Fibril Aggregates Formed by a Glatiramer-Mimicking Random

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Fibril Aggregates Formed by a Glatiramer-Mimicking Random Copolymer of Amino Acids Jingjing Lai,† Wenxin Fu,‡ Lin Zhu,§ Ruohai Guo,† Dehai Liang,*,§ Zhibo Li,*,‡ and Yanbin Huang*,† †

Key Laboratory of Advanced Materials (MOE), Department of Chemical Engineering, Tsinghua University, Beijing 100084, China Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China § College of Chemistry & Molecular Engineering, Peking University, Beijing 100871, China ‡

S Supporting Information *

ABSTRACT: Amyloid formation is now considered a universal and intrinsic property of all proteins, irrespective of their sequences. Therefore, it is interesting to see whether random copolymers of amino acids can also form amyloid aggregates. Here we use a copolymer of 4 amino acids, mimicking the clinically used drug Glatiramer, and demonstrate that it does form amyloid-like fibrils in the aqueous solution despite its random sequence structure. The fibrillar aggregates show an alanine-rich β-sheet secondary structure, proving the high tolerance of amyloid aggregates to the sequence irregularity in poly(amino acid)s, and suggesting the potential application of random copolymers as amyloid materials.

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INTRODUCTION Amyloid is the fibrillar aggregate with the characteristic cross-β sheet structure formed by protein or peptide.1,2 This fibrillar aggregate was initially found in proteins related to diseases such as Alzheimer’s disease, Type II diabetes, and Parkinson’s disease, but now is considered a universal and intrinsic property of all proteins.2,3 Non-disease-related proteins,3 homo-αpolyamino acids,4 and even non-α-polyamino acids5 are found to be able to form amyloid-like fibrils. So, it is tempting to think that formation of amyloid or amyloid-like fibrils is mainly driven by the interchain multivalent hydrogen bonds formed between the amide groups along the backbone, and the side group might just act to stabilize or destabilize the cross-β sheet structure.6 Therefore, amyloid formation can tolerate a certain extent of differences in side group sequences, which means even within one fibril, the side group sequences of the comprising species could be different. For example, MacPhee et al.6 found that two unrelated short peptides, TTR10‑19 and TTR105‑115, which are derived from the sequence of the human plasma protein transthyretin, are able to form mixed amyloid fibrils. There are also cross-seeding cases where fibrils of one peptide can be added to accelerate the amyloid aggregation of another peptide.7 These findings are significant both for the better understanding of conformational disease8,9 and for the development of amyloid-based materials.10,11 In this respect, a random copolymer of amino acids, which has normal peptide backbone but disordered sequence of side groups in every single chain, will be an interesting model to study how much the amyloid formation can tolerate the irregularity in the sequence structure of poly(amino acid)s. © 2014 American Chemical Society

Here we use a Glatiramer-mimicking random copolymer (GmRC) of four amino acids as a model molecule. Glatiramer acetate is an FDA-approved drug currently used to treat multiple sclerosis. It is a random copolymer of glutamic acid, lysine, alanine, and tyrosine with the molar ratio of 14%:34%:43%:9%, and has a molecular weight of about 5000−9000. 12 Our GmRC polymer (Scheme 1) was Scheme 1. Chemical Structure of GmRC

synthesized by N-carboxyanhydride (NCA) ring-opening polymerization and the molar ratio of the four amino acids is 16%:36%:40%:8% as calculated from 1H NMR (Figure S1) and the average molecular weight is 6500 (GPC).

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EXPERIMENTAL SECTION

Copolymer Synthesis. The amino acids were purchased from GL Biochem (Shanghai) Ltd. All solvents were purchased from Sinopharm Chemical Reagent Co., and used after purification by columns of activated alumina. Other reagents were purchased from Aladdin Received: April 28, 2014 Revised: May 30, 2014 Published: June 2, 2014 7221

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Figure 1. Images of aggregates from 2.5 mg/mL GmRC solution, incubated at 60 °C: (A) pH 12, 2 days; (B) pH 7, 6 days using AFM; (C) pH 12, 140 days; (D) pH 7, 180 days, using TEM (scale bar: 1μm). Industrial Inc. The detailed preparations of NCA monomers were reported elsewhere.13 N-Carboxyanhydrides of tyrosine (0.65 g, 3.09 mmol), alanine (1.61 g, 13.91 mmol), γ-benzyl glutamate (1.2 g, 4.56 mmol), and trifluoroacetyllysine (3 g, 11.14 mmol) were added into a Shlenk flask with magnetic stirring bar and then were evacuated and purged with argon three times. 120 mL dioxane was added under argon purge to dissolve these four monomers. Then the polymerization was initiated by the addition of 0.015% diethylamine. The reaction mixture was stirred at room temperature for 24 h and then poured into the mixture of 45 mL acetone and 240 mL water. The precipitated copolymer was filtered, washed with water, and dried (94.7% yield). The removal of the γ-benzyl blocking groups from the glutamate residue was carried out by treating the protected copolymer with 33% hydrobromic acid in glacial acetic acid at room temperature for 6−12 h with stirring, and the removal of trifluoroacetyl groups from the lysine residues was carried out in 120 mL water and 52 mL 40% tetrabutylammonium hydroxide in water for 24 h at room temperature. Finally, the mixture’s pH value was adjusted to 3−4 by adding acetic acid to a glatiramer acetate solution and dialyzed using a 3000 Da membrane to remove the low-molecular-weight impurities, and then the resulting solution was concentrated and lyophilized to give GmRC as a white solid (60% of yield). Sample Preparation. The copolymer was dissolved in deionized water, and concentrated sodium hydroxide and hydrochloric acid solutions were used to adjust pH to the set values. The solution was filtered through a 220 nm syringe filter (PES) before incubation at 60 °C. Atomic Force Microscope (AFM). AFM was performed with a Shimadzu SPM-9500 in the tapping mode. The sample solution was dropped onto a freshly prepared mica surface and air-dried for more than 24 h before observation. Transmission Electron Microscope (TEM). TEM were performed by a Hitachi H-7650B (Hitachi, Japan) transmission electron

microscope at an accelerating voltage of 120 kV. The carbon-coated copper grids were immersed in the sample solution for about 5 min, washed with water, and air-dried. Dynamic Light Scattering (DLS). DLS experiments were performed on a Brookhaven Instrument equipped with a BI-200SM goniometer as specified in our previous work.5 Gel Permeation Chromatograph (GPC). Analytical GPC was performed on a Waters HPLC system equipped with a UV−vis detector (Waters 2489). Samples were separated on an Asahipak GS520 HQ and GS-320 HQ column (with a guard column) using Tris· HCl buffer (50 mM Tris·HCl, 150 mM NaCl, pH = 7.4) as the mobile phase (25 °C, flow rate of 0.5 mL/min). Interferon-α and green fluorescent protein were used to quantify the elution volumemolecular weight relationship. Congo Red (CR) Binding.14 CR working solution was prepared by adding excess of CR into a solution of 80% ethanol:20% milli-Q water with a saturating amount of NaCl, and filtered through a 220 nm syringe filter before use. About 20 μL of sample was dried on a glass slide, and then CR working solution was added. After staining for a few seconds, the sample was blotted up and observed with a polarized light microscope. Thioflavin T (ThT) Binding.14 ThT was added to 10 mM PBS to prepare a 16 μg/mL working solution on the day of analysis, and filtered through a 220 nm syringe filter before use. About 1 mL working solution was used as the control solution, and then 20 μL of the polymer sample solution was added to another 1 mL ThT solution and stirred before measurement. The fluorescence spectra were collected on a Hitachi F-7000 fluorescence spectrometer with excitation at 450 nm and emission wavelength range of 420−480 nm. Circular Dichroism (CD). The sample solution was diluted to 0.1 mg/mL before analysis. CD spectra were collected on a Pistar π-180 instrument at 20 and 60 °C in a cuvette with a 0.1 cm path length. The spectra were recorded at a 0.5 nm interval from 260 to 190 nm. The 7222

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Figure 2. Size (hydrodynamic radius, Rh, determined at 30°) distribution of the aggregates formed in GmRC solution at selected time points (A), and the time dependence of the excess scattered intensity (denoted as (I − I0)/It, with It, I, and I0 denoting the scattered intensity from the standard, the solution, and the solvent, respectively) (B). The sample at 2.5 mg/mL and pH 12 is incubated at 60 °C.

Figure 3. CR-stained GmRC aggregates observed under natural light (A) and showed apple-green birefringence under the polarized light (B). Scale bars: 500 μm.

14 days. These large precipitates were stable at 60 °C during the whole observation period and even so after several months, and TEM showed that they were the clusters of fibrils (Figure 1C,D). The same visible precipitates were also obtained in a shorter time of about 2 days by adding a small amount of preformed fibrils as “seed”. The characterization results below were mainly from the seeded pH 12 group because it was easier to obtain enough amount of samples for detailed characterization. DLS was used to monitor the aggregation process of the GmRC solution at pH 12 after heating to 60 °C. Figure 2A shows the size (Rh) distribution at selected time points. In the very beginning (at 3 min), the Rh shows a bimodal distribution. The fast mode with size of about 2 nm is attributed to the diffusion of single GmRC chains, and the peak around 100 nm is attributed to the diffusion of the aggregates of GmRC. After incubation for 9 h, a new diffusive mode corresponding to an aggregate with the average size above 1 μm is observed, which maybe correspond to the fibril formation observed under AFM (Figure 1A and B). The formation of the larger aggregate and its corresponding growth result in a sharp increase in the excess scattered intensity (Figure 2B). As incubation proceeds, the aggregate above 1 μm starts to precipitate. At 230 h, its fraction greatly decreases (Figure 2A) and the excess scattered intensity drops to a very low level (Figure 2B). In the meantime, the precipitate in the container bottom can be observed by the naked eye.

fraction of secondary structures was calculated using the Selcon3 algorithm with reference set 4 (190−240 nm) at the DichroWeb server.15,16 Fourier Transform Infrared Spectroscopy (FTIR). The sample solution was dried on a polyimide film for FTIR experiments. The spectra were recorded at room temperature in a Nicolet 6700 FTIR spectrometer with MCT-A detector. The peak fitting is calculated with Gaussian functions. X-ray Diffraction (XRD). The sample solution was dried on a polyimide film for 2D-XRD experiments. The X-ray pattern was then collected using a Rigaku R-Axis Spider instrument with a Mo target at wavelength of 0.708 Å. The X-ray beam is set perpendicular to the films. The 1D pattern was converted from 2D diffraction pattern and followed by background removal.

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RESULTS AND DISCUSSION The GmRC was dissolved in Mili-Q water to prepare solutions at 2.5 mg/mL. Concentrated sodium hydroxide solution was used to adjust the pH to desired values (pH 7−12). After 2 days’ incubation at pH 12 and 60 °C, the solutions remained apparently clear, but fibril aggregates were observed under AFM. The fibrils were unbranched with the height of about 5− 20 nm and the length up to the micrometer scale (Figure 1A and SI Figure S2), which was similar to the typical amyloid fibrils.17 Similar fibrils were also observed in samples incubated at pH 7 and 60 °C (Figure 1B), but a longer incubation time of about 6 days was required. The number of fibrillar aggregates kept growing as incubation proceeds, and the solutions became cloudy with large precipitates visible to the naked eye after 7− 7223

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The fibrillar precipitates were collected by centrifugation and stained with Congo red, and showed apple-green birefringence under polarized light (Figure 3), which is characteristic of typical amyloid fibrils.14 In addition, when added to ThT solution, these precipitates resulted in an increase of ThT fluorescence intensity at about 490 nm when excited at 450 nm (Figure 4), which is also typical to the amyloid fibrils.14

Figure 5. FTIR spectra of GmRC: (A) without any treatment and (B) films collected from pH 12 solutions, dotted line: experimental data; solid line: peak-deconvolution fitting. The untreated GmRC powder shows amide I at 1645 cm−1, suggesting that its secondary structure is mainly α-helix. After incubation, besides the peaks at 1645 cm−1, slight increases at 1680 cm−1 and 1620 cm−1 are also observed, suggesting the appearance of β-sheet structure.

Figure 4. ThT fluorescence spectra before and after mixing with the GmRC fibrils (excitation: 450 nm). Dashed line: ThT solution; Solid line: ThT solution + GmRC fibrils.

The secondary structure of GmRC aggregates was analyzed by FTIR spectroscopy. The collected precipitates show an increase of IR absorption at 1620 cm−1, indicating more βstructure components than the fresh sample17 (Figure 5). CD also gives similar results that GmRC solution is mainly α-helix conformation at room temperature, but at 60°, the increase of the β-structure component is observed, along with a decrease of α-helix structure (SI Figure S3 and Table S1). However, the increase of β-structure component here is not as significant as in typical amyloid peptides.17 Considering the random arrangement of amino acids along GmRC chains, it’s reasonable to think that only part of the chains formed the β-sheet structure, while other parts remain α-helix or unstructured. The collected precipitates were then used for 2D X-ray diffraction characterization. As shown in Figure 6, the strongest diffraction peak corresponds to a distance of 4.4 Å. This diffraction distance is frequently reported in alanine-rich amyloid fibrils18,19 and the β-form crystal structure of poly(Ala), where the adjacent β-strands would be arranged in an orthogonal (or pseudo-orthogonal) double lattice, resulting in a closer distance of 4.4 Å, instead of the 4.7 Å interchain distance in typical amyloid fibrils.20,21 GPC was used to detect the possible degradation of the GmRC polymers during incubation. As shown in Figure S4, the average molecular weight of the samples did not change much within 7 days, but the peak was broadened, indicating both degradation and cross-linking. The cross-linking might be physical as in oligomer aggregates and/or the chemical crosslinking via the interchain dityrosine formation, as shown in the fluorescence spectra (Figure S5). Therefore, we have demonstrated that the Glatiramermimicking random copolymer of 4 amino acids also forms amyloid-like fibril aggregates in solution. To the best of our

knowledge, the only other study in the literature on random copolymer amyloid fibril was by Colaco and coworkers,22 who showed that a copolymer of glutamic acid and alanine formed fibril aggregates in the acidic condition. According to the results of FTIR, GmRC fibrils may contain less β-structure component than typical amyloids, and this can be explained with the “fuzzy coat” model of protein amyloid, where the β-sheet segments form the main stem of fibrils, while the other parts of the polymer chains could be in random coil or α-helix state flanking around the stem (Figure 7).23,24 For the random copolymers of amino acids, there may be no two polymer chains with the same sequence structure, and it would be difficult for all the segments to be arranged in the regular β-sheet structure. However, exactly because of its random sequence, each polymer chain is a library of peptide sequences and may contain peptide blocks of the same sequence, which may serve as the stem blocks of the amyloid-like fibrils. In the case of GmRC, as the XRD pattern suggested, an alanine-rich peptide seemed to be the building block for the amyloid stem. Since it is usually thought that the building block for the main stem requires about 6 amino acids,25,26 we calculated the probability to have a 6-alanine (-AAAAAA-) sequence in a single chain (Figure 8, details of the calculation can be found in the Supporting Information). For a GmRC chain of 60 residues (MW ∼ 6500) with 40 mol % of alanine residues, the probability of a chain with a block of 6 consecutive alanine residues is about 0.13. However, studies showed that some peptide with fewer than 6 amino acids can also form amyloids,27,28 and if we relax the requirement to 5 consecutive alanine residues for the stem block, this probability 7224

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Figure 6. (A) 2D X-ray diffraction pattern of GmRC films, obtained from pH 12 seeding samples. (B) Corresponding 1D diffraction. The strongest peak is corresponding to a distance of 4.4 Å, which is reported to be the main chain spacing in alanine-rich β-fibrils.

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CONCLUSIONS In summary, we demonstrated that a Glatiramer-mimicking random copolymer of 4 amino acids is able to form amyloidlike fibrillar aggregates, supporting that the amyloid aggregate structure has high tolerance of sequence irregularity. Recently, amyloid-forming polymers have been proposed for use as biomaterials with interesting mechanical and function properties,29−32 and our results suggest that random copolymers of amino acids might also be included in this material category. Moreover, with appropriate amino acid types and molar ratio, random copolymers may provide libraries of peptide sequences to recognize the target peptides without sequence-design, and may further modify their amyloid fibril formation, i.e., for promotion, recognition or inhibition of amyloid fibrils.

Figure 7. “Fuzzy coat” structure of GmRC fibrils. A peptide block of the same sequence forms the β-stem, and the other part is in random coil or α-helix state flanking around the stem.

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectrum, AFM morphology and height measurement, circular dichroism studies, degradation studies using GPC, fluorescence studies for the detection of dityrosine, calculation of probability. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

Figure 8. Probability of finding k or more consecutive A units in a chain with n constituent units when the fraction of A unit is 0.4.

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ACKNOWLEDGMENTS This work was sponsored by the National Natural Science Foundation of China (Project No. 21074064 to Y.H.). We thank Professor Weiping Gao Group (Tsinghua University) for GPC studies.

will increase to 0.30 in the case of GmRC. Furthermore, the aforementioned mixed fibrils and cross-seeding fibrils are evidence that the neighboring chains in the amyloid fibril are not required to have exactly the same sequence, so other alanine-rich segments would also be possible to incorporate into the stem. By combining all these factors, a significant fraction of the GmRC chains can possess the short consecutive alanine or alanine-rich peptide block, and these similar blocks will build amyloid-like stem.

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REFERENCES

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