©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The -Helical to -Strand Transition in the Amino-terminal Fragment of the Amyloid -Peptide Modulates Amyloid Formation (*)

(Received for publication, November 1, 1994)

Claudio Soto (§) Eduardo M. Castaño (1) Blas Frangione (1) Nibaldo C. Inestrosa (¶)

From the Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile and the Department of Pathology, New York University, Medical Center, New York, New York 10016

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Amyloid-beta peptide (Abeta) consists of a hydrophobic C-terminal domain (residues 29-42) that adopts beta-strand conformation and an N-terminal domain (amino acids 10-24) whose sequence permits the existence of a dynamic equilibrium between an alpha-helix and a beta-strand. In this paper we analyzed the effect of the alternate N-terminal conformations on amyloid fibril formation through the study of the analogous Abeta peptides containing single amino acidic substitutions. The single mutation of valine 18 to alanine induces a significant increment of the alpha-helical content of Abeta, determined by Fourier transform infrared spectroscopy and circular dichroism and dramatically diminishes fibrillogenesis, measured by turbidity, thioflavine T binding, Congo red staining, and electron microscopic examination. In hereditary Dutch cerebral hemorrhage with amyloidosis (a variant of Alzheimer's disease), the substitution of glutamine for glutamic acid at position 22 decreased the propensity of the Abeta N-terminal domain to adopt an alpha-helical structure, with a concomitant increase in amyloid formation. We propose that Abeta exists in an equilibrium between two species: one ``able'' and another ``unable'' to form amyloid, depending on the secondary structure adopted by the N-terminal domain. Thus, manipulation of the Abeta secondary structure with therapeutical compounds that promote the alpha-helical conformation may provides a tool to control the amyloid deposition observed in Alzheimer's disease patients.


INTRODUCTION

Alzheimer's disease (AD) (^1)is the most common form of dementia in adults. AD is characterized neuropathologically by amyloid deposition in the form of neuritic plaques and congophilic angiopathy as well as by the formation of neurofibrillary tangles(1) . The main component of amyloid is the 4.3-kDa amyloid beta-peptide (Abeta), which is part of a much longer precursor protein (APP) codified in chromosome 21(2) .

Amino acid sequence analyses of the Abeta peptide by the Chou-Fasman (3) and Garnier-Osguthorpe-Robson (4) methods indicate that the probability of finding beta-strand conformation in Abeta is high within the C-terminal region after residue 28. The region between amino acids 10 and 24 presents a high and similar probability to display alpha-helix or beta-strand conformation(5) . There are also two probable beta-turns between residues 6 and 8 and between residues 24 and 29. Using synthetic peptides and spectroscopic techniques, the secondary structure assignments obtained by predictive methods have been confirmed(6, 7, 8, 9) .

While the hydrophobic segment in the C-terminal domain of Abeta invariably adopts a beta-strand structure in aqueous solutions, independently of pH or temperature conditions, the N-terminal domain can show different conformations and solubilities depending on environmental conditions(8, 9, 10) . In fact, the N-terminal domain exists as a soluble monomeric alpha-helical structure at pH 1-4 and pH greater than 7. However, at pH 4-7 it rapidly precipitates into an oligomeric beta-sheet structure. Furthermore, it adopts an alpha-helical conformation in a membrane-mimicking solvent that promotes intramolecular hydrogen bonding(8) . Thus, Abeta exists in two alternative conformations depending on the secondary structure adopted by the N-terminal domain. These alternative conformations have different solubility properties and may determine changes in the rate of amyloid fibril formation. We had hypothetized that the structure adopted by the N-terminal domain of Abeta is important in amyloid fibril formation(5) . In the present work we have evaluated this possibility by analyzing the ability of Abeta sequences containing single mutations in its N-terminal domain to form amyloid fibrils. Specifically we studied amyloid formation using peptides containing the mutation of valine to alanine at residue 18, and glutamic acid to glutamine at position 22 (Dutch variant of Abeta).


EXPERIMENTAL PROCEDURES

Predicted Secondary Structure

The alpha-helix, beta-sheet, and beta-turn propensities for different sequences of Abeta peptide were calculated by the Chou and Fasman secondary structure prediction algorithm(3) , using the program Protylze version 3.01 from Copyright.

Peptide Synthesis and Characterization

Fragment 1-40 of the Abeta wild-type peptide (SP40) was obtained from Sigma. Peptide 1-40, containing a valine to alanine mutation in position 18 (SP40A), was synthesized by Chiron Corp. Inc., Emeryville, CA. The Dutch variant of Abeta (SP40Q) was synthesized by using solid-phase techniques at the Center for the Analysis and Synthesis of Macromolecules, State University of New York, Stony Brook, NY. All peptides were purified by reverse-phase high performance liquid chromatography, and their purity was evaluated by amino acid sequence analysis.

Preparation of the Peptide Samples

Stock solutions of the peptides were prepared by dissolving them in 50% acetonitrile. The concentration of the stock solution was determined by amino acid composition analysis on a Waters Pico-Tag amino acid analyzer, after hydrolyzing the samples under reduced pressure in the presence of 6 M HCl for 20 h at 110 °C. Next, peptide aliquots were lyophilyzed and resuspended in the buffer used in the aggregation or amyloid detection assay.

Fourier Transform Infrared Spectroscopy

IR spectra were collected in a Perkin-Elmer System 2000 FT-IR operated at 2 cm resolution. The peptide solutions (20 mg/ml) were prepared in 25 mM HEPES disolved in ^2H(2)O, pD 7.5. Electrode readings were uncorrected for deuterium effects. Sample aliquots (20 µl) were placed in demountable cells containing CaF(2) windows separated by 50-µm Teflon spacers. The trifluoroacetic acid (used in high performance liquid chromatography purification of the peptides) was removed as described by Fraser et al.(11) .

Circular Dichroism Measurements

CD spectra were obtained with a Jasco spectropolarimeter, model J-720 at room temperature in a 0.1-cm path-length cell. Double distilled and deionized water and TFE (spectroscopy grade) were used as solvents. Spectra were recorded at 1-nm intervals over the wavelength range 180 to 260 nm. Results are expressed in terms of molar ellipticity () in units of degrees cm^2 dmol. All the spectra were obtained by subtracting buffer base-line spectra and smoothed by using the algorithm provided by Jasco. The approximate percent of alpha-helical content was determined using the 208 nm absorption (12) according to the following equation: % alpha-helix = ([] - 4000/-33000 - 4000) times 100.

Aggregation Studies

Lyophilized aliquots of the peptides were resuspended in 0.1 M sodium acetate, pH 5.0. At various times, the aggregation was detected via turbidity measurements at 405 nm as described previously by Jarrett et al.(13) .

Amyloid Detection in Suspension

Amyloid quantification in suspension was performed by the thioflavine T method(14, 15) , with a few modifications. In short, 1.5 µM thioflavine T (ThT) in a buffer of 50 mM glycine, pH 9.5, was added to peptide aliquots previously incubated in 0.1 M Tris-HCl, pH 7.5. Immediately thereafter, the fluorescence was monitored at excitation 435 nm and emission 490 nm on a Hitachi 200 spectrofluorometer. A time scan of fluorescence was performed, and three values after the decay reached a plateau (280, 290, and 300 s) were averaged after subtracting the background fluorescence of 1.5 µM ThT. The mean ± standard deviation for three separated experiments made in duplicated is shown in Fig. 2B and Fig. 4B.


Figure 2: Aggregation and amyloid formation by SP40 and SP40A. The aggregation level obtained with different concentrations of SP40 (squares) and SP40A (circles) was studied (A). The peptides were incubated for 48 h at 25 °C in 0.1 M sodium acetate buffer, pH 5.0, at various peptide concentrations, from 1 µg/ml to 4 mg/ml. The aggregation was measured by the turbidity at 405 nm, as described in ``Experimental Procedures''. The value shown represent the mean between 2 different experiments. The amyloid formation was quantified through the ThT method (B). Aliquots of each peptide in a concentration of 1 mg/ml (0.25 mM) were incubated for 15 h in 0.1 M Tris-HCl, pH 7.5, at room temperature. Then the amyloid formation was quantified as described under ``Experimental Procedures''. The protein concentration for BSA and ubiquitin was 1 mg/ml. The graph shows the fluorescence emission, in arbitrary units, of ThT bound to the amyloid formed in the presence of the peptides indicated in the x axis. The value shown correspond to the average ± standard deviation of three different experiments made in duplicated.




Figure 4: Aggregation and amyloid formation by SP40 and SP40Q. This figure was performed under the same experimental conditions described in Fig. 2. In the turbidity measurement (A) the time of incubation was 24 h. In the fluorescence studies (B) the incubation time was 15 h. Both experiments were performed in a peptide concentration of 1 mg/ml. The mean ± standard deviation of three different experiments made in duplicated is shown.



Congo Red Staining

Amyloid detection by Congo red was performed as described previously(16) .

Electron Microscopy

For fibril formation, the peptides (1-2 mg/ml) were incubated in phosphate-buffered saline for 5 days at room temperature. The 300-mesh Formvar-coated nickel grids were floated on the peptide solutions, air-dried, and negatively stained with uranyl acetate. The specimens were viewed with a Philips EM-300 electron microscope at 100 kV.


RESULTS

To study the relation between the secondary structure of Abeta and its ability to form amyloid fibers, we designed a mutation that does not produce a significant change in the ionic or hydrophobic properties of Abeta but is sufficient to increase the propensity of the N-terminal domain of Abeta to adopt the alphahelical conformation. A peptide in which valine at position 18 of Abeta was replaced by alanine (SP40A) was synthesized. This modification considers that valine at position 18 is the residue that has the highest probability value to exist as a beta-strand in the N-terminal region of Abeta. Valine is an amino acid that destabilizes the alpha-helix, whereas alanine is a very good helix-former in aqueous solution. The above statement is supported by thermodynamic considerations(17, 18, 19) , by comparative studies of the helix-forming tendency in synthetic peptides(20) , and by statistical surveys(3, 4) . In fact, by comparative analysis of the secondary structure of both peptides through FT-IR, we found that the modified peptide contains a significant amount of alpha-helical structure (Fig. 1A). A quantitative analysis of the spectra gave the following percentage of secondary structure for SP40A: 32.8% of alpha-helix, 31.3% of beta-sheet, 24.7% of random coil, and 11.2% of beta-turn, while the percentage of secondary structure for the control peptide, containing the 1-40 sequence of Abeta (SP40), was: 1.1% of alpha-helix, 60.4% of beta-sheet, 31.4% of random coil, and 7.1% of beta-turn. The higher content of alpha-helix in the modified peptide was additionally found through CD studies in the presence of 20% TFE (Fig. 1B). The alpha-helical content for SP40 and SP40A in this solvent was 16.6% and 40.9%, respectively. The higher level of alpha-helix obtained in the last experiment may be due to the presence of TFE, which is a solvent that stabilizes the alpha-helical conformation (21) .


Figure 1: Fourier transform infrared and Circular dichroism studies of wild type and mutant Abeta peptides. In A is shown the FT-IR spectra taken under the following conditions; peptide aliquots of 20 mg/ml (20 µl) prepared in 25 mM HEPES (disolved in ^2H(2)O), pD 7.5, were placed in CaF(2) cells and the spectra were collected at 2 cm of resolution taking 500 scans. The spectra obtained with SP40 (dashedline) and SP40A (solidline) are shown. The arrows indicate the position of the band in the amide I region corresponding to the alpha-helix (approx1653 cm), beta-sheet (approx1626 cm), beta-turn (approx1696 cm), and random coil (approx1644 cm) structures. In B the CD spectra of SP40 (solid line), SP40A (dashed line), and SP40Q (dotted line) is shown. Aliquots of peptides were disolved in 20% TFE prepared in 10 mM sodium phosphate, pH 7.4. The peptide concentration was 0.15 mg/ml in a final volume of 0.3 ml. The spectra were recorded at room temperature as described under ``Experimental Procedures.''



The comparative study of the solubility properties of SP40 and SP40A was performed by means of turbidimetric measurements. After 48 h of incubation, SP40A became aggregated to a lesser extent than SP40 at equal concentration (Fig. 2A). Thus, at a peptide concentration of 4.0 mg/ml, SP40A exhibits 80% less aggregation than the control. However, an increase in the turbidity indicates only aggregation, but not necessarily amyloid formation. To semi-quantify the amount of amyloid formed under each condition, we used a novel method based on the fluorescence emission by thioflavine T (ThT) bound to amyloid(14, 15) . ThT binds specifically to amyloid and this binding produces a shift in the emission spectra and a fluorescence enhancement proportional to the amount of the amyloid formed(15) . The ability to form amyloid, measured by the ThT method, was compared between SP40 and SP40A. The modified peptide showed very little fluorescence in comparison with SP40 at a concentration of 1 mg/ml (Fig. 2B). To further evaluate the ability of SP40A to form amyloid, we performed staining with Congo red. Light microscopic examination after Congo red staining showed that only SP40 (1 mg/ml), incubated 5 days, displayed a significant green birefringence under polarized light, while the modified peptide exhibited only a slight green birefringence under the same conditions (data not shown). Furthermore, the presence and the morphology of the fibrils formed by both peptides was studied by electron microscopy. Both SP40 (Fig. 3A) and SP40A (Fig. 3B) were able to form amyloid fibrils with the typical features described by these fibers(7, 16) . Although the morphology of the amyloid fibers was identical, the amount of this structure obtained in the electron microscopic grids was higher in SP40 than in SP40A, which strengthened the results described above.


Figure 3: Electron micrographs of negative-stained preparations of fibrils assembled from SP40 and SP40A. Aliquots of both peptides, SP40 (A) and SP40A (B), were adsorbed onto 300-mesh Formvar-coated grids and negative-stained with 2% uranyl acetate. The specimens were viewed for fibrils with a Phillips electron microscope. Magnification, times 66,000.



In order to additionally study the influence of the secondary structure of the N-terminal domain on the rate of amyloid formation, we analyzed the effect produced by the glutamine/glutamic acid substitution at residue 22 of Abeta. This substitution is found in hereditary cerebral hemorrhage with amyloidosis, Dutch type(22) . Previous studies with synthetic peptides have shown accelerated fibril formation in a 28-residue peptide homologous to the Dutch variant Abeta(23) . Our CD studies indicates that the Dutch mutation diminishes the level of alphahelical conformation in comparison to the wild-type human peptide (Fig. 1B). In fact, the alpha-helical content of a synthetic peptide containing the sequence 1-40 of Abeta but bearing the Dutch mutation (SP40Q), in the presence of 20% TFE was only 5.9%, in comparison with the 16.6% present in SP40. This result is in agreement with previous studies showing a higher amount of beta-sheet in the Dutch variant than the normal Abeta obtained by FT-IR and CD spectroscopy(24, 25) . To evaluate the effect of this change in the secondary structure, we compared the ability to form amyloid between SP40 and a SP40Q. The turbidity measurements showed that SP40Q became aggregated at a higher level than SP40 (Fig. 4A) after 24 h of incubation. The amyloid quantification using the ThT method showed that SP40Q formed 100% more amyloid than SP40 (Fig. 4B) after 15 h of incubation. This result indicates that the 1-40 peptide of the Dutch variant Abeta has accelerated amyloid fibril formation, as was previously shown for shorter peptides consisting of residues 1-28 and 21-28(23) . No differences were detected in the morphology of the amyloid fibrils formed for SP40Q, in comparison with SP40 (data not shown), which is consistent with previous studies(23, 24) .


DISCUSSION

In the present work we demonstrate that a single mutation of valine 18 to alanine produces a homologous Abeta peptide that is less able to form amyloid in vitro. This substitution does not significantly modify either the ionic or the hydrophobic properties of Abeta. However, this modification increases the alpha-helical content of Abeta as was observed through FT-IR and CD spectroscopy. Considering the position of the substitution and the analysis by secondary structure prediction, it is widely possible that the change in conformation occurs in the N-terminal domain of Abeta, which in SP40A would adopt mainly an alpha-helical structure. By contrast, in the Dutch variant of Abeta, the glutamine for glutamic acid substitution at residue 22 diminishes the propensity of the N-terminal region of Abeta to adopt the alpha-helical conformation concomitantly with an increase in amyloid formation. These findings suggest the existence of a correlation between the secondary structure of the N-terminal domain (amino acids 10-24) of Abeta and its ability to form amyloid fibrils.

In view of the foregoing results, we postulate that Abeta exists in an equilibrium between two alternative species in solution: one able to form amyloid, which presents a beta-strand conformation in its N-terminal domain; and another one unable to form amyloid, which adopts an alpha-helical structure in this region (Fig. 5). In fact, our recent studies have shown that after long incubation times, the non-sedimentable peptide fractions contain a large amount of alpha-helical and random coil structure, completely different to the structure of the sedimentable fraction, which presents almost completely a beta-sheet conformation. Recently, it has been proposed that in another type of amyloidosis the conversion of an alpha-helix into a beta-strand is a feature of the transformation of the normal cellular prion proteins into the pathological scrapie prion proteins(26) .


Figure 5: Schematic representation of the conformational equilibrium between alternative structures for Abeta, indicating some possible regulators of the transition. The structure shown at left should be the soluble form of Abeta, which is released by normal cells and is detected as a soluble entity in cerebrospinal fluid from normal and AD individuals(32, 33) . On the other hand, the structure shown at right should be the form of Abeta able to form aggregates(34, 35) , once the effective local concentration allows interactions between chains.



The existence of an equilibrium between alternatives conformations for Abeta is additionally supported by a recent study of the tridimensional structures of Abeta and its Dutch variant through NMR(27) . Furthermore, the finding that the aggregated Abeta is in equilibrium with a non-sedimentable form of the peptide (7, 28) supports the idea that in solution there are two species of Abeta differing in their abilities to form amyloid. When the N-terminal segment of Abeta adopts a beta-strand conformation, Abeta may exist as an anti-parallel beta-sheet capable of interacting with other peptides forming a cross-beta supersecondary structure. This structure has been proposed for the amyloid fibril, based on its x-ray diffraction pattern(29, 30) .

It has been shown previously that Abeta analogs in which hydrophobic residues (phenylalanine 19 and 20) were substituted by non-hydrophobic amino acids exhibit a markedly increased solubility as determined by sedimentation assays(8) . Accordingly, it was proposed that the formation of aggregates depends upon a hydrophobic effect that leads to intra- and intermolecular interactions between hydrophobic parts of Abeta. Although the effect obtained with SP40A could be explained by modifications in the hydrophobic properties induced by the substitution, this appears unlikely because the putative hydrophobicity changes in this case are small. Another study showing the effect of Abeta(1-28) with alanine substituted by lysine at position 16 has been reported previously(31) . In that case, the modified peptide formed beta-pleated sheet assemblies that were dissimilar to those formed by non-modified Abeta(1-28) and enhanced the packing of the sheets. However, in that work, the effect of the substitution in the aggregation kinetics was not studied.

Our data show that the N-terminal domain of Abeta may be important for modulation of peptide aggregation and amyloid fibril formation. This is an alternative view to the current model in which the determinant role for Abeta aggregation is placed on its C-terminal segment(6, 7, 13, 28) .

Our hypothesis may explain how the same amino acid sequence can exist in a soluble (32, 33) and an insoluble form(34, 35) . In fact, small perturbations in the conformational equilibrium could determine big changes in the ability of Abeta to form amyloid fibrils. These modifications of the equilibrium shown in Fig. 5could, for example, be caused by local pH changes, alterations of environmental hydrophobicity, or binding to other proteins, which could act as pathological chaperones such as proteoglycans (36) and apolipoprotein E (37) . The factors that eventually produce alterations in the conformational equilibrium of Abeta could be important risk factors in the development of AD, and could explain why not all persons shown the disease.

Finally, if the amyloid deposition is sensitive to the secondary structure adopted by the N-terminal domain of Abeta, as proposed in this work, compounds that promote the alpha-helical conformation in this region could prevent amyloid fibril formation. In fact, in the presence of 1,1,1,3,3,3-hexafluoro-2-propanol, an alpha-helix promoting solvent, Abeta is completely soluble up to a concentration of 40 mg/ml (28) . Thus, the hypothesis proposed in this paper opens a potential new avenue to search for therapeutic agents that can promote alpha-helix formation in the N-terminal domain of Abeta and could stop or delay the advance of AD.


FOOTNOTES

*
This work was supported in part by grants from FONDECYT (Grant 1940694 to N. C. I. and Grant 3930010/93 to C. S.), Sandoz Foundation for Gerontological Research (to C. S.), and National Institutes of Health Grants AG10953 and AG05891 (to B. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a postdoctoral fellowship from Fundación Andes de Chile.

To whom correspondence should be addressed: Molecular Neurobiology Unit, Catholic University of Chile, P. O. Box 114-D, Santiago, Chile. Fax: 56-2-2225515.

(^1)
The abbreviations used are: AD, Alzheimer's disease; Abeta, amyloid beta-peptide; APP, amyloid precursor protein; ThT, thioflavine T; FT-IR, Fourier transform infrared spectroscopy; TFE, 2,2,2-trifluorethanol.


ACKNOWLEDGEMENTS

We are grateful to Dr. Jorge Garrido from the Department of Cellular and Molecular Biology of the Catholic University of Chile, for aid with the electron microscope. We also appreciate the collaboration of Dr. Octavio Monasterio and Patricio Rodriguez from the Department of Biology of the Faculty of Sciences, University of Chile, for help with the FT-IR studies. Finally, we thank Drs. Thomas Wisniewski, Jaime Alvarez, and Hugo L. Fernández for helpful comments on the manuscript.


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