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Does the Cytotoxic Effect of Transient Amyloid Oligomers from Common Equine Lysozyme in Vitro Imply Innate Amyloid Toxicity?

2004; Elsevier BV; Volume: 280; Issue: 8 Linguagem: Inglês

10.1074/jbc.m407273200

1083-351X

Mantas Mališauskas, Johan Östman, Adas Darinskas, Vladimir Zamotin, Evaldas Liutkevičius, Erik Lundgren, Ludmilla A. Morozova‐Roche,

Enzyme Structure and Function

In amyloid diseases, it is not evident which protein aggregates induce cell death via specific molecular mechanisms and which cause damage because of their mass accumulation and mechanical properties. We showed that equine lysozyme assembles into soluble amyloid oligomers and protofilaments at pH 2.0 and 4.5, 57 °C. They bind thioflavin-T and Congo red similar to common amyloid structures, and their morphology was monitored by atomic force microscopy. Molecular volume evaluation from microscopic measurements allowed us to identify distinct types of oligomers, ranging from tetramer to octamer and 20-mer. Monomeric lysozyme and protofilaments are not cytotoxic, whereas the oligomers induce cell death in primary neuronal cells, primary fibroblasts, and the neuroblastoma IMR-32 cell line. Cytotoxicity was accessed by ethidium bromide staining, MTT reduction, and TUNEL assays. Primary cultures were more susceptible to the toxic effect induced by soluble amyloid oligomers than the neuroblastoma cell line. The cytotoxicity correlates with the size of oligomers; the sample incubated at pH 4.5 and containing larger oligomers, including 20-mer, appears to be more cytotoxic than the lysozyme sample kept at pH 2.0, in which only tetramers and octamers were found. Soluble amyloid oligomers may assemble into rings; however, there was no correlation between the quantity of rings in the sample and its toxicity. The cytotoxicity of transient oligomeric species of the ubiquitous protein lysozyme indicates that this is an intrinsic feature of protein amyloid aggregation, and therefore soluble amyloid oligomers can be used as a primary therapeutic target and marker of amyloid disease. In amyloid diseases, it is not evident which protein aggregates induce cell death via specific molecular mechanisms and which cause damage because of their mass accumulation and mechanical properties. We showed that equine lysozyme assembles into soluble amyloid oligomers and protofilaments at pH 2.0 and 4.5, 57 °C. They bind thioflavin-T and Congo red similar to common amyloid structures, and their morphology was monitored by atomic force microscopy. Molecular volume evaluation from microscopic measurements allowed us to identify distinct types of oligomers, ranging from tetramer to octamer and 20-mer. Monomeric lysozyme and protofilaments are not cytotoxic, whereas the oligomers induce cell death in primary neuronal cells, primary fibroblasts, and the neuroblastoma IMR-32 cell line. Cytotoxicity was accessed by ethidium bromide staining, MTT reduction, and TUNEL assays. Primary cultures were more susceptible to the toxic effect induced by soluble amyloid oligomers than the neuroblastoma cell line. The cytotoxicity correlates with the size of oligomers; the sample incubated at pH 4.5 and containing larger oligomers, including 20-mer, appears to be more cytotoxic than the lysozyme sample kept at pH 2.0, in which only tetramers and octamers were found. Soluble amyloid oligomers may assemble into rings; however, there was no correlation between the quantity of rings in the sample and its toxicity. The cytotoxicity of transient oligomeric species of the ubiquitous protein lysozyme indicates that this is an intrinsic feature of protein amyloid aggregation, and therefore soluble amyloid oligomers can be used as a primary therapeutic target and marker of amyloid disease. The molecular basis of the pathogenicity of amyloid aggregates is a central theme in understanding the causes of a wide range of amyloid-related diseases, including Alzheimer's, Parkinson's, prion diseases, type II diabetes, and familial amyloidotic polyneuropathy (1Dobson C.M. Nature. 2003; 426: 884-890Crossref PubMed Scopus (3894) Google Scholar, 2Hammarstrom P. Schneider F. Kelly J.W. Science. 2001; 293: 2459-2462Crossref PubMed Scopus (257) Google Scholar, 3Goldberg M.S. Lansbury Jr., P.T. Nat. Cell Biol. 2000; 2: 115-119Crossref PubMed Scopus (124) Google Scholar, 4Andersson K. Olofsson A. Nielsen E.H. Svehag S.E. Lundgren E. ) Biochem. Biophys. Res. Commun. 2002; 294: 309-314Crossref PubMed Scopus (104) Google Scholar). There is a striking difference between the amounts of amyloid depositions in various types of amyloid disorders. In systemic lysozyme amyloidosis, for example, the deposits can grow to kilogram quantities in the liver (5Pepys M.B. Hawkins P.N. Booth D.R. Vigushin D.M. Tennent G.A. Soutar A.K. Totty N. Nguyen O. Blake C.C. Terry C.J. Feest G. Zalin A.M. Hsuan J.J. Nature. 1993; 362: 553-557Crossref PubMed Scopus (576) Google Scholar, 6Harrison R.F. Hawkins P.N. Roche W.R. MacMahon R.F. Hubscher S.G. Buckels J.A. Gut. 1996; 38: 151-152Crossref PubMed Scopus (60) Google Scholar). In neurodegenerative diseases, by contrast, there is no clear correlation between the amount of amyloid deposition and the clinical severity of disease. Significant cognitive impairment of Alzheimer's patients was observed without noticeable amyloid deposits in the brain, although the level of readily soluble amyloid oligomeric assemblies in the Alzheimer's brain was found to be greatly elevated (7Lee K.W. Lee S.H. Kim H. Song J.S. Yang S.D. Paik S.G. Han P.L. J. Neurosci. Res. 2004; 76: 572-580Crossref PubMed Scopus (72) Google Scholar, 8Gong Y. Chang L. Viola K.L. Lacor N.P. Lambert M.P. Finch C.E. Krafft G.A. Klein W.L. Proc. Natl. Acad. Sci. U. S. 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S. A. 2000; 97: 4221-4226Crossref PubMed Scopus (320) Google Scholar), SH3 domain, or HypF-N protein, have also been found to be cytotoxic, which implies a common mechanism for cytotoxicity of misfolded proteins (11Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Crossref PubMed Scopus (2183) Google Scholar). By contrast, it has been shown that the mature amyloid fibrils of amyloid β peptide and transthyretin are not toxic (4Andersson K. Olofsson A. Nielsen E.H. Svehag S.E. Lundgren E. ) Biochem. Biophys. Res. Commun. 2002; 294: 309-314Crossref PubMed Scopus (104) Google Scholar, 12Hoshi M. Sato M. Matsumoto S. Noguchi A. Yasutake K. Yoshida N. Sato K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6370-6375Crossref PubMed Scopus (451) Google Scholar). It is still not evident, however, which particular amyloid structures induce cell death by specific molecular mechanisms and which play the role of "inert" material and cause disease due to their quantity or mechanical properties. In our research, we addressed this problem by subjecting well defined oligomeric intermediates of equine lysozyme (EL) 1The abbreviations used are: EL, equine lysozyme; AFM, atomic force microscopy; ThT, thioflavin-T; EtBr, ethidium bromide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TUNEL, transferase-mediated dUTP nick-end-labeling; SPIP, Scanning Probe Image Processor; SH, Src homology. to the toxicity assays on different cell types. EL belongs to an extended family of structurally related proteins, chicken-type lysozymes, and α-lactalbumins. It possesses a calcium-binding site similar to α-lactalbumins and a bacteriolytic enzymatic activity like conventional lysozymes. It combines also the structural and folding properties of both subfamilies and is viewed as an evolutionary bridge between them (13Morozova-Roche L.A. Jones J.A. Noppe W. Dobson C.M. J. Mol. Biol. 1999; 289: 1055-1073Crossref PubMed Scopus (67) Google Scholar, 14Morozova-Roche L.A. Arico-Muendel C.C. Haynie D.T. Emelyanenko V.I. Van Dael H. Dobson C.M. J. Mol. Biol. 1997; 268: 903-921Crossref PubMed Scopus (71) Google Scholar, 15Morozova L.A. Haynie D.T. Arico-Muendel C. Van Dael H. Dobson C.M. Nat. Struct. Biol. 1995; 2: 871-875Crossref PubMed Scopus (123) Google Scholar). In the 1990s, it was found that amyloidogenic mutants of human lysozyme are involved in systemic amyloidosis (6Harrison R.F. Hawkins P.N. Roche W.R. MacMahon R.F. Hubscher S.G. Buckels J.A. Gut. 1996; 38: 151-152Crossref PubMed Scopus (60) Google Scholar), whereas wild-type human lysozyme also is able to form amyloid fibrils in vitro (16Morozova-Roche L.A. Zurdo J. Spencer A. Noppe W. Receveur V. Archer D.B. Joniau M. Dobson C.M. J. Struct. Biol. 2000; 130: 339-351Crossref PubMed Scopus (292) Google Scholar). Oligomers of human α-lactalbumin (HAMLET) appeared to be toxic to immature and tumor cells (11Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Crossref PubMed Scopus (2183) Google Scholar). Recently, we have demonstrated that, in contrast to lysozymes and α-lactalbumins (16Morozova-Roche L.A. Zurdo J. Spencer A. Noppe W. Receveur V. Archer D.B. Joniau M. Dobson C.M. J. Struct. Biol. 2000; 130: 339-351Crossref PubMed Scopus (292) Google Scholar, 17Goers J. Permyakov S.E. Permyakov E.A. Uversky V.N. Fink A.L. Biochemistry. 2002; 41: 12546-12551Crossref PubMed Scopus (205) Google Scholar), EL forms morphologically very distinct linear and annular protofilaments, depending on calcium ion concentrations and pH (18Malisauskas M. Zamotin V. Jass J. Noppe W. Dobson C.M. Morozova-Roche L.A. J. Mol. Biol. 2003; 330: 879-890Crossref PubMed Scopus (104) Google Scholar). In the present study, the soluble amyloid oligomers of EL were produced under fibrillation conditions prior to development of the filamentous structures, and their toxicity was assessed in comparison with the monomeric and fibrillar species. This analysis revealed that toxicity depends on the size of soluble amyloid oligomers. Protein Samples—Holo-EL was purified from horse milk as described previously (19Noppe W. Hanssens I. De Cuyper M. J. Chromatogr. A. 1996; 719: 327-331Crossref PubMed Scopus (46) Google Scholar). Protein concentration was determined by absorbance measurements on a Beckman UV-VIS spectrophotometer at 280 nm using an extinction coefficient, E1% = 23.5. To produce amyloid structures, EL was incubated at a 20 mg/ml (1.36 mm) concentration in 20 mm glycine (pH 2.0) or 10 mm sodium acetate (pH 4.5) buffers with 0.2% sodium azide at 57 °C, as described previously (18Malisauskas M. Zamotin V. Jass J. Noppe W. Dobson C.M. Morozova-Roche L.A. J. Mol. Biol. 2003; 330: 879-890Crossref PubMed Scopus (104) Google Scholar). Amyloid Assays—Thioflavin-T (ThT) binding assay was performed as described previously (16Morozova-Roche L.A. Zurdo J. Spencer A. Noppe W. Receveur V. Archer D.B. Joniau M. Dobson C.M. J. Struct. Biol. 2000; 130: 339-351Crossref PubMed Scopus (292) Google Scholar). Fluorescence of ThT was measured on a FluoroMax-2 spectrofluorometer (JOBIN YVON/PSEX Instruments) using excitation at 440 nm, emission between 450–550 nm, and setting the excitation and emission slits at 5 nm. Congo red binding assay was performed as described by Klunk et al. (20Klunk W.E. Pettegrew J.W. Abraham D.J. J. Histochem. Cytochem. 1989; 37: 1273-1281Crossref PubMed Scopus (574) Google Scholar). Absorbance spectra of the reaction solution were collected together with negative controls containing dye and protein separately, subtracting from the former the signals associated with the absorption of the dye and the scattering contribution from the fibrils. Atomic Force Microscopy (AFM)—AFM measurements were performed on a PicoPlus AFM (Molecular Imaging) in a tapping mode using a 100-μm scanner. Acoustically and magnetically driven cantilevers were used for imaging under both ambient and liquid conditions. Both cantilevers have etched silicon probes of the TESP model with diameters of 10 nm and less (Digital Instruments). In the acoustic mode, we applied a resonance frequency in the 170–190 kHz range, a scan rate of 1 Hz, and a resolution of 512 × 512 pixels. In magnetic mode, the cantilevers operated at a resonance frequency of ∼25 kHz, a scan rate of 1–2 Hz and a resolution of 256 × 256 or 512 × 512 pixels. Height, amplitude, and phase data were collected simultaneously. Images were flattened and plane adjusted. The scanning of samples was performed in trace and retrace to avoid the scan artifacts. The scanner was calibrated by measuring atomic steps on a highly orientated pyrolytic graphite in the z-axis and using a standard 1-μm calibration grid (Molecular Imaging) in the xy plane. For ambient imaging, amyloid samples (200 μg/ml) were deposited on the surface of freshly cleaved mica (GoodFellow) for 5 s, washed three times with 250 μl of MilliQ water, and dried at room temperature. For imaging in fluid, the samples were diluted to 50–80 μg/ml, placed on the mica for 10 min, washed three times with 200 μl of buffer solution, and a final 300-μl solution was added to the open liquid cell. The short adsorption time of the amyloid species on the mica substrate (compared with hours and days of the incubation period) and the high dilution of the samples ensured that aggregation was not triggered by the mica surface. Graphite was used as a substrate in control measurements. Topographical images of soluble amyloid oligomers and fibrils were essentially the same under all conditions. Measuring Molecular Dimensions—The dimensions of protein species were measured in multiple cross-sections of the same particle in AFM height images using PicoPlus software (Molecular Imaging). The distribution of the z-heights of particles was also evaluated by applying the grain analysis module of Scanning Probe Image Processor (SPIP) software (Image Metrology). In the latter, the heights of all species above the threshold surface set at the noise level were measured. To build an average height distribution, 3–4 areas on the mica surface of 1 × 1-μm size, with 1000–4000 particles in each, were subjected to the SPIP grain analysis in each sample; the experiments were repeated three times. Because of adhesion forces, the molecular species were spread on the mica surface, giving larger lateral measurements compared with vertical dimensions. Generally, an AFM tip also contributes to the broadening effect because of its specific geometry. To evaluate the tip geometry and the accuracy of our measurements, we used the tip deconvolution module of the SPIP software and performed measurements on the reference samples, such as carbon nanotubes with ∼1-nm diameters and spherical latex particles with ∼1.5-nm diameters. Processing images with the SPIP tip deconvolution module indicated that the shape of the tip did not produce topological artifacts. Measurements of the reference samples confirmed the previous conclusions (21Schneider S.W. Larmer J. Henderson R.M. Oberleithner H. Pflg ̈ers Arch. 1998; 435: 362-367Crossref PubMed Scopus (208) Google Scholar, 22Geisse N.A. Wasle B. Saslowsky D.E. Henderson R.M. Edwardson J.M. J. Membr. Biol. 2002; 189: 83-92Crossref PubMed Scopus (24) Google Scholar) that the diameter at the half-maximal height of the individual protein particle treated as a spherical cap sufficiently compensates for the AFM-induced broadening of the xy dimensions. The volume of each particle was derived from Equation 1, VAFM=(πh/6)(3r2+h2)(Eq. 1) where h is the particle height, and r is the radius at half-height (21Schneider S.W. Larmer J. Henderson R.M. Oberleithner H. Pflg ̈ers Arch. 1998; 435: 362-367Crossref PubMed Scopus (208) Google Scholar). The molecular volume of monomeric EL was estimated using, Vm=(Mo/No)(V1+dV2)(Eq. 2) where Mo is the protein molecular weight, No is Avogadro's number, d is the extent of protein hydration (0.4 mol H2O/mol protein), and V1 and V2 are the partial specific volumes of protein (0.74 cm3 g–1) and water (1 cm3 g–1) molecules, respectively (21Schneider S.W. Larmer J. Henderson R.M. Oberleithner H. Pflg ̈ers Arch. 1998; 435: 362-367Crossref PubMed Scopus (208) Google Scholar). The number of EL monomers in oligomeric species was determined by the following ratio. n=VAFM/Vm(Eq. 3) Cell Cultures—Primary neural cell cultures, containing both neurons and glial cells, and primary mouse embryonic fibroblasts were isolated from 9–12.5 gestation day embryos BALB/c mice, according to the procedure described previously (23Dorman D.C. Bolon B. Morgan K.T. Toxicol. Appl. Pharmacol. 1993; 122: 265-272Crossref PubMed Scopus (27) Google Scholar, 24Piras G. El Kharroubi A. Kozlov S. Escalante-Alcalde D. Hernandez L. Copeland N.G. Gilbert D.J. Jenkins N.A. Stewart C. Mol. Cell. Biol. 2000; 20: 3308-3315Crossref PubMed Scopus (169) Google Scholar). The cells were cultivated as monolayers in Dulbecco's modified Eagle's medium with 1000 mg/liter d-glucose, 110 mg/liter sodium pyruvate, heat-inactivated 10% fetal calf serum, 2 mml-glutamine, 100 units/ml penicillin/streptomycin (Biological Industries) in 5% CO2 at 37 °C. The medium was changed everyday. IMR-32 cells were grown in Dulbecco's modified Eagle's medium supplemented with 20 mm Hepes, 6% fetal bovine serum, 2.4 mm l-glutamate, and 1% PEST in a humidified incubator containing 5% CO2 and at 37 °C. The cell viability was assessed after 48 h of incubation with the amyloid structures, as it has been shown previously that the cell metabolic response to added compounds requires hours or days (4Andersson K. Olofsson A. Nielsen E.H. Svehag S.E. Lundgren E. ) Biochem. Biophys. Res. Commun. 2002; 294: 309-314Crossref PubMed Scopus (104) Google Scholar, 11Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Crossref PubMed Scopus (2183) Google Scholar, 25Watjen W. Haase H. Biagioli M. Beyersmann D. Environ. Health Perspect. 2002; 110: 865-867PubMed Google Scholar, 26Roher A.E. Chaney M.O. Kuo Y.M. Webster S.D. Stine W.B. Haverkamp L.J. Woods A.S. Cotter R.J. Tuohy J.M. Krafft G.A. Bonnell B.S. Emmerling M.R. J. Biol. Chem. 1996; 271: 20631-20635Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). In all cytotoxicity assays, the control measurements were performed after 24 h of incubation with amyloid, and the cell viability was decreased; however the effect was less pronounced than after 48 h. Cell Staining with Ethidium Bromide (EtBr)—Three days after isolation, the primary cells were confluent. They were diluted three times after treatment with 0.05% trypsin and 0.53 mm EDTA (Invitrogen). Both primary cell cultures and IMR-32 cells were seeded into the cell culture plates (96-well; BD Biosciences) with ∼10,000 cells/well in 200 μl of medium. At confluence after 24 h, the medium was changed, and aliquots of EL containing amyloid structures were added to a final concentration of 5–50 μm. In the control experiments, the cells were incubated in the presence of 30 μm amyloid incubation buffer or with 15 μm freshly dissolved monomeric EL. The cells were incubated in the presence of EL amyloid samples for 48 h and then detached as described above and suspended in the medium. 500-μl aliquots of the cell suspensions in primary mouse embryonic fibroblast medium were stained with 5 μl of 20 mg/ml EtBr for 10 min at 4 °C. Cell viability was estimated using a fluorescence-activated cell sorter (FACS) caliber flow cytometer (Bd Biosciences) according to the procedure described in Ref. 27Arata T. Oyama Y. Tabaru K. Satoh M. Hayashi H. Ishida S. Okano Y. Environ. Toxicol. 2002; 17: 472-477Crossref PubMed Scopus (24) Google Scholar. The viability was estimated by counting 10,000 events using the Cell Quest program (FACS caliber manual). Inhibition of MTT Reduction—IMR-32 cells were plated at a density of 10,000 cells/well in 96-well plates. The aliquots of EL amyloid were added to the wells (100 μl) at various final EL concentrations (see Fig. 5), and the cells were incubated for 48 h. 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) labeling reagent was added to each well, and the samples were incubated for a further 4 h. 100 μl of solubilization mixture (10% SDS, 0.01 m HCl) were added to each well, and the samples were incubated overnight. Absorbance of blue formazan at 570 nm was measured with a plate reader (28Datki Z. Juhasz A. Galfi M. Soos K. Papp R. Zadori D. Penke B. Brain Res. Bull. 2003; 62: 223-229Crossref PubMed Scopus (139) Google Scholar). Labeling of Free DNA Ends—IMR-32 cells were cultured in 48-well plates (Nunclon). EL amyloid aliquots were added to the final concentration of 14 and 64 μm, and the slides were further incubated for 48 h. DNA fragmentation was detected by a transferase-mediated dUTP nick-end-labeling (TUNEL) method using a fluorescent staining kit according to the manufacturer's instructions (Roche Diagnostics). Similar aliquots of the incubation buffers (pH 2.0 and 4.5) were added to IMR-32 cells in control experiments. Stained cells were analyzed by flow cytometry as described above. Structural Characterization of EL Fibrillation—The kinetics of EL self-assembly was monitored using ThT fluorescence assay (Fig.1), which serves as a marker for amyloid (17Goers J. Permyakov S.E. Permyakov E.A. Uversky V.N. Fink A.L. Biochemistry. 2002; 41: 12546-12551Crossref PubMed Scopus (205) Google Scholar). Protein aliquots were diluted into a buffer containing ThT after differing periods of incubation. Both samples of EL, incubated at pH 2.0 and 4.5 at 57 °C, were characterized by short lag phases of 3 and 12–14 h, respectively, which correspond to the initiation of nucleation (29Harper J.D. Lansbury Jr., P.T. Annu. Rev. Biochem. 1997; 66: 385-407Crossref PubMed Scopus (1429) Google Scholar). This was followed by an increase of fluorescence intensity reaching a plateau level with 10- and 7.5-fold increases of ThT dye fluorescence bound to EL amyloid incubated for ∼120 and ∼200 h at pH 2.0 and pH 4.5, respectively (Fig. 1). EL-aggregated structures were analyzed by AFM. Representative images of EL species are shown in Fig. 2, and their height distribution histograms are presented in Fig. 3. Protein molecules from freshly prepared EL samples adsorbed on the mica surface, which was manifested in a narrow log-normal distribution of z-heights with a mean value of 0.4 nm in both cases of pH 4.5 and pH 2.0 (Fig. 3, a and b). The molecular volumes of these species were evaluated by Equation 1 using their z-heights and diameters at half-height, determined by both the grain analysis SPIP software and by multiple crosssection measurements of individual particles. The results are presented in Table I. Both methods gave a good agreement with the calculated volume of EL monomer using Equation 2.Fig. 3Molecular dimensions of EL determined by AFM. In each protein sample, 3–4 areas of 1 × 1-μm size, with 1000–4000 particles in each area, were subjected to SPIP software grain analysis. The distribution of z-heights in the freshly prepared EL samples at pH 4.5 (a) and pH 2.0 (b) immediately after protein dissolving and after 72 h of incubation at pH 4.5 and 57 °C (c) and after 24 h at pH 2.0 and 57 °C (d) are shown. The histograms showing the difference between the population of oligomers in the samples c–a (e), d–b (f), and d–c(g) are presented here. The formation of larger species manifested by the appearance of additional maxima with larger z-height values. The data were fit to log-normal functions, as shown in the inset to panel a for the freshly dissolved EL and in panel h for the oligomers formed at pH 4.5 and 57 °C after 72 h. To compare the samples, all distributions were normalized to the total number of particles in the scanned field.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IMolecular dimensions of EL-soluble amyloid oligomers determined by AFMStructuresnhDiameterVmNo. of monomersNo. of monomersaThe stoichiometry of oligomers were derived from AFM data using the SPIP grain analysis module.nmnmnm30.490.39 ± 0.0615.7 ± 2.4238.3 ± 11.61 ± 0.51 ± 0.50.780.70 ± 0.1420.96 ± 0.8122.5 ± 9.44 ± 0.55 ± 21.1101.05 ± 0.11422.33 ± 1.46207.9 ± 26.88 ± 18 ± 32.182.03 ± 0.22427.85 ± 2.83584.3 ± 119.021 ± 425 ± 7a The stoichiometry of oligomers were derived from AFM data using the SPIP grain analysis module. Open table in a new tab During the growth phase (Fig. 1), the larger round-shaped oligomers were formed in the samples (Fig. 2a) characterized by broader distributions of z-heights (Fig. 3, c and d). They gave rise to elongated linear and annular (Fig. 2, d–f) protofibrils with a "bead-on-string" morphology. Subsequently the protofilaments with an ∼2-nm z-height emerged in the samples (Fig. 2, b and c); the cross-section of a typical protofilament is presented in Fig. 2c. The protofilaments were rather short, 30–300 nm in length, and there were both straight and curved sections, whereas some of them were locked into circular structures (Fig. 2b). Similar protofilaments were observed previously at pH 4.5 and 57 °C in the presence of 10 mm CaCl2 (18Malisauskas M. Zamotin V. Jass J. Noppe W. Dobson C.M. Morozova-Roche L.A. J. Mol. Biol. 2003; 330: 879-890Crossref PubMed Scopus (104) Google Scholar). After incubation periods of 72 h (pH 4.5) and 24 h (pH 2.0), respectively, both EL samples displayed the same increase in ThT fluorescence and were characterized by the formation of round-shaped oligomers, whereas the protofilaments have not yet been observed. These samples were subjected further to the cytotoxicity assays to compare the effect induced by monomeric, oligomeric, and fibrillar species of EL. The z-height histograms of the species produced at these time points were characterized by a decrease of the main peak concomitantly with the appearance of a pronounced right-hand shoulder corresponding to progressive enlargement of oligomers. In the pH 4.5 sample (Fig. 3c), a second peak centered at the z-height of ∼2.1 nm became apparent. The fraction of oligomers with a characteristic z-height of ∼0.7 nm was determined in both samples at pH 4.5 and 2.0 by subtracting from the histograms at given time points those of freshly dissolved EL (Fig. 3, e and f). In addition, in the sample incubated at pH 4.5 compared with the sample at pH 2.0, there is a fraction with a z-height of ∼1.1 (Fig. 3g), identified by the difference between the corresponding distributions shown in Fig. 3, c and d. The control experiments, with a significantly smaller number of particles were carried out using manual cross-section analysis (Table I) to confirm the values obtained from larger screenings by the SPIP grain analysis module (Fig. 3). The volumes of oligomers were calculated by Equation 1 using the linear parameters of oligomers determined in both types of measurements. The stoichiometry of oligomers was calculated using Equation 3 and presented in Table I. Both approaches gave consistent results. The distribution of oligomers in the pH 4.5 sample (Fig. 3c) was well fit by a sum of log-normal functions with the means of z-height equal to ∼0.4, ∼0.7, ∼1.1, and ∼2.1 nm (χ2 = 0.002) (Fig. 3h), although the best fit of the distribution of the pH 2.0 sample was achieved using only two log-normal functions with the means of 0.4 and 0.7 nm (χ2 = 0.005). The soluble amyloid oligomers described above (Fig. 2a) were characterized by a 2–3-nm long wavelength shift of Congo red spectra compared with a 5–6-nm shift caused by the fibrillar structures formed subsequently. Apart from single round-shaped oligomers in both samples, we also observed characteristic annular protofibrils with 4- or 5-fold symmetry (Fig. 2, d–f). They consisted of segments in which z-heights and molecferringular volumes were similar to the dimensions of the individual round-shaped species with z-heights of 0.7–1.2 nm present in the same samples (Fig. 2a). The control AFM imaging of the amyloid structures was performed immediately after their transfer from the incubation buffers to serum-free culture medium and in a 48-h incubation in serum-free culture medium at 37 °C. Under these conditions, the morphology and the cross-sectional dimensions of the oligomers, protofibrils, and protofilaments did not change, as shown in the representative images in Fig. 2a, inset, and Fig. 2, d–f). To confirm the stability of the oligomeric structures in the physiological solutions, population analysis using the SPIP software was also performed and compared with the manual measurements in the cross-sections. Immediately after trans to the culture medium, the distributions of oligomeric species in both the pH 4.5 and 2.0 samples remained identical to those shown in Fig. 3. After 48 h of incubation in the culture medium, the positions of all of the main peaks in the oligomeric distributions did not change compared with those shown in Fig. 3; however, large amorphous aggregates were observed in the same samples. Similarly, amorphous aggregation was also prompted in the sample of monomeric EL during the same length of incubation in the serum-free culture medium; however, these aggregates were found to be