©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Alzheimer -Peptide Fibril Formation by Serum Amyloid P Component (*)

(Received for publication, July 10, 1995; and in revised form, August 22, 1995)

Sabina Janciauskiene (1) Pablo García de Frutos (2) Erik Carlemalm (3) Björn Dahlbäck (2) Sten Eriksson (1)(§)

From the  (1)Department of Medicine and (2)Clinical Chemistry, Lund University, University Hospital, S-20502 Malmö, Sweden and the (3)The Electronmicroscopy Unit, University Hospital, Lund, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A 39-43-amino acid residue-long fragment (beta-peptide) from the amyloid precursor protein is the predominant component of amyloid deposits in the brain of individuals with Alzheimer's disease. Serum amyloid P component (SAP) is present in all types of amyloid, including that of Alzheimer's disease. We have used an in vitro model to study the effects of purified SAP on the fibril formation of synthetic Alzheimer beta-peptide 1-42. SAP was found to inhibit fibril formation and to increase the solubility of the peptide in a dose-dependent manner. At a 5:1 molar ratio of Abeta1-42 peptide to SAP, fibril formation was completely inhibited, and approximately 80% of the peptide remained in solution even after 4 days of incubation. At lower SAP concentrations, e.g. at peptide to SAP ratio of 1000:1, short fibrillar like structures, lacking amyloid characteristics, were formed. These structures frequently contained associated SAP molecules, suggesting that SAP binds to the polymerizing peptide in a reaction which prevented further fibril formation.


INTRODUCTION

Serum amyloid P component (SAP) (^1)is a calcium-dependent lectin, the best defined specificity of which is 4,6-cyclic pyruvate acetal of beta-D-galactose. The SAP pentamer consists of five identical 25-kDa subunits each of 204 amino acids, the three-dimensional structure of which was determined recently(1) . Each subunit was found to be constructed from multiple antiparallel beta-strands arranged in two sheets, and the tertiary fold was remarkably similar to that of the legume lectins. It binds to DNA, to chromatin, and to glycosaminoglycans such as heparin, heparan, and dermatan sulfate, which are frequently associated with amyloid deposits (2, 3) . It is also present in glomerular basal membranes and associated with elastic fibers(4) . In many SAP-ligand interactions, phosphorylated and/or sulfated groups are involved(5, 6, 7, 8) . After calcium-dependent self-aggregation, or when bound to chromatin, SAP may trigger complement activation(9, 10, 11) . In addition, SAP also inhibits the complement regulatory function of C4b-binding protein, a protein to which it is complexed in blood(12, 13) . Despite the numerous properties ascribed to SAP, its physiological function is largely unknown. SAP is a universal constituent of amyloid deposits, including plaques from Alzheimer's disease (AD), amorphous beta deposits, and neurofibrillar tangles(14) .

The predominant component of amyloid deposits in the brain of individuals with AD is a 39-43-amino acid-long peptide (Abeta-peptide) which is a proteolytic product of the amyloid precursor protein(15, 16) . Recently, it was shown that the Abeta1-42 and Abeta1-43 forms are specifically found in all kinds of AD plaques, indicating that those forms are critically important in AD pathology(17) . beta-Amyloid-related peptides are secreted by cultured cells and are normally present in the cerebrospinal fluid(18) . The local fibril formation is probably a multistep process which is influenced by the rate of Abeta-peptide production and also involves conformational changes of the peptide. The presence of amyloid-associated proteins such as alpha(1)-antichymotrypsin (ACT) and apolipoprotein E (apoE) appears to enhance fibril formation(19, 20, 21, 22) . ACT is present only in amyloid plaques from AD, while a specific allele of apoE (apoE4) has been associated with late onset AD. It has been suggested that ACT and apoE may act as pathological chaperones promoting fibril formation(22) . Although SAP is a universal constituent of all types of amyloid, its physiological role in fibril formation is not known.

SAP binds to preformed amyloid fibrils and to Alzheimer Abeta1-40 peptide in vitro in a calcium-dependent reaction (23, 24) . It has been suggested that SAP protects amyloid from proteolytic degradation in vivo by binding to fibrils and masking fibrillar conformation(1, 25) . We now wish to report that SAP inhibits amyloid fibril formation from the Alzheimer Abeta1-42 peptide in an in vitro model.


EXPERIMENTAL PROCEDURES

Materials

Alzheimer's peptide (>98% pure) and alpha(1)-antitrypsin C-terminal peptide (amino acids 358-394; >90% pure) were purchased from Saveen (Copenhagen, Denmark). DEAE-Sephacel and heparin-Sepharose were from Pharmacia Biotech (Uppsala, Sweden).

Purification of SAP

SAP was prepared from human plasma by barium-citrate absorption followed by chromatography on DEAE-Sephacel and heparin-Sepharose, as described previously(26) . The purified protein gave a single band of 25 kDa in SDS-polyacrylamide gel electrophoresis. Three different batches of SAP were tested, and they gave similar results. The concentration of SAP was determined by absorbance at 280 nm using an extinction coefficient of 18.2(27) .

Turbidity Assay

The Abeta1-42 (20 µl of a 40 µM solution) was mixed with different amounts of SAP in 15 mM Tris/HCl, 150 mM NaCl, pH 7.4 (TBS; final volume 500 µl) and incubated at 37 °C. The final concentration of Abeta1-42 was kept constant at 1.6 µM, SAP concentrations being 1.6 nM (1000:1), 16 nM (100:1), 32 nM (50:1), and 320 nM (5:1); Abeta1-42:SAP ratios are given in parentheses. At different times, the light scattering at 400 nm was measured and compared with controls not containing the peptide. Presented results represent the mean of two experiments.

Electron Microscopy

Samples were applied to carbon-coated copper grids, negatively stained with 2% (v/w) uranyl acetate and examined in a JEOL 200CX electron microscope. The results shown are representative of samples taken from each test solution.

Radioassay

The Abeta1-42 peptide was labeled with I using the lactoperoxidase method according to Thorell and Larson (28) and stored in TBS containing 0.02% NaN(3). The radiolabeled peptide was mixed with unlabeled peptide to a final specific activity of approximately 3 mCi/mmol. Twenty µl of the peptide stock solution (40 µM) was incubated with increasing concentrations of purified SAP in 100 µl of TBS. The final concentration of the peptide was kept constant at 8 µM, SAP concentrations being 8 nM (1000:1), 40 nM (200:1), 80 nM (100:1), 160 nM (50:1), 800 nM (10:1), and 1.6 µM (5:1); Abeta1-42:SAP ratios are given in parentheses. The amount of peptide remaining in solution at the different time points was measured in a counter after centrifugation (10 min at 13,000 times g) and filtration through 0.2 µm filters (Millipore). In control experiments, peptide Abeta1-42 was incubated alone or in the presence of bovine serum albumin at Abeta1-42 to bovine serum albumin molar ratios of 5:1 and 1000:1 under standard experimental conditions. No influence on Abeta1-42 solubility by bovine serum albumin was found. In a control experiment, the radioactivity remaining in solution was determined after a 1-h centrifugation of the samples at 120,000 times g in an Aerofugue (Beckman).


RESULTS AND DISCUSSION

To examine the effect of SAP on Abeta1-42 fibril formation, an in vitro model was used in which the Alzheimer Abeta1-42 peptide spontaneously adopts abeta-pleated sheet conformation and forms elongated, approximately 7-8 nm thick, fibrils. The fibril formation of the Abeta1-42 peptide was monitored by light-scattering and electron microscopy (Fig. 1). In the absence of SAP, the light scatter increased to a maximum after 72 h (Fig. 1A), and elongated fibrils, having amyloid characteristics(29) , were observed in the electron micrographs (Fig. 1B). The addition of SAP resulted in a dose-dependent inhibition of fibril formation. At a peptide to SAP ratio of 5:1, there was almost no increase in light scattering (Fig. 1A), and the fibril formation was completely inhibited (Fig. 1B). A distinct inhibitory effect was observed also at the highest molar ratios of peptide over SAP. Even at a peptide to SAP ratio of 1000:1, the solubility of the peptide was increased as compared to the control, and short, flexible fibrils were formed as revealed by electron microscopy (Fig. 1D) and as reflected by a slight but significant increase in light scatter (Fig. 1A). These fibrils were 1-2 nm thicker than those formed by Abeta1-42 alone. Moreover, they did not exhibit green birefringence after Congo red staining, which is characteristic of amyloid fibrils.


Figure 1: Inhibition of fibril formation from Alzheimer Abeta1-42 peptide by SAP. Synthetic Alzheimer peptide Abeta1-42 was incubated for 4 days at 37 °C in the presence of increasing concentrations of SAP (the peptide to SAP molar ratios were 5:1 bullet, 50:1 circle, 100:1 , and 1000:1 box. , Abeta1-42 alone as described under ``Experimental Procedures.'' In the absence of SAP, the peptide polymerized into extended fibrils, a process which was monitored by light scattering at 400 nm (A) or by electron microscopy after a 48-h incubation (B, C, and D): B, Abeta1-42 alone; C, Abeta1-42 and SAP, 5:1 ratio; D, Abeta1-42 and SAP, 1000:1 ratio. A dose-dependent inhibition of fibril formation was observed with both techniques, and, at a peptide to SAP ratio of 5:1, the fibril formation was essentially completely inhibited. At a peptide to SAP ratio of 50:1 up to 1000:1, short fibrils (120 nm long, 10-12 nm thick) were formed which yielded intermediate light scattering. In the electron micrographs, it was noteworthy that SAP molecules often appeared associated with the short fibrils. In the upper panels of B, C, and D, bars represent 200 nm, whereas, in the bottom panels, bars represent 100 nm.



Although the precise mechanism of the molecular interaction between SAP and Abeta1-42 has not been elucidated, the changes in fibril morphology suggest that SAP affects the packing mechanism of Abeta1-42 and disturbs the typical uniformity of the fibrils. SAP is unique in its ability to inhibit fibril formation at all concentrations reported in this study. The amyloid-associated proteins apoE and ACT have been reported either to inhibit or to stimulate the beta-peptide amyloid fibril formation. At a low ratio between the Abeta1-42 peptide and apoE (1000:1), a significant delay in the onset of amyloid fibril formation was observed, whereas under other conditions, apoE stimulated fibril formation(20, 22, 30) . Similar results have been reported for ACT(19, 21) .

To study the effect of SAP on the overall solubility of Abeta1-42 peptide, the amount of radiolabeled peptide remaining in solution after centrifugation and filtration was measured at various times of incubation (Fig. 2). After a 96-h incubation, the solubility of Abeta1-42 peptide reached its minimum with 20% of radioactivity remaining in solution. Addition of increasing amounts of SAP resulted in increased solubility demonstrating that SAP may be able to prevent Abeta1-42 aggregation. To ensure that the experimental conditions (10-min centrifugation at 13,000 times g and filtration through 0.2-µm filters) were sufficient to remove small aggregates of Abeta1-42 peptide, a second experiment was performed in which the peptide:SAP mixtures after a 72-h incubation were centrifuged at 120,000 times g for 1 h. The results confirmed the ability of SAP, at an Abeta1-42:SAP ratio of 5:1, to keep essentially all the peptide in solution (89%) for up to 72 h. At Abeta1-42:SAP ratios of 100:1 and 1000:1, the amounts of peptide remaining in solution were 81 and 60%, respectively. In this experiment, 54% of the peptide remained soluble in the absence of SAP.


Figure 2: Effect of SAP on solubility of Alzheimer Abeta1-42 peptide. The radiolabeled Alzheimer Abeta1-42 peptide (final concentration 8 µM) was incubated with increasing concentrations of purified SAP in 100 µl of TBS. The amount of peptide remaining in solution was determined as described under ``Experimental Procedures.'' A, the soluble peptide was determined after centrifugation (10 min at 13,000 times g) and filtration through 0.2-µm filters of the samples. The peptide to SAP molar ratios were 5:1 bullet, 10:1 up triangle, 50:1 circle, 100:1 , 200:1 , 1000:1 box, and Abeta1-42 alone . Each point represent the mean of two experiments.



Direct binding between SAP and the Abeta1-42 peptide was demonstrated by nondenaturing agarose gel electrophoresis, but, in contrast to the complexes between ACT and the Abeta1-42 peptide(21) , complexes between SAP and the beta-peptide were not stable in SDS (results not shown). Recently, it has been shown that SAP is able to bind to Abeta1-40 immobilized in microtiter plates(24) . Together, these data suggest that SAP is able to bind Abeta1-42 under nondenaturing conditions.

The inhibitory effect of SAP on Alzheimer Abeta1-42 peptide fibril formation appears to reflect a general ability of SAP to inhibit amyloid fibril formation. In support for this concept, SAP was found to inhibit the formation of fibrils from a beta-pleated sheet containing peptide derived from alpha(1)-antitrypsin(31) . Complete inhibition of fibril formation was observed at a peptide to SAP ratio of 5:1. Even at a peptide to SAP ratio of 1000:1, a clear attenuation of fibril formation was observed (Fig. 3). It was noteworthy that at this latter experimental condition, the morphology of the aggregated peptide was different from that observed in the experiment using Abeta1-42 (compare Fig. 1D and 3C). Instead of short flexible fibers, dense aggregates of fibers were observed.


Figure 3: Inhibition of fibril formation from alpha(1)-antitrypsin C-terminal peptide by SAP. Synthetic alpha(1)-antitrypsin C-terminal peptide was incubated at 37 °C (final concentration 12 µM) in the presence of increasing concentrations of SAP as described for Abeta1-42 under ``Experimental Procedures.'' In the absence of SAP, the peptide polymerized into extended fibrils as observed by electron microscopy (A). No fibrils were observed at a peptide to SAP ratio of 5:1 (B). At a peptide to SAP ratio of 1000:1, a few number of dense clusters of fibrils were observed (C). It was noteworthy that SAP molecules often appeared associated with the fibrils, as observed in Fig. 1(arrow). In the upper and lower panels, bars represent 200 and 100 nm, respectively.



Our present data suggest SAP both to impede the seeding process, which initiates fibril formation(32) , and to inhibit fibril growth by binding to the peptide and thus preventing peptide-peptide polymerization. Both processes presumably involve binding of SAP to beta-pleated sheet structures formed by the polymerizing peptide. If the physiological function of SAP is to inhibit amyloid fibril formation, this process is probably imbalanced in AD, either due to changes in the metabolism of the beta-peptide or to the presence of other SAP ligands in the plaques, such as glycosaminoglycans which would inhibit SAP function. Moreover, in such pathological situations, the attachment of SAP to amyloid fibrils may lead to increased resistance to proteolysis (1, 25) . SAP has been extremely well conserved through evolution (33) and no deficiency of SAP has been described, suggesting SAP to have important functions. The ability of SAP to inhibit pathological deposition of amyloid-forming peptides may be such a function.


FOOTNOTES

*
This work was supported by the Ernhold Lundström, Anna and Edwin Berger Foundations, and the Swedish Medical Research Council through Grant B96-03X-07143-12A. 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: Wallenberg Laboratoriet, Universitetsjukhuset, S-20502 Malmö, Sweden. Fax: 46-40-33-62-08.

(^1)
The abbreviations used are: SAP, serum amyloid P component; ACT, alpha(1)-antichymotrypsin; apoE, apolipoprotein E; AD, Alzheimer's disease.


REFERENCES

  1. Emsley, J. White, H. E., O'Hara, B. P., Oliva, G., Srinivasan, N., Tickle, I. J., Blundell, T. L., Pepys, M. B., and Wood, S. (1994) Nature 367, 338-345 [CrossRef][Medline] [Order article via Infotrieve]
  2. Snow, A. D., and Wight, T. N. (1989) Neurobiol. Aging 10, 481-497 [CrossRef][Medline] [Order article via Infotrieve]
  3. Stenstad, T., Magnus, J. H., Syse, K., and Husby, G. (1993) Clin. Exp. Immunol. 94, 189-195 [Medline] [Order article via Infotrieve]
  4. Breathnach, S. M., Melrose, S. M., Bhogal, B., De Beer, F. C., Dyck, R. F., Tennent, G., Black, M. M., and Pepys, M. B. (1981) Nature 293, 652-654 [Medline] [Order article via Infotrieve]
  5. Hamazaki, H. (1987) J. Biol. Chem. 262, 1456-1460 [Abstract/Free Full Text]
  6. Pepys, M. B., and Butler, P. J. G. (1987) Biochem. Biophys. Res. Commun. 148, 308-313 [Medline] [Order article via Infotrieve]
  7. de Beer F. C., Baltz, M. L., Holford, S., Feinstein, A., and Pepys, M. B. (1981) J. Exp. Med. 154, 1134-1149 [Abstract/Free Full Text]
  8. Christner, R. B., and Mortensen, R. F. (1994) J. Biol. Chem. 269, 9760-9766 [Abstract/Free Full Text]
  9. Breathnach, S. M., Kofler, H., Sepp, N., Ashworth, J., Woodrow, D., Pepys, M. B., and Hinter, H. (1989) J. Exp. Med. 170, 1433-1438 [Abstract]
  10. Hicks, P. S., Saunero-Nava, L., DuClos, T. W., and Mold, C. (1992) J. Immunol. 149, 3689-3694 [Abstract/Free Full Text]
  11. Ying, S., Gewurz, A. T., Jiang, H., and Gewurz, H. (1993) J. Immunol. 150, 169-176 [Abstract/Free Full Text]
  12. García de Frutos, P., and Dahlbäck, B. (1994) J. Immunol. 152, 2430-2437 [Abstract/Free Full Text]
  13. Schwalbe, R., Dahlbäck, B., and Nelsestuen, G. L. (1990) J. Biol. Chem. 265, 21749-21757 [Abstract/Free Full Text]
  14. Perlmutter, L. S., Barrón, E., Myers, M., Saperia, D., and Chui, H. C. (1995) J. Comp. Neurol. 352, 92-105 [Medline] [Order article via Infotrieve]
  15. Roher, A. E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10836-10840 [Abstract]
  16. Iwatuso, T., Odaka, A., Suzuki, N., Mizusawa, H., Nukina, N., and Ihara, Y. (1994) Neuron 13, 45-53 [Medline] [Order article via Infotrieve]
  17. Gravina, S. A., Ho, L., Eckman, C. B., Long, K. E., Otvos, L., Younkin, L. H., Suzuki, N., and Younkin, S. G. (1995) J. Biol. Chem. 270, 7013-7016 [Abstract/Free Full Text]
  18. Seubert, P., Vigo-Pelfrey, C., Esch, F., Lee, M., Dovey, H., Davis, D., Sinha, S., Schlossmacher, M., Whaley, J., Swindlehurst, C., McCormack, R., Wolfert, R., Selkoe, D., Lieberburg, I., and Schenk, D. (1992) Nature 359, 325-327 [CrossRef][Medline] [Order article via Infotrieve]
  19. Eriksson, S., Janciauskiene, S., and Lannfeldt, L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2313-2317 [Abstract]
  20. Ma, J., Yee, A., Brewer, B. H., Das, S., and Potter, H. (1994) Nature 372, 92-94 [CrossRef][Medline] [Order article via Infotrieve]
  21. Gallo, G., Wisniewski, T., Choi-Miura, N., Ghiso, J., and Frangione, B. (1994) Am. J. Pathol. 145, 526-530 [Abstract]
  22. Sanan, D. A., Weisgraber, K. H., Rusell, S. J., Mahley, R. W., Huang, D., Saunders, A., Schmechel, D., Wisniewski, T., Frangione, B., Roses, A. D., and Strittmatter, W. J. (1994) J. Clin. Invest. 94, 860-869 [Medline] [Order article via Infotrieve]
  23. Pepys, M. B., Dyck, R. F., deBeer, F. C., Skinner, M., and Cohen, A. S. (1979) Clin. Exp. Immunol. 38, 284-293 [Medline] [Order article via Infotrieve]
  24. Hamazaki, H. (1995) J. Biol. Chem. 270, 10392-10394 [Abstract/Free Full Text]
  25. Tennent, G. A., Lovat, L. A., and Pepys, M. B. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 763-767 [Abstract]
  26. Schwalbe, R., Dahlbäck, B., and Nelsestuen, G. L. (1991) J. Biol. Chem. 266, 12896-12901 [Abstract/Free Full Text]
  27. Hamazaki, H. (1989) Biochim. Biophys. Acta 998, 231-235 [Medline] [Order article via Infotrieve]
  28. Thorell, J. I., and Larson, S. M. (1978) Radioimmunoassay and Related Techniques , The C. V. Mosby Co., St. Louis, MO
  29. Pepys, M. B. (1995) in Samter's Immunologic Diseases (Frank, M. M., Claman, H. N., and Unanue, E. R., eds) Vol. II, pp. 637-657, Little, Brown and Co., Boston
  30. Evans, K. C., Berger, E. P., Cho, C. G., Weisgraber, K. H., and Lansbury, P. T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 763-767 [Abstract]
  31. Janciauskiene, S., Carlemalm, E., and Eriksson, S. (1995) Biol. Chem. Hoppe-Seyler 376, 415-423 [Medline] [Order article via Infotrieve]
  32. Jarret, J. T., Berger, E. P., and Lansbury, P. T. (1993) Biochemistry 32, 4693-4697 [Medline] [Order article via Infotrieve]
  33. Rubio, N., Sharp, P. M., Rits, M., Zahedi, K., and Whitehead, A. S. (1993) J. Biochem. (Tokyo) 113, 277-284 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.