©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Interaction between Apolipoprotein E and Alzheimers Amyloid -Peptide Is Dependent on -Peptide Conformation (*)

(Received for publication, November 30, 1995; and in revised form, February 21, 1996)

Adam A. Golabek (1) (3) Claudio Soto (1) Tikva Vogel (2) Thomas Wisniewski (1)(§)

From the  (1)Department of Neurology, New York University Medical Center, New York, New York 10016, (2)Biotechnology General Ltd., Rehovot, Israel, and (3)Department of Neuropathology, Medical Research Center, Polish Academy of Sciences, Warsaw, Poland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

An important feature of Alzheimer's disease (AD) is the cerebral deposition of amyloid. The main component of the amyloid is a 39-44-amino acid residue protein called amyloid beta (Abeta), which also exists as a normal protein in biological fluids, known as soluble Abeta. A major risk factor for late-onset AD is the inheritance of the apolipoprotein (apo) E4 isotype of apoE. How apoE is involved in the pathogenesis of AD is unclear; however, evidence exists for a direct apoE/Abeta interaction. We and others have shown that apoE copurifies with Abeta from AD amyloid plaques and that under certain in vitro conditions apoE promotes a beta-sheet structure in Abeta peptides. Currently we document the high affinity binding of Abeta peptides to both human recombinant apoE3 and -E4 with a K of 20 nM. This interaction is greatly influenced by the conformational state of the Abeta peptide used. Furthermore, we show that the fibril modulating effect of apoE is also influenced by the initial secondary structure of the Abeta peptide. The preferential binding of apoE to Abeta peptides with a beta-sheet conformation can in part explain the copurification of Abeta and apoE from AD amyloid plaques.


INTRODUCTION

Amyloid deposition is one of the hallmarks of the Alzheimer's disease (AD). (^1)The major protein component of the amyloid, which is deposited in the form of senile plaques (SP) and cerebrovascular amyloid angiopathy, is a 39-44-residue protein, called amyloid beta-peptide (Abeta)(1, 2, 3, 4, 5, 6, 7) . Abeta is derived by proteolytic processing from a large transmembrane precursor termed beta-amyloid precursor protein(8, 9, 10, 11) . In both parenchymal and vascular amyloid deposits, there is heterogeneity at both the N and C termini of the peptide that is greater for the peptide isolated from SP amyloid (2, 3, 6, 12) . Immunohistochemical and biochemical studies suggest that the major initially deposited Abeta consists of Abeta 42/43 species (13, 14, 15, 16) . Both Abeta 40 and 42/43, as well as numerous shorter Abeta fragments, have a propensity to self-aggregate and form fibrils in vitro(17, 18, 19, 20) . However, it has been documented that longer Abeta pep-tides, extending beyond Val-40, display a higher propensity for aggregation and fibrillogenesis(20) . This leads to the hypothesis that Abeta 42/43 is critical for the formation of SP (21) , although this hypothesis has recently been questioned(22) . In 1992, a soluble form of Abeta, termed sAbeta, was found in cerebrospinal fluid and plasma of both healthy and AD patients(23, 24, 25, 26) . The circulating form of Abeta contains mostly Abeta 1-40 but also includes Abeta 1-42/1-28(27) . Thus, the fibrillogenic Abeta peptide can exist in both a soluble form and as a fibril in SP. These data raise the question of what factors determine the aggregation and fibrillogenesis of Abeta in vivo.

In vitro studies have shown that amyloid-like fibril formation by Abeta peptides is dependent on several factors such as the length of Abeta(20) , pH(28) , and metal ions(29, 30) . Recent data disclosed that a group of compounds, collectively termed as amyloid-associated proteins, could also significantly modulate the process of Abeta fibrillogenesis. Apolipoprotein E (apoE) is one of the proteins shown in vivo to be associated with Abeta amyloid and other cerebral and systemic amyloids(31, 32) . It was proposed that apoE could play a role of ``pathological chaperone''-promoting aggregation/fibrillogenesis of Abeta peptide, possibly through propagation of a beta-sheet conformation(31, 33, 34, 35) .

ApoE in humans is expressed in three common isoforms of E2, E3, E4 that differ by the presence or absence of cysteine at two amino acid residues, 112 and 158(36) . Genetic studies have shown that inheritance of apoE4 is a significant risk factor for late onset AD, decreasing the age of onset and the duration of disease(37, 38) . Although apoE4 is neither necessary nor sufficient to cause AD, apoE4 carriers appear to have a higher amyloid load(39) . There are a number of ways by which apoE may be involved in the pathogenesis of AD. For example, a differential effect on neurite outgrowth of apoE3 and -E4 has been demonstrated(40, 41, 42) . However, several pieces of evidence also suggest a direct apoE/Abeta interaction. Under in vitro conditions, apoE hydrophobically binds Abeta, forming complexes resistant to SDS, as well as organic and chaotropic solvents(38, 43, 44) . Initially, a striking difference between apoE3 and apoE4 binding to Abeta was found, showing that apoE4 binds Abeta peptide more avidly(45) , but a recent study has demonstrated that this is dependent on whether apoE is associated with lipids(46, 47) . In addition to binding Abeta, apoE promotes fibril formation by Abeta(33, 34, 35) , and other synthetic amyloidogenic peptides(48) , under the conditions used in several studies; however, some investigators (49) have not found apoE to promote Abeta fibrillogenesis. Recent biochemical studies have indicated that apoE is complexed in vivo to the amyloid in both AD senile plaques (44, 50) and systemic amyloid deposits of serum amyloid A and immunoglobulin light chain(51) . These in vivo and in vitro data suggest a potentially important role of apoE in amyloidogenesis in vivo. However, the nature of apoE interaction with Abeta remains to be clarified. The aim of this study was to analyze the sequential and structural determinants of the Abeta/apoE interaction.


EXPERIMENTAL PROCEDURES

Synthetic Peptides and Proteins

The following commercial and custom-synthesized lots of Abeta peptides were used as follows: from Sigma, Abeta 1-40 (lot no. 93H49502, referred to as S1-40), Abeta 12-28 (lot no. 42H38602), Abeta 1-16 (lot no. 124H00271), Abeta 10-20 (lot no. 14H08951), Abeta 25-35 (lot no. 74H0715); from Bachem (King of Prussia, PA), Abeta 1-40 (lot no. 506063, B1-40). In the Center for Analysis and Synthesis of Macromolecules (SUNY, Stony Brook, NY) the following peptides were synthesized: Abeta 1-40 (C1-40), Abeta 1-40Q (Gln for Glu substitution at residue 22), and Abeta 1-40G (Gly for Glu substitution at residue 22). Abeta 1-40 (Y1-40) and Abeta 1-42 were also synthesized at W. M. Keck Foundation, New Haven, CT. All peptides were purified on a semipreparative 1 times 25-cm C4 RP-HPLC column with the linear gradient of 0-80% acetonitrile in 0.1% trifluoroacetic acid at flow rate of 2 ml/min. Purity of the peptides was verified by analytical RP-HPLC and mass spectrometry. Stock solutions of the peptides were made at 2 mg/ml in 50% acetonitrile, 0.1% trifluoroacetic acid and quantitated by amino acid analysis on a Waters AccQ-Tag System (Millipore Corp., Milford, MA). Nonamyloidogenic and amyloidogenic conformers (Abeta and Abeta) of Abeta C1-40 were obtained as described(52) . Briefly, peptide was dissolved at 1 mg/ml in 100 mM Tris, pH 7.4, and incubated for 1 week. After centrifugation at 15,000 times g for 10 min, pellet Abeta was dissolved in 50% acetonitrile and quantitated by amino acid analysis. Peptide from the supernatant Abeta was quantitated by analytical HPLC. Escherichia coli expressed human recombinant apoE3 was obtained from BioTechnology General (Israel, lot 738-1)(53) . Human recombinant apoE2, apoE3, and apoE4 were also purchased from PanVera Corp. (Madison, WI) and apoE3 and apoE4 from Calbiochem.

Secondary Structure Analysis

Secondary structure of the Abeta peptides was analyzed by circular dichroism. The peptides were dissolved in 10 mM phosphate buffer, pH 7.4, at a concentration of 0.15-0.20 mg/ml and centrifuged to remove precipitated/undissolved material. The CD spectrum was recorded using a 0.1-cm path length cell, on a Jasco 720 spectropolarimeter (Jasco, Japan) controlled by a 486 IBM computer and the Jasco software. Five consecutive readings at 1-nm bandwidth, response time of 0.25 s, and resolution at 1 nm were taken from each sample and averaged, base line-subtracted, and noise-reduced. Results are expressed in terms of molar ellipticity (). Spectra were analyzed with the Lincomb algorithm(54) . In addition, after CD measurements, all Abeta peptides were subject to gel filtration on a Superose 12 HR column (Pharmacia Biotech Inc.) to assess the aggregation state. The column was run in 50 mM Tris, pH 7.4, 150 mM NaCl, at 0.4 ml/min, with the absorbance of eluted fractions measured at 220 nm.

Solid-phase Binding Studies

The interaction of Abeta and apoE was evaluated by enzyme-linked immunosorbent solid phase assay, adapted from Matsubara et al.(55) . Standard microtiter plates Immulon-2 (Dynatech Lab, Chantilly, VA) were coated with Abeta C1-40 peptide at a concentration of 400 ng/well/100 ml in 50 mM carbonate/bicarbonate buffer, pH 9.0, for 2.5 h at 37 °C. Under these conditions 10 ng/well of the peptide bound to the plate. The remaining sites on the plate were blocked with 1% bovine serum albumin and 1% gelatin in PBS, overnight at 4 °C. After washing in PBS, apoE was applied (overnight at 37 °C) at a concentration of 0-200 nM in 0.1% bovine serum albumin in PBS. Following incubation, the plates were washed three times in PBS, and bound apoE was evidenced with monoclonal anti-apoE antibody 3D12 (Biodesign, Kennebunk, ME) applied at a dilution of 1:3000 in PBS for 2.5 h at room temperature. Following three PBS washes, a secondary antibody, goat anti-mouse alkaline phosphatase-linked (BioSource, Camarillo, CA), was applied at a dilution of 1:3000 in PBS for 45 min at room temperature. After washing, alkaline phosphatase was revealed with p-nitrophenyl phosphate in diethanolamine buffer (Bio-Rad) for 20 min. The reaction was terminated with 0.4 M sodium hydroxide and quantitated at 405 nm on a 7520 Microplate Reader (Cambridge Technology, Watertown, MA). Data were analyzed by a nonlinear regression fit algorithm by GraphPad Prism v 1.1 (GraphPad Software, San Diego, CA). Controls for nonspecific binding included binding of apoE to wells without peptide and omission of the primary and/or secondary antibodies.

To quantitate the relative affinity of different Abeta analogs and its different lots, competitive inhibition studies were performed. Plates were coated with Abeta C1-40 in the same manner as for the saturation curve for apoE. Following blocking, a saturating amount of apoE was added (150 nM) along with serial dilutions of different Abeta peptides at a concentration of 0-50 µM, in a volume of 100 µl. This allowed for competition between the peptide immobilized and peptides in solution for apoE binding and the calculation of IC values.

Fibrillogenesis

To quantitate the affect of apoE on different fragments of Abeta and different lots of full-length peptide, we used a Thioflavin T assay(56, 57) .

Abeta peptides were dissolved at 250-1000 µM, with or without 2.5 µM apoE (peptide:apoE molar ratio 100-400:1), in 100 mM Tris, pH 7.4, in 30-µl aliquots. After 3 days of incubation, samples were diluted to 2 ml in 50 mM glycine, 2 µM Thioflavin T (Sigma), pH 9.2, and were read on Hitachii F-2000 spectrofluorometer at excitation and emission wavelengths 435 and 485 nm, respectively. Time scans were performed, and values at 280, 290, and 300 s were averaged to give the final reading.


RESULTS

Incubation of serial dilutions of recombinant apoE3 with wells coated with Abeta 1-40 revealed a dose-dependent binding that reached saturation at 150 nM (Fig. 1.). Based on our previous data of 1:1 stoichiometry of apoE to Abeta binding (58, 59) , K(D) was calculated as 20 nM ± 2 (S.D.). The affinity of recombinant apoE4, apoE3, and apoE2 to Abeta under these conditions was in a similar range (data not shown). Therefore, all the following experiments were performed on the most frequent apoE isoform in humans, apoE3. These experiments were performed on delipidized apoE; however, different states of lipid association may affect the K(D) of the binding of apoE with Abeta.


Figure 1: Saturation curve for apoE binding to immobilized Abeta. Abeta C1-40 was immobilized at 400 ng/well and incubated in the presence of serial dilutions of apoE (0-150 nM). Bound apoE was revealed by using monoclonal antibody 3D12. Using nonlinear regression fit, the K value of this interaction was calculated as 20 nM. Plotted data represent mean values (±S.D.) from three independent experiments performed in duplicate.



To characterize the binding site of apoE on Abeta 1-40, we used a competitive inhibition assay using different Abeta fragments, which were allowed to interact with apoE in solution in the presence of immobilized full-length peptide. The ability of Abeta 1-16, 10-20, 12-28, and 25-35 to inhibit binding of apoE to Abeta 1-40 was tested. None of these short Abeta peptides, which together span almost the entire Abeta molecule, were able to significantly inhibit this interaction (Fig. 2A). Since we found that sequence determinants are not decisive for Abeta-apoE binding, we have looked for conformational aspects of this interaction.


Figure 2: Inhibition of apoE and Abeta 1-40 interaction by using different shortened and full-length Abeta peptides. A, inhibition by peptides Abeta 12-28 (), Abeta 25-35 (up triangle), and Abeta C1-40 (). B, comparison of the inhibitory effect of some full-length Abeta peptides, Abeta Y1-40 (), Abeta C1-40 (up triangle), Abeta 1-42 (circle), and Abeta B1-40 (). IC values for these inhibitions are presented in Table 1. Plotted data represent mean values (±S.D.) from three independent experiments done in duplicate.





To evaluate the influence of the secondary structure of Abeta on the affinity to apoE, we did inhibition studies using Abeta 1-42, Abeta 1-40Q, and Abeta 1-40G, as well as different lots of Abeta 1-40. The greater propensity for aggregation of Abeta 1-42 (20) as well as peptides with a substitution of Gln for Glu at residue 22 of Abeta (found in a familial variant of AD) (60, 61, 62) and Abeta 1-40G (Gly for Glu substitution also at residue 22 found in another familial type of AD) (63) has been previously demonstrated and appears to result from their higher initial beta-sheet content(64) . The lot-to-lot variability in fibrillogenesis of different synthetic Abeta preparations may also be related to their initial secondary structure(65) . We also have examined the affinity to apoE of two major Abeta conformers, Abeta and Abeta(52) . Varying lots of Abeta and Abeta analogs, as well as isolated Abeta conformers, had quite different abilities to inhibit the binding of apoE to Abeta immobilized to the plate (Fig. 2). Calculated IC values for this inhibition varied from 315 nM to above 10 µM. Hence, a severalfold difference in the relative affinity to apoE was found for Abeta peptides sharing identical or very similar chemical structure (Table 1). The highest relative affinities for apoE was shown by Abeta, followed by Abeta 1-40Q and Abeta 1-42. Abeta, Abeta S1-40, and Abeta Y1-40 showed the lowest affinities for apoE. In addition, the affinity of some of these peptides to apoE3 was directly measured by immobilizing them on the plate and obtaining saturation curves. This also revealed severalfold differences in the calculated K(D) value (data not shown). To evaluate the influence of the secondary structure of these different Abeta peptides on their relative affinity to apoE, we have performed CD analyses of these peptides. Abeta B1-40, 1-40Q, 1-40G, and 1-42 displayed predominantly beta-pleated sheet spectra with a minimum at 215 nm and a maximum at 195 nm, whereas Abeta S1-40, C1-40, and Y1-40 showed predominantly random coil structure with a minimum at 190 nm (as shown partially in Fig. 3). By gel filtration all these Abeta peptides were >90% monomeric or dimeric (data not shown). As presented in Table 1, the affinity to apoE of the peptides used appears to correlate with their secondary structure; peptides with the highest beta-pleated sheet content, such as Abeta and Abeta 1-40Q, displayed the greatest relative affinities for apoE3, whereas Abeta S1-40, Y1-40, and Abeta, with the lower content of beta-sheet, were the weaker inhibitors of the Abeta/apoE interaction.


Figure 3: CD analysis of full-length Abeta peptides. Peptides were dissolved at 0.15-0.20 mg/ml in 10 mM sodium phosphate buffer, centrifuged, and their spectra recorded on Jasco 720 spectropolarimeter. The CD spectra are labeled 1-4; corresponding to peptides Abeta 1-42, Abeta 1-40Q, Abeta Y1-40, and Abeta C1-40, respectively. Obtained spectra were fitted by using the Lincomb algorithm(54) . Resulting estimations of beta-sheet content are shown in Table 1.



Once having established the secondary structure of the Abeta peptides used and their binding affinities to apoE, we correlated these data with the known modulatory effect of apoE on the fibrillogenesis of Abeta peptides(33, 34, 35, 58) . We performed incubations of these Abeta peptides in the presence or absence of apoE3 and measured the amount of fibril formation using a Thioflavin T assay(56, 57) . ApoE3 enhanced the fibrillogenesis of all full-length Abeta peptides, although to a variable extent. Fibrillogenesis of the peptides that showed an initial random coil structure was stimulated the most, whereas fibrillogenesis of the peptides with an initial high beta-sheet content was stimulated the least (Fig. 4). The highest modulatory effect of apoE was observed for Abeta 12-28, which suggests that this peptide constitutes a target sequence for apoE effect in Abeta. None of the other shortened Abeta peptides showed a significant increase in fibrillogenicity upon coincubation with apoE.


Figure 4: Thioflavin T fluorescence of Abeta peptides incubated in the presence and absence of apoE. Peptides Abeta 1-16, 12-28, 10-20, and 25-35 were incubated at 2 mg/ml and full-length peptides at 1 mg/ml with or without 2.5 µM of apoE (apoE:Abeta molar ratio 1:100-400) in 30-µl aliquots for 3 days. The fluorescence of Thioflavin T (2 µM) was measured at an excitation of 435 nm and and an emission of 485 nm(56, 57) . The mean value (±S.D.) from three independent experiments is plotted.




DISCUSSION

By using solid-phase assay we have shown that delipidated, recombinant apoE saturably binds to Abeta to give a final K(D) value of 20 nM, indicating a high affinity binding. A similar K(D) value has recently been reported between human purified, delipidated apoE3 and Abeta1-40, using surface plasmon resonance(72) . However, in normal physiological fluids apparently little sAbeta exists in complex with apoE(59) . ApoE under normal conditions exists mainly in lipid particles, and under these conditions the affinity of binding of apoE to Abeta may be lower. Furthermore, the calculated affinity of Abeta 1-40 to another apolipoprotein-apoJ is 10 times higher than the value we found for apoE/Abeta binding(55) . This is in agreement with the major binding protein of sAbeta under normal conditions being apoJ(59, 66) . Additionally, we were able to correlate the affinity of apoE binding to Abeta with the secondary structure of the peptide. We show that apoE has a greater affinity for Abeta peptides with a high beta-sheet content, whereas binding to the mainly random coil peptides is low. Consistent with these in vitro findings, it has recently been shown that Abeta in senile plaques, which is predominantly beta-sheet, is complexed to apoE and apoE carboxyl fragments(44, 50) .

Furthermore, we show that the modulatory effect of apoE on Abeta fibrillogenesis is dependent on the initial secondary structure of the peptide. Fibrillogenesis of Abeta peptides with a high content of beta-sheet is only slightly stimulated, whereas fibrillogenesis of the peptides with a random coil initial secondary structure is stimulated severalfold. The distinct behavior of these Abeta peptides with the same amino acid sequence may be one explanation for different laboratories obtaining varying results in Abeta/ApoE fibrillogenesis experiments. We have identified residues 12-28 of Abeta as a most probable target for this stimulatory effect; fibrillogenesis of this peptide was stimulated by apoE about 10-fold. This portion of Abeta has already been shown to be important for the adoption of a beta-pleated sheet structure(28, 67) , and a recent NMR-based study has shown that Abeta residues 10-35 are required for the binding of Abeta peptides to authentic amyloid plaques(68) . By interacting with this sequence, apoE could mimic the effect of known mutations associated with FAD that are found within this region and which tend to increase Abeta fibrillogenicity(60, 61, 62, 63) . Additionally, other factors that have been shown to promote Abeta fibril formation, such as metal cations (Zn, Al), as well as heparan sulfate proteoglycans, also bind in this region(29, 30) . This portion of the Abeta protein may serve as a potential target for the designed compounds to interfere with the initial random coil to beta-sheet conformational change that occurs in amyloidogenesis.

Late-onset AD has been linked to the presence of the apoE4 isotype, with individuals with the 4 allele having a greater Abeta load (37, 38, 39) . Under the conditions studied, we did not find major differences in the binding between the three apoE isotypes and Abeta 1-40. When we used human-purified, delipidated apoE3 for these same binding experiments to Abeta 1-40, a slightly different K(D) of 15 nM was observed (data not shown). Therefore, we cannot rule out that some variations in the binding of three apoE isotypes to Abeta peptides will be found when other apoE preparations are used. Alternatively, distinct binding affinities of apoE isotypes to Abeta peptides may not be related to the linkage of apoE4 to late-onset AD.

To explain the conformational requirements of Abeta for apoE binding and its modulatory effect, we propose that apoE in the process of Abeta fibrillogenesis displays chaperone-like activity, modulating Abeta molecules to adopt or stabilize a pathological beta-sheet secondary structure. Secondary structure predictions and direct measurements show that apoE contains about 30% beta-sheet, with the beta-sheet content being slightly increased in the absence of lipids(69, 70) . Some of these beta-sheet stretches could form hydrophobic pockets, easily accessible for small hydrophobic peptides. We propose that a beta-sheet motif of apoE acts as an instructor for the random coil Abeta peptides. In vitro we have recently shown that apoE can act to increase the beta-sheet content of Abeta peptides(73) . Although beta-sheet-structured Abeta binds apoE with high affinity, this interaction has little effect on fibrillogenesis. The critical interaction in AD is the low affinity binding between apoE and the random coil sAbeta that leads to the greatest modulation of fibril formation.

Our previous (58) and current data show that the aggregation of poorly fibrillogenic Abeta peptides, even in the presence of apoE, do not proceed to the level observed for more amyloidogenic peptides. Fibrillogenesis of the initially beta-sheet-rich peptides studied here is not significantly affected by the presence of apoE. This can be explained on the basis of the molar ratio of beta-sheet and random coil peptide. An excess of beta-sheet conformers will change the equilibrium of the bound and free apoE, sequestering most of the templates. Fibrillogenesis of these peptides, if not inhibited by other factors, proceeds until it reaches a high level, predetermined by their specific initial secondary structure. This model incorporates the concepts of pathological chaperone (31, 33) and conformational mimicry previously proposed by us(71) .

In summary, we show the preferential binding of apoE to Abeta peptides with a high beta-sheet content. This could in part explain the reported in vivo complexing of Abeta with apoE within senile plaques, while at the same time little sAbeta is found complexed to apoE in biological fluids. Our findings underscore the importance of the conformation of Abeta peptides in the pathogenesis of AD.


FOOTNOTES

*
This research was supported by National Institutes of Health Grants AG00542, AG05891, and NS30455, as well as the Metropolitan Life Award to Dr. B. Frangione. 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.

§
To whom correspondence should be addressed: Dept.of Neurology, TH 427, New York University Medical Center, 550 First Ave., New York, NY 10016. Tel.: 212-263-7993; Fax: 212-263-6751; Thomas.Wisniewski{at}MCFPO.med.nyu.edu.

(^1)
The abbreviations used are: AD, Alzheimer's disease; SP, senile plaques; Abeta, amyloid beta-peptide; RP-HPLC, reverse phase-high performance liquid chromatography; PBS, phosphate-buffered saline; sAbeta, soluble Abeta.


ACKNOWLEDGEMENTS

We thank Dr. Blas Frangione for support and helpful discussions.


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