Positively Charged Chitosan and N-Trimethyl ... - ACS Publications

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Positively Charged Chitosan and N‑Trimethyl Chitosan Inhibit Aβ40 Fibrillogenesis Haiyang Liu,† Bimlesh Ojha,† Clifford Morris,† Mengting Jiang,† Ewa P. Wojcikiewicz,‡ Praveen P. N. Rao,§ and Deguo Du*,† †

Department of Chemistry and Biochemistry and ‡Department of Biomedical Science, Florida Atlantic University, Boca Raton, Florida 33431, United States § School of Pharmacy, Health Sciences Campus, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada S Supporting Information *

ABSTRACT: Amyloid fibrils, formed by aggregation of improperly folded or intrinsically disordered proteins, are closely related with the pathology of a wide range of neurodegenerative diseases. Hence, there is a great deal of interest in developing molecules that can bind and inhibit amyloid formation. In this regard, we have investigated the effect of two positively charged polysaccharides, chitosan (CHT) and its quarternary derivative N-trimethyl chitosan chloride (TMC), on the aggregation of Aβ40 peptide. Our aggregation kinetics and atomic force microscopy (AFM) studies show that both CHT and TMC exhibit a concentration-dependent inhibiting activity on Aβ40 fibrillogenesis. Systematic pH-dependent studies demonstrate that the attractive electrostatic interactions between the positively charged moieties in CHT/TMC and the negatively charged residues in Aβ40 play a key role in this inhibiting activity. The stronger inhibiting activity of TMC than CHT further suggests the importance of charge density of the polymer chain in interacting with Aβ40 and blocking the fibril formation. The possible interactions between CHT/TMC and Aβ40 are also revealed at the atomic level by molecular docking simulation, showing that the Aβ40 monomer could be primarily stabilized by electrostatic interactions with charged amines of CHT and quaternary amines of TMC, respectively. Binding of CHT/TMC on the central hydrophobic core region of Aβ40 peptide may be responsible for blocking the propagation of the nucleus to form fibrillar structures. These results suggest that incorporation of sugar units such as D-glucosamine and N-trimethyl-D-glucosamine into polymer structural template may serve as a new strategy for designing novel antiamyloid molecules.

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small molecules as inhibitors of Aβ amyloid formation.15,16 In addition, recent studies have also reported a number of polymeric macromolecules as modulators of Aβ fibrillogenesis, either facilitating the rate of fibril formation or inhibiting this self-assembly process.17−19 Although still at an early stage of development, these polymeric molecules can provide a novel scaffold for designing new molecules as modulators of protein aggregation pathway. Furthermore, the physiological environment, wherein Aβ aggregation happens, is crowded with a large variety of macromolecules. Many biological macromolecules, for example, proteins, nucleic acids, and polysaccharides, such as proteoglycans and glycosaminoglycans (GAGs), are polyelectrolytes whose repeating units contain ionizable groups. These charged molecular chains can play a critical role in determining structure, stability, and the interactions of various molecular assemblies.20,21 For example, proteoglycans and GAGs are associated with a variety of amyloid deposits in tissues. The colocalization of these molecules and amyloid deposits is a pathological feature in amyloidogenesis.22,23 Some GAGs, such as heparin sulfate and heparin polyanions, have

INTRODUCTION Progression of protein misfolding and anomalous aggregation events that form amyloid fibrils is closely related to a number of devastating amyloid diseases, such as Alzheimer’s, Parkinson’s, and Creutzfeldt−Jakob diseases.1 In Alzheimer’s disease (AD), the extracellular amyloid plaques are composed primarily of amyloid-β (Aβ), a mixture of peptides ranging in length from 39 to 43 amino acid residues out of which the most abundant forms are 40 and 42, derived by proteolytic cleavage of transmembrane region of amyloid precursor protein (APP). The aggregated amyloids consist of a characteristic cross-βsheet fibrillar structure with β-strands perpendicular to the fibril axis.2,3 The mechanistic details of protein self-assembly have been studied extensively over the past decades.4−8 Protein fibrillogenesis generally is a multistep nucleated event that goes through multiple forms including oligomers, protofibrils, and fibrils.4,9−11 Each of these aggregates has characteristic molecular conformations and different degrees of toxicity to the neuronal cells.12−14 One plausible way to ameliorate the neurotoxicity of Aβ in AD is to inhibit the amyloidogenesis of Aβ, thus, preventing the formation of toxic aggregated species. In the past decade, numerous pharmacologic approaches aimed at inhibiting Aβ amyloid formation have led to the discovery of a variety of © 2015 American Chemical Society

Received: May 5, 2015 Revised: June 30, 2015 Published: June 30, 2015 2363

DOI: 10.1021/acs.biomac.5b00603 Biomacromolecules 2015, 16, 2363−2373

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Their inhibition activity is sensitive to the pH values of the environment, suggesting the critical role of electrostatic interactions in Aβ40−polymer interactions. The plausible interactions in Aβ40−polymer complex at atomic level are also described based on the molecular docking studies.

been found to play an important role in facilitating the initiation of protein aggregation24−26 and affect the resulting amyloid morphology and neurotoxicity.27−29 Despite the ubiquitous presence of protein−polymeric macromolecule interactions in a physiological environment, the detailed mechanistic effect of polymeric macromolecules on protein self-assembly is still largely unclear. Chitosan (CHT; Scheme 1) is a versatile natural polysaccharide obtained by deacetylation of chitin. It contains

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EXPERIMENTAL SECTION

Materials. All chemical reagents were obtained from commercial suppliers and used without further purification, unless otherwise mentioned. Chitosan (low molecular weight, 75−85% deacetylated, molecular weight 50−190 kDa, #448869) was purchased from SigmaAldrich. TMC was synthesized by following a literature procedure.35 The 1H NMR spectra of TMC were acquired on a Varian UnityINOVA 400 spectrometer. The solution of chitosan was prepared by thoroughly mixing appropriate amount of solid chitosan in aqueous solution containing 0.25% acetic acid. The solution of TMC was prepared in water. Synthesis and Purification of Aβ40. Aβ40 was synthesized on a PS3 solid phase peptide synthesizer (Protein Technologies Inc., Woburn, MA) using Fmoc chemistry. The crude peptide was purified by high-performance liquid chromatography (HPLC) using a C18 reverse phase column. After purification, the peptide was lyophilized to obtain power samples. The molecular weight of Aβ40 was verified by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The Aβ40 peptide utilized in the kinetic aggregation assay was monomerized as described previously.36 Briefly, lyophilized Aβ40 powder was dissolved in aqueous NaOH solution (2 mM) and the pH was adjusted to 11 by using 100 mM NaOH solution followed by sonicating the solution for 1 h in an ice-cold water bath. The resulting solution was filtered through 0.22 μm filter and kept on ice before use. The concentration of Aβ40 stock solution was determined by tyrosine (Tyr) UV absorbance at 280 nm (ε280 = 1280 cm−1 M−1). Kinetic Aggregation Assay of Aβ40 Using Thioflavin T (ThT). The aggregation kinetics followed by ThT fluorescence were measured as described previously.37 Briefly, monomerized Aβ40 peptide solution was diluted to a final concentration of 5 μM in 50 mM phosphate buffer (150 mM NaCl) of pH 7.4, 50 mM acetate buffer (150 mM NaCl) of pH 4.0, or 50 mM phosphate buffer (150 mM NaCl) of pH 8.5. The solution also contained ThT with a final concentration of 20 μM. A total of 100 μL of solution was then transferred into a well of a 96-well microplate (Costar black, clear bottom). The plate was sealed with a microplate cover and loaded into a Gemini SpectraMax EM fluorescence plate reader (Molecular Devices, Sunnyvale, CA), where it was incubated at 37 °C. The fluorescence (excitation at 440 nm, emission at 485 nm) was measured from the bottom of the plate at 10 min intervals, with 5 s of agitation before each reading. For the assays with CHT or TMC, appropriate amounts of polysaccharide samples were dissolved in water (TMC) or 0.25% aqueous acetic acid (CHT) to obtain the desired concentrations. A particular amount of polysaccharide solution was added to Aβ40 solution (final concentration 5 μM) either in 50 mM phosphate buffer (150 mM NaCl) of pH 7.4, 50 mM acetate buffer (150 mM NaCl) of pH 4.0, or 50 mM phosphate buffer (150 mM NaCl) of pH 8.5, each containing 20 μM ThT. The solution was then mixed on a vortex for 5 s and pipetted into the plate reader (100 μL/well) for the kinetic assay. The kinetic parameter lag time (tlag) was extracted by fitting the kinetic data using the following equation:38

Scheme 1. Structures of Chitosan (CHT) and N-Trimethyl Chitosan (TMC)

a linear copolymer structure of β(1 → 4) linked 2-amino-2deoxy-D-glucopyranose units and residual 2-acetamido-2-deoxyD-glucopyranose units. Since it is readily available, biodegradable, biocompatible, and versatile, the use of CHT and its derivatives as part of drug delivery systems has increased substantially in recent years.30−33 The CHT molecule possesses a polymeric backbone that primarily consists of hydrophilic functional groups. In contrast to commonly studied polyanionic GAGs, CHT is a linear cationic base biopolymer with a pKa of ∼6.5 and carries positive charges in an acidic environment. The charge density in CHT chain is dependent on the solution pH and the degree of deacetylation of CHT molecule. The physicochemical properties of CHT can be further enhanced by modifying the side chain groups, for example, introducing permanently charged moieties in the polymer chain. This makes CHT a favorable model system to study the effects of structural properties of polysaccharides on their modulating activities of protein aggregation. Recently, Valle-Delgado and co-workers reported that CHT can interfere with the fibril formation of Aβ42 peptide.34 However, the detailed mechanistic effects of CHT on Aβ amyloidogenesis still remain to be elucidated. In the present work, we systematically studied the effect of CHT on the kinetics and the morphology of Aβ40 aggregation using a combination of spectroscopic and imaging methods coupled with molecular docking simulation. A quaternary derivative of CHT, N-trimethyl chitosan (TMC; Scheme 1), was also studied. TMC contains a permanent positive charge in the repeating unit of the sequence, regardless of the pH value. This allows to rigorously assess the role of electrostatic forces in directing the interaction with Aβ peptide and the regulating mechanism of the polymers on Aβ40 fibrillogenesis. In this study, we used Aβ40 since it exhibits a more moderate aggregation propensity than Aβ42, and therefore is considered as an appropriate model peptide for studying the dynamics of protein aggregation. Our findings reveal that both CHT and TMC can inhibit the aggregation of Aβ40 peptide, with TMC exhibiting a stronger inhibiting activity compared to CHT.

F=

Fmax (1 + ve−k(t − tm))1/ v

where F is the fluorescence intensity at time t, Fmax is the maximum steady-state fluorescence, tm is the point of the maximum elongation rate, and v represents the asymmetry of the curve. The lag time (tlag) is the time at which the tangent at tm crosses the initial baseline and is given by tm − (1 + v)/k. Atomic Force Microscopy (AFM). An aliquot of the peptide solution (20 μL) was adsorbed onto the surface of fresh mica (8 × 8 mm) for 5 min. The liquid was wicked off by absorption into filter 2364


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Biomacromolecules paper. Salt and unbound materials were washed in triplicate by 20 μL of Milli-Q H2O and removed by filter paper absorption. The samples were dried overnight before measurement. AFM images were recorded in tapping mode using an Asylum Research MFP 3D Bio AFM system with MikroMasch NSC15/AI BS cantilevers. Molecular Docking Studies. The 3D structures of fully deacetylated CHT, 50% deacetylated CHT and TMC (containing either 100% quaternary amines or 50% quaternary and 50% tertiary amines), each consisting of 14-glucopyranose units (14-mer) were built using the software program Discovery Studio (DS)-structure based design version 4.0 (Accelrys/BIOVIA, San Diego, U.S.A.). The energy minimization was done at pH 7.4 using 1000 steps each of steepest descent followed by conjugate gradient minimization protocols (0.1 and 0.01 kcal/mol) using CHARMm force field and an implicit solvent function GBSW.39 The NMR solution structure of full length Aβ40 monomer was obtained from PDB file: 2M9S.40 The bound ligands were removed, hydrogens were added, the N- and Cterminals were capped with acetyl and amide groups, respectively, and appropriate protonation states were assigned using CHARMm force field at pH 7.4. Any bad contacts were removed by carrying out 500 steps, each of steepest descent, followed by conjugate gradient minimization protocol (0.1 and 0.01 kcal/mol) using CHARMm force field and an implicit solvent function GBSW using SHAKE constraints. The polylelectrolytes were docked to Aβ monomer using the CDOCKER tool in DS and CHARMm force field after creating a 30 Å binding site sphere around Aβ monomer. This was carried out with 2000 heating steps, target temperature of 700 K, and cooling temperature of 300 K with 5000 cooling steps to generate 10 docked poses of fully deacetylated CHT, 50% deacetylated CHT and TMC (100 or 50% quaternarized), respectively. The poses were ranked using CDOCKER interaction energy (kcal/mol). The best pose obtained was further analyzed by considering the polar and nonpolar contacts with Aβ monomer. The polyelectrolytes-peptide binding energy in kcal/mol was calculated using the equation, energy of binding (Ebinding) = energy of complex (Eligand−enzyme) − energy of ligand (Eligand) − energy of receptor (Eenzyme) using the implicit solvent model GBSW in DS.

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RESULTS AND DISCUSSION Effect of CHT on Aβ40 Fibrillogenesis. The effect of CHT on aggregation kinetics of Aβ40 was monitored by using the fluorescence of ThT. The fluorescent dye ThT interacts with β-sheet-rich structures in amyloid fibrils leading to an increase in the fluorescence intensity in the vicinity of 480 nm.41 As shown in Figure 1A, the aggregation kinetics of Aβ40 peptide exhibit a typical sigmoidal appearance containing a lag phase associated with nucleation, a fast growth phase linked to the elongation and propagation of fibrils, and a final stationary phase. The half time (t50) of the growth phase of the Aβ40 amyloidogenesis under the current condition was approximately 15 h, where t50 is defined as the time at which the fluorescence intensity reaches the midpoint between the preand postaggregation baselines. The CHT used in our experiments is known to be present in 75−85% deacetylated form. This CHT was initially dissolved in aqueous acetic acid solution and then added to the Aβ40 sample solution (containing 0.25% acetic acid in the final solution) for ThT fluorescence measurement. An untreated Aβ40 sample solution containing the same amount of 0.25% acetic acid was used as a control to compare the effect of chitosan on Aβ40 aggregation kinetics. Addition of 0.25% acetic acid only made a negligible change in Aβ40 aggregation kinetics. As shown in Figure 1A,B, the presence of CHT dramatically decreased the ThT fluorescence intensity at the final plateau phase of Aβ40 aggregation in a dose-dependent manner. Adding CHT into preformed Aβ40 fibril solution with

Figure 1. (A) Effect of CHT on the aggregation kinetics of Aβ40 (5 μM), followed by ThT fluorescence at 37 °C in pH 7.4 phosphate buffer (50 mM Na-phosphate, 150 mM NaCl). (B) Relative ThT fluorescence intensity of the stationary phase of Aβ40 (5 μM) amyloidogenesis in the absence or presence of different concentrations of CHT. ThT fluorescence intensity was measured at 40 h in kinetic aggregation assays at 37 °C. The data are reported as mean ± standard deviation of triplicate results. (C) Lag time of the aggregation kinetics of Aβ40 in the absence or presence of CHT. The data are reported as mean ± standard deviation of triplicate results.

ThT did not lead to decrease of ThT fluorescence intensity (Figure S1), suggesting little effect of CHT on ThT fluorescence and lack of competitive binding of CHT to the ThT interaction site of Aβ40 fibrils. Treatment with a higher amount of CHT, for example, 0.5 mg/mL, results in negligible ThT fluorescence (Figure 1A,B), suggesting a strong inhibition of Aβ40 fibril formation. The lag phase of the aggregation trace is moderately elongated in the presence of low amount of CHT (Figure 1C), for example, with the lag time changed from 11.9 to 14.8 h (∼24% delay) in the presence of 0.05 mg/mL CHT (molar ratio of Aβ40 and sugar units ∼ 1:59). This suggests that a low amount of CHT does not influence the initial nucleation reactions dramatically; instead, it may prevent the elongation of nucleus intermediates to form fibrils. With higher concentrations (e.g., 0.5 mg/mL), CHT may directly interact with the Aβ40 monomer, thus, blocking the nucleation process at the early stage of the aggregation pathway. 2365


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Biomacromolecules The inhibitory activity of CHT on Aβ40 fibrillogenesis was further confirmed by AFM imaging results. In the absence of chitosan, Aβ40 aggregated to form long and curvy amyloid fibrils after a six-day incubation on a Speci-Mix aliquot mixer at 37 °C (Figure 2A). In the presence of 0.002 mg/mL CHT,

negatively charged acidic amino acid residues (3 Asp and 3 Glu residues). The inhibitory activity of CHT therefore could be reasonably attributed to the attractive electrostatic interactions between CHT and the negatively charged residues of Aβ40 peptide. Effect of TMC on Aβ40 Fibrillogenesis. To further investigate the mechanistic details of the inhibitory effect of CHT on Aβ40 fibrillogenesis and ascertain the role of charge interactions in the inhibitory activity, we studied the effect of TMC, a derivative of CHT, on Aβ40 amyloid formation. TMC contains positively charged quaternary nitrogens with a trimethyl substitution (N-trimethyl group) in the repeating units of the sequence, unlike the amine groups in CHT. Therefore, regardless of the pH value, TMC is a positively charged polymer. The charge density of TMC is determined by the level of N-trimethylation. The NMR spectrum of the TMC product shows a peak at 3.25 ppm attributed to N-trimethyl group, and a peak at 2.95 ppm attributed to N-dimethyl group (Figure S2). This is consistent with other recently reported results.35,44 The yield of trimethylation is about twice of dimethylation. It should be noted that the degree of Omethylation side reaction will also affect the lipophilicity of the TMC material. Our NMR data show that O-methylation (peak at 3.33 ppm, Figure S2) occurrence is much lower compared to N-trimethylation (∼40% of degree of trimethylation), which is consistent with other literature reports.44 The N-trimethylation was identified as the major reaction. Comparison of peak ratios suggests that the degree of quaternization is approximately 52%. The product shows better solubility in water than CHT, which could be more suitable for biological applications.45 The kinetic effect of TMC on the aggregation of Aβ40 peptide was also examined using the ThT fluorescence assay. As shown in Figure 3, the presence of TMC also dramatically decreases the ThT fluorescence intensity at the plateau phase of aggregation in a dose-dependent manner, suggesting its ability to inhibit Aβ fibril formation. The presence of 0.05 mg/mL or higher concentrations of TMC eliminated the ThT fluorescence, suggesting a strong inhibition activity at these concentrations. Moreover, although the fluorescence intensity of ThT is strongly influenced by TMC concentration, the lag time of the aggregation kinetic traces is not much affected at lower concentrations (0.0005 and 0.005 mg/mL measured) compared to the lag time in the absence of TMC (Figure 3B). This suggests that, similar as CHT, low amount of TMC does not significantly influence the nucleation reactions along the Aβ40 aggregation pathway. However, at higher concentrations (e.g., 0.05 and 0.5 mg/mL), TMC affected all phases of aggregation, indicating its ability to directly interact with Aβ monomer and prevent its self-assembly. Furthermore, our results reveal that TMC exhibits stronger inhibitory activity on Aβ40 aggregation compared to that of CHT (Figure 3C). The inhibitory activity of TMC on Aβ40 fibrillogenesis was also confirmed by AFM imaging. As depicted in Figure 4, with a low concentration of 0.002 mg/mL of TMC, Aβ40 formed shorter and less curvy fibrils, with a height of ∼5−7 nm, which is similar to the fibrils formed in the absence of TMC. At a concentration of 0.02 mg/mL of TMC, almost no fibrils were observed, with only some spherical intermediate structures presented (Figure 4C). A further increase in TMC concentration (0.2 mg/mL) led to the inhibition of both prefibrillar intermediates and fibrils (Figure. 4D). Moreover, in comparison to the AFM imaging results of CHT (Figure 4 vs Figure 2), one can see that TMC shows a stronger inhibiting activity

Figure 2. Tapping mode AFM images of Aβ40 amyloids formed in the absence (A) and presence (B−D) of different concentrations of CHT. The Aβ40 (20 μM) samples in pH 7.4 phosphate buffer (50 mM Naphosphate, 150 mM NaCl) were incubated on a speci-mix aliquot mixer at 37 °C for 6 days before acquiring images. (E) Height of the fibrils of Aβ40 in the absence or presence of 0.02 mg/mL CHT (n = 20).

Aβ40 still formed fibrillar structures, with shorter fibrils observed and less fibrils formed overall (Figure 2B). With 0.02 mg/mL CHT, the amount of fibrils was dramatically reduced, with mainly less curly and shorter fibrils of 200−600 nm length being formed (Figure 2C). The height of these shorter fibrils was approximately 5−8 nm, similar to those formed in the absence of CHT (Figure 2E). More significantly, at an increased concentration of CHT (0.2 mg/mL), no mature fibrils were observed (Figure 2D). Only some small amounts of spherical structures were present. These spherical intermediates have been reported with negligible ThT response due to the lack of β-sheet rich structures.42 The AFM imaging results are consistent with the ThT aggregation kinetics results. Taken together, our results demonstrate that CHT is an effective inhibitor of Aβ40 fibrillogenesis. Interestingly, a just published paper by Dai et al. showed that chitosan oligosaccharides (COS) used in their research can inhibit the aggregation of Aβ42 peptide and attenuate Aβ-mediated neurotoxicity.43 This is in good agreement with our results, and suggests that chitosan based polysaccharides may be an appropriate scaffold for developing molecules as inhibitors of Aβ amyloidogenesis. A variety of GAGs with negatively charged groups, including carboxylic acid or sulfate groups, have been reported to accelerate the amyloidogenesis of proteins including Aβ.24,25 Thus, the inhibitory activity of CHT on Aβ40 aggregation is intriguing. Although containing unbranched polysaccharide backbone structure comparable with GAGs, CHT is a polymeric amino sugar and contains appreciable positive charge under physiological condition. This can likely be a critical factor for its inhibiting ability. Aβ40 peptide contains a few of 2366


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Figure 4. Tapping mode AFM images of Aβ40 amyloids formed in the absence (A) and presence (B−D) of different concentrations of TMC. The Aβ40 (20 μM) samples in pH 7.4 phosphate buffer (50 mM Naphosphate, 150 mM NaCl) were incubated on a speci-mix aliquot mixer at 37 °C for 6 days before acquiring images. (E) Height of the fibrils of Aβ40 in the absence or presence of 0.002 mg/mL TMC (n = 20).

(N+Me3). As shown in Figure S3, the presence of additional CHT over a concentration range of 0.0005−0.5 mg/mL has little impact on Aβ40 aggregation kinetics. There is no observable difference in the ThT intensity either in the absence or the presence of different amounts of CHT (Figure 5A). AFM imaging also shows that, in the presence of CHT, Aβ40 fibrils still formed at pH 8.5 (Figure S4). These results demonstrate that CHT does not inhibit the aggregation of Aβ40 under this condition. In contrast, TMC still behaves as an effective inhibitor to block the Aβ40 amyloid formation, as depicted in Figure S3. At 0.005−0.5 mg/mL of TMC, the ThT fluorescence is dramatically decreased in a dose-dependent manner (Figure 5A). Furthermore, almost no fibrils were observed when Aβ40 was coincubated with 0.02 mg/mL TMC (Figure S4). The results are in agreement with the assumption that the positive charge in the CHT/TMC polymeric chain is critical in directing the interactions between Aβ40 peptide and the polysaccharide molecule. Uncharged amine group in CHT under basic conditions makes it less effective in interacting with the Aβ40 peptide, thus, losing the inhibiting ability. Effect of CHT and TMC on the Fibrillogenesis of Aβ40 at Acidic pH. We finally investigated the effect of CHT and TMC on the aggregation kinetics of Aβ40 under acidic conditions, that is, at pH 4.0. At this pH value, both CHT and TMC contain positive charges with comparable charge density, as the amine group in CHT is completely protonated at this pH value. The ThT kinetic study showed that Aβ40 still aggregates steadily at pH 4.0, while a shorter lag phase suggested a faster aggregation rate (Figure S5). Interestingly, at this pH value, both polymers show little effect on the aggregation of Aβ40 (Figures 5B and S5). The side chains of acidic amino acids (e.g., Asp and Glu) in Aβ40 will be present largely in un-ionized forms at pH 4.0, which can impair the attractive electrostatic interactions with CHT and TMC. Moreover, the pH value of 4.0 is well below the pI of Aβ40 (∼5.6). The net charge of the peptide at pH 4.0 becomes positive (∼+4), which is opposite to the net negative charge at pH 7.4 (∼−3). Therefore, it could

Figure 3. (A) Effect of TMC on the aggregation kinetics of Aβ40 (5 μM) followed by ThT fluorescence at 37 °C in pH 7.4 phosphate buffer (50 mM Na-phosphate, 150 mM NaCl). (B) Lag time of the aggregation kinetics of Aβ40 in the absence or presence of different concentrations of TMC. The data are reported as mean ± standard deviation of triplicate results. (C) Comparison of the ThT fluorescence intensity of the stationary phase of Aβ40 (5 μM) amyloidogenesis in the absence or presence of different concentrations of CHT and TMC, respectively. The data are reported as mean ± standard deviation of triplicate results.

on Aβ40 aggregation than CHT, in accord with the kinetic results. One of the critical structural differences between CHT and TMC is the presence of permanent positively charged amine groups in TMC. Thus, the higher positive charge density in TMC may be responsible for its stronger inhibiting activity than that of CHT. Effect of CHT and TMC on Aβ40 Fibrillogenesis at Basic pH. In order to verify the role of the positive charges in CHT and TMC on their inhibitory activity on Aβ40 aggregation, we performed similar kinetic experiments under a higher pH value of 8.5. At this pH condition, the amine group in CHT is mostly neutralized (NH2), while the TMC molecule still maintains the positive charge on the repeating units 2367


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protonated D-glucosamine groups underwent electrostatic interactions (distance < 5 Å) with COO− groups of acidic amino acids E3, D7, and D23, respectively. In addition, one of the NH3+ groups underwent cation−π interaction (distance < 4.5 Å) with an aromatic ring of F20. Other interactions include polar contacts of hydroxyl groups (OH and CH2OH) of CHT with backbones of R5, H6, and G9. This study reveals that polar interactions contribute significantly to the ability of CHT to bind Aβ40 monomer which can prevent Aβ aggregation and β-sheet assembly (Ebinding = −23.10 kcal/mol). The binding interactions of 50% deacetylated CHT at pH 7.4 shows that the presence of acetyl groups (MeCO) in N-acetyl-D-glucosamine units made the polyelectrolyte molecule more lipophilic compared to fully deacetylated CHT and promotes selfaggregation in polar solvents. As seen in Figure 6B, 50% deacetylated CHT adopts a compact, rigid U-shaped conformation unlike fully deacetylated CHT when bound to Aβ40 due to the presence of N-acetyl groups that exhibit a tendency to undergo van der Waal’s interaction with nonpolar regions of CHT. At the N-terminal 50% deacetylated CHT was in close proximity to amino acid residues 2AEFR5 and 8SGY10, whereas at the C-terminal it was interacting with amino acids 18 VFFAEDVGSNKG29. In general, binding of 50% deacetylated CHT to Aβ40 was largely through polar contacts. However, nonpolar contacts also played a role due to the presence of Nacetyl groups unlike fully deacetylated CHT. The presence of N-acetyl groups in 50% deacetylated CHT also increases the conformational flexibility due to increase in the number of rotatable bonds. This is reflected by the binding energy (Ebinding = −7.74 kcal/mol), which shows that the 50% deacetylated CHT-Aβ monomer complex is less stable compared to fully deacetylated CHT-Aβ monomer complex. These studies indicate that CHT binding to Aβ is dependent on the protonation state of D-glucosamine unit, and the amine protonation promotes polar interactions with Aβ40 to form stable complex.47 Binding interaction of TMC (with 100% N+Me3 groups) with Aβ40 monomer was also studied. In contrast to CHT, TMC is known to carry a permanent positive charge irrespective of the pH values due to the presence of quaternary amine groups. As depicted in Figure 7A, at the N-terminal TMC underwent polar contacts with 3EFRHDS6 and Y10, whereas at the C-terminal it was in contact with 18 VFFAEDVGSNK28 and I31. TMC exhibited a horseshoe conformation with linear ends and made greater number of polar and nonpolar contacts with Aβ40 monomer compared to 50% deacetylated CHT. The positively charged quaternary nitrogens of TMC (N-trimethyl-D-glucosamine) underwent interactions (distance < 5 Å) with acidic amino acids E3 and D7, Y10, and backbones of R5, H6, and N27. The methyl groups of N+Me3 underwent nonpolar contacts with aromatic rings of F4, F20, and Y10. In addition, the CH2OH and OH groups of TMC underwent polar contacts with R5 and S26. The presence of methyl groups (N+Me3) in TMC makes it much more lipophilic compared to fully deacetylated and 50% deacetylated CHT. Its superior in vitro antiaggregation activity is in accord with its higher binding affinity (Ebinding = −26.02 kcal/mol) compared to 50% deacetylated CHT (Ebinding = −7.74 kcal/mol). These studies show that better antiaggregation properties demonstrated by fully deacetylated CHT and TMC is due to the presence of positive charge present which limits their conformational flexibility after binding to Aβ40 monomer. Furthermore, previous work from Franca and co-

Figure 5. (A) Comparison of the ThT fluorescence intensity of the stationary phase of Aβ40 (5 μM) amyloidogenesis in pH 8.5 phosphate buffer (50 mM Na-phosphate, 150 mM NaCl) in the absence or presence of different concentrations of CHT and TMC, respectively. (B) Comparison of the ThT fluorescence intensity of the stationary phase of Aβ40 (5 μM) amyloidogenesis in pH 4.0 acetate buffer (50 mM Na-acetate, 150 mM NaCl) in the absence or presence of different concentrations of CHT and TMC, respectively. The data are reported as mean ± standard deviation of triplicate results.

also be argued that unfavorable repulsive electrostatic interactions between the overall positively charged Aβ40 and CHT/TMC hinder their interactions,46 which leads to the loss of inhibition on Aβ40 fibrillogenesis. In principle, it supports the hypothesis that polar interactions, especially electrostatic interactions in this case, play a critical role in stabilizing the hypothesized Aβ40-CHT/TMC complex to prevent Aβ40 fibril formation. Molecular Docking Studies of Fully Deacetylated CHT, 50% Deacetylated CHT, and TMC with Aβ40. The possible binding interaction of CHT and TMC with Aβ40 was investigated by conducting molecular docking studies, in order to provide insights into the interaction details at molecular level. Models of CHT and TMC containing 14- monosaccharide units were built using the modeling software Discovery Studio (Accelrys/BIOVIA). Two models of CHT were built including fully deacetylated CHT and 50% deacetylated CHT to understand the role of acetylation on Aβ40 binding. Acetylation of the amine group in CHT will neutralize the charge in this group. The binding mode of fully deacetylated CHT with Aβ40 monomer is shown in Figure 6A. It binds in an extended conformation and was oriented in a perpendicular fashion across Aβ40 monomer. CHT underwent a number of polar interactions with Aβ40 in this binding mode. At the N-terminal, CHT was in contact with amino acids 1 DAEFRHDSGY10 whereas at the C-terminal CHT was interacting with 18VFFAEDVG25 (Figure 6A). At pH 7.4, the 2368


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Figure 6. Binding modes of fully deacetylated CHT (A) and 50% deacetylated CHT (B) with full length Aβ40 monomer (PDB id: 2M9S).

quaternary nitrogens underwent an electrostatic interaction with the side chain of D23 (distance = 4.6 Å) and also formed a hydrogen bond with OH group of sugar units (CH2OH, distance = 3.2 Å). In addition, the backbone CO of S26 underwent a hydrogen bonding interaction with OH group of CH2OH (distance = 2.4 Å). Overall, it was clear that unlike fully quaternarized TMC, the binding interactions of Aβ40 and TMC with 1:1 ratio of quaternary and tertiary amines comprise more nonpolar contacts. Calculation of binding energy (Ebinding = −17.13 kcal/mol) also indicates that it exhibits reduced affinity to Aβ40 compared to fully quaternarized TMC, although it is still better at binding to Aβ40 compared to CHT. Interactions between CHT/TMC and Aβ40. A number of previous studies on a series of polyanionic GAGs suggest that these negatively charged polyelectrolytes promote the fibrillogenesis of a series of amyloidogenic proteins.25,50−53 Favorable attractive electrostatic interactions were speculated to play a

workers has shown that in aqueous environment fully deacetylated CHT predominantly exists as a monomer with reduced tendency to aggregate.48,49 This suggests that increased lipophilicity of both 50% deacetylated CHT and TMC can promote self-aggregation which could be the actual species responsible for their antiamyloid properties at higher concentrations. Binding interactions of TMC containing 1:1 ratio of quaternary and tertiary amines (50% N+Me3 and 50% NMe2 groups) show that it was oriented perpendicular to the axis of Aβ40 monomer (Figure 7B). Unlike the fully quaternarized TMC (Figure 7A), it underwent a less number of contacts with the Aβ40 monomer. At the N-terminal, it was in contact with R5, A2, and D1, whereas at the C-terminal it was in contact with 20FAEDVGS26 region. Fewer electrostatic interactions were seen between the positively charged quaternary amines and amino acids with carboxylic acid side chains. One of the 2369


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Figure 7. Binding modes of fully quaternarized TMC (A) and TMC with 1:1 ratio of quaternary and tertiary amines (B) with full length Aβ40 monomer (PDB id: 2M9S).

dominating role in the interplay of GAGs with amyloidogenic proteins.25 Calamai and co-workers reported that the sulfated structure of heparin and in particular the O-sulfo groups of heparin are critical for accelerating protein gelsolin amyloidogenesis.24 CHT and its derivative TMC studied here, in contrast, contain positively charged moieties in their structures. Unlike polyanionic GAGs, such as heparin and heparin sulfate, which accelerate Aβ40 fibril formation,54 our results demonstrate that the positively charged polysaccharide CHT and TMC show inhibiting activities on Aβ40 amyloid formation under appropriate experimental conditions. Aβ40 peptide itself is an amphipathic polyelectrolyte, and the overall net charge and the charge distribution along the sequence are highly dependent on pH (Figure 8A). The peptide therefore can interact with CHT/TMC through electrostatic interactions and weak polar and van der Waals interactions. A proposed mechanism with electrostatic interactions as the driving force in determining the inhibitory activity is summarized in Figure 8B. While TMC is a polysaccharide with permanent positive

charge, the charge property of CHT is pH-dependent. In addition, the net charge of Aβ40 peptide at pH 7.4 is essentially negative, whereas at pH 4.0, Aβ40 becomes positively charged (Figure 8A,B). This electrostatic interaction-driving hypothesis supports the results that the antiaggregation activity by CHT and TMC observed at pH 7.4 is superior to that seen under acidic pH, and CHT losses its inhibiting activity at pH 8.5 whereas TMC does not. Furthermore, since TMC has a higher charge density compared to partially protonated CHT (∼10% amine groups protonated at pH 7.4), it likely results in stronger electrostatic interactions between TMC and Aβ40, in comparison to that in CHT−Aβ40 complex, therefore, leading to a stronger inhibitory capability for TMC. The molecular docking results provide atomic-level details of plausible interactions between CHT/TMC and Aβ40, further demonstrating the importance of polar interactions especially electrostatic interactions in stabilizing CHT/TMC−Aβ40 complex. Moreover, it is well-known that the 17LVFFA21 hydrophobic core segment of Aβ acts as a critical nucleation 2370


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polysaccharide backbone structure in CHT and TMC, besides their polycationic nature, is indispensable for their inhibiting activity on Aβ40 amyloidogenesis. This polysaccharide backbone may form suitable conformations and direct a feasible binding on critical regions such as the central hydrophobic core in Aβ40. Furthermore, understanding which stage(s) of Aβ aggregation process CHT/TMC could interact with is critical. GAGs have been reported to act at the earliest nucleation stage of fibril formation of Aβ40 and Aβ42, instead of simply involved in the later aggregation toward fibril formation.54 For CHT and TMC, one possibility is that, similar as GAGs, they can bind to the natively unstructured monomers and interfere with the very early nucleation step in the amyloid formation cascade. However, although this would be quite reasonable at high concentration of CHT/TMC condition, our results show that the presence of moderate concentrations of CHT/TMC does not dramatically change the lag time of Aβ40 aggregation. Cohen and co-workers recently reported that Aβ peptide aggregation kinetics could be dominated by a fibril-catalyzed secondary nucleation mechanism.60 Our results suggest that this secondary nucleation reaction in Aβ amyloidogenesis is not influenced markedly by these polymers. Instead, the affinity of CHT/TMC on the aggregation intermediates formed on the amyloidogenesis pathway may interfere with the subsequent propagation to form fibrillar structures.

Figure 8. (A) Charge distribution of Aβ40 (only charge rich region D1-G29 shown) at pH 7.4. The acidic residues are highlighted in blue color. (B) Schematic representation of the effect of low concentration of CHT and TMC on Aβ40 fibrillogenesis under different pH conditions.

center in Aβ aggregation, and determines Aβ aggregation propensity to a large extent.55,56 Interestingly, both CHT and TMC can interact favorably with the 18VFFAEDVG25 region of Aβ40 monomer, as seen with modeling experiments. CHT can interact with D23 through electrostatic interaction and with F20 through cation−π interaction. The methyl groups of N+Me3 in TMC can also form nonpolar contacts with aromatic rings of F20. Binding of CHT and TMC at this crucial region will very likely prevent self-assembly and elongation of Aβ40 to form β-sheet rich fibrils. GAGs have been also reported to bind to the Aβ sequence, specifically in the 13HHQK16 region.57,58 Recently, Madine and co-workers studied heparin−Aβ40 fibril interaction using solid-state NMR spectroscopy, and their results did not show direct interaction between heparin and the central hydrophobic core of Aβ40.27 It is likely that the difference of the eventual binding site on Aβ40 is a key factor that leads to distinct effects of CHT/TMC and heparin (and other GAGs) on Aβ40 aggregation. Besides the electrostatic interactions, other interactions such as van der Waals interactions can also contribute to the interplay between the polymers and Aβ40. As suggested in the molecular docking studies, the hydrophobic affinity between the hydrophobic side chains of the amino acid residues and the alkyl (methyl) moieties on amine groups in TMC could also contribute to the enhanced antiamyloid activity of TMC. Manipulating the electronic and hydrophobic properties of the structural moieties in CHT-based oligosaccharide molecules, e.g., incorporating the units of D-glucosamine and N-trimethylD-glucosamine into CHT oligosaccharide structural template, may be used as a new strategy for designing novel antiamyloid molecules for therapeutic purpose.59 Interestingly, Assarsson and co-workers recently reported that a group of positively charged polymers including poly lysine and poly(ethylenimine) accelerate the aggregation of Aβ42.18 The accelerating effect of these polymers could be attributed to the locally increased Aβ42 concentration near the polymers, which favors the association of the peptide.18 In comparison to these polymers, our results suggest that the

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CONCLUSION

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ASSOCIATED CONTENT

In summary, our study demonstrates that the polysaccharide CHT and its derivative TMC can inhibit the amyloidogenesis of Aβ40. The attractive electrostatic interactions between the positively charged moieties in CHT/TMC and the negatively charged residues in Aβ40 are implicated to play a dominating role in the inhibiting activity. The stronger inhibiting activity of TMC in comparison of that of CHT further suggests the importance of charge density of the polymer chain in interacting with Aβ40 and blocking the fibril formation. The plausible interactions between CHT/TMC and Aβ40 are also revealed at the atomic level based on molecular docking simulation. These findings suggest that positively charged polysaccharides, such as CHT and TMC, can serve as useful chemical tools for mediating mechanistic aggregation properties of Aβ40 and other aggregation-prone proteins. Design of CHTbased oligosaccharides with appropriate positively charged moieties may lead to discovery of novel inhibitor molecules for blocking protein amyloidogenesis.

S Supporting Information *

1

H NMR spectrum of TMC, aggregation kinetics under acidic and basic pH conditions, and the AFM images at basic pH. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00603.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 2371


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ACKNOWLEDGMENTS The authors gratefully thank the start-up fund from Florida Atlantic University and NSERC-Discovery RGPIN 03830-2014 (PPN) for supporting the current research.

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