Báo cáo khoa học: Protein oligomerization induced by oleic acid at the solid–liquid interface – equine lysozyme cytotoxic complexes potx

Protein oligomerization induced by oleic acid at the solid–liquid interface – equine lysozyme 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 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 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 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 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 oleic acid 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 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) 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 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 -). Protein oligomerization induced by oleic acid 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 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 ⁄ P i 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 1 H 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 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 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 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) equine lysozyme 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. Protein oligomerization induced by oleic 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 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 1.0 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 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 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 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. Protein oligomerization induced by oleic acid K. Wilhelm et al. 3980 FEBS Journal 276 (2009) 3975–3989 ª 2009 The Authors Journal compilation ª 2009 FEBS 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 85 255 426 596 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 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 l M equine lysozyme (left) and 596 l M oleic acid (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. Protein oligomerization induced by oleic 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 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 + + + + + + + + Sepharose matrix Sepharose matrix Sepharose matrix A D BC Sepharose matrix 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). Protein oligomerization induced by oleic acid 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 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 ⁄ turn and 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 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 the protein 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 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 1 H 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- K. Wilhelm et al. Protein oligomerization induced by oleic 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 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 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 oligomerization induced by oleic acid 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 Protein oligomerization induced by oleic acid References 1 Uversky VN (2003) Protein folding revisited A polypeptide chain at the 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 induced by oleic and linoleic acids Clin Nutr 25, 100 4–1 014 Protein oligomerization induced by oleic acid 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 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... 3985 Protein oligomerization induced by oleic acid 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 Protein oligomerization induced by oleic acid K Wilhelm et... & Privalov PL (1995) The unfolding thermodynamics of c-type lysozymes: a calorimetric study of the heat denaturation of equine lysozyme 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 Oleic acid 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 the complexes 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 equine lysozyme in the molten globule state used as a reference The number of equine lysozyme molecules... (2005) Does the cytotoxic effect of transient amyloid oligomers from common equine lysozyme 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