Expansion of Polyglutamine Induces the Formation of Quasi-aggregate in the Early Stage of Protein Fibrillization
2003; Elsevier BV; Volume: 278; Issue: 36 Linguagem: Inglês
10.1074/jbc.m209852200
ISSN1083-351X
AutoresMotomasa Tanaka, Yoko Machida, Yukihiro Nishikawa, Takumi Akagi, Tsutomu Hashikawa, Tetsuro Fujisawa, Nobuyuki Nukina,
Tópico(s)Protein Structure and Dynamics
ResumoWe examined the effects of the expansion of glutamine repeats on the early stage of protein fibrillization. Small-angle x-ray scattering (SAXS) and electron microscopic studies revealed that the elongation of polyglutamine from 35 to 50 repeats in protein induced a large assembly of the protein upon incubation at 37 °C and that its formation was completed in ∼3 h. A bead modeling procedure based on SAXS spectra indicated that the largely assembled species of the protein, quasi-aggregate, is composed of 80 to ∼90 monomers and a bowl-like structure with long and short axes of 400 and 190 Å, respectively. Contrary to fibril, the quasi-aggregate did not show a peak at S = 0.21 Å–1 corresponding to the 4.8-Å spacing of β-pleated sheets in SAXS spectra, and reacted with a monoclonal antibody specific to expanded polyglutamine. These results imply that β-sheets of expanded polyglutamines in the quasi-aggregate are not orderly aligned and are partially exposed, in contrast to regularly oriented and buried β-pleated sheets in fibril. The formation of non-fibrillary quasi-aggregate in the early phase of fibril formation would be one of the major characteristics of the protein containing an expanded polyglutamine. We examined the effects of the expansion of glutamine repeats on the early stage of protein fibrillization. Small-angle x-ray scattering (SAXS) and electron microscopic studies revealed that the elongation of polyglutamine from 35 to 50 repeats in protein induced a large assembly of the protein upon incubation at 37 °C and that its formation was completed in ∼3 h. A bead modeling procedure based on SAXS spectra indicated that the largely assembled species of the protein, quasi-aggregate, is composed of 80 to ∼90 monomers and a bowl-like structure with long and short axes of 400 and 190 Å, respectively. Contrary to fibril, the quasi-aggregate did not show a peak at S = 0.21 Å–1 corresponding to the 4.8-Å spacing of β-pleated sheets in SAXS spectra, and reacted with a monoclonal antibody specific to expanded polyglutamine. These results imply that β-sheets of expanded polyglutamines in the quasi-aggregate are not orderly aligned and are partially exposed, in contrast to regularly oriented and buried β-pleated sheets in fibril. The formation of non-fibrillary quasi-aggregate in the early phase of fibril formation would be one of the major characteristics of the protein containing an expanded polyglutamine. Amyloid is a fibrillar deposit observed in various proteins associated with human neurodegenerative diseases including Alzheimer's diseases, Creutzfeldt-Jakob diseases, and polyglutamine diseases. Amyloid has a "cross-β structure," with β-strands perpendicular to, and backbone hydrogen bonds parallel to, the fibril axis (1Sunde M. Blake C. Adv. Protein Chem. 1997; 50: 123-159Crossref PubMed Google Scholar, 2Jimenez J.L. Guijarro J.I. Orlova E. Zurdo J. Dobson C.M. Sunde M. Saibil H.R. EMBO J. 1999; 18: 815-821Crossref PubMed Scopus (443) Google Scholar). Recent in vitro studies have revealed that an intermediate termed protofibril is transiently formed prior to the formation of the amyloid fibrils of amyloid-β peptide and α-synuclein, the depositions of which in brain are hallmarks of Alzheimer's disease and Parkinson's disease, respectively (3Harper J.D. Wong S.S. Lieber C.M. Lansbury P.T. Chem. Biol. 1997; 4: 119-125Abstract Full Text PDF PubMed Scopus (621) Google Scholar, 4Harper J.D. Lieber C.M. Lansbury Jr., P.T. Chem. Biol. 1997; 4: 951-959Abstract Full Text PDF PubMed Scopus (411) Google Scholar, 5Walsh D.M. Lomakin A. Benedek G.B. Condron M.M. Teplow D.B. J. Biol. Chem. 1997; 272: 22364-22372Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar, 6Harper J.D. Wong S.S. Lieber C.M. Lansbury Jr., P.T. 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Stefani M. Nature. 2002; 416: 507-511Crossref PubMed Scopus (2128) Google Scholar) showed that non-disease-related proteins might generally form globular intermediates prior to the formation of amyloid fibrils and that the intermediates induce cellular toxicity. Thus, studies on the earliest phase of protein fibrillization, in particular the oligomerization and nucleation processes, are of great importance for identifying therapeutic targets as well as for understanding the molecular mechanism of fibril formation. In addition, structural characterization of such globular precursors of amyloid fibril would be important to reveal origins of the cellular toxicity of the amyloid-forming proteins.Amyloid fibrils are also observed in the aggregates of proteins bearing an expanded polyglutamine (12Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Hollenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar, 13McGowan D.P. van Roon-Mom W. Holloway H. Bates G.P. Mangiarini L. Cooper G.J. Faull R.L. Snel R.G. Neuroscience. 2000; 100: 677-680Crossref PubMed Scopus (78) Google Scholar). Expansion of polyglutamine to more than ∼35 repeats in certain proteins induces the formation of intranuclear inclusions that are characteristic of polyglutamine diseases such as Huntington's disease and hereditary spinocerebellar ataxia (14Cummings C.J. Zoghbi H.Y. Annu. Rev. Genomics Hum. Genet. 2000; 1: 281-328Crossref PubMed Scopus (280) Google Scholar). Although the protein aggregate including amyloid fibril might be closely involved in the cellular dysfunction that causes polyglutamine diseases (15Zoghbi H.Y. Orr H.T. Annu. Rev. Neurosci. 2000; 23: 217-247Crossref PubMed Scopus (1082) Google Scholar), little is known about the effects of the expansion of glutamine repeats on the aggregation mechanism of the causative proteins. Although Poirier et al. (16Poirier M.A. Li H. Macosko J. Cai S. Amzel M. Ross C.A. J. Biol. Chem. 2002; 277: 41032-41037Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar) recently reported spheroids and protofibrils as precursors in the fibrillization of the N-terminal fragment of mutant huntingtin containing 44 glutamine repeats, an early phase of the fibrillization process of the protein has not been fully characterized. In particular, structural and physico-chemical properties of such precursors have remained unclear.We have recently designed sperm whale myoglobin (Mb 1The abbreviations used are: Mb, myoglobin; WT, wild type; SAXS, small-angle X-ray scattering; MOPS, 4-morpholinepropanesulfonic acid; SVD, singular value deconvolution; DAMMIN, dummy atom minimization.1The abbreviations used are: Mb, myoglobin; WT, wild type; SAXS, small-angle X-ray scattering; MOPS, 4-morpholinepropanesulfonic acid; SVD, singular value deconvolution; DAMMIN, dummy atom minimization.) mutants in which a varying length of glutamine repeats was inserted (17Tanaka M. Morishima I. Akagi T. Hashikawa T. Nukina N. J. Biol. Chem. 2001; 276: 45470-45475Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Although the expansion of glutamine repeat in general renders the protein insoluble in water, we successfully established preparation of the mutant Mbs containing 50 (Mb-Gln50) or 35 (Mb-Gln35) glutamine repeats on a large scale, which is required for their structural analysis. 50 glutamine repeats inserted into protein are pathological, whereas 35 glutamine repeats lie between pathological and non-pathological status in polyglutamine diseases. In our previous study (17Tanaka M. Morishima I. Akagi T. Hashikawa T. Nukina N. J. Biol. Chem. 2001; 276: 45470-45475Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), Mb-Gln50 and Mb-Gln35, but not a mutant Mb bearing shorter, 12 glutamine repeats (Mb-Gln12), reacted with a monoclonal 1C2 antibody specific for an expanded polyglutamine (18Trottier Y. Lutz Y. Stevanin G. Imbert G. Devys D. Cancel G. Saudou F. Weber C. David G. Tora L. et al.Nature. 1995; 378: 403-436Crossref PubMed Scopus (581) Google Scholar), suggesting that the expanded polyglutamines in Mb-Gln50 and Mb-Gln35 form a characteristic structure similar to that of the native proteins that cause polyglutamine diseases. In addition, the tendency to form amyloid fibrils was highly dependent on the length of glutamine repeats inserted into Mb. Mb-Gln50 and Mb-Gln35 formed amyloid fibrils under physiological conditions rapidly and slowly, respectively, whereas Mb-Gln12 and Mb wild-type (Mb-WT) did not form any amyloid fibrils under the same conditions (17Tanaka M. Morishima I. Akagi T. Hashikawa T. Nukina N. J. Biol. Chem. 2001; 276: 45470-45475Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). We suggest, by these observations, that Mb-Gln50 and Mb-Gln35 would be appropriate to investigate the effects of the expansion of glutamine repeats on the fibrillization process of polyglutamine-bearing proteins. To elucidate the formation mechanism of fibril would be crucial for better comprehension of the molecular basis for polyglutamine diseases.In the present study, we applied small-angle x-ray scattering (SAXS) to examine and compare the early stage of fibril formation of the mutant Mbs, because SAXS is a powerful technique for quantitative detection of smaller particles with accurate time dependence, compared with light scattering. We revealed that the expansion of polyglutamine from 35 to 50 repeats in a mutant Mb induced a large assembly of the protein in the early phase of fibrillization. The formation of a largely assembled species of Mb-Gln50 was confirmed by electron microscopy. We further examined structural properties of β-sheets of expanded polyglutamines in the Mb-Gln50 assembled species and determined its low-resolution structure based on SAXS spectra.EXPERIMENTAL PROCEDURESProtein Expression and Purification—Recombinant wild-type and mutant Mbs were expressed in TB1 Escherichia coli and purified as reported previously (17Tanaka M. Morishima I. Akagi T. Hashikawa T. Nukina N. J. Biol. Chem. 2001; 276: 45470-45475Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The concentration of ferric Mbs was determined by the extinction coefficient of 157 mm–1 · cm–1 at the Soret band in the UV-visible spectra. The sample solutions of Mbs in 10 mm MOPS buffer at pH 7.0 were centrifuged at 25,000 × g for 30 min, before incubation at 37 °C, to remove any pre-existing precipitates.Congo Red Binding Assay—We incubated 100 μl of 1.86 mg/ml Mb-Gln50 and Mb-Gln35 and 4.49 mg/ml Mb-Gln35, Mb-Gln12 and Mb-WT solutions at 37 °C. We also incubated the supernatant fraction (1.86 mg/ml) of a Mb-Gln50 solution, which was pre-incubated at 37 °C for 6 h at 37 °C. The pellet of the Mb-Gln50 solution, which was pre-incubated for 6 h, was resuspended with 100 μl of 10 mm MOPS buffer (pH 7.0) and seeded into fresh 1.86 mg/ml Mb-Gln50 and 4.49 mg/ml Mb-Gln35 solutions (10% (v/v)). At appropriate time points, 5 μl of the protein solutions was added to 10 μm Congo red in 10 mm MOPS buffer (pH 7.0) and incubated at room temperature for 30 min. UV-visible spectra of these samples were measured with a Shimadzu 2400-PC spectrophotometer. Congo red bound to fibril was determined using the following equation: Congo red (μmol/l) = A 540/25,295 – A 480/46,306 (8Hartley D.M. Walsh D.M. Ye C.P. Diehl T. Vasquez S. Vassilev P.M. Teplow D.B. Selkoe D.J. J. Neurosci. 1999; 19: 8876-8884Crossref PubMed Google Scholar).Small-angle X-ray Scattering (SAXS)—The measurement of SAXS was performed by synchrotron radiation of RIKEN structural biology beamline I (BL45XU) at Spring-8 in Harima, Japan (19Fujisawa T. Inoue K. Oka T. Iwamoto H. Uruga T. Kunmasaka T. Inoko Y. Yagi N. Yamamoto M. Ueki T. J. Appl. Crystallogr. 2000; 33: 797-800Crossref Scopus (166) Google Scholar). The sample-to-detector distance was 1 or 2.2 m, which was calibrated using meridional diffraction of dried chicken collagen. The temperature of the sample was maintained at 37 °C by an incubator, and the sample cell and stage were also set to 37 °C. Using an x-ray image intensifier and cooled CCD detector (XR-II+CCD) (20Ameyama Y. Ito K. Yagi N. Asano Y. Wakabayashi K. Ueki T. Endo T. Rev. Sci. Instrum. 1995; 66: 2290-2294Crossref Scopus (126) Google Scholar), each scattering profile was collected for 1 s during which no radiation damage was found. The data were normalized to the intensity of the incident beam, and the buffer was subtracted. The radius of gyration (R g) was determined by the Guinier approximation: I(S) = I(0) exp(–4π2 Rg 2 S 2/3), where S and I(0) are the momentum transfer and intensity at the zero scattering angle, respectively, with fitting ranges of S 2 (Å–2) from 5 × 10–6 to 40 × 10–6. S is defined as S = 2sinθ/λ, where 2θ and λ are the scattering angle and the x-ray wavelength, respectively (21Guinier A. Fournet G. Small-angle Scattering of X-rays. Wiley, New York1955Google Scholar). The S range used for R g determination satisfied the condition 2π SR g < 1.3. The distance distribution function, P(r), was calculated by GNOM, which uses an indirect Fourier transform method (22Svergun D.I. Semenyuk A.V. Feigin L.A. Acta Crystallogr. A. 1988; 44: 244-250Crossref Scopus (272) Google Scholar). The maximum dimension, D max, was determined from the first zero cross-point of the P(r) function (21Guinier A. Fournet G. Small-angle Scattering of X-rays. Wiley, New York1955Google Scholar). The cross-sectional radius of gyration, R c, was determined by fitting the data points, using the equation I(S)S = I c (0)exp(–π2 R c2 S 2/2) over S2 ranges (Å–2) from 2 × 10–6 to 10 × 10–6 and from 8 × 10–6 to 23 × 10–6 for oligomer and quasi-aggregates, respectively. The spectral analysis by singular value deconvolution (SVD) was performed using SPECFIT (version 3.0, Spectrum Software Associates, Marlborough, MA), using the S versus S 2×I(S) (Kratky) plot of the data.Construction of Low-resolution Model—Low-resolution particle shapes were restored from SAXS intensity profiles using a bead modeling procedure of DAMMIN (23Svergun D.I. Biophys. J. 1999; 76: 2879-2886Abstract Full Text Full Text PDF PubMed Scopus (1709) Google Scholar). In the dummy atom minimization (DAMMIN), a protein molecule was approximated by densely packed small spheres (dummy atoms). Minimization was performed by the simulated annealing method (22Svergun D.I. Semenyuk A.V. Feigin L.A. Acta Crystallogr. A. 1988; 44: 244-250Crossref Scopus (272) Google Scholar), starting from the dummy atoms placed at random coordinates within the search sphere, sphere of diameter D max. The detailed procedure of DAMMIN has been described previously (24Fujisawa T. Kostyukova A. Maeda Y. FEBS Lett. 2001; 498: 67-71Crossref PubMed Scopus (33) Google Scholar). The stability of the model was checked by repeating the minimization 10 times in different runs. The three-dimensional models were displayed using the program VMD.Electron Microscopy and Immunoblotting—The negative staining for the 1.86 mg/ml of sample of Mb-Gln50 and the 4.49 mg/ml of samples of Mb-Gln35, Mb-Gln12, and Mb-WT was performed as reported previously (17Tanaka M. Morishima I. Akagi T. Hashikawa T. Nukina N. J. Biol. Chem. 2001; 276: 45470-45475Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), and the images were recorded on a LEO 912AB electron microscope (LEO, Cambridge, UK) normally at a magnification of 63,000 × g. For immunoelectron microscopy of Mb, the copper grid was treated with a polyclonal Mb (1:250–500, Chemicon) or 1C2 antibody (1:50–100, Chemicon), followed by a 5-nm gold-conjugated rabbit or mouse secondary antibody, respectively (1:200, Amersham Biosciences). Each 1.86 mg/ml of sample of Mb-Gln50 with various incubations (0, 3, 10, 24, and 72 h) at 37 °C was also processed to immunoblotting by the Mb (1:5000) and 1C2 (1:2000) antibodies.X-ray Diffraction Experiment—The sample-to-detector distance was 30 cm, which was calibrated using meridional diffraction of dried chicken collagen. Mb-Gln50 and Mb-Gln35 fibrils grown from ∼5 mg/ml of solution in 10 mm MOPS buffer at pH 7.0 were collected by brief centrifugation and mounted onto the last guard slit with a width of 0.6 mm. The sample-to-detector distance was 30 cm. The sample holder was vacuumized to remove the scattering of air, and the fibrils were exposed to x-ray radiation operating at ∼90 mA. Data were collected at room temperature for 30 s on a modified RIGAKU R-Axis IV2+ imaging plate detector. 2Y. Nishikawa and T. Fujisawa, unpublished results.Size Exclusion Chromatography—A 1.86 mg/ml of solution of Mb-Gln50 and 4.49 mg/ml of solutions of Mb-Gln35 and Mb-Gln12 were incubated at 37 °C, and 40 μl of each sample at 3, 10, and 24 h was centrifuged at 25,000 × g for 20 min. The supernatant was analyzed by size exclusion chromatography using the Superdex-200 in the SMART system (Amersham Biosciences). The running buffer was 10 mm MOPS containing 150 mm NaCl at pH 7.0, and the flow rate was 40 μl/min.RESULTSFormation of Quasi-aggregate by Expanded Polyglutamine—We examined the fibril formation of mutant Mbs by a Congo red binding assay. We incubated separate solutions (1.86 mg/ml) of Mb-Gln50 and Mb-Gln35 at 37 °C and determined the amount of Congo red bound to fibril, using UV-visible spectroscopy (8Hartley D.M. Walsh D.M. Ye C.P. Diehl T. Vasquez S. Vassilev P.M. Teplow D.B. Selkoe D.J. J. Neurosci. 1999; 19: 8876-8884Crossref PubMed Google Scholar). The time-dependent profiles showed that the fibril formation of Mb-Gln50 was initiated in ∼50 h, whereas Mb-Gln35 did not clearly exhibit fibrillization within 200 h (Fig. 1A). We then increased the concentration of a Mb-Gln35 solution from 1.86 mg/ml to 4.49 mg/ml and examined the fibril formation by the Congo red binding assay. The 4.49 mg/ml of solution of Mb-Gln35 formed fibrils slowly with a lag time of ∼150 h (Fig. 1A). Contrary to Mb-Gln50 and Mb-Gln35, 4.49 mg/ml of solutions of Mb-Gln12 and Mb-WT did not show the binding to Congo red upon incubation >200 h (Fig. 1A), indicating no fibril formation as reported previously (17Tanaka M. Morishima I. Akagi T. Hashikawa T. Nukina N. J. Biol. Chem. 2001; 276: 45470-45475Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). We also verified that the lag time of fibrillization did not change when we used mutant Mbs of different sample preparations.Because Mb-Gln50 and Mb-Gln35 formed fibrils under the physiological condition, we examined the early phase of fibrillization of the mutant Mbs by SAXS. We incubated separate solutions (1.86 mg/ml) of the mutant Mbs at 37 °C and measured their SAXS spectra with various incubation times. The Guinier plot of Mb-Gln50 showed an increase in the intensity at S 2 = ∼1 × 10–3 Å–2 upon incubation at 37 °C, suggesting formation of an assembled species of the mutant Mb (data not shown). We plotted the scattering profiles of Mb-Gln50 as S versus S 2 × I(S) (Kratky plot) to clarify the protein assembly. As shown in Fig. 1B, an intensity of the peak at S = 1.4 × 10–3 Å–1 was clearly increased with incubation times. The peak showed a maximum scattering intensity in ∼3 h of incubation, and the intensity was constant until 27 h (Fig. 1C). Because a peak at the extremely small-angle region (S = 1.4 × 10–3 Å–1) corresponds to a large-sized particle, our result indicated that a largely assembled Mb-Gln50 was formed in the early time of the lag phase (∼50 h) of fibrillization. Hereafter, we referred such a largely assembled species of protein, which was formed in the early stage of the Mb-Gln50 fibrillization, as a quasi-aggregate. In contrast to Mb-Gln50, SAXS spectra of a 1.86 mg/ml of solution of Mb-Gln35 did not change upon incubation at 37 °C for more than 20 h, suggesting an absence of such an assembled species of protein as observed for Mb-Gln50 (data not shown). Thus, we measured SAXS spectra of a 4.49 mg/ml of solution of Mb-Gln35 (Fig. 1D), which facilitated the fibril formation as shown in Fig. 1A. However, the peak at S = 1.0 × 10–3 Å–1 did not increase with incubation times (Fig. 1E), which confirmed the absence of an assembled species of protein in the early stage of the fibrillization for Mb-Gln35. In addition, we did not detect any peak at the extremely small-angle region in SAXS spectra for Mb-Gln12 and Mb-WT upon incubation at 37 °C over 50 h, indicating absence of any largely assembled species of protein (data not shown). The reliability of time-dependent SAXS spectra was verified by repeating measurements with mutant Mbs of different sample preparations and with different incubation times.To gain more physico-chemical insights into the formation of the quasi-aggregate of Mb-Gln50, we measured time-dependent SAXS spectra at various temperatures (25, 29, 33, and 37 °C) for Mb-Gln50. The formation of quasi-aggregates was greatly affected by temperature, and representative scattering profiles at 25 °C and 33 °C are shown in Fig. 2, A and B. The scattering intensity at S = 1.4 × 10–3 Å–1 was plotted against incubation times, and the formation rate of the quasi-aggregate was calculated. We found that these plots were best fitted by a single exponential (Fig. 2C), indicating that the formation of the quasi-aggregate follows the first-order kinetics. We also show an Arrhenius plot of the formation rate of the Mb-Gln50 quasi-aggregate in Fig. 2D. The activation energy for the formation of the quasi-aggregate was calculated from the slope of the Arrhenius plot (22.8 ± 1.8 kcal/mol). Furthermore, we examined the effects of the Mb-Gln50 quasi-aggregate on fibrillization of Mb-Gln50 or Mb-Gln35 by the Congo red binding assay. Elimination of the quasi-aggregate, which accumulated upon incubation at 37 °C for 6 h, in the Mb-Gln50 sample prolonged a lag time of fibrillization from ∼50 to ∼70 h (Fig. 1A). On the other hand, addition of the Mb-Gln50 quasi-aggregate into a fresh Mb-Gln50 or Mb-Gln35 solution as a seed eliminated or shortened the lag time, respectively (Fig. 1A).Fig. 2Temperature-dependent formation of Mb-Gln50 quasi-aggregate. Representative S versus S 2 × I(S) (Kratky) plots at 25 °C(A) and 33 °C (B) are shown. C, the plots of the scattering intensity at S = 1.4 × 10–3 Å–1 against the incubation time at 25 °C (•), 29 °C (▪), 33 °C (▴), and 37 °C (▾) were best fitted by a single exponential, which allowed determination of the formation rate of the quasi-aggregate at various temperatures. D, an Arrhenius plot (ln(k app) versus K –1) for the formation rate of the quasi-aggregate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We ascertained the formation of quasi-aggregate by electron microscopy. We carried out the negative staining for mutant Mbs, which had been incubated at 37 °C for 3 h, and observed them by electron microscopy. A typical electron micrograph is shown in Fig. 3A. We clearly detected particles with a length of ∼20–45 nm in the Mb-Gln50 sample, whereas such particles were not observed for Mb-Gln35, Mb-Gln12, and Mb-WT samples (data not shown). This electron micrograph suggested that the observed particles for Mb-Gln50 correspond to the quasi-aggregate, which was indicated in the SAXS experiment for Mb-Gln50 (Fig. 1B), because the formation of quasi-aggregate was completed in 3 h (Fig. 1B). We further performed an immunoelectron microscopic study for the quasi-aggregate with a polyclonal Mb and a monoclonal 1C2 antibody (18Trottier Y. Lutz Y. Stevanin G. Imbert G. Devys D. Cancel G. Saudou F. Weber C. David G. Tora L. et al.Nature. 1995; 378: 403-436Crossref PubMed Scopus (581) Google Scholar). Although some of the quasi-aggregates initiate to assemble each other by further incubation during the immunostaining procedure, the quasi-aggregate reacted with both of the Mb and 1C2 antibodies (Fig. 3, B and C). We also investigated the reactivity of these antibodies to the Mb-Gln50 fibrils that were formed by incubation at 37 °C for 7 days. The Mb antibody reacted with the fibrils along the fibers (Fig. 3D), whereas the 1C2 antibody recognized only ends and branch sites of the fibrils (Fig. 3E). We further investigated this observation by the following immunoblotting experiment. Each sample of Mb-Gln50 with various incubations (0, 3, 10, 24, and 72 h) were separated by SDS-PAGE and processed for immunoblotting by the Mb and 1C2 antibodies. We focused on the gel top in the immunoblot, because the quasi-aggregate and/or fibril can be detected at the gel top. We found a clear difference in the immunoreactivity to the gel top in 72 h between the Mb and 1C2 antibodies. Although the Mb antibody stained the gel top in 72 h, the reactivity of the 1C2 antibody to the gel top was obviously decreased in 72 h (Fig. 3F).Fig. 3Electron micrographs and an immunoblot of Mb-Gln50 quasi-aggregate and fibril. Negative staining was performed with 2% sodium phosphotungstic acid. A, Mb-Gln50 after incubation at 37 °C for 3 h. B–E, immunoelectron microscopy was also carried out for the Mb-Gln50 after 3 h incubation and for the Mb-Gln50 fibrils (after 7 days) with a polyclonal Mb or a monoclonal 1C2 antibody (anti-expanded polyglutamine). F, immunoblot of Mb-Gln50 after incubation at 37 °C for 0, 3, 10, 24, and 72 h by Mb and 1C2 antibodies. The immunoblots at the gel top were shown. G and H, typical electron micrographs of the Mb-Gln50 after incubation at 37 °C for 3 and 5 days, respectively. Arrows indicate the association or connection of fibrils to clusters of quasi-aggregates. I, a close-up view of the lower arrow site in H. Scale bar, 100 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Furthermore, we followed the morphological changes of Mb-Gln50 upon incubation at 37 °C for 1 to ∼7 days, using electron microscopy. The quasi-aggregates of Mb-Gln50 were associated with each other to form largely assembled clusters, but fibrils were not observed at 1 day (data not shown), whereas fibrils were clearly detected at 3 days. We display typical electron micrographs of Mb-Gln50 at 3 or 5 days in Fig. 3, G–I. In the electron micrographs, we frequently observed the fibrils that were connected to and associated with clusters of the quasi-aggregates (arrow, Fig. 3, G–I).Models of Mb-Gln 50 Monomer, Oligomer, and Quasi-aggregates—To gain more structural information on the quasi-aggregate, we attempted to perform SVD analysis for the time-dependent SAXS spectra. Because the SVD analysis required more data points in the first h of protein fibrillization, we measured SAXS of Mb-Gln50 (1.86 mg/ml), again in shorter incubation intervals from 1 to 60 min at 37 °C (Fig. 4A). The resultant 11 scattering profiles were analyzed using SPECFIT. The SVD analysis showed two species, and the scattering profiles were best fitted by single exponentials. This analysis together with the presence of an isoscattering point at S = 4.4 × 10–3 Å–1 in the SAXS spectra (Fig. 4A) indicated that this reaction follows the first-order kinetics. Therefore, we deconvoluted the time-dependent SAXS profiles of Mb-Gln50 into two spectra, which displayed the scattering profiles of the initial (0 min) and final (60 min) states of Mb-Gln50. In the first-order kinetic scheme, the scattering profile of the final state (60 min) corresponded to that of the quasi-aggregate. We calculated Kratky plots of the two states of Mb-Gln50 and show them in Fig. 4B. From the scattering profiles, we calculated R g values of the initial and final states of Mb-Gln50 (68 and 146 Å, respectively). We realized that the initial state (0 min) was not a monomer but an oligomer, because the R g of 68 Å was larger than that of the Mb-Gln50 monomer (27 Å) (25Tanaka M. Machida Y. Nishikawa Y. Akagi T. Morishima I. Hashikawa T. Fujisawa T. Nukina N. Biochemistry. 2002; 41: 10277-10286Crossref PubMed Scopus (25) Google Scholar). We found, by SAXS and dynamic light scattering analyses, that the Mb-Gln50 monomer is rarely isolated as a complete monomeric state and that some oligomerized species are formed during the purification. From the SAXS spectra, we also calculated cross-sectional radii of gyration, R c, of the Mb-Gln50 oligomer (30 Å) and quasi-aggregate (92 Å). In addition, D max values of the oligomer (213 Å) and quasi-aggregate (463 Å) were determined by the pair distance distribution functions using GNOM. We found that these values were not changed by different data sets, confirming the reliability of the scattering profiles of the Mb-Gln50 oligomer and quasi-aggregate. We summarized the structural parameters of the Mb-Gln50 monomer, oligomer, and quasi-aggregate in Table I.Fig. 4Molecular models of Mb-Gln50 oligomer and quasi-aggregate. A, time-dependent S versus S 2 × I(S) (Kr
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