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BIOPHYSICS

Vol. 292, No. 1, January, pp. 199-205, 1992

Amyloid-Like Properties of a Synthetic Peptide Corresponding to the Carboxy Terminus of ,&Amyloid Protein Precursor Claudia B. Caputo,‘p* Paul E. Fraser,?,?: Irene Evangelista

Sobel,* and Daniel A. Kirschnert$

*ICI Pharmaceuticals Group, Wilmington, Delaware 19897; tNeurology Research, Children’s Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115; and $Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115

Received June 28, 1991, and in revised form September 18, 1991

A synthetic peptide whose sequence corresponds to the 20 carboxy-terminal amino acids of @-amyloid protein precursor (APP) was found to form fibrils in uitro. These fibrils showed birefringence in polarized light when stained with Congo red, fluoresced when bound with thioflavin S, were resistant to proteases, and had across,f3 conformation. By contrast, peptides with other sequences from the intracellular domain of APP and a peptide corresponding to this entire domain did not exhibit the full range of &amyloid properties. These results suggest that a fragment from the C-terminus of the /3-amyloid protein precursor could bind to intraneuronal paired helical filaments and account for some of its amyloid-like properties. (0 1992 Academic Press, Inc.

Alzheimer’s disease is characterized by the presence of large numbers of senile plaques and neurofibrillary tangles in the hippocampus and neocortex. Senile plaques are extracellular lesions which sometimes contain an extracellular core deposit of P/A4 amyloid, comprised of a peptide of up to 43 amino acids (1). This amyloid peptide is also deposited in the meninges and its sequence is known (2). It appears to be derived from a larger protein precursor, referred to as fl-amyloid protein precursor (APP)’ (3). Synthetic peptides that correspond to part or all of the /?/A4 sequence spontaneously form fibrils in vitro which have optical propert.ies and X-ray diffraction patterns indicative of a P-pleated sheet conformation (4-9). These findings suggest that the P/A4 amyloid deposits in senile plaques may accumulate as the result of proteolytic 1 To whom correspondence should be addressed. * Abbreviations used: APP, /3-amyloid protein precursor; PHFs, paired helical filaments; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid. 0003.9861/92 rights

of reproduction

METHODS Sources of proteins and peptides.

The following peptides were synthesized: SP14 (amino acids 611-624 of APP695), QKLVFFAEDVGSNK; SP12 (amino acids 613-624 of APP695), LVFFAEDVGSNK; C-APP (amino acids 676-695 of APP695), KMQQNGYENPTYKFFEQMQN; APP2 (amino acids 649-668 of APP695), KKKQYTSIHHGVVEVDAAVT; and APP3 (amino acids 662-681 of APP695), EVDAAVTPEERHLSKMQQNG. Synthetic peptides were purified by reverse-phase HPLC. Purity and sequence identity were determined by amino acid sequencing. AL24 (amino acids 647-695 of APP695) was expressed in Escherichia coli and purified as described elsewhere (10). The locations of these peptides in the precursor protein are shown in Fig. 1. Microtubules were isolated from pig brain by two cycles of assembly as previously described (11) and neurofilaments were isolated from rat brain as previously described (12). Proteolysis. Peptides were dissolved directly in solutions of chymotrypsin or porcine pancreatic elastase in 0.08 M Tris buffer, pH 7.8, with 1.0 mM CaC12 or V8 protease in 0.1 M phosphate buffer, pH 7.8, and incubated for one h at 37°C. Proteins were mixed with the proteases under the same conditions. Proteolysis of microtubles and neurofilaments was confirmed by loading 10 pg of protein onto 4-20% gradient SDSPAGE gels, electrophoresing them at 40 mA, and staining the gels with pro-blue. 199

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processing of the precursor to generate an amyloid peptide containing the P/A4 sequence. The sequence of APP suggests that it may be a transmembrane protein (3). Proteolysis of the precursor to release the P/A4 sequence would also release one or more fragments that contain the extracellular domain as well as one or more fragments containing the transmembrane and intracellular domains. It is not clear what the subsequent fate of the fragment containing the carboxy terminus would be and whether it would remain within the neuron. However, another amyloid structure accumulates within neurons as paired helical filaments (PHFs). The complete composition of these PHF amyloid fibrils is unknown, as is the basis for their amyloid properties. We investigated whether peptides corresponding to APP sequences from the intracellular domain displayed amyloidlike properties.

in any

form

reserved.

200

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transmembrane

extracellular PIA4

intracellular

-

C

Njp ‘sp12’

’

APPP ’

sp14

’

’ APP3

C-APP

’

’

ALZ4

FIG. 1. Schematic diagram of APP695 illustrating the locations of the sequences for the peptides used in this study; see Methods section for the amino acid sequences of these peptides.

Electron microscopy. Peptides (except ALZ4) were dissolved in water at 10 mg/ml, incubated at 37”C, pipetted onto carbon and formvarcoated grids, and stained with 2% uranyl acetate. To observe assemblies from APPS, it was necessary prior to negative staining to expose the grids to glutaraldehyde vapor, a procedure that we found stabilized preformed assemblies. ALZ4 was analyzed at 0.2 mg/ml in 0.1% TFA or at 4 mg/ml in water or was treated with 8 M urea and then dialyzed to remove the urea. Tuhulin samples were fixed in 1% glutaraldehyde in assembly buffer (11) prior to staining. Neurofilaments were prepared at 2 mg/ml in 25 mM Tris buffer, pH 7. Specimens were examined with a Zeiss EM-1OA electron microscope with an accelerating voltage of 60 kV or a JEOL 100-S electron microscope at 80 kV. Micrographs were recorded at 25,000 or 50,000X magnification. Light microscopy. Samples were smeared on glass slides, fixed in 95% ethanol, stained with 0.05% Congo red in 50% glycerol, and observed under polarized light or stained with 0.01% thioflavin S in 50% glycerol and observed with fluorescent light using a Reichert Jung Polyvar light microscope equipped with a V2 filter. All samples were also observed by phase microscopy to verify sample locations on each slide. Samples were prepared by gradual deX-ray diffraction analysis. hydration of about 10 mg per milliliter peptide solutions in siliconized glass capillaries while in a permanent magnet of 2 T field strength. Such an external magnetic field has been shown to orient fibrillar assemblies (8, 13). Collection of diffraction patterns and analysis of them were carried out as previously described (8, 9).

RESULTS

Abundant fibrils were present in the Fibril formation. C-APP samples (Fig. 2, left panels). These fibrils were about 70 A wide, were uniform in width, and occassionally showed a central groove suggestive of constituent filaments. The fibrils were often curved. In some preparations, the C-APP fibrils showed extensive lateral aggregation (Fig. 2). When APP2 preparations on grids were fixed by glutaraldehyde vapor before negative staining, two kinds of fibrils were observed: some were about 40 A in diameter, and others were about twice as thick (Fig. 3). Little or no lateral aggregation of APP2 fibrils was apparent. Unlike the thin fibrils formed by C-APP and APPS, assemblies formed by SP12 (not shown) and SPl4 were flattened or twisted ribbons (Fig. 2); however, under certain conditions, e.g., at pH t3, SP14 does form narrow amyloid-like fibrils (9). APP3 also failed to form fibrils, although it occasionally formed sheet-like structures. ALZ4 did not form fibrils under the conditions that were used here to generate C-APP fibrils. The C-APP sequence conProteolytic susceptibility. tains one to six sites for cleavage by each protease. Even

ET AL.

when lyophilized SP14 and C-APP peptides were added directly to the protease solutions, no noticeable changes in the appearance or abundance of fibrils were observed for either peptide (Fig. 2). However, both microtubules and neurofilaments were substantially degraded by these proteases under the same conditions, with no detectable intact protein remaining on SDS-PAGE gels after any of the protease treatments. C-APP Stainingproperties of syntheticpeptidefibrils. fibrils showed green birefringence under polarized light following staining with Congo red and fluoresced after thioflavin S staining (Fig. 4). Both SP12 and SP14 also showed such birefringence and fluorescence, while APP2 and APP3 and the cytoskeletal fibrils all failed to show either birefringence or fluorescence. X-ray diffraction analysis. Patterns from SP14 and APPl were highly oriented, while those from APP2 and APP3 were only slightly oriented or completely unoriented, respectively (Fig. 5). SP14 showed a prominent sharp reflection at 4.8 A on the meridian and a diffuse band at 10.5 A on the equator, which correspond to the hydrogen-bonding distance between P-strands and the spacing between pleated sheets, and which are characteristic of the cross-0 conformation. The other reflections on the equator arise presumably from the face-to-face packing of the ribbon-like assemblies during drying. The other wide-angle reflections indexed according to a pseudo-orthogonal lattice similar to that of P-keratin, as previously described (5, 8). C-APP also showed a prominent hydrogen-bonding spacing at 4.8 A on the meridian, but only a very weak intersheet spacing at about 8.9 A. The strongest scatter from C-APP was at small angles around the central beam stop, and the asymmetric shape of this scatter indicates that the diffracting units are elongated in the hydrogenbonding direction along the fiber axis. The 20°-30” fanning of the equatorial scatter from the center toward the edges of the film, and the weak layer-line at about 120 A and centered about 40 A off the meridian suggest that the axis defined by the H-bonded polypeptide chains is tilted or twisted lo”-15” relative to the fiber axis. The partially oriented patterns from APP2 were also consistent with a cross-p conformation; however, H-bond spacings at both 4.8 and 4.7 A were present. This apparent heterogeneity may be accounted for by slightly different packing of P-strands in the single and double fibrils seen in the electron micrographs. The broad, diffuse bands centered at about 10 and 4.4 A in samples from APP3 indicate an amorphous-type pattern suggesting no regular secondary structure. To record X-ray patterns from ALZ4 required exposure times considerably longer than those for the other peptides. The weak patterns showed circularly symmetric broad rings centered at 10 and 4.6 A spacing, and sharp rings at 42 and 4.2 A. While the broad reflections are

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‘. ‘.

FIG. 2. Electron micrographs of C-APP (A-C) and SP14 (D-F) peptides before (A,B,D,E) and after (C,F) porcine pancreatic elastase treatment. Samples were negatively stained with 2% uranyl acetate. Magnification, 75,000X. B shows lateral aggregation of C-APP fibrils. The central groove suggestive of constituent filaments can be seen by tilting the figure and viewing obliquely along the fibril. Bar, 1000 A.

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FIG. 3. Electron micrograph of APP2 showing the presence of two sizes of fibrils (arrows and arrowheads). aldehyde vapor and negatively stained with 2% uranyl acetate. Bar, 1000 A.

consistent with a P-conformation, the sharp rings are suggestive of scatter from residual lipids in the sample. High-performance thin-layer chromatography for polar and neutral lipids confirmed the presence of a complex mixture of neutral lipids in the ALZ4 sample (data not shown). TABLE Summary

Sample

Congo red”

/3-Amyloid SP14 SP12 C-APP APPZ APP3 ALZ4

+ + t + -

Thioflavin S + + + + -

I

of Observations

Morpholog$ 40- to 90-A fibrils Ribbons, sheets Slabs/sheets 70- to 80-A fibrils 40- and 80-A fibrils Sheets Amorphous

Conformation’ cross-p Cross-p P cross-p Cross-p Amorphous Bd

’ “+” indicates sample showed green birefringence after staining with Congo red and viewed through crossed polarizers, or fluorescence after staining with Thioflavin S; “-” indicates absence of these features. b Morphology assessed by electron microscopy; measurements made from electron micrographs. ’ Assessed by X-ray diffraction from partially dried samples. d Characteristics of hydrogen-bond spacing and intersheet spacing were present but extremely weak.

The sample was fixed with glutar-

DISCUSSION

C-APP assemblies resemble fibrils from known amyloid sequences based on the properties studied here. They are similar in dimensions and appearance, show proteolytic resistence, exhibit birefringence with Congo red staining, fluoresce with thioflavin S staining, and have a cross-p conformation. While APP2 had a fibrillar morphology and cross-p conformation similar to those of P-amyloid (14) and /3-amyloid homologues (4-6,8,9), it did not show the characteristic tinctorial properties. The SP14 samples examined here showed Congo red birefringence and a cross-p structure, but assembled into ribbons rather than amyloid-like fibrils. Our findings emphasize that in evaluating whether synthetic peptides or protein fragments form amyloid-like structures, one must consider all three properties, i.e., tinctorial, morphological, and conformational (Table I). Surprisingly, a peptide corresponding to the entire intracellular domain of the precursor of 49 amino acids failed to form fibrils, even though it contained the entire sequence of the C-APP peptide. This observation suggests that perhaps only a proteolytic fragment of the intracellular domain of APP is capable of P-pleated sheet formation. Proteolytic processing is known to generate other amyloid peptides from precursor proteins including not only the P/A4 precursor but also for example prion protein

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of C-APP (A-C) and SP14 (D-F) peptides stained with Congo red and viewed by polarized (A,D) and cross-polarizeL FIG. 4. Birefringence (B,E) light, and fluorescence of thioflavin S-stained peptides (C,F). Peptides at 10 mg/ml in water were smeared on coverslips and fixed in ethanol prior to staining.

in the brain (15, 16), islet amylid polypeptide precursor, (17) and Bence Jones proteins (18). PHFs display amyloid properties similar to those which were observed for C-APP (14, 19-21). Recent immuno-

logical data suggest that the carboxy terminus of APP may also be associated with PHFs in tangles (22) and in the dystrophic neurites of plaques (23,24). Although it is not known how these APP fragments are bound to PHFs,

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FIG. 5. X-ray diffraction patterns from (A) SP14, (B) C-APP, (C) APPZ, (D) APP3, and (E) ALZ4. Arrowheads indicate the hydrogen-bond spacing at 4.7-4.8 A, and arrows mark the intersheet spacing at 9-11 A. Exposure times for (A-D) were 20 to 24 h and the exposure time for (E) was 53 h. In (E), note the sharp small angle scatter at 42 A, which is suggestive of lipids (open arrowhead).

from the present results it seems possible that these APP fragments could contribute to the P-pleated sheet conformation found for PHFs. Such a conformation may underlie their high degree of insolubility and resistence to proteolysis, which are probably important biologically since they may lead to accumulation of amyloid in tissues. The presence of P/A4 in PHFs has been the subject of several studies. A sequence corresponding to P/A4 was obtained from a PHF-enriched preparation in one study and an antibody to this sequence reacted with tangles in situ (25). However, in several other studies P/A4 antibodies failed to react with tangles (26-29). P/A4 epitopes seem to be located on material that adheres to the PHF but may not be an integral part of the PHF structure itself (30, 31). 7 protein is known to be present in PHFs but by itself has not been demonstrated to form fibrils or exhibit amyloid-like properties. For example, T is soluble and proteolytically susceptible (32) until it is bound to or exists as PHFs. Perhaps T may also form P-pleated sheets by interacting with other proteins in the PHFs. Our present

results suggest that fragments from the carboxy terminus of APP, which likely remain in the cytosol after /3-amyloid fragments are released into the extracellular space, may also contribute to the amyloid-like nature of PHFs. Such fragments may be derived from proteolytic processing steps that are the same as or different from those that generate PIA4 fragments. Such an association between PHFs and APP would provide a direct biochemical link between the two major amyloid deposits of Alzheimer’s disease. ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Andrea Klika, William Brunner, and Jack T. Nguyen. We thank Dr. Clay Scott for providing pig brain microtubules, Drs. Peter Barth and David Blowers for providing ALZ4 peptide, Dr. Craig Thornber for providing the synthetic peptides, and Dr. Hideyo Inouye for helpful discussions. The research was supported in part by the American Health Assistance Foundation (D.A.K.), NIH-NIA Grant AGO8572 (D.A.K.), NIH-NICHD Center Grant HD-18655 (Children’s Hospital), and the Medical Research Council of Canada (P.E.F.).

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