Interaction of the Heparin-Binding Consensus Sequence of β-Amyloid

Feb 12, 2016 - the interaction of heparin and heparin-derived oligosaccharides with a model peptide for the heparin- and HS-binding domain of Aβ pept...
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Interaction of the Heparin-Binding Consensus Sequence of β‑Amyloid Peptides with Heparin and Heparin-Derived Oligosaccharides Khanh Nguyen and Dallas L. Rabenstein* Department of Chemistry University of California, Riverside, Riverside, California 92521, United States ABSTRACT: Alzheimer’s disease (AD) is characterized by the presence of amyloid plaques in the AD brain. Comprised primarily of the 40- and 42-residue β-amyloid (Aβ) peptides, there is evidence that the heparan sulfate (HS) of heparan sulfate proteoglycans (HSPGs) plays a role in amyloid plaque formation and stability; however, details of the interaction of Aβ peptides with HS are not known. We have characterized the interaction of heparin and heparin-derived oligosaccharides with a model peptide for the heparin- and HS-binding domain of Aβ peptides (Ac-VHHQKLV-NH2; Aβ(12−18)), with mutants of Aβ(12−18), and with additional histidinecontaining peptides. The nature of the binding interaction was characterized by NMR, binding constants and other thermodynamic parameters were determined by isothermal titration calorimetry (ITC), and relative binding affinities were determined by heparin affinity chromatography. The binding of Aβ(12−18) by heparin and heparin-derived oligosaccharides is pH-dependent, with the imidazolium groups of the histidine side chains interacting site-specifically within a cleft created by a trisaccharide sequence of heparin, the binding is mediated by electrostatic interactions, and there is a significant entropic contribution to the binding free energy as a result of displacement of Na+ ions from heparin upon binding of cationic Aβ(12− 18). The binding constant decreases as the size of the heparin-derived oligosaccharide decreases and as the concentration of Na+ ion in the bulk solution increases. Structure−binding relationships characterized in this study are analyzed and discussed in terms of the counterion condensation theory of the binding of cationic peptides by anionic polyelectrolytes.

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INTRODUCTION Amyloids are extracellular fibrillar protein deposits that cause or contribute to the pathogenesis of some 20 disorders, including Alzheimer’s disease (AD), which is characterized by the presence of amyloid plaques in the AD brain.1−4 The 40- and 42-residue β-amyloid (Aβ) peptides Aβ(1−40) and Aβ(1−42), formed by β- and γ-secretase cleavage of amyloid precursor protein (APP), are major components of AD amyloid plaques.5−8 Aβ(1−40) and Aβ(1−42) convert from soluble, random coil or α-helical conformations to insoluble, aggregated β-sheet structures during the formation (β-amyloidosis) of βamyloid plaques.9−11 Proteoglycans, including heparan sulfate proteoglycans (HSPGs), colocalize with Aβ peptides in AD amyloid plaques. HSPGs are ubiquitous on cell surfaces and in the extracellular matrix,12 and there is experimental evidence that the heparan sulfate (HS) of HSPG plays a role in amyloid plaque formation and stability.11,13−19 HS and the closely related glycosaminoglycan heparin bind tightly to Aβ peptides,14,20,21 and both catalyze in vitro the transition to β-sheet structure and amyloid fibril formation.11,14,15,22 Aβ(1−42) activates microglia to kill neurons, via a mechanism that involves binding to the HS of membrane surface HSPG,16 and binding to HS of cell-surface HSPGs of wild-type Chinese hamster ovary (CHO-WT) cells mediates Aβ peptide internalization and toxicity.12 © XXXX American Chemical Society

HS and heparin are linear polysaccharides composed of repeating uronic acid-(1 → 4)-glucosamine disaccharide units (Figure 1).23,24 The uronic acid is α-L-iduronic acid (IdoA) or β-D-glucuronic acid (GlcA), both of which can be 2-O-sulfated (IdoA(2S) and GlcA(2S)). The glucosamine (GlcN) is Nacetylated (GlcNAc) or N-sulfated (GlcNS), both of which can be 6-O-sulfated (GlcNAc(6S) and GlcNS(6S)). Heparin and HS differ in the abundance and sequence of possible repeating disaccharides. The structure of heparin is largely accounted for by repeating sequences of the trisulfated disaccharide sequence IdoA(2S)-(1 → 4)-GlcNS(6S) (e.g., bovine lung and porcine mucosal heparin have on average 2.8 and 2.5 sulfate groups, respectively, per disaccharide), while HS has a domain structure consisting of blocks of highly sulfated, heparin-like disaccharides (NS domains), blocks with little or no sulfation (NA domains), and partially N-sulfated, partially N-acetylated (NS/ NA) transition domains.23 HS and heparin bind peptides via electrostatic interaction of their dense negative charge (Figure 1) with positively charged guanidinium, ammonium, and, at low pH, imidazolium groups on the side chains of the basic amino acids arginine, lysine, and Received: December 14, 2015 Revised: February 10, 2016

A

DOI: 10.1021/acs.jpcb.5b12235 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

that has the sequence of the HS and heparin-binding domain of Aβ(1−40) and Aβ(1−42). A second goal of the research was to characterize more generally the interaction of the imidazolium side chain of peptide histidyl residues with heparin. While histidyl side chains participate less than those of arginine and lysine in binding of peptides and proteins by heparin and HS due to their lower pKAs (∼6.7), the protonated imidazolium side chains of histidyl residues of a number of peptides and proteins bind to heparin and/or HS, including histidine-rich glycoprotein,29−31 human and bovine platelet factor 4,32,33 mouse mast cell proteases 6 and 7 (mMCP-6 and mMCP-7),34−36 human mast cell chymase,37 selenoprotein P,38 granulocyte-macrophage colony-stimulating factor (GM-CSF),39,40 synthetic histidine-rich antimicrobial peptides,41 insulin-like growth factor binding protein,42 vascular endothelial growth factor (VEGF),43 and apolipoprotein serum amyloid A.44 Binding of these peptides and proteins by heparin or HS via their histidine side chains alters their activity. For example, the activity of chymase, which is stored in the secretory granules of human mast cells in its fully active form, is regulated by binding to and release from mast cell heparin at the low pH (∼5.5) of the granule and the higher pH of extracellular space, respectively.37 Mast cell granules also contain histamine, which binds to the granule heparin via its imidazolium group at the pH of the granule.23,45−47 We report the results of studies of the binding of the histidine-containing peptides in Table 1, including Aβ(12−18),

Figure 1. Monosaccharide building blocks of heparin and HS (top) and the fully sulfated heparin-derived tetrasaccharide. The Δ4,5unsaturated uronic acid residue (ΔUA(2S)) at the nonreducing end of the tetrasaccharide is produced from IdoA and GlcA residues upon cleavage of the glycosidic bonds of heparin by heparinase.

histidine, respectively.24−27 The β-amyloid peptides Aβ(1−40) and Aβ(1−42) contain one arginine (R5), two lysines (K16 and K28), and three histidines (H6, H13, and H14). Histidines 13 and 14, together with valine 12, glutamine 15, lysine 16, and leucine 17, form a cluster of amino acids (VHHQKL) that has the pattern of the linear XBBXBX consensus heparin-binding motif, where X and B represent hydropathic (either neutral or hydrophobic) and basic amino acid residues, respectively.25 The histidine residues at positions 13 and 14 are critical for the binding of Aβ peptides by heparin, as indicated by the pH dependence of the binding affinity (tightly at pH 4.0 and essentially no binding at pH 8.0) and the loss of binding (both at pH 4.0 and 8.0) when the histidines are replaced by serines in model peptides.14−16 It has been proposed that one or both positively charged histidine residues in the HHQK cluster bind to the anionic sulfate groups of HS and heparin and that the interaction is ionic in nature.14,15 However, the details of how the Aβ peptides bind to HS and heparin are not known. It also is noteworthy that HS is a universal component of amyloid fibrils formed in other amyloid disorders; the HS binding sites that have been identified in amyloid polypeptides require at least one histidine residue for full binding activity, and acidic pH favors the formation of the amyloid fibrils.1 Despite the importance of the role that binding of Aβ peptides and other amyloid peptides by HS may play in facilitating the formation of and/or stabilizing amyloid fibril aggregates, little is known about the nature of the binding interaction. The primary goal of the research reported in this paper was to characterize the mechanism, including the thermodynamics, of the interaction of the HHQK cluster of Aβ(1−40) and Aβ(1−42) with HS. Lindahl et al. found that HS structures containing IdoA(2S) residues and GlcNS(6S) residues, i.e., the fully sulfated, heparin-like NS domains of HS, are critical for the binding of Aβ monomers by HS.20 Thus, we have used heparin and heparin-derived oligosaccharides as a proxy for HS.1,28 We report the results of studies of the interaction of heparin and heparin-derived oligosaccharides with the model peptide Ac-VHHQKLV-NH2 (Aβ(12−18))

Table 1. Tetrasaccharide-Ligand Binding Constants and Heparin Affinity Chromatography Retention Timesa ligand no.

ligand

1 2 3 4 5 6 7 8 9 10

Ac-VHHQKLV-NH2 Ac-VHAQKLV-NH2 Ac-VAHQKLV-NH2 FRHDSGY GHK GHG HG GH histidine histamine

binding constant,b KB (M−1) 4250 331e 285e 329 1860 257 171 87 118 2400

pDc

± 120

6.0

± ± ± ± ± ± ±

5.0 6.0 6.0 5.7 6.0 5.7 6.0

30 40 11 4 5 4 140

HAC retention timed (min) 38.6 19.8 18.6 25.8 34.7 16.0 13.7 12.8 11.4 35.7

a At 25 °C. bDetermined by fitting chemical shift-ligand concentration data at constant tetrasaccharide concentration, except as noted. cpD at which the upfield displacement of the C3H resonance of the internal GlcNS(6S) residue was a maximum and at which the binding constant was determined. dAt pH 5.6. eEstimated from HAC retention time using the linear relationship between log KB and retention time.

by heparin and heparin-derived oligosaccharides. The pH dependence of the binding interactions was characterized by NMR, binding constants were determined by NMR and by isothermal titration calorimetry (ITC), and relative binding affinities were determined by heparin affinity chromatography (HAC).

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EXPERIMENTAL SECTION Materials. FMOC (9-fluorenylmethoxycarbonyl)-protected amino acids and trifluoroacetic acid (TFA) were obtained from Chem-Impex International Inc. N,N′-Dicyclohexylcarbodiimide (DCC), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium

B

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The Journal of Physical Chemistry B hexafluorophosphate (HBTU), and N-methyl-2-pyrrolidone (NMP) were obtained from Applied Biosystems. Sigma-Aldrich supplied triisopropylsilane (TIPS), α-cyanol-4-hydroxycinnamic acid (CHCA), piperidine (PIP), sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4(TMSP), and N,N′-diisopropylcarbodimide (DIPCDI). Rink amide 4-methylbenzhydrylamine (MBHA) resin (0.56 mmol/g) was purchased from NovaBiochem. Peptides 4 and 5−8 were obtained from American Peptide and Sigma-Aldrich, respectively. Low molecular weight heparins (molecular weights of 3000, 6000, and 12 000 Da) were obtained from Sigma-Aldrich. pH was measured directly in the NMR tubes using a microelectrode obtained from Microelectrodes, Inc. Peptide Synthesis and Purification. Peptides 1−3 in Table 1 were synthesized on an Applied Biosystems ABI-433A peptide synthesizer using solid-phase Fmoc peptide synthesis methodology. Peptides were cleaved from the resin, and side chains were deprotected with a cleavage reagent composed of 88% TFA, 4.2% H2O, 5.8% phenol, and 2% TIPS by volume. The resin was removed by filtration, and the filtrate was diluted with water. After extraction three times with methyl tert-butoxy ether (MTBE), the aqueous phase was lyophilized and crude peptides were obtained. The crude peptides were purified by HPLC on a Vydac 10 mm × 250 mm C18 semiprep column (5 μm particles, 300 Å pore size) using a Varian HPLC and an acetonitrile−water gradient containing 0.1% TFA. The detector was set at 215 nm. Peptide identities were confirmed by MALDI-TOF mass spectrometry. Preparation of Heparin-Derived Tetrasaccharide. The tetrasaccharide in Figure 1 was prepared by depolymerization of porcine intestinal mucosal heparin with heparinase I (EC 4.2.2.7).48 Heparin and heparinase I were both obtained from Sigma Chemical Co. One gram of heparin was dissolved in 50 mL of pH 7 solution containing 100 mM sodium acetate, 30 mM calcium acetate, and 0.02% sodium azide. 250 units of heparinase I was added, and depolymerization was monitored by measuring the absorbance at 232 nm. When the absorbance reached a constant value, the oligosaccharide mixture was concentrated by lyophilization. To obtain size uniform fragments, the mixture was separated by gravity flow size exclusion chromatography on a 3 × 200 cm Bio-Gel P6 column using a 0.5 M NH4HCO3 eluent. The tetrasaccharide in Figure 1 was isolated from the size-uniform tetrasaccharide fraction by strong anion HPLC with a Dionex 500 ion chromatography system equipped with a Dionex semipreparative scale CarboPac PA 1 column. The tetrasaccharide was eluted with a linear gradient of 70 mM pH 3 phosphate buffer (solvent A) and 70 mM pH 3 phosphate buffer containing 2 M NaCl (solvent B) at a flow rate of 3.3 mL/min. Measurement of Binding Constants by NMR. The chemical shift of the C3H proton of GlcNS(6S) changes upon binding of Aβ(12−18) and the other ligands in Table 1 by the tetrasaccharide. The first step in determining a binding constant (KB) was to obtain chemical shift-pD data for C3H of the tetrasaccharide in solution with the binding peptide. The pD at which there is maximum displacement of the chemical shift of the resonance for the C3H proton from that of free tetrasaccharide was taken to be the pD at which there was maximum binding. The binding constant was determined at this pD by measuring the chemical shift of the C3H proton as a function of the added peptide concentration at constant tetrasaccharide concentration. All chemical shifts were

corrected for the slight pD dependence of the chemical shift of the TMSP resonance using the equation δ = δobs − 0.019/(1 + 105.0 − pD)

(1)

Binding constants were calculated by a nonlinear least-squares fit of the observed C3H chemical shift (δobs) as a function of the total peptide concentration to the equation δobs = ff δf + fc δc

(2)

where f f and fc are the fractional concentrations of the tetrasaccharide in the free and complexed forms, respectively, δf is the chemical shift of the C3H proton of the tetrasaccharide free in solution at the pD of maximum binding, and δc is the chemical shift of the C3H proton of the tetrasaccharide− peptide complex, which is unknown but calculated in the nonlinear least-squares fitting of the data. f f and fc are expressed as follows: f f = [T]/[Tt] and fc = 1 − [T]/[Tt], where [T] is the concentration of the tetrasaccharide in the free form and [Tt] the total concentration of tetrasaccharide. [Tt] is expressed as follows: [Tt] = [T] + ((KB[T][HAt])/(1 + KB[T])). The parameters KB and δc were determined by nonlinear leastsquares fitting of the chemical shift data to eq 2. Heparin Affinity Chromatography (HAC). HAC experiments were performed on a Dionex 500 HPLC system using two HiTrap heparin HP columns (2.5 × 0.7 cm i.d., 1 mL column volume) in series. Peptides were eluted with a linear gradient, starting with 100% mobile phase A (20 mM sodium phosphate buffer, pH 5.6) and adding mobile phase B (20 mM sodium phosphate buffer, pH 5.6, with 1.0 M NaCl) at 0.5%/ min at a flow rate of 0.6 mL/min. Peptide solutions were prepared in mobile phase A. Isothermal Titration Calorimetry (ITC). ITC experiments were conducted at 25 °C on a Microcal VP-ITC MicroCalorimeter. Oligosaccharide and peptide solutions were prepared in 20 mM pH 5.6 sodium acetate buffer at 0−50 mM NaCl. The peptide solution was placed in the sample cell, and the oligosaccharide titrant solution was loaded into the syringe injector. Titrant was added in 5 μL increments, with a 240 s delay between injections. Heat of reaction was measured after each addition of titrant. Calorimetric titration data were fit to give the reaction stoichiometry (N), the binding constant (KB), and the binding enthalpy (ΔH), using the Origin 5.0 nonlinear least-squares program supplied with the Microcal VPITC. ΔS was calculated from KB and ΔH. The reported values for KB are the average of triplicate measurements. Titration data were corrected for the heat of dilution using a blank titration of titrant into buffer solution. NMR Measurements. One- and two-dimensional 1H NMR spectra were measured on a 500 MHz Varian Unity Inova spectrometer at a temperature of 25 °C. Samples were in D2O solution. Chemical shifts are reported relative to the methyl resonance of TMSP at 0.000 ppm. The residual HOD resonance was suppressed in most experiments using a selective saturation pulse applied during the relaxation delay. Twodimensional TOCSY, ROESY, BASHD-TOCSY, and BASHDROESY spectra were measured with standard pulse sequences. TOCSY and ROESY spectra were acquired using a spectral width of 5 kHz in both dimensions, 8K or 16K data points were collected in the t2 dimension, and 128−512 increments were used in the t1 dimension, depending on the necessary digital resolution. 64 transients were coadded at each t1, and 64 dummy scans were run prior to acquisition. A mixing time of C

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The Journal of Physical Chemistry B

model peptide that has the sequence of the second histidinecontaining region of Aβ peptides (Aβ(4−10)). The tetrasaccharide was prepared by heparinase depolymerization of heparin, which introduces the 4,5-unsaturated uronic acid (ΔUA) residue at the nonreducing end. The tetrasaccharide is fully sulfated, with three sulfate groups per disaccharide unit. Characterization of Tetrasaccharide-Aβ(12−18) Binding by NMR. The interaction of Aβ peptides with heparin and HS is pH-dependent, with binding taking place in the pH range where the histidyl side chains are protonated.14−16 To characterize the binding of Aβ(12−18) by the tetrasaccharide in Figure 1, 1H NMR spectra were measured as a function of pD for the tetrasaccharide free in solution and in solution with the Aβ(12−18) peptide, the two mutants of Aβ(12−18), and the other peptides and ligands in Table 1. The chemical shift of the C3H proton of the internal GlcNS(6S) residue is plotted as a function of pD in Figure 2 for the tetrasaccharide (A) free in

120−300 ms was used for TOCSY and ROESY experiments. Shifted sine bell and Gaussian apodization were applied in the F1 and F2 dimensions, respectively. BASHD-TOCSY and BASHD-ROESY spectra were measured with the same parameters, with the exception that the spectral width in the F1 dimension was less, as needed to cover a specific band of resonances. NMR samples were prepared by dissolving peptide and tetrasaccharide in 100% D2O at various pD values and varying ratios of peptide to tetrasaccharide in 20 mM NaCl. The sample volume was 320 μL with TMSP added as a chemical shift reference. Solution pD was measured directly in the NMR tube using a Micro-Combination pH microelectrode and adjusted to the desired pD by adding 0.1 M DCl and 0.1 M NaOD solutions. pD values were obtained by using the equation pD = pHreading + 0.4 to correct for the deuterium isotope effect.49 5 mm Shigemi NMR tubes were used for all NMR measurements to reduce the sample volume to 320 μL and to improve suppression of the residual HOD resonance. Calculation of the Concentration of Aβ(12−18) Bound by Heparin. The concentration of Aβ(12−18) bound by heparin was calculated as a function of pH by accounting for the multiple, competing heparin-Aβ(12−18) binding equilibria and the acid dissociation equilbria for the heparin carboxylic acid groups and the imidazolium groups of Aβ(12−18). The calculations included the following heparin-Aβ(12−18) binding reactions: Hep + H2P ⇆ Hep−H2P; Hep + HAP ⇆ Hep−HAP; and Hep + HBP ⇆ Hep−HBP, where Hep represents the carboxylate form of heparin, H2P represents Aβ(12−18) with both imidazole side chains and the lysine side chain protonated, and HAP and HBP represent Aβ(12−18) with the imidazole side chains of His-13 and His-14, respectively, protonated and the lysine side chain protonated. The calculations also included the following acid/base equilibria: HHep ⇆ Hep + H+; H2P ⇆ HAP + H+; H2P ⇆ HBP + H+; HAP ⇆ P + H+; and HBP ⇆ P + H+, where HHep represents the carboxylic acid form of heparin. Equilibrium constant expressions for the multiple equilibria were combined to solve for the concentrations of Hep−H2P, Hep−HAP, and Hep−HBP as a function of pH.

Figure 2. pD dependence of the chemical shift of the C3H proton of the internal GlcNS(6S) residue of the tetrasaccharide in solutions containing (A) only the tetrasaccharide and (B) 0.505 mM tetrasaccharide and 4.74 mM Aβ(12−18). Both solutions contained ∼20 mM Na+.

solution and (B) in solution with Aβ(12−18). Exchangeaveraged resonances were observed for peptide and tetrasaccharide in B, indicating fast exchange of both between their free and bound forms on the NMR time scale. The pD dependence of the chemical shift of the C3H proton of free tetrasaccharide over the pD range 3−6 reflects titration of the carboxylic acid groups of the ΔUA(2S) and IdoA(2S) residues. pKA(D2O) values of 4.17 ± 0.02 and 4.69 ± 0.02 were obtained by fitting the chemical shift-pD data to a diprotic acid model.50 pKA(D2O) values of 4.17 ± 0.01 and 4.66 ± 0.01 were also determined for the carboxylic acid groups of the ΔUA(2S) and IdoA(2S) residues, respectively, by fitting chemical shift− pD titration curves for the C4H proton of the ΔUA residue and C5H proton of the IdoA(2S) residue to a monoprotic acid model.50 pKA(H2O) values of 3.02 and 3.44 have been reported previously for the carboxylic acid groups of the tetrasacharide.51 The pKA values were determined in D2O solution by 13C NMR, at a tetrasaccharide concentration of 0.081 M, and were converted to pKA(H2O) values. When the 13C values are converted back to pKA(D2O) values and the higher ionic strength of the solutions used in the 13C measurements (∼1.30 M vs ∼0.040 M) are accounted for, the values determined by 13 C NMR and those determined by 1H NMR are in good agreement. The large upfield shift of the resonance for the C3H proton in the presence of the Aβ(12−18) peptide indicates binding of the peptide by the tetrasaccharide. The pD dependence of the

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RESULTS The goal of this research was to characterize the thermodynamic and mechanistic aspects of the interaction of the Aβ peptide consensus sequence VHHQKL with heparin. In previous research on the binding of histamine by heparin, the imidazolium ring of histamine in the diprotonated form was shown by 1H NMR to interact site-specifically with a cleft formed by an IdoA(2S)−GlcNS(6S)−IdoA(2S) triad sequence, as evidenced by the change in chemical shift of the resonance for the C3H proton of the GlcNS(6S) residue.46,47 It was found by molecular modeling that with the imidazolium ring in the binding cleft the C3H proton is in the shielding cone of the imidazolium ring causing an upfield shift of its resonance.46 The carboxylate group of the IdoA(2S) residue at the reducing end of the triad and the sulfamido group of the GlcNS(6S) residue are essential for site-specific binding.46 In the first part of the research, we used this 1H NMR signature of site-specific binding of the imidazolium ring to investigate the nature of the interaction of the Aβ(12−18) model peptide, and the other ligands listed in Table 1, with the heparin-derived tetrasaccharide shown in Figure 1. Peptides 2 and 3 are mutants of Aβ(12−18) in which His-14 and His-13, respectively, were substituted by alanine, while peptide 4 is a D

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The Journal of Physical Chemistry B chemical shift is identical to that observed in our study of the binding of histamine by heparin and heparin-derived oligosaccharides.46 The resonance shifts upfield as the pD is increased from 3, reaching a maximum upfield shift at pD ∼ 5.8. This is the pD range over which the carboxylic acid group of the IdoA(2S) is titrated, consistent with participation of this carboxylate group of the ΔUA(2S)−GlcNS(6S)−IdoA(2S) triad in binding to the imidazolium ring.46 As the pD is increased beyond pD ∼ 5.8, the resonance shifts back downfield, reaching the chemical shift of the free tetrasaccharide at pD ∼ 8. Over this pD range, the imidazolium groups are titrated (pKA ∼ 6.7), indicating the histidyl side chains in the imidazolium form interact site-specifically with the tetrasaccharide. Similar chemical shift−pD titration curves were observed for the C3H proton of the internal GlcNS(6S) residue of the tetrasaccharide in solution with each of the histidine-containing peptides and other ligands listed in Table 1, in each case reaching a maximum upfield displacement in the pD 5−6 range. However, the magnitude of the upfield displacement of the C3H resonance was different for each of the peptides, indicating different degrees of tetrasaccharide−ligand binding. Binding constants were determined by measuring the chemical shift of the C3H resonance as a function of the ligand concentration at constant tetrasaccharide concentration, at the pD of maximum upfield displacement of the C3H resonance. Chemical shift data used for determination of the tetrasaccharide-Aβ(12−18) binding constant are presented in Figure 3. A binding constant of 4250 ± 120 was obtained by

Figure 4. Binding isotherm for the interaction of Aβ(12−18) with 12 kDa heparin. Aβ(12−18) (0.404 mM in the cell) was titrated with 0.600 mM heparin (in the syringe) in 20 mM sodium acetate buffer and 25 °C.

frame represent the heat released after each injection of heparin. The bottom frame shows the integrated heat released, after correction of the titration data in the top frame for heat of dilution. The line through the points represents the nonlinear least-squares fit of the data to obtain the binding constant, ΔH, and the number of Aβ(12−18) ligands binding per heparin molecule. The results are reported in Table 2. Also reported in Table 2 are binding parameters determined for binding of the listed ligands by heparin. The binding constants for ligands 7−9 in Table 1 are too small to be determined by ITC. Aβ peptides bind to the heparin-like NS domains of HS.20 To investigate the dependence of Aβ peptide binding on size of the heparin-like domain, binding constants were also determined by ITC for the interaction of Aβ(12−18) with smaller heparin-derived oligosaccharides. The results are reported in Table 3. The results indicate a significant entropic contribution to the binding free energy for each heparin oligomer. As discussed below, the entropic contribution is due, in part at least, to the release of Na+ counterions from the heparin oligomers upon binding of positively charged Aβ(12− 18). To investigate this further, binding constants were measured by ITC for the interaction of Aβ(12−18) with heparin as a function of the Na+ ion concentration. The results are reported in Table 4. There is a significant decrease in the binding constant as the sodium ion concentration increases. This is discussed in terms of polyelectrolyte theory in the Discussion section. Characterization of Heparin-Aβ(12−18) Binding by Heparin Affinity Chromatography. Relative binding strengths were also determined by HAC. The Aβ(12−18) peptide and the other ligands listed in Table 1 were injected separately onto heparin affinity columns, as described in the Experimental Section. Ligands were eluted using a linear gradient of low-salt to high-salt buffers at pH 5.6. Initially, the

Figure 3. Chemical shift of the C3H proton of the internal GlcNS(6S) residue of the tetrasaccharide as a function of the concentration of Aβ(12−18) at pD 5.99 ± 0.05. The concentration of tetrasaccharide was held constant at 0.149 mM. Nonlinear least-squares fit of the data yielded a binding constant of 4250 ± 120. [Na+] = ∼20 mM.

fitting the data by the method described in the Experimental Section. Tetrasaccharide−ligand binding constants determined using C3H chemical shift data are reported in Table 1. Determination of the Heparin-Aβ(12−18) Binding Constant by ITC. To better understand the mechanism of interaction of Aβ peptides with HS, binding constants and other thermodynamic parameters were determined by ITC for the interaction of Aβ(12−18) and several ligands from Table 1 with heparin and heparin-derived oligosaccharides. The ITC method for determining binding constants is based on measurement of heat of reaction during the titration of one binding partner with the other binding partner. Binding constants were determined by titration of Aβ(12−18) and other ligands with heparin at pH 5.63. ITC titration data for determination of the binding constant of Aβ(12−18) with heparin are shown in Figure 4. The negative peaks in the upper E

DOI: 10.1021/acs.jpcb.5b12235 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B Table 2. Binding Constants and Thermodynamic Parameters for the Binding of Selected Ligands by Heparina,b ligand

binding constant KB (M−1)

Ac-VHHQKLV-NH2 Ac-VHAQKLV-NH2 Ac-VAHQKLV-NH2 FRHDSGYd GHK GHG histamine

15200 584c 487c 1210 4620 368 5880

N

ΔH (cal/mol)

ΔS (cal/(mol °C))

± 300

21.8 ± 0.1

−2860 ± 10

9.6 ± 0.2

± ± ± ±

16.8 18.7 15.2 13.7

60 150 19 100

± ± ± ±

−2830 −1910 −1290 −3430

0.6 0.2 0.6 0.1

± ± ± ±

10 8 5 9

4.6 10.4 7.4 5.7

± ± ± ±

0.1 0.6 0.3 0.1

12 kDa heparin. bDetermined by ITC at pH 5.63 in 20 mM sodium acetate buffer and 25 °C, except as noted. cEstimated from HAC retention time and the linear relationship between log KB and retention time. dKB, N, ΔH, and ΔS were determined to be 2700 ± 130, 15.0 ± 0.4, −1960 ± 10, and 9.1 ± 0.1, respectively, at pH 4.65. a

Table 3. Binding Constants and Thermodynamic Parameters for the Binding of Aβ(12−18) by Heparin and Heparin-Derived Oligosaccharidesa KB (M−1)

heparin oligosaccharide heparin sodium salt, 12 000 Da low molecular weight heparin, 6000 Da low molecular weight heparin, 3000 Da tetrasaccharide, 1332 Da a

15200 12000 10200 3650

± ± ± ±

ΔH (cal/mol)

N

300 190 260 270

21.8 10.7 4.8 1.9

± ± ± ±

−2860 −2960 −2790 −3240

0.1 0.04 0.03 0.1

± ± ± ±

10 8 11 30

ΔS (cal/(mol °C)) 9.6 8.7 9.0 5.5

± ± ± ±

0.2 0.1 0.1 0.2

Determined by ITC at pH 5.63 in 20 mM sodium acetate buffer and 25 °C.

Table 4. Binding Constants and Thermodynamic Parameters for the Binding of Aβ(12−18) with Heparin as a Function of the Sodium Ion Concentrationa [Na+]total (M) 0.020 0.025 0.030 0.040 0.050 0.060 0.070 a

KB (M−1) 15200 11400 7400 5420 3580 2270 2040

± ± ± ± ± ± ±

300 150 90 50 50 30 44

ΔH (cal/mol)

N 21.8 19.3 17.7 16.4 16.4 17.1 14.4

± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.1 0.1 0.2

−2860 −3040 −3280 −3060 −2700 −2870 −2540

± ± ± ± ± ± ±

10 6 6 5 5 5 6

ΔS (cal/(mol °C)) 9.6 8.4 6.7 6.8 7.2 5.7 6.6

± ± ± ± ± ± ±

0.2 0.0 0.1 0.1 0.1 0.2 0.1

Determined by ITC at pH 5.63 in 20 mM sodium acetate buffer plus added NaCl at 25 °C.

C3H proton of the central GlcNS(6S) residue reaches a maximum, and then the resonance shifts back to the chemical shift of the free tetrasaccharide as the imidazolium side chains of His-13 and His-14 are titrated. These results indicate an electrostatic component to the binding of Aβ peptides, as both the negatively charged carboxylate groups of heparin and the positively charged histidyl side chains of Aβ(12−18) are essential for binding. The upfield displacement of the chemical shift of the C3H resonance of the internal GlcNS(6S) residue indicates site-specific binding of at least one of the imidazolium groups of Aβ(12−18) by the tetrasaccharide.46,47 Based on the NMR and molecular modeling results for binding of diprotonated histamine by heparin, the carboxylate group of the IdoA(2S) and the sulfamido group of the internal GlcNS(6S) residue are essential for the site-specific binding of the imidazolium groups of Aβ(12−18) by the tetrasaccharide.46 In addition, the 2-O-sulfate group of IdoA(2S) and the 6O-sulfate group of GlcNS(6S) contribute to the binding affinity through the polyelectrolyte effect. Heparin is a polyelectrolyte, which is fundamental to its interaction with peptides.52 In the counterion condensation theory (CCT) of polyelectrolytes, heparin is modeled as a linear chain of anionic sites.53−55 The density of its anionic sites is so high that its negative charge density is partially neutralized by counterions. In CCT, above a critical charge density, sufficient counterions condense around heparin to reduce its

ligands bind to the heparin affinity column when the mobile phase is the low-salt buffer. As the salt concentration is increased, the ligand is released from the heparin-affinity column by ion exchange; the higher the concentration of salt at which a peptide elutes, i.e., the longer the retention time, the tighter the heparin−ligand binding. The retention times for Aβ(12−18) and the other ligands are reported in Table 1. The relationships between the retention times (RT) in Table 1 and binding constants in Tables 1 and 2 are linear: log KB = 0.051(RT) + 1.45 and log KB = 0.066(RT) + 1.46. The binding constants listed in Tables 1 and 2 for peptides 2 and 3 were estimated using these relationships and their retention times.

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DISCUSSION The results in Figure 2 show that the binding of Aβ(12−18) by the tetrasaccharide is strongly pD-dependent, with some binding at low pD, maximum binding at pD ∼ 6, and no binding at pD > 8, consistent with the pH dependence reported for the binding of Aβ peptides by heparin.13−15 As the pD is increased from low pD, the two carboxylic acid groups of the tetrasaccharide are titrated with pKA(D2O) values of 4.17 and 4.69 to give the negatively charged carboxylate groups that were found to be essential for both the binding of Aβ peptides by HS and the site-specific binding of the imidazolium group of histamine by heparin.20,46 As the pD is increased further, displacement of the chemical shift of the resonance for the F

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The Journal of Physical Chemistry B net charge density to a critical threshold value.54,56 CCT predicts that at this threshold value the fraction of a monovalent counterion condensed per anionic charge on heparin is 0.59,57 independent of salt concentration in the bulk solution; experimental values of 0.58 and 0.63 have been reported.58,59 Central to the CCT model of the binding of cationic peptides by heparin is the release into bulk solution of a number of condensed Na+ counterions equal to the number of cationic groups of the peptide that penetrate the counterion condensation layer, with the result that the net linear charge density per anionic charge remains invariant at the threshold value.52,56,60 When the concentration of Na+ in the bulk solution is less than its concentration in the counterion condensation layer, calculated to be 0.38 M for heparin by CCT, release of Na+ ions to bulk solution provides an entropic contribution to the free energy of the binding reaction.56 The negative of the slope of a plot of log KB vs log[Na+] is equal to the number of Na+ ions released per peptide bound.53 The negative of the slope of such a plot for the data in Table 4 is 1.68. In an extension of the formulation for the dependence of KB on bulk salt concentration, the slope of a log KB vs log[Na+] plot is equal to −ZΨ, where Z is the number of ionic interactions the peptide makes with heparin and Ψ is the apparent fraction of a Na+ ion condensed per anionic charge, reported to be 0.8 for heparin, which includes the fraction of a Na+ ion condensed per anionic site (0.59) and the screening effect of condensed Na+ ions on the interaction of residual heparin anionic charges.60−62 According to this treatment of the experimental data in Table 4, the cationic sites on Aβ(12−18) make on average two charge contacts with anionic sites on heparin, presumably a carboxylate group of an IdoA(2S) residue and a sulfamido group of a GlcNS(6S) residue, as discussed above. The thermodynamic parameters reported in Table 3 provide additional information about the nature of the interaction of Aβ(12−18) with heparin oligosaccharides. The 12 kDa, 6 kDa, 3 kDa, and 1332 Da heparin oligosaccharides are composed of approximately 19, 10, 5, and 2 repeating disaccharides, respectively. The number of Aβ(12−18) peptides bound per oligosaccharide (N) is essentially equal to the number of dissacharides in each of the oligosaccharides, suggesting a binding stoichiometry of one Aβ(12−18) peptide per disaccharide segment. The results in Table 3 also indicate a strong dependence of the binding constant on the size of the heparin oligosaccharide, decreasing as the size of the oligosaccharide decreases. This also is the case for the heparin binding constants reported in Table 2 as compared to the tetrasaccharide binding constants reported in Table 1 for the same ligands. The dependence of magnitude of the binding constant on size of the oligosaccharide can be accounted for in terms of CCT. In the CCT theory of ligand binding by a polyelectrolyte, the release of Na+ ions from the counterion condensation volume provides a driving force for the binding reaction through an entropic contribution to the binding free energy when the Na + concentration in the counterion condensation volume is greater than its concentration in the bulk solution. However, the concentration of condensed Na+ ions is predicted by CCT to be less at each end of a heparin oligosaccharide, and thus the extent of binding of Aβ(12−18) and the entropic contribution

to the binding free energy is predicted to be less at the two end segments.54,55 The equilibrium charge fraction f can be predicted as a function of the distance s from the end of the oligosaccharide with the equation f (s) = (1/2zξ)(1 − (ln κb)/(ln s /b))

(3)

where b, the linear distance between charge sites, is 0.254 nm for heparin, ξ is the bare charge density (ξ = lB/b where lB, the Bjerrum length, is 0.71 nm), z is the valence of the counterion, κ−1 (nm) is the Debye screening length (= 0.304I−1/2), and s is the distance from the end of the oligosaccharide.54 The fractional charge neutralization as a function of distance along the oligosaccharide is 1 − f(s). The calculated effective charge fraction per site is plotted in Figure 5 for the four oligosaccharides in Table 3.

Figure 5. Calculated effective charge fraction along the length of (a) the tetrasaccharide and (b, c, and d) the 3, 6, and 12 kDa heparin oligosaccharides, respectively. The effective charge fraction is shown for only half the 12 kDa oligosaccharide; the effective charge fraction for the other half is the mirror image of that shown.

The effective charge fraction, and thus the extent of Na+ condensation, is strongly dependent on position along the length of the oligosaccharide, particularly along the end segments. For the 12 and 6 kDa oligosaccharides, the effective charge fraction decreases up to site 12 and then remains essentially constant up to site P − 12, where P is the total number of anionic sites, beyond which it then mirrors the site number dependence at the other end. Twelve anionic sites corresponds to three repeating disaccharides. Thus, the number of Na+ ions condensed onto a three-disaccharide segment at each end of the 12 and 6 kDa oligosaccharides is significantly less than onto their interior disaccharides. The end segments represent 32 and 60% of the total sites on the 12 and 6 kDa oligosaccharides. The 3 kDa oligosaccharide and the tetrasaccharide are both shorter than two end segments, and thus Na+ condensation onto these two oligosaccharides is less than onto the two larger oligosaccharides, particularly for the tetrasaccharide. Averaged over all sites, the fractional condensation of Na+ ions per charge site is calculated to be 0.62, 0.56, 0.46, and 0.23 for the 12, 6, and 3 kDa oligosaccharides and the tetrasaccharide, respectively. (Note the agreement of the 12 kDa value with the values of 0.58 and 0.63 determined experimentally for heparin.58,59) As the concentration of condensed Na+ decreases, the binding reaction will shift to the left and the binding constant will decrease. Thus, the measured binding constants are predicted to decrease as the end segments represent an increasing fraction of the total oligosaccharide, and the average fractional condensation G

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The Journal of Physical Chemistry B

The pH dependence of the binding of Aβ(12−18) (Figure 2) indicates the protonated histidine side chains are critical for binding of HHQK by the tetrasaccharide. This pH dependence raises questions about the physiological relevance of the binding. In considering this, first it should be noted that the local pH might be less than neutral in the AD brain.63 It has been reported that the pH in the frontal cortex and caudate nucleus of post-mortem brains from cases of Alzheimer’s disease was 6.66 ± 0.05 and 6.56 ± 0.05, respectively. Also, acidosis in the brain, where the pH value is less than 6.6, has been correlated with Alzheimer’s disease.64,65 Second, using the pKA(D2O) values of 6.6 and 6.5 reported for His-13 and His14, respectively, of Aβ(1−28),66 some 29% and 24% of the two histidine side chains of Aβ peptides are predicted to be in the critical protonated imidazolium form at neutral pD. This is consistent with the chemical shift data in Figure 2 which indicate that a significant fraction of Aβ(12−18) interacts with the tetrasaccharide at neutral pH. Using the binding constants in Table 2, we can estimate the extent of binding of Aβ(12−18) by heparin at neutral pH. The binding constant for Aβ(12−18) in Table 2 is in terms of the total concentrations of the various free heparin, free peptide and complexed species present at pH 5.63. Using this “conditional” binding constant, the pKAs for His-13 and His14 of Aβ(1−28),66 and a pKA(D2O) of 5.26 for the carboxylic acid groups of heparin in 0.025 M Na+ solution,57 a value of 2.7 × 104 is estimated for a pH-independent binding constant in terms of the concentration of carboxyl deprotonated heparin and the Aβ(12−18) species with both histidine side chains and the lysine side chain protonated. In a similar way, pHindependent values of 910 and 750 are estimated for the heparin binding constants of ligands 2 and 3, respectively, in terms of the species with their respective histidine side chains protonated. Using these binding constants, the pKAs for the two histidine side chains of Aβ(1−28), and the pKA for the carboxylic acid groups of heparin, the fraction of Aβ(12−18) complexed by heparin in a solution containing 1 mM Aβ(12− 18) and 1 mM heparin was calculated as a function of pH. The results are plotted in Figure 6. The pH dependence of the extent of binding parallels that shown qualitatively by the chemical shift data in Figure 2 for the binding of Aβ(12−18) by the tetrasaccharide. Of particular significance is the finding that

decreases, as is found experimentally for the binding constants reported in Table 3. Also, the entropic contribution to the binding free energy decreases as the size of the oligosaccharide decreases, in agreement with the decreasing calculated fractional condensation of Na+ per charge site as the size decreases. Although the smaller heparin-derived oligosaccharides in Table 3 are models for the heparin-like NS domains of HS, it should be noted that binding to similar sized NS domains in HS is likely stronger than reported in Table 3. As discussed above, the extent of binding is strongly influenced by the reduced Na+ condensation at the two end segments in each oligosaccharide. However, the extent of Na+ condensation is predicted to be higher at the end segments of a NS domain of HS because it will be flanked at each end by charged NS/NA transition domains. As a result, the Na+ condensation on each side of a NS|NS/NA junction interface is predicted to be greater than on the end segments of an isolated heparin-derived oligosaccharide, as shown by the calculations by Manning of charge densities on each side of a junction interface between segments of single-strand and double-strand DNA.54 The calculations show that the charge density on each side of the interface quickly approaches that of the internal segments. Binding constants for the binding of Aβ(12−18) by the tetrasaccharide and by heparin in Tables 1 and 2 and the HAC retention time for Aβ(12−18) in Table 1 are significantly larger than for the other ligands, including those for ligands 2 and 3 in which His-14 and His-13, respectively, are replaced by alanine. This is as predicted since Aβ(12−18) has the sequence of the XBBXBX consensus heparin-binding motif.25 However, the number of Na+ ions released upon binding of Aβ(12−18) by heparin, as indicated by the slope of the log KB vs log[Na+] plot, suggests that not all three cationic sites of Aβ(12−18) penetrate the counterion condensation layer. If so, the large difference between the binding constants in Table 1 for Aβ(12−18) and ligands 2 and 3 in which His-14 and His-13, respectively, have been replaced by alanine suggests it is the two histidine imidazolium groups of Aβ(12−18) that penetrate the counterion condensation layer and not the ammonium group of Lys-16. Indeed, although ligands 2 and 3 have net charges of +2 at the pH at which their binding constants are estimated, the magnitude of their binding constants are of similar magnitude to those ligands in Table 1 that have a net charge of +1 at pH ∼ 6. After Aβ(12−18), the ligand with the next largest binding constant is histamine; the strength of the histamine−heparin binding is perhaps not surprising since nature stores histamine bound to heparin in mast cells.45 The growth hormone GHK, with one imidazolium group and two ammonium groups, has a relatively large binding constant with both the tetrasaccharide and heparin. However, the binding constants are much smaller for the other ligands that contain a single histidine, including ligand 4, the model peptide for the second histidine-containing domain of Aβ peptides, consistent with previous reports that the Arg-5 and His-6 residues of Aβ peptides are not critical for HSPG binding and the conformational transition to β-sheet structure.14 While ligand 4 also contains an Arg residue that can bind heparin through its positively charged guanidinium group, it also contains the negatively charged carboxylate side chain of the Asp residue, which likely decreases the strength of the binding as evidenced by the significantly larger binding constant at pH 4.65 (Table 2) when the aspartic acid carboxlylate group is largely protonated.

Figure 6. Calculated percent Aβ(12−18) bound to heparin as a function of pH in a solution containing 1 mM Aβ(12−18) and 1 mM heparin. H

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for the concentrations used in the calculations some 58% of the Aβ(12−18) is bound to heparin at neutral pH. The extent of binding will be different for different concentrations, increasing as the ratio of the concentration of heparin to that of Aβ(12− 18) increases. These results indicate that even though the histidine side chains of free Aβ(12−18) are largely neutral at neutral pH, binding of the protonated imidazole side chains by heparin is sufficiently strong to compete with acid dissociation of the free imidazolium groups, thus shifting the apparent acid dissociation equilibrium in favor of protonation of the histidine side chains. For these conditions, some 45% and 10% of Aβ(12−18) are bound to heparin at pH 4 and 8, respectively, in qualitative agreement with the pH dependence of the binding of Aβ peptides observed by heparin affinity chromatography.14−16 The results reported here indicate a strong dependence of the extent of binding of Aβ(12−18) by heparin on the Na+ concentration due to the polyelectrolyte effect. The apparent acidity of the carboxylic acid groups of heparin also depends strongly on the Na+ concentration due to heparin being a polyelectrolyte, as discussed previously.57 Specifically, the apparent pKA decreases as the Na+ concentration increases. For example, pKA(D2O) decreases from 5.71 to 4.45 as the Na+ is increased from 0.011 to 0.20 M.57 A pKA(H2O) = 3.13 has also been reported for the IdoA(2S) carboxylic acid groups of heparin.51 This value was determined by 13C NMR at a significantly higher heparin concentration and thus a significantly higher Na+ concentration. When the higher Na+ concentration is accounted for, the lower pKA determined by 13 C NMR is as predicted by the counterion condensation theory of polyelectrolytes.

Article

AUTHOR INFORMATION

Corresponding Author

*(D.L.R.) E-mail [email protected]; Tel (951) 8273585; Fax (951) 827-2435. Present Address

K.N.: Robinson Pharma, Inc. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the University of California, Riverside. Funding for the Varian Inova 500 spectrometer was provided in part by NSF-ARI Grant 9601831.

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REFERENCES

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CONCLUSIONS In this work, the mechanism of interaction of histidinecontaining peptides with heparin was characterized, with a focus on a model peptide, Aβ(12−18), for the heparin and HS binding domain of Aβ peptides. Binding constants were determined for the binding of Aβ(12−18), mutants of Aβ(12−18), and other histidine-containing peptides by the tetrasaccharide ΔUA(2S)−GlcNS(6S)−IdoA(2S)−GlcNS(6S) and by 12, 6, and 3 kDa heparin oligosaccharides. The interaction of the histidine side-chain imidazolium groups of each of the peptides studied with heparin oligosaccharides is site-specific, as indicated by the upfield shift induced by the imidazolium ring current in the resonance for the C3H proton of the central GlcNS(6S) residue of the tetrasaccharide. The extent of binding of Aβ(12−18) is strongly dependent on: the size of the oligosaccharide, decreasing as the size decreases; pH, with maximum binding around pH 5−6, where the heparin carboxylic acid groups are ionized and the histidine side-chain imidazole groups are protonated; and the concentration of Na+ in bulk solution, decreasing as the concentration of Na+ increases. The extent of binding is also dependent on peptide sequence, with maximum binding for the Aβ(12−18) peptide, which has the heparin-binding XBBXBX consensus amino acid sequence. The pH dependence indicates binding is mediated by electrostatic interactions. The dependence of the extent of binding on size of the heparin oligosaccharide and concentration of Na+ was shown to be consistent with the CCT of the binding of cationic ligands by anionic polyelectrolytes. The structure−binding relationships characterized in this work should provide useful insight for the design of oligosaccharide therapeutic agents for Alzheimer’s disease.4 I

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