossberg

A, Storm P, Trulsson M, Persson F, Hun Mok K &

Svanborg C (2009) Can misfolded proteins be benefi-

cial? The HAMLET case Annals Med 41, 162–176.

58 Cheon M, Chang I, Mohanty S, Luheshi LM, Dobson

CM, Vendruscolo M & Favrin G (2007) Structural

reorganisation and potential toxicity of oligomeric

species formed during the assembly of amyloid fibrils.

PLoS Comput Biol 3, 1727–1738.

59 Zhu Y, Schwarz S, Ahlemeyer B, Grzeschik S, Klumpp

S & Krieglstein J (2005) Oleic acid causes apoptosis and

dephosphorylates Bad. Neurochem Int 46, 127–135.

60 Fernanda Cury-Boaventura M, Cristine Kanunfre C,

Gorjao R, Martins de Lima T & Curi R (2006) Mecha-

nisms involved in Jurkat cell death induced by oleic and

linoleic acids. Clin Nutr 25, 1004–1014.

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 molecu-

lar profiling of steatosis induction in immortalized

human hepatocytes. Lab Invest 87, 792–806.

62 Gill CI, Boyd A, McDermott E, McCann M, Servili M,

Selvaggini R, Taticchi A, Esposto S, Montedoro G,

McGlynn H et al. (2005) Potential anti-cancer effects of

virgin olive oil phenols on colorectal carcinogenesis

models in vitro. Int J Cancer 117, 1–7.

63 Noppe W, Haezebrouck P, Hanssens I & De Cuyper M

(1999) A simplified purification procedure of alpha-lact-

albumin from milk using Ca(2+)-dependent adsorption

in hydrophobic expanded bed chromatography. Biosep-

aration 8, 153–158.

64 LeVine H III (1993) Thioflavine T interaction with syn-

thetic Alzheimer’s disease beta-amyloid peptides: detec-

tion of amyloid aggregation in solution. Protein Sci 2,

404–410.

65 Wu D, Chen A & Johnson CS (1995) An improved dif-

fusion-ordered spectroscopy experiment incorporating

bipolar-gradient pulses. J Magn Reson Ser A 115, 260–

264.

66 Christodoulou J, Larsson G, Fucini P, Connell SR,

Pertinhez TA, Hanson CL, Redfield C, Nierhaus KH,

Robinson CV, Schleucher J et al. (2004) Heteronuclear

NMR investigations of dynamic regions of intact

Escherichia coli ribosomes. Proc Natl Acad Sci USA

101, 10949–10954.

67 Tsuge H, Ago H, Noma M, Nitta K, Sugai S & Miy-

ano M (1992) Crystallographic studies of a calcium

binding lysozyme from equine milk at 2.5 A resolution.

J Biochem 111, 141–143.

Supporting information

The following supplementary material is available:

Fig. S1. Effect of ELOA on cell viability.

Doc. S1. WST-1 (water soluble tetrazolium) assay.

This supplementary material can be found in the

online article.

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Protein oligomerization induced by oleic acid at the solid-liquid interface - equine lysozyme cytotoxic complexes

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