Influence of trehalose on human islet amyloid polypeptide fibrillation and aggregation

Abstract

Abnormal denaturation and aggregation of human amylin or islet amyloid polypeptide (IAPP) into amyloid fibrils has been implicated in the pathogenesis of type 2 diabetic mellitus. Trehalose, a super-hydrophilic molecule, has been shown to prevent denaturation of biomolecules when they are under environmental stress. In this work, we sought to investigate the effects of trehalose on the fibrillation and aggregation of human IAPP (hIAPP) by using circular dichroism spectrum, thioflavin-T fluorescence spectrum, dynamic light scattering, transmission electronic microscopy, atomic force microscopy and quartz crystal microbalance. We demonstrated that (1) the conformation of hIAPP changed from α-helix to β-sheet, followed by fibrillation and aggregation, (2) a low dose of trehalose (under 100 mM) inhibited or delayed the conformation transition of hIAPP and (3) a high dose (more than 500 mM) induced the conformation transition, and promoted the fibrillation and aggregation of hIAPP. These findings are in agreement with the hypothesis of the water replacement and volume exclusion effect on the proteins. The lower concentration of trehalose could replace the water molecules surrounding the hIAPP, and interact with proteins through hydrogen bonding, leading to a reduction in the protein interaction itself, and therefore inhibiting or delaying the protein fibrillation and aggregation. In contrast, the higher concentration of trehalose might interact with itself to form macromolecular clusters, acting as a crowding agent, leading to the hIAPP molecules being excluded by the trehalose clusters and interacting between each other, and therefore promoting the hIAPP fibrillation and aggregation.

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Introduction

Misfolding of proteins is an important pathological process of many diseases including neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's and prion diseases^1,2 and type 2 diabetic mellitus (T2DM).^3–5 T2DM, also called adult-onset diabetes, is the most common form, by approximately 90% among diabetes cases.

Human amylin or islet amyloid polypeptide (IAPP), a 37-residue polypeptide synthesized in pancreatic β-cells in the islets and stored in the insulin secretory granules, is co-secreted with insulin in response to the stimuli in both healthy and diabetic individuals.^6,7 The amyloid plaques exist in T2DM patients, but are absent in healthy people.⁸ Under specific conditions, human IAPP (hIAPP) is likely to form folded β-sheet fibrils or oligomers that can damage the membrane permeability, leading to the apoptosis of β-cells and eventually T2DM disease.^9,10 Therefore, inhibiting the hIAPP fibrillation and aggregation has been considered to be an effective way to prevent and treat T2DM.

The evidence shows that specific amino acids in hIAPP, such as His18, Ser20, Phe23 and Ile26, which are different from the amino acids in non-amyloidogenic rat IAPP (rIAPP), are critical for hIAPP aggregation.^11–15 Furthermore, partial hIAPP sequences such as hIAPP (22–27), i.e. sequence of NFGAIL, and hIAPP (20–29), i.e. sequence of SNNFGAILSS, can form insoluble amyloid and accelerate the aggregation of hIAPP.^16,17 Recently, some small molecules such as trehalose, curcumin, epigallocatechin gallate (EGCG), salvianolic acid B have been investigated for their potential functions in prevention and treatment of neurodegenerative diseases and T2DM.^18–23

Trehalose, a super-hydrophilic molecule with many hydroxyl groups (Fig. 1), exists in many creatures. This disaccharide is synthesized naturally to protect biomolecules from denaturation when cells are exposed to environmental stress such as heat and cold.^24,25 The trehalose has special properties including high glass transition temperature, the polymorphs which exist both in the crystalline as well as amorphous states, and the strong interaction with water.^26–29 The mechanism of trehalose stabilizing protein has been studied by FTIR²⁴ and in situ micro-Raman spectroscopy.³⁰ Trehalose has been demonstrated to be effective in inhibiting protein misfolding and aggregation in vitro in amyloid beta (Aβ) peptide,³¹ α-synuclein (AS),^32,33 myelin basic protein,³⁴ β-hairpin,³⁵ insulin,³⁶ and in vivo model of Alzheimer's disease, Huntington's disease and prion disease.^37–39 The process of amyloid formation is considered to be similar between hIAPP and Aβ peptides.⁴⁰ However, the effect of trehalose on hIAPP has not been reported. Thus, we herein sought to investigate the effect of trehalose on hIAPP, a T2DM related peptide.

Fig. 1 Molecular structure of trehalose.

In the present work, the fibrillation and aggregation of hIAPP and the effect of trehalose on hIAPP behaviors were investigated by circular dichroism (CD) spectrum, thioflavin-T (ThT) fluorescence spectrum, dynamic light scattering (DLS), transmission electronic microscopy (TEM), atomic force microscopy (AFM) and quartz crystal microbalance (QCM). The mechanism was discussed as well.

Materials and methods

Chemicals and peptides

Synthetic hIAPP was purchased from Chinese Peptide Company with purity higher than 95%. Trehalose and thioflavin-T (ThT) were purchased from Sigma, USA. Hexafluoroisopropanol (HFIP) was purchased from Aladdin Industrial Corp. Phosphate buffer solution (PBS) was purchased from Sangon biotechnology, China.

Sample preparation

hIAPP was freshly dissolved in HFIP and sonicated for 2 min, and then placed in oven at 37 °C for 2 hours to ensure the hIAPP dissolved completely. The stock solution was stored at 4 °C. For kinetic investigation, the stock solution was filtered through 0.22 μm cellulose acetate filters. The filtered hIAPP solution was diluted into PBS (20 mM, pH = 7.4) to give a final concentration of 8 μM for circular dichroism (CD) measurement, and 16 μM for the other measurements. Trehalose was added at concentration of 0, 50, 250 mM for CD measurement, 80, 100, 200, 250, 300, 400, 500 mM for ThT fluorescence and DLS measurement, and 0, 100, 500 mM for other analysis (QCM and AFM).

Circular dichroism (CD) spectrum

For analysis of hIAPP conformation, CD measurement was performed on MOS-450 spectrometer with a 1 cm path length quartz cell. CD spectra were recorded from 190 to 250 nm at a step of 0.25 nm by two scans with 5 mL min⁻¹ nitrogen flow during the measurement. A background spectrum was subtracted from the collected data.

Thioflavin-T (ThT) fluorescence spectrum

For detection of hIAPP β-sheet conformation, ThT fluorescence experiments were performed on Varioskan Flash (Thermo, USA) with a 96-well fluorescence plate reader. The ThT fluorescence intensity was measured every 1 min for 2 hours with excitation wavelength at 450 nm and emission wavelength at 485 nm. 800 μM peptide stock solution in HFIP was diluted into PBS buffer to give a final concentration of 16 μM peptide, 16 μM ThT and 2% HFIP. The data from triplicate wells were averaged, and the experiment was repeated for three times.

Dynamic light scattering (DLS)

For analysis of the particle size, DLS measurement was performed on the Zetasizer Nano instrument (Malvern Instruments, UK). 1 mL hIAPP PBS buffer solution in a quartz cell of 1 cm path length was measured by a He–Ne laser of 633 nm with a detector angle of 90°.

Transmission electronic microscopy (TEM)

For observation of hIAPP morphology, 10 μl hIAPP PBS buffer solutions were dropped on a glow 400-mesh carbon-coated copper grid after two hours incubation. The sample was washed with distilled water and then stained with 2% (w/v) uranyl acetate for 2 min. The grid was observed under a transmission microscope (Tecnai G2 20 TWIN, FEI, USA).

Atomic force microscopy (AFM)

For observation of hIAPP morphology, AFM images were acquired within size of 5 × 5 μm in a tapping mode on Multimode 8 (Bruker, Germany) under ambient conditions. 10 μl samples during incubation for 0 and 40 min were dropped on the freshly cleaved mica, rinsed with deionized water, and then dried in vacuum overnight.

Quartz crystal microbalance (QCM)

QCM is an alternative technique to be used to probe the protein aggregation recent years.⁴¹ hIAPP peptide was covalently immobilized on a gold electrode surface for the pre-treatment, meanwhile 16 μM hIAPP was pre-incubated for 20 min to produce the seed fibrils which was observed by AFM. The pre-treated gold electrode was immersed in the hIAPP solutions with seed fibrils for 40 min for the hIAPP fibrillation and aggregation on the electrode. The experiment was performed on QMC Q-sense E4 (Sweden) at 37 °C in accordance with the published method.⁴¹

Results and discussion

Kinetics of conformation transition, fibrillation and aggregation of hIAPP

CD measurement was used to investigate the conformation transition of hIAPP in solution. CD spectrum of hIAPP shows two negative peaks at 206 and 222 nm (Fig. 2A), indicating a dominant α-helical structure at the beginning of incubation. The positive peak at 199 nm increased, meanwhile, the negative peak at 206 nm red-shifted along with 222 nm blue-shifted gradually during incubation, indicating a conformational transition from dominant α-helix to a mixed structure of α-helix and β-sheet which showed a typical negative peak at 215 nm as well as a positive peak at 194 nm. There are also some reports suggesting that the conformation transition of hIAPP from random coil to β-sheet is easy.²⁰

Fig. 2 The conformation transition of hIAPP as incubation time probed by CD spectra (A) and ThT fluorescence (B). The aggregate particle sizes of hIAPP detected by DLS (C), and the fibrils morphology of hIAPP observed by TEM (D).

ThT is a fluorescent dye that can selectively associate with β-sheet protein, resulting in its fluorescence enhancement, therefore generally used to probe the hydrophobic β-sheet conformers.⁴² ThT fluorescence intensity is enhanced when ThT molecules bound to the amyloid fibrils.⁴³ It is found in Fig. 2B that ThT fluorescence intensity shows a sigmoidal curve as hIAPP incubation time increased. The process includes a lag phase during which the β-sheet oligomer species were formed, a growth phase during which fibrils were elongated quickly, and a steady phase.¹²

DLS is a sensitive and powerful tool to detect the particle size distribution of the assembled protein. This technique has been used to investigate the aggregation of insulin and hIAPP.^44,45 Fig. 2C shows the change of aggregation particle sizes as hIAPP incubation. It is found that sizes of hIAPP increased gradually as incubation time increased via a CONTIN analysis, and the light scattering intensity even went out of the limited range after 30 min incubation (data not shown).

Fig. 2D is TEM image of hIAPP aggregate morphology, showing the typical fibrils formed by hIAPP after two-hour incubation.

Effects of trehalose on conformation, fibrillation and aggregation of hIAPP

Fig. 3A shows the influence of trehalose on hIAPP conformation by ThT fluorescence assays. It is found that trehalose has no fluorescence intensity at 485 nm. The low dose of trehalose at concentration from 80 to 100 mM increased the lag time, indicating that trehalose inhibited or delayed the conformation transition of hIAPP from α-helix to β-sheet. However, the high dose of trehalose at concentration from 300 to 500 mM shortened the lag time, indicating that high dose of trehalose promoted the formation of β-sheet conformers.

Fig. 3 (A) Effect of trehalose at various concentrations on the change of β-sheet conformer of hIAPP detected by ThT fluorescence spectrum after 2 hours incubation in the presences of 0 mM (black squares), 80 mM (red circles), 100 mM (blue up-triangles), 200 mM (dark cyan down-triangles), 250 mM (magenta left-triangles), 300 mM (dark yellow right-triangles), 400 mM (navy diamonds), 500 mM (wine pentagons) trehalose. The pink hexagon curve is ThT fluorescence spectrum of 500 mM trehalose. (B) Effect of trehalose on the Z-average size of hIAPP detected by DLS measurement during hIAPP incubation with various concentration of trehalose for 15 min. (C) Effect of 0, 100 and 500 mM trehalose on the Z-average size of hIAPP incubated for 1 min. (D) Effect of trehalose at various concentrations on conformation transition of hIAPP after 40 min incubation detected by CD spectrum.

DLS experiment was also used to determine the influence of trehalose on hIAPP aggregation. Fig. 3B shows Z-average value of hIAPP during 15 min incubation. The results demonstrated that when the concentration of trehalose was lower than 100 mM, the particle sizes of hIAPP were almost unchanged, however, when the concentration of trehalose was increased from 300 to 500 mM, the particle sizes of hIAPP increased significantly, as also observed clearly in Fig. 3C where hIAPP were incubated with and without 100 mM and 500 mM trehalose, respectively, for 1 min.

In CD experiments, the concentration of hIAPP was reduced to 8 μM, because 16 μM is too high for the conformation analysis of hIAPP, and the concentrations of low and high dose trehalose were reduced to 50 mM and 250 mM, respectively (Fig. 3D). The spectrum of hIAPP treated by 250 mM trehalose shows predominantly a typical β-sheet structure, and the β-sheet content in various samples is in order as trehalose concentration of 250 > 0 > 50 mM based on the intensity at 199 nm after hIAPP incubation along with trehalose for 40 min. The results suggested that high dose (250 mM) of trehalose accelerated the conformation transition of hIAPP from α-helix to β-sheet, while low dose of trehalose delayed the transition process. The result is consistent with that of ThT fluorescence assays as in Fig. 3A.

The morphologies of hIAPP incubated with and without trehalose were observed by AFM in Fig. 4. Fig. 4A-1 and A-2 are the images of 16 μM hIAPP after 0 and 40 min incubation without trehalose, and Fig. 4B-1, B-2, C-1 and C-2 are that with 100 mM and 500 mM trehalose present, respectively. It is found that at beginning, the images of hIAPP without and with 100 mM trehalose show few fibrils, while that of hIAPP with 500 mM trehalose shows many aggregates, which demonstrates that 500 mM trehalose promoted the formation of hIAPP oligomers that are crucial for the formation of amyloid fibril. After 40 min incubation of hIAPP, many fibrils were formed in all samples, but the amount of fibrils with 100 mM trehalose present is less than that without trehalose, and much less than that with 500 mM trehalose. The sample with 100 mM trehalose incubated for 40 min formed short fibrils shown in Fig. 4B-2, while the sample with 500 mM trehalose formed large amount of the amyloid plagues shown in Fig. 4C-2. AFM images demonstrates again that low dose of trehalose inhibited or delayed the formation of hIAPP amyloid fibrils, while high dose of trehalose promoted the formation of β-sheet oligomers and amyloid fibrils.

Fig. 4 AFM images of hIAPP in the absence (A-1 and A-2) and presence of 100 mM (B-1 and B-2), 500 mM (C-1 and C-2) trehalose incubated for 0 (A-1, B-1 and C-1) and 40 (A-2, B-2 and C-2) min, respectively. The inserted image in C-2 is the enlarged image of the violet area.

QCM experiment was further used to study the influence of various concentration of trehalose on the aggregation of hIAPP. Fig. 5A shows that at state I, PBS eluent was not adsorbed on the protein-treated gold electrode, and at state II, the trehalose eluent was adsorbed on the electrode with |ΔF| = 105 Hz, while at state III, the trehalose adsorbed on the electrode was completely washed out by PBS eluent with |ΔF| = 0 Hz, indicating that the trehalose cannot be immobilized on the protein-treated gold electrode. Fig. 5B shows that when hIAPP, hIAPP with 100 mM trehalose, and hIAPP with 500 mM were used as eluents at state II, hIAPP was immobilized on the electrode in amount order as eluents of hIAPP ≅ hIAPP with 100 mM trehalose ≪ hIAPP with 500 mM with |ΔF| about 140, 250, 1050 Hz, respectively. However, when the eluent was replaced by PBS at state III, partial proteins were washed out of the electrode, and the amount of proteins still remained on the electrodes was in order as eluents of hIAPP ≅ hIAPP with 100 mM trehalose ≪ hIAPP with 500 mM with |ΔF| about 130, 210, 770 Hz, respectively, indicating that high dose (500 mM) of trehalose much efficiently promoted the hIAPP aggregation immobilized on the electrode, while low dose (<100 mM) of trehalose did not. The result is basically consistent to the results from ThT fluorescence and DLS analysis.

Fig. 5 QCM measurement on the hIAPP pre-treated gold electrode. (A) State I, II, and III with eluents of PBS, 500 mM trehalose and PBS again, respectively; (B) state I with eluent of PBS; state II with eluent of hIAPP (black curve), hIAPP with 100 mM trehalose (red curve), and hIAPP with 500 mM trehalose (blue curve), respectively; state III with eluent of PBS again.

In general, trehalose has been demonstrated to be effective in the protection of proteins from the denaturation in different extent depending on the nature of the protein. However, most of those studies focused on the inhibiting effect of trehalose on the fibrillation and aggregation of amyloid peptides within a narrow range.^31,33 Here, we showed that trehalose performed different roles on the hIAPP at different concentrations. When the concentration is lower than 100 mM, trehalose behaved as an inhibitor, which delayed the transition of hIAPP from α-helix to β-sheet (ThT fluorescence and CD spectrum) and prevented the formation of hIAPP amyloid fibrils (DLS measurement and AFM observation). The result from QCM experiment also revealed that when the concentration was higher than 500 mM, trehalose accelerated the conformation transition of hIAPP from α-helix to β-sheet and promoted the formation of hIAPP amyloid fibrils. Thus, it should be attentional to apply the proper therapeutic concentrations in different diseases with different proteins.

Possible mechanism of trehalose influences on conformation, fibrillation and aggregation of hIAPP

Based on our findings, there are two possible mechanisms of action for trehalose influencing on the hIAPP conformation. One is water replacement hypothesis,²⁴ i.e., the trehalose substitutes the water molecules around hIAPP molecules when the trehalose concentration is low, leading to the hIAPP being far away from each other, and inhibiting the hIAPP aggregation. The other is the trehalose cluster acting as a macromolecular crowding agent which exclude the proteins⁴¹ when the trehalose concentration is high, i.e., the trehalose itself form the supermolecule cluster, leading to the proteins crowded together and self-assembled into the fibrils and aggregates as showed in Fig. 6.

Fig. 6 Schematic processes of trehalose effect on the hIAPP fibrillation and aggregation based on the QCM experimental technique.

Conclusions

By using the QCM experimental technique along with CD spectrum, ThT fluorescence, DLS measurement, AFM and TEM observation, the processes of hIAPP and the effect of trehalose on the hIAPP fibrillation and aggregation were schematically proposed in Fig. 6. When low dose of trehalose are present in the protein, the trehaloses replace the water around proteins for interaction with the proteins, inhibiting or delaying the hIAPP fibrillation and aggregation. While high dose of trehalose present in the proteins, trehalose can form the supermolecular cluster and even despoil water from hIAPP, resulting in the proteins excluded by trehalose cluster volume, and then fibrillated and aggregated. We conclude that lower dose of trehalose inhibits or delays, while higher dose of trehalose promotes the hIAPP fibrillation and aggregation.

Acknowledgements

The work was supported by the Natural Science Foundation of China (No. 21074025, 21374022, 81374032). Senior Visiting Scholar Foundation of Key Laboratory in Fudan University (No. 15FGJ03) and Construction project of the key disease of Shanghai combination of Chinese traditional and western medicine: Metabolic syndrome (No. zxbz2012-01).

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