Aggregation Properties of the Peptide Fragments Derived from the 17

Dec 29, 2009 - A. Doria 6, 95125 Catania, Italy ... region of human amylin (hIAPP17-29), was modified by grafting a hydrophilic PEG chain in order to ...
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J. Phys. Chem. B 2010, 114, 705–713

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Aggregation Properties of the Peptide Fragments Derived from the 17-29 Region of the Human and Rat IAPP: A Comparative Study with Two PEG-Conjugated Variants of the Human Sequence Antonino Mazzaglia,† Norberto Micali,‡ Luigi Monsu` Scolaro,§ Francesco Attanasio,| Antonio Magrı´,| Giuseppe Pappalardo,*,| and Valentina Villari*,‡ CNR-Istituto per lo Studio dei Materiali Nanostrutturati, c/o Dipartimento di Chimica Inorganica, Chimica Analitica e Chimica Fisica dell’UniVersita´ di Messina, S.ta Sperone 31, I-98166, Messina, Italy, CNR-Istituto per i Processi Chimico-Fisici, S.ta Sperone C.da Papardo, Faro Superiore, I-98158, Messina, Italy, Dipartimento di Chimica Inorganica, Chimica Analitica e Chimica Fisica dell’UniVersita´ di Messina, S.ta Sperone 31, I-98166 and C.I.R.C.M.S.B., Messina, Italy, and CNR-Istituto di Biostrutture e Bioimmagini, Viale A. Doria 6, 95125 Catania, Italy ReceiVed: September 1, 2009; ReVised Manuscript ReceiVed: NoVember 30, 2009

The amyloidogenic amino acid sequence Ac-VHSSNNFGAILSS-NH2, corresponding to the 17-29 peptide region of human amylin (hIAPP17-29), was modified by grafting a hydrophilic PEG chain in order to obtain a novel class of peptides to be used as models to study the aggregation process of the full-length IAPP. The amphiphilic feature of the pegylated peptide fragment at the N-terminus (PEG-N-hIAPP17-29) drives the aggregation process toward stable micellar clusters without fibrillogenesis, despite the presence of β-sheet interaction between peptides at pH values higher than 4.0. The hIAPP17-29-C-PEG, in which the PEG moiety is linked to the C-terminus, does not possess analogous amphiphilic character and the ability of PEG in forming H-bonds with the solvent overcomes that of the peptide chain, thereby causing peptide flocculation. The comparison with the unmodified hIAPP17-29 and the rat’s peptide sequence Ac-VRSSNNLGPGLPPNH2(rIAPP17-29) revealed the crucial role of hydrogen bonding between peptide and solvent in determining the aggregate structure and preventing fibril formation, as well as the non-negligible effect of a small amount of organic solvent in the aqueous solution which affects the aggregation process and rate. Introduction Amyloid formation is a pathological process underlying several devastating diseases including Alzheimer’s disease, prion-related diseases, familial amyloidoses, and type 2 diabetes.1-5 As a result of recent evidence suggesting that soluble low molecular weight (LMW) aggregates of amyloidogenic proteins are the primary cause of neurotoxicity,6 it has become urgent to understand the molecular mechanisms governing the stepwise process of amyloid formation including the early events leading to the formation of small oligomers, followed by assembly into soluble protofibrils first and insoluble fibrils afterward. It is also necessary to explore the nature of external conditions, including pH and solvents, that can drive conformational fluctuations in monomeric peptides, making them prone to aggregation. The islet amyloid polypeptide (IAPP, also called amylin) is the major component of the amyloid depositions in pancreatic islets commonly observed in type 2 diabetes patients.7,8 The physiological role of amylin is unknown,9 though its aggregation in vivo as β-pleated sheets and subsequent deposition in the islets of Lagerhans is likely to be an aberrant process and has been heavily implicated in the degeneration of β-cells.10,11 In particular, it has been shown that aggregates of human IAPP (hIAPP) can disrupt the integrity of the β-cell’s membrane, * To whom correspondence should be addressed. E-mail: [email protected] (G.P.); [email protected] (V.V.). † CNR-ISMN. ‡ CNR-IPCF. § UNIME. | CNR-IBB.

allowing entry of Ca2+ ions into the cell either through the formation of small pores or complete disruption of the membrane.12-15 For these reasons, much interest is currently devoted to the elucidation of the mechanisms underlying IAPP’s amyloid formation and particularly into the sequential and structural determinants governing its aggregation. To get insights into the mechanism of aggregation by structure-aggregation-activity studies might not only shed more light on the structural parameters, which play key roles in the process, but also lead to the discovery of compounds capable of interfering with fibrillogenesis. It is hopefully expected that these new compounds might have therapeutic potential in the treatment of the disease.16-19 Among the experimental approaches reported in the literature to study the biophysical properties of the full length hIAPP (whose primary sequence is composed by 37 amino acids: 1KCNTATCAT10QRLANFLVHS20SNNFGAILSS30TNVGSNTY), the use of shorter peptide sequences derived from fibrillogenic regions of the parent polypeptide was revealed to be a valid one.19-22 Such an approach is based on the use of peptide models that efficiently reproduce the conformational polymorphism displayed by the full length IAPP. According to this hypothesis, peptides corresponding to residues 8-20, 10-19, and 20-29, of human IAPP, have been identified as “hot spots” of amyloid formation.23 Some of us have recently proposed the hIAPP17-29 fragment as a useful peptide model able to recapitulate most of the structural features of full length hIAPP.24 This is because

10.1021/jp908436s  2010 American Chemical Society Published on Web 12/29/2009

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Figure 1. One letter code of the amino acid sequence of the hIAPP 17-29 (a) and rIAPP (b) peptides. Chargeable amino acids are indicated by an asterisk, and hydrophilic and hydrophobic residues, by superscribed and subscribed lines, respectively. Parts c and d show the schematic representation of PEG-N-hIAPP17-29 and hIAPP17-29-CPEG, respectively.

hIAPP17-29 encompasses the histidyl residue at position 18 whose protonation state has been shown to be important for human IAPP’s aggregation rate and fibril formation.25 However, the hIAPP17-29 peptide model was revealed also to be particularly prone to self-aggregation and this prevented a deeper characterization of the morphology of the aggregates in aqueous solution. Noteworthy, our previous studies carried out on metal complexes with amyloid peptides demonstrated that PEG conjugation can improve the solubility of the peptide chain without affecting its conformational properties.26 On the basis of this result, we expect that the covalent binding of a PEG moiety to the hIAPP17-29 would result in a more soluble peptide model which in turn may lead to an in-depth investigation of the molecular events associated with its aggregation process. In this paper, we report the synthesis and spectroscopic results of two novel PEG-conjugated peptides homologous to the 1729 amino acid sequence of the human IAPP. The PEG moiety has been covalently attached either to the N-terminus or C-terminus to afford two isomeric peptides that differ only for the position of the attached PEG. The conformational properties of the PEG peptides have been comparatively investigated by means of CD spectroscopy, whereas the morphology and size of the aggregates as well as the time dependent aggregation rate were determined by means of static and dynamic light scattering and fluorescence experiments, respectively. Furthermore, the results were compared with those obtained from the measurements carried out on the underivatized hIAPP17-29 and rIAPP17-29 (rat amylin) using the same experimental conditions. Experimental Section Materials. All N-Fmoc-protected amino acids and FmocNH-(PEG)11-COOH were purchased from Novabiochem (Switzerland); 2-(1-H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and N-hydroxybenzotriazole (HOBT) were from Inbios; N,N-diisopropylethylamine (DIEA), piperidine, triisopropylsilane (TIS), and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich; N,N-dimethylformamide (DMF) was obtained from Lab-Scan. 1,1,1,3,3,3-Hexafluoro2-propanol (HFIP) was purchased from Fluka and glycine from Carlo Erba. All other chemicals were of the highest available grade and were used without further purification. Synthesis and Purification of the Peptides. The peptides Ac-PEG11-VHSSNNFGAILSS-NH2 (PEG-N-hIAPP17-29) and Ac-VHSSNNFGAILSS-PEG11-NH2 (hIAPP17-29-C-PEG) (see Figure 1) were synthesized on a Pioneer (Applied Biosystem) peptide synthesizer using standard 9-fluorenylmethoxycarbonyl

Mazzaglia et al. (Fmoc) chemistry. The use of a 5-(4′-Fmoc-aminomethyl-3′,5dimethoxyphenol) valeric acid (PAL-PEG) resin (with a substititon level of 0.22 mEq/g) provided peptides with an amidated C-terminus. All amino acid residues and Fmoc-NH-(PEG)11COOH were introduced according to the TBTU/HOBT/DIEA activation method. A 4-fold amino acid excess was used for each coupling cycle, whereas N-terminal acetylation of the PEGconjugated peptides was performed by treating the fully assembled and protected peptide resins (after removal of the N-terminal Fmoc group) with a solution containing acetic anhydride (6% v/v) and DIEA (5% v/v) in DMF. Peptides were cleaved from the resin using a TFA/H2O/TIS (95%/2.5%/2.5%, v/v/v) mixture. The crude peptides were precipitated with cold diethyl ether and then lyophilized. Purification of the peptides was carried out by preparative reversed-phase high performance liquid chromatography (rp-HPLC) on a Varian Prepstar 200 Model SD-1 chromatography system equipped with a Prostar photodiode array detector with detection at 222 nm. The peptides were eluted with a binary solvent system (with solvent A ) 0.1% TFA in water and solvent B ) 0.1% TFA in acetonitrile) using a Vydac C18 250 × 22 mm (300 Å pore size, 10-15 µm particle size) column, at a flow rate of 10 mL/min. Analytical RP-HPLC analyses were performed on a Waters 1525 instrument equipped with a Waters 2996 photodiode array detector with detection at 222 nm. The peptide samples were analyzed using gradient elution with solvents A and B using a Vydac C18 250 × 4.6 mm (300 Å pore size, 5 µm particle size), run at a flow rate of 1 mL/min. PEG-N-hIAPP17-29. The peptide was purified according to the following protocol: from 0 to 5 min isocratic elution in 85% of A, then linear gradient from 15 to 40% B over 20 min, finally isocratic elution in 40% B from 25 to 30 min (Rt ) 17.40 min). The peptide fractions containing the desired product were collected and lyophilized. Peptide purity (higher than 95%) was checked by analytical rp-HPLC. Sample identity was confirmed by ESI-MS [Obsd m/z: (M + H)+ 1973.3; (M + 2H)2+ 987.3; Calcd for C86H145N19O33 ) 1972.02]. hIAPP17-29-C-PEG. The peptide was purified according to the following protocol: from 0 to 5 min isocratic elution in 80% of A, then linear gradient from 20 to 30% B over 20 min, finally isocratic elution in 30% B from 25 to 30 min (Rt ) 21.78 min). The peptide fractions containing the desired product were collected and lyophilized. Peptide purity (higher than 95%) was checked by analytical rp-HPLC. Sample identity was confirmed by ESI-MS [Obsd. m/z: (M + H)+ 1973.3; (M + 2H)2+ 987.4; Calcd for C86H145N19O33 ) 1972.02]. The unconjugated peptides hIAPP17-29 and rIAPP17-29 have been synthesized and purified as reported elsewhere.24 Sample Preparation. For the light scattering measurements, synthetic peptides hIAPP17-29 and rIAPP17-29 were dissolved in HFIP at a concentration of 3.6 × 10-3 M to prepare two stock solutions. These solutions were diluted in aqueous buffers at different pH to a concentration of 5.0 × 10-5 M. All of the buffers were prepared with glycine at 5.0 × 10-2 M in pure microfiltered (0.2 µm pore size) water, adjusting the pH at 4.0 with HCl and at 7.0 and 8.0 with NaOH, respectively. No filtration was carried out in the solutions in order to not affect sample concentration and size distribution. The dilution procedure was performed very carefully and slowly, taking care to act always under the same conditions, since aggregation is dramatically dependent also on the sample agitation. It was reported27 that, for example, the rate of amyloid formation of IAPP is faster in stirred samples than in unstirred ones. Moreover, it was shown that dissolving lyophilized

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β-amyloid peptide directly into buffer or diluting with buffer a concentrated stock solution prepared in an organic solvent affects the fibril concentration28 and that different organic solvents in the stock solution give rise to different aggregate morphologies.29 After gently injecting the amount of stock solution in the buffer, the cuvette was slowly turned upside down only once. This procedure ensured the reproducibility of the light scattering results. We believe that the reported dependence on sample agitation is due to the rate of evaporation of the organic solvent during the mixing between stock solution and buffer. With the used mixing procedure, the aggregation was slow enough to be monitored by light scattering (at least in most solutions). The PEG variants PEG-N-hIAPP17-29 and hIAPP17-29-CPEG were studied at the same concentration and pH values as the unmodified peptides. Solutions have also been prepared by dissolving PEG with a molecular weight of 600 Da (whose polymerization degree is close to 11, that used in the PEG-conjugated variants) at 4.0 × 10-5, 4.0 × 10-4, and 2.0 × 10-3 M, respectively, in buffer at pH 4.0 and by adding hIAPP17-29 (5.0 × 10-5 M), according to the mixing procedure described above. For the circular dichroism measurements, the stock solution was diluted in water to obtain a final peptide concentration of 1.0 × 10-5 M and the pH range investigated was between 4.0 and 10.0. The sample for thioflavine-T (Th-T) assay was obtained from identical stock solutions of the hIAPP17-29, PEG-N-hIAPP1729, and hIAPP17-29-C-PEG, prepared by dissolving 5 mg/mL of the peptide fragments in neat HFIP. Experiments were performed by diluting 0.50 µL of peptide stock into 2.25 mL of 5 × 10-2 M citrate/phosphate buffer, pH 8.0 or pH 4.0 containing Th-T. Final solutions resulted 8.8 × 10-5 M in Th-T content and contained 5.1 × 10-5 M peptide in 2% HFIP. Buffer solutions were filtered with a 0.2 µm filter. Method Light Scattering Measurements. Light scattering experiments were performed by using a Nd:YAG laser source (λ ) 532 nm) at a power of 100 mW, linearly polarized orthogonally to the scattering plane, and a homemade computer controlled goniometric apparatus which collected the scattered light in a pseudo-cross-correlation mode30 (through two cooled R943-02 photomultipliers at the same scattering angle). The temperature was controlled by a homemade water-circulating apparatus at 25 ( 0.01 °C. The scattered light, collected in a self-beating mode, was analyzed by a MALVERN 4700 correlator to build up the normalized intensity autocorrelation function:31,32

g2(Q, t) )

〈I(Q, 0)I(Q, t)〉 〈I(Q)〉2

(1)

where Q is the exchanged wavevector whose absolute value is |Q| ) (4πn/λ) sin(θ/2) (θ being the scattering angle, n the refractive index of the solution, and λ the wavelength of light in vacuum). For scattered electric fields obeying Gaussian statistics, the Siegert’s relation holds:

g2(Q, t) ) 1 + a|g1(Q, t)| 2

(2)

where a is a constant depending on the experimental setup and 0)Es(Q, t)〉/〈I(Q)〉 is the normalized scattered g1(Q, t) ) 〈E*(Q, s electric field autocorrelation function. For diffusing monodisperse spherical scatterers with radius R, the normalized scattered electric field autocorrelation function takes a simple exponential form, g1(Q, t) ) exp(-Γt). Under the condition QR , 1, Γ is related to the collective diffusion coefficient, D, by the relation Γ ) DQ2. However, if the diffusing spherical scatterer is rigid, the latter relation is fulfilled also for QR > 1. For large particles, correlation functions can contain other information regarding the coupling between translation and rotational motions, so that the relation Γ ) DQ2 is not valid even for dilute solutions. The role of this coupling into Γ is taken into account by an additional coupling parameter,33-35 γ ) Q2(D| - D⊥)/Θ, where D| and D⊥ are the sideways and lengthways translational diffusion coefficients and Θ the rotational diffusion coefficient, the latter being obtained by the depolarized correlation function. At small γ, the particle can reorient many times before diffusing over a distance close to Q-1 and then coupling vanishes. Unlike rigid spherelike particles, for which D| - D⊥ ≈ 0, in the case of rigid rod-shaped (or ellipsoidal) particles,36,37 the rotational and translational diffusions can be correlated. For long particles QL > 3 (L being the length of the rod), even in dilute solutions, the difference in the lengthways and sideways translational diffusion can play an important role. However, for γ < 5, correlation functions furnish, with a good approximation, the translational diffusion coefficient of rigid rods. For polydisperse scatterers, the field autocorrelation function may be expressed as the Laplace transform of a continuous distribution G(Γ) of decay rates, each related to differently sized diffusing entities: g1(Q, t) ) ∫G(Γ) exp(-Γt) dΓ. In the case of monomodal distribution of decay rates, the mean diffusion coefficient 〈D〉 ) 〈Γ〉/Q2 (where 〈Γ〉 ) ∫ΓG(Γ) dΓ is the mean decay rate) can be obtained from the standard cumulant analysis:31,32

ln|g1(Q, t)| ) -〈Γ〉t + 1/2!µ2t2 - 1/3!µ3t3 + ...

(3)

with µn being the moments of the distribution G(Γ). The polydispersity index is related to the second moment µ2 (variance) as µ2/〈Γ〉2 (for values lower than 0.3). At infinite dilution, the Einstein-Stokes relation, RH ) kBT/ (6πηD), can be used to obtain the hydrodynamic radius RH of the scatterers from their diffusion coefficient (kB being the Boltzmann’s constant, T the absolute temperature, and η the solvent viscosity). In the case of rod-like particles, the hydrodynamic radius represents the radius of an equivalent sphere having that measured diffusion coefficient. For the static light scattering measurements, the scattered intensity of the solvent is subtracted from that of the solutions; then, the obtained angular profile is normalized by the scattered intensity of toluene used as a reference sample. The normalized scattered intensity can be written as31,32,38

I(Q) ) NKMwcP(Q)S(Q)

(4)

with P(Q) and S(Q) being the normalized form factor and structure factor, respectively, N the aggregation number, Mw the molecular weight of the molecule, c the mass concentration, and K the optical constant.32

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The form factor is related to the spatial Fourier transform of the particle mass distribution all over the volume of the particle, and its mathematical expression derived for homogeneous spherical particles with radius R and thin rigid rods with length L is, respectively,

P(Q) )

[

P(Q) )

2 QL

]

3 (sin(QR) - QR cos(QR)) (QR)3

2

2 QL dx - [ sin ∫0QL sin(x) x QL ( 2 )]

(5)

(6)

For polydisperse systems, the form factor is obtained as an average weighted by the size distribution function. It can be considered that, for the solutions investigated, the concentration is low enough that S(Q) can be approximated to unity. Circular Dichroism. The CD spectra were obtained at 25 °C under a constant flow of N2 on a Jasco J-810 spectropolarimeter which had been calibrated with an aqueous solution of (1R)-(-)-10-camphorsulfonic acid, ammonium salt.39 The CD spectra were recorded in the UV region (190-260 nm) using a 1 mm path length cuvette. The spectra represent the average of 8-20 scans. CD intensities are expressed as mean residue ellipticity (deg cm2 dmol-1). Thioflavin-T-assay. Fluorescence emission spectra of Th-T undergo a red shift upon incorporation into β-sheet amyloid structures.40,41 Fluorescence was monitored as a function of time in a 1.0 cm path length quartz cuvette using a Spex Fluorolog-2 (mod. F-111) spectrofluorimeter. The measurements were carried out using, as a control, the time dependence of the fluorescence of thioflavine-T solutions without the peptide at both pH values. The sample mixtures were monitored over a 70 min period at an excitation wavelength of 442 nm, and the emission was 485 nm. Both excitation and emission bandwidths were set to 2 nm. The Th-T fluorescence was also monitored over the time (4000 s) for PEG600 alone under identical experimental conditions as those used for the PEG-conjugated variants. We did not observe any Th-T fluorescence increase with respect to the control experiment (fluorescence of Th-T alone).

Figure 2. Size distribution function of hIAPP17-29 at pH 7.0 at different time delays: at t ) 20 min (filled circles) and at t ) 40 min (open circles).

Figure 3. Mean hydrodynamic radius evolution of the aggregated form of hIAPP17-29 at pH 7.0, as obtained from the mean value of the main peak of the distribution of Figure 2.

Results Human 17-29 Fragment. The aggregation process of hIAPP17-29 at pH 8.0 gives quick rise to micrometric aggregates, within a couple of minutes from sample preparation. The rate of aggregation is moderately slower at pH 7.0 and the formed aggregates, presumably prefibrillar assemblies, have a hydrodynamic radius of about 60 nm with a high polydispersity (30%). Although at the beginning of the aggregation the main contribution to the correlation function comes from these smaller aggregates, also micrometric aggregates are present, as shown by the size distributions of Figure 2. After about 30 min, all hIAPP17-29 is involved in aggregates having micrometric size (see Figures 2 and 3). This quick evolution, toward larger aggregates, did not allow for an investigation of their morphology through static light scattering measurements. In the acidic solution (at pH 4.0), hIAPP17-29 does not form any aggregated species. Rat 17-29 Fragment. The comparison with the corresponding fragment of rat amylin, rIAPP17-29, shows that it undergoes aggregation only at pH 8.0 and that the rate of the process is significantly slower than that of the human analogue. Figure 4 displays the mean decay rate of the correlation functions at

Figure 4. Dependence of the correlation function decay rate of rIAPP17-29 on the exchanged wavevector at different time delays; the inset reports the size distribution at a delay of t ) 290 min. Dashed lines represent the linear fit furnishing the value of the diffusion coefficient.

different time delays; after the first 5 h, in which aggregate dynamics vary very slowly, the aggregation process stabilizes, at least up to about 10 h. From an inspection of Figure 4, 〈Γ〉 appears to depend reasonably on the squared exchanged wavevector, indicating that the aggregated species are rigid and that the size distribution is narrow. From the slope of the linear fit of Figure 4, the diffusion coefficient can be extracted and the hydrodynamic radius of the aggregates, evaluated through

Comparative Study with Two PEG-Conjugated Variants

Figure 5. Time evolution of the mean hydrodynamic radius of rIAPP17-29 at pH 8.0.

Figure 6. Scattered intensity profile of rIAPP17-29 at pH 8.0; the continuous curve is the fit according to the thin rigid rod model (eq 6). The straight line indicates the Q-1 power law typical of an infinitely long thin rigid rod.

the Einstein-Stokes relation, is about 60 nm (with a size polydispersity of 10%). The evolution of the hydrodynamic radius in the whole investigated time range is shown in Figure 5. During the time in which dynamical properties are stable, the intensity profile shown in Figure 6 is measured. The theoretical form factor which best fits experimental data is that of a thin rigid rod (eq 6) with L ) 350 ( 50 nm. Although QL . 1, the coupling between rotation and translation seems to be small enough to guarantee the Q2 dependence of the correlation function relaxation rate. The radius of gyration of a thin rigid rod is Rg ) L/(12)1/2, so that for the rat amylin it is Rg ≈ 100 nm. By taking into account that, for a rod, the ratio Rg/RH g 2, we believe that dynamic and static light scattering agree in suggesting that rIAPP17-29 at pH 8.0 aggregates to form rod-like structures, which do not evolve toward micrometric amyloid fibrils. PEG-Conjugated Peptides. The PEG-conjugated peptides display different properties depending on which side of the peptide chain the PEG moiety is linked to. Freshly prepared hIAPP17-29-C-PEG solution forms clusters with a hydrodynamic radius of about half a micrometer, as indicated by the value of the mean relaxation rate 〈Γ〉, but rapidly (within 10 min) they evolve toward micrometric objects making the solution very turbid, independently of pH. On the other hand, the aggregation process of the PEG-NhIAPP17-29 variant is more stable and the formed aggregates slightly depend on the pH. In the freshly prepared solution,

J. Phys. Chem. B, Vol. 114, No. 2, 2010 709 within 10 min, the aggregate hydrodynamic radius takes a value of about 110 nm at pH 8.0 and pH 7.0 and of about 95 nm at pH 4.0. Within 30 min, aggregates reach a hydrodynamic radius of about 170 nm at pH 8.0 and pH 7.0 and about 140 nm at pH 4.0 and stop growing. Data on the hydrodynamic radius of the aggregated human fragment and its PEG-conjugated variants are summarized in Table 1. Figure 7 displays the Q2 dependence of the correlation function mean decay rate, despite the condition QRH > 1, indicating that aggregates are rigid. The intensity profile of this variant of the human peptide (see Figure 8) suggests that the formed aggregates take an almost spherical shape with radius R ≈ 160 nm for all of the pH values (and polydispersity of 20%) and a homogeneous mass distribution, as also indicated by the value R/RH ≈ 1. It is worth noting that, whereas the aggregate’s size does not seem to depend on pH markedly, the scattered intensity strongly depends on it. Therefore, by considering that the aggregate molecular weight, Maggr ) NMw, does not change (size remaining the same), the zero-Q value of the scattered intensity is related to the number 2 density of aggregates, Naggr (I(0) ∝ NaggrMaggr ), which decreases at pH 4.0. In order to investigate the effect of PEG conjugation on the conformational behavior of the studied peptides, the CD spectra of the PEG-N-hIAPP17-29 and hIAPP17-29-C-PEG were recorded in aqueous solution at different pHs (Figure 9). Analogous CD experiments were previously carried out on the unpegylated peptide hIAPP17-29 and rIAPP17-29. The recorded CD spectra displayed profiles attributable to β-sheet or β-turn conformation and unstructured peptide chain, respectively, for hIAPP17-29 and rIAPP17-29.24 In the case of PEG-N-hIAPP1729, the CD spectra give evidence of a pH dependent conformational transition. In particular, the peptide chain is mainly unstructured at pH 4.0 and 5.0, as indicated by major negative ellipticity below 200 nm, while it sharply changes to an ordered conformation, as demonstrated by the CD profiles observed in the 6.0-10.0 pH range that show composite negative and positive dichroism at 222 and 205 nm, respectively. Moreover, a less intense negative band is always observed below 200 nm in the CD spectra collected in the 7.0-10.0 pH range. This feature might be assigned either to residual random coil peptide conformation or to the red-shifted negative band of type II β-turn, that is usually predicted to occur below 190 nm.42 A major event observed in these experiments is related to the evident effect on the peptide conformation due to the deprotonation of the histidine residue. We determined the pKa of the histidine’s imidazole in this peptide by a potentiometric titration, and a value of 6.37 was found. Such a value nicely falls within the pH range where the conformational transition occurs. As far as CD spectra of hIAPP17-29-C-PEG are concerned, it should be pointed out that, with the exception of the spectrum recorded at pH 4.0, for which the negative signal is at 220 nm, all curves are characterized by a negative band at 224 nm and a positive one at 204 nm in the whole pH range investigated. Furthermore, the peptide conformation is only slightly affected by the protonation state of histidine. Such a conformational behavior strongly recalls that observed for the underivatized hIAPP previously reported.24 Another apparent difference between the two derivatives is related to their solubility in aqueous solution. Indeed, although in both cases the CD experiments were carried out using the same peptide concentrations (1.0 × 10-5 M), the solubility of PEG-N-hIAPP17-29 was estimated in the order of millimolar concentration, whereas hIAPP17-29-C-PEG is around 1 order of magnitude less soluble.

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TABLE 1: Time Dependence of the Hydrodynamic Radius (Expressed in nm) of the Rat and Human Fragment and PEG-Conjugated Variantsa

a

pH

sample

5 min

20 min

40 min

300 min

4.0 4.0 4.0 4.0 7.0 7.0 7.0 7.0 8.0 8.0 8.0 8.0

rIAPP17-29 hIAPP17-29 PEG-N-hIAPP17-29 hIAPP17-29-C-PEG rIAPP17-29 hIAPP17-29 PEG-N-hIAPP17-29 hIAPP17-29-C-PEG rIAPP17-29 hIAPP17-29 PEG-N-hIAPP17-29 hIAPP17-29-C-PEG

no aggr no aggr 95 500 no aggr 70 110 500 48 micrometric 110 500

no aggr no aggr

no aggr no aggr 140

no aggr no aggr 140

no aggr 60

no aggr 1500 170

no aggr 170 59

170

170

The experimental error is (5%.

Figure 7. Q2 dependence of the correlation function decay rate of N-PEGhIAPP17-29 variant at pH 4.0 (stars), at pH 7.0 (circles), and at pH 8.0 (squares). Dashed lines represent the linear fit furnishing the value of the diffusion coefficient.

shows a sharp increase of thioflavine-T fluorescence over the time monitored, indicating the presence of amyloid-like structures. For the PEG-conjugated variants, a β-sheet formation at both of the pHs studied is revealed, as monitored by an increase in Th-T fluorescence intensity over the time course of the assay. In particular, the hIAPP17-29-C-PEG sample shows at both pHs an abrupt increase in the Th-T binding signal with no large differences between the two pH values. On the contrary, the PEG-N-hIAPP17-29 sample shows a pH dependence that induces a slight fluorescence enhancement of thioflavine-T at pH 4.0, over the time monitored, and a more prominent increase of thioflavine-T fluorescence signal at pH 8.0, thereby suggesting that for this variant a smaller amount of peptide is involved in β-sheets. PEG-Added Solutions. Finally, in order to study the effect of the PEG chain on the aggregation process, aqueous solutions of the fragment hIAPP17-29 have been prepared at pH 4.0, and PEG600 was added to the solution in different molar ratios with respect to the amylin human fragment. At 1:1 molar ratio between hIAPP17-29 and PEG600, no significant differences in the correlation function appear with respect to the hIAPP1729 solution in the absence of PEG600. On the contrary, at 1:50 molar ratio, extended aggregation drives the system toward the formation of micrometric clusters. At 1:10 molar ratio, a detectable aggregation kinetics gives rise to clusters with a mean hydrodynamic radius of about 35 nm, which, after 10 min, grows to about 60 nm, as shown in Figure 11. Afterward, the size keeps on growing and reaches micrometric values after about 10 h. Discussion

Figure 8. Scattered intensity profile of PEG-N-hIAPP17-29 at pH 8.0 (squares) and at pH 4.0 (stars). The continuous lines represent the best fit with the polydisperse homogeneous sphere model.

The thioflavine-T binding assays were used to investigate whether or not ordered β-sheet aggregates are present in the aqueous solutions of the studied peptides. The Th-T fluorescence as a function of incubation time is shown in Figure 10 for samples of hIAPP17-29, hIAPP17-29-C-PEG, and PEG-NhIAPP17-29, at 25 °C and at a pH of 4.0 and 8.0. All of the kinetics traces reveal an increase in the fluorescence signal without a lag time, except for the hIAPP17-29 at pH 4.0 in which the low fluorescence gain with respect to control is consistent with the absence of β-sheet structures in agreement with light scattering results. Conversely, hIAPP17-29 at pH 8.0

The occurrence that rat amylin does not cause amyloidosis both in vivo and in vitro43,44 induced us to perform a comparison between the fragments hIAPP17-29 and rIAPP17-29. The use of such fragments instead of the parent IAPP proteins is based on previous results24 showing that the hIAPP17-29 fragment can reproduce some of the key properties of the full-length protein. Indeed, the hIAPP17-29 sequence encompasses both the His residue and the highly amyloidogenic sequence NFGAIL. This fact allows for an investigation on the role of the electrostatic and solvent interaction in preventing extensive aggregation of the polypeptide chains, in addition to the study of the different morphologies of the formed aggregates. Despite the fact that many parameters, in principle, can affect the resulting aggregate concentration, size, and morphology, it is known that, in the wild type amylin, His18 is responsible for the pH dependence of the aggregation process as well as for

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Figure 9. Far-UV CD spectra of PEG-N-hIAPP17-29 (A) and hIAPP17-29-C-PEG (B) in aqueous solution at different pHs. Following the arrow direction: pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0. [PEG-N-hIAPP17-29] ) [hIAPP17-29-C-PEG] ) 5.0 × 10-5 M.

Figure 10. Kinetics of hIAPP17-29, hIAPP17-29-C-PEG, and PEGN-hIAPP17-29 fibrillation in 5.0 × 10-2 M citrate-phosphate buffer, at 25 °C, pH 4.0 and 8.0. The Th-T fluorescence is shown as a function of incubation time. (a) Th-T; (b) hIAPP17-29, pH 4.0; (c) PEG-NhIAPP17-29, pH 4.0; (d) PEG-N-hIAPP17-29, pH 8.0; (e) hIAPP1729-C-PEG, pH 4.0; (f) hIAPP17-29-C-PEG, pH 8.0; (g) hIAPP17-29, pH 8.0.

Figure 11. Size distributions of hIAPP17-29 in the presence of PEG600 at 1:10 molar ratio at t ) 0 (circles) and after 10 min (squares). In the inset, the corresponding scattered intensity correlation functions are reported.

the aggregation rate.25 The light scattering results here reported show that the aggregation kinetics is triggered at physiologic and basic pH as a consequence of the deprotonation of the histidine’s imidazole ring; therefore, the lack of electrostatic

repulsion would allow for a closer approach of peptides and the consequent aggregation, which occurs as faster as higher is the pH. On the other hand, the intensity profile of the rIAPP17-29 fragment at pH 8.0 proves that, above the physiologic pH, the rat fragment aggregates to form submicrometric rod-like structure without causing fibrillogenesis. This occurrence can be directly related to the absence of amyloidogenesis in rats, likely because of the presence of prolines which unfavor interactions between peptides. With the aim of gaining more details on the role of the fragment 17-29 in the aggregation process and to design more soluble variants of the model peptide27,45-47 (as well as new materials48-50), the hIAPP17-29 conjugated with a PEG chain linked to the N-terminus of the fragment has been studied under the same experimental conditions. The occurrence that the formed aggregates are more stable and possess a different morphology can be lead back to the net separation between the hydrophobic (hIAPP17-29) and hydrophilic (PEG) part of the molecule. The structure of the aggregates, suggested by the form factor of Figure 8, seems to indicate that molecules self-organize to form micellar clusters. The decreased absolute scattered intensity at pH 4.0, along with the constancy of the aggregate size, can be attributed to the decreased number of aggregates due to the repulsion between adjacent protonated His18. Such an event increases the surface area of the hydrophilic part and tends to break the aggregates, thereby shifting the equilibrium toward the monomeric form. This result perfectly agrees with what is observed by CD spectra and Th-T assays, i.e., that most of the peptides do not form β-sheets, despite some aggregates still being present. A very different result is obtained when the PEG chain is linked to the C-terminus of the fragment; in this case, the system lacks solubility and the peptide chains interact by forming β-sheet structures and the solution flocculates. The comparison of both PEG-conjugated variants with the hIAPP17-29 fragment, which was shown to not aggregate at pH 4.0 because of the electrostatic repulsion, suggests that the polymer chain must play an important role in the aggregation process of these variants. By considering that at the investigated concentration PEG-PEG aggregation is not expected to occur, due to its highly favored interaction with water, the PEG chain might compete with the peptide for H-bond interactions with the solvent.51-53 It is known, in fact, that PEG is able to bind water

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molecules in its neighborhood inducing on them a structuring effect;54 then, the decrease of H-bonds available for facilitating hydration (and hence solubilization) of the peptide seems to favor the hydrophobic interaction between peptides themselves giving rise to the aggregates, stabilized by the β-sheet structure. In order to validate this hypothesis and to exclude that PEG aggregation phenomena are due to the presence of HFIP in the buffer, PEG600 was prepared in water solution at pH 4.0 (at the same concentration as in the sample at 1:10 molar ratio) without hIAPP17-29. The lack of aggregation phenomena in this sample confirms that the aggregation of the PEG-NhIAPP17-29 is due to the competition between the peptide part and the polymer part of the variant fragment in forming H-bonds with the solvent. This occurrence favors hydrophobic interactions between peptides, and the system self-assembles into sphere-like structures also when His18 is protonated, even if the number of aggregates strongly decreases at pH 4.0. It has to be also considered that in the survived aggregates under acidic conditions the electrostatic repulsion hampers hydrogen bonding between peptides and hence the β-sheet structure.55 On the other hand, in the hIAPP17-29-C-PEG variant, a net separation between hydrophobic and hydrophilic parts is missing, due to the closer position of the most hydrophobic residues (Ile and Leu) to the PEG chain side. Therefore, the increased hydrophobicity of the peptide (induced by the lower availability of H-bonds in the solvent) gives rise to fast and extended aggregation with consequent flocculation. In summary, the presence of HFIP in the solution favors the solubilization of the peptide via H-bonds (HFIP possesses a high tendency to form H-bonds) and the presence of PEG as a competitor of H-bonds destabilizes the system and gives rise to aggregation. These results indicate that the presence of HFIP in the buffer cannot be neglected and suggest the reason why different solvents used to prepare the amylin stock solution affect the aggregation properties and the rate of fibril formation.29 Moreover, from the experimental results reported in this work, it turns out that, besides the presence of His18 and the higher tendency of serine to form β-sheets than proline (the latter being present in the rat amylin), the delicate role of the hydrogen bond between the hydrophilic residues of amylin and the solvent must also be considered in order to investigate correctly the aggregation processes driving to fibrils. Conclusions The process underlying the amyloid formation in vitro is certainly the result of complex mechanisms involving electrostatic and H-bond interactions, peptide sequence structure, as well as external conditions including pH. However, the comparative spectroscopic study of human and rat IAPP17-29 model fragments and some variants of the human sequence in aqueous solution sheds light on some peculiarity of IAPP17-29 selfaggregation. In particular, it has been shown that self-aggregation occurs in the analogous fragment of rat’s IAPP17-29 as well by increasing the pH to 8.0 and that the formed aggregates possess a rod-like structure without evolving to micrometric fibrils. The variant of the human fragment bearing a PEG moiety at the N-terminus of the peptide chain is responsible for hindering the fibril formation (despite the presence of a certain fraction of β-sheet structure at pH higher than 4.0) and for giving rise to stable aggregates, likely micellar clusters. The C-terminated variant, on the other hand, does not possess an analogous amphiphilic character and the high tendency of the PEG chain to form a H-bond deprives the peptide of hydrogen bonding with the solvent, thus resulting in extended β-sheet structures.

Mazzaglia et al. In view of these results, a balance between the protonated groups of the fragment and the tendency to form a β-sheet structure is crucial for amyloid formation, with the interactions between peptide and solvent playing an important role in determining the aggregate structure. Moreover, it must be taken into account that the organic solvent present in the aqueous solution (even in very small amount) takes part in the aggregation process influencing its rate. The evidence reported in this work is consistent with the kinetics of amyloid formation and may contribute to explaining the lack of experimental reproducibility, in terms of the kinetics aggregation and cell toxicity, observed in the literature for amyloid peptides. Our results propose the PEG-conjugated peptides, especially the N-terminus one, as good models to study the molecular phenomena associated with IAPP oligomerization. In this regard, these peptides are being used to perform in vitro biological studies and to develop a model of IAPP’s cytotoxicity in cultured cell lines. Acknowledgment. This work was supported by MIUR FIRB RBIN04L28Y and FIRB RBNE03PX83. References and Notes (1) Cohen, F.; Kelly, J. Nature 2003, 426, 905–909. (2) Chiti, F.; Dobson, C. M. Annu. ReV. Biochem. 2006, 75, 333–366. (3) Hardy, J.; Selkoe, D. Science 2002, 297, 353–356. (4) Prusiner, S. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 13363–13383. (5) Westermark, P.; Wernsted, C.; Wilander, E.; Sletten, K. Biochem. Biophys. Res. Commun. 1986, 140, 827–831. (6) Selkoe, D. J. BehaV. Brain Res. 2008, 192, 106–113. (7) Westermark, P.; Wernsted, C.; Wilander, E.; Hayden, D. W.; O’Brien, T. D.; Johnson, K. H. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 3881–3885. (8) Cooper, G. J.; Willis, A. C.; Clark, A.; Turner, R. C.; Sim, R. B.; Reid, K. B. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 8628–8632. (9) Robertson, R. P.; Harmon, J. S. FEBS Lett. 2007, 581, 3743–3748. (10) Konarkowska, B.; Aitken, J. F.; Kistler, J.; Zhang, S.; Cooper, G. J. S. FEBS J. 2006, 273, 3614–3624. (11) Ritzel, R. A.; Meier, J. J.; Lin, C.-Y.; Veldhuis, J. D.; Butler, P. C. Diabetes 2007, 56, 65–71. (12) Kayed, R.; Sokolov, Y.; Edmonds, B.; McIntire, T.; Milton, S.; Hall, J.; Glabe, C. J. Biol. Chem. 2004, 279, 46363–46366. (13) Mirzabekov, T.; Lin, M.; Kagan, B. J. Biol. Chem. 1996, 271, 1988– 1992. (14) Demuro, A.; Mina, E.; Kayed, R.; Milton, S.; Parker, I.; Glabe, C. J. Biol. Chem. 2005, 280, 17294–17300. (15) Mattson, M.; Goodman, Y. Brain Res. 1995, 676, 219–224. (16) Yan, L.; Tatarek-Nossol, M.; Velkova, A.; Kazantzis, A.; Kapurniotu, A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2046–2051. (17) Evers, F.; Jeworrek, C.; Tiemeyer, S.; Weise, K.; Sellin, D.; Paulus, M.; Struth, B.; Tolan, M.; Winter, R. J. Am. Chem. Soc. 2009, 131, 9516– 9521. (18) Gotz, J.; Ittner, L. M.; Lim, Y. A. Cell. Mol. Life Sci. 2009, 66, 1321–1325. (19) Potter, K. J.; Scrocchi, L. A.; Warnock, G. L.; Ao, Z.; Younker, M. A.; Rosenberg, L.; Lipsett, M.; Verchere, C. B.; Fraser, P. E. Biochim. Biophys. Acta 2009, 1790, 566–574. (20) de Groot, N.; Pallares, I.; Aviles, F.; Vendrell, J.; Ventura, S. BMC Struct. Biol. 2005, 5, 18. (21) Tenidis, K.; Waldner, M.; Bernhagen, J.; Fischle, W.; Bergmann, M.; Weber, M.; Merkle, M.; Voelter, W.; Brunner, H.; Kapurniotu, A. J. Mol. Biol. 2000, 295, 1055–1071. (22) Zanuy, D.; Ma, B.; Nussinov, R. Biophys. J. 2003, 84, 1884–1894. (23) Kajava, A.; Aebi, U.; Steven, A. J. Mol. Biol. 2005, 348, 247– 252. (24) Pappalardo, G.; Milardi, D.; Magrı`, A.; Attanasio, F.; Impellizzeri, G.; La Rosa, C.; Grasso, D.; Rizzarelli, E. Chem.sEur. J. 2007, 13, 10204– 10215. (25) Abedini, A.; Raleigh, D. Biochemistry 2005, 44, 16284–16291. (26) Damante, C. A.; Osz, K.; Nagy, Z.; Pappalardo, G.; Grasso, G.; Impellizzeri, G.; Rizzarelli, E.; Sovago, I. Inorg. Chem. 2008, 47, 9669– 9683. (27) Abedini, A.; Meng, F.; Raleigh, D. P. J. Am. Chem. Soc. 2007, 129, 11300–11301. (28) Shen, C.; Murphy, R. Biophys. J. 1995, 69, 640–651.

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