Endogenous Proteins Controlling Amyloid β-Peptide Polymerization
1999; Elsevier BV; Volume: 274; Issue: 23 Linguagem: Inglês
10.1074/jbc.274.23.15990
ISSN1083-351X
AutoresBernd Bohrmann, Lars O. Tjernberg, Pascal Kuner, Sonia Poli, Bernard Levet‐Trafit, Jan Näslund, Grayson Richards, Walter Huber, Heinz Döbeli, Christer Nordstedt,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoWe report that certain plasma proteins, at physiological concentrations, are potent inhibitors of amyloid β-peptide (Aβ) polymerization. These proteins are also present in cerebrospinal fluid, but at low concentrations having little or no effect on Aβ. Thirteen proteins representing more than 90% of the protein content in plasma and cerebrospinal fluid were studied. Quantitatively, albumin was the most important protein, representing 60% of the total amyloid inhibitory activity, followed by α1-antitrypsin and immunoglobulins A and G. Albumin suppressed amyloid formation by binding to the oligomeric or polymeric Aβ, blocking a further addition of peptide. This effect was also observed when the incorporation of labeled Aβ into genuine β-amyloid in tissue section was studied. The Aβ and the anti-diabetic drug tolbutamide apparently bind to the same site on albumin. Tolbutamide displaces Aβ from albumin, increasing its free concentration and enhancing amyloid formation. The present results suggest that several endogenous proteins are negative regulators of amyloid formation. Plasma contains at least 300 times more amyloid inhibitory activity than cerebrospinal fluid. These findings may provide one explanation as to why β-amyloid deposits are not found in peripheral tissues but are only found in the central nervous system. Moreover, the data suggest that some drugs that display an affinity for albumin may enhance β-amyloid formation and promote the development of Alzheimer's disease. We report that certain plasma proteins, at physiological concentrations, are potent inhibitors of amyloid β-peptide (Aβ) polymerization. These proteins are also present in cerebrospinal fluid, but at low concentrations having little or no effect on Aβ. Thirteen proteins representing more than 90% of the protein content in plasma and cerebrospinal fluid were studied. Quantitatively, albumin was the most important protein, representing 60% of the total amyloid inhibitory activity, followed by α1-antitrypsin and immunoglobulins A and G. Albumin suppressed amyloid formation by binding to the oligomeric or polymeric Aβ, blocking a further addition of peptide. This effect was also observed when the incorporation of labeled Aβ into genuine β-amyloid in tissue section was studied. The Aβ and the anti-diabetic drug tolbutamide apparently bind to the same site on albumin. Tolbutamide displaces Aβ from albumin, increasing its free concentration and enhancing amyloid formation. The present results suggest that several endogenous proteins are negative regulators of amyloid formation. Plasma contains at least 300 times more amyloid inhibitory activity than cerebrospinal fluid. These findings may provide one explanation as to why β-amyloid deposits are not found in peripheral tissues but are only found in the central nervous system. Moreover, the data suggest that some drugs that display an affinity for albumin may enhance β-amyloid formation and promote the development of Alzheimer's disease. Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid β-peptide; BSA, bovine serum albumin; CSF, cerebrospinal fluid; HSA, human serum albumin; PrP, prion protein. 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid β-peptide; BSA, bovine serum albumin; CSF, cerebrospinal fluid; HSA, human serum albumin; PrP, prion protein. is associated with the accumulation of a specific form of amyloid in the brain parenchyma and in meningocerebral blood vessels (1Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 120: 885-890Crossref PubMed Scopus (4202) Google Scholar, 2Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 122: 1131-1135Crossref PubMed Scopus (1254) Google Scholar, 3Selkoe D.J. J. Biol. Chem. 1996; 271: 18295-18298Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar). The primary components of the amyloid are polymers of a short peptide derived through proteolytic processing of a ubiquitous transmembrane protein (4Tanzi R.E. Gusella J.F. Watkins P.C. Bruns G.A. St. George-Hyslop P. Van Keuren M.L. Patterson D. Pagan S. Kurnit D.M. Neve R.L. Science. 1987; 235: 880-884Crossref PubMed Scopus (1221) Google Scholar, 5Kang J. Lemaire H.G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3943) Google Scholar) termed the β-amyloid precursor protein. The amyloid β-peptide is usually referred to as the Aβ. It is present in two principal variants (2Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 122: 1131-1135Crossref PubMed Scopus (1254) Google Scholar,6Naslund J. Schierhorn A. Hellman U. Lannfelt L. Roses A.D. Tjernberg L.O. Silberring J. Gandy S.E. Winblad B. Greengard P. Norstedt C. Terenius L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8378-8382Crossref PubMed Scopus (369) Google Scholar), one that contains 40 amino acid residues (Aβ1–40), and one C-terminally extended variant that contains 42 amino acid residues (Aβ1–42). The longer variant has been suggested to be of major importance in the pathogenesis of AD because it has a greater tendency to form amyloid fibrils in vitro and possibly also in vivo (7Jarrett J.T. Berger E.P. Lansbury Jr., P.T. Biochemistry. 1993; 32: 4693-4697Crossref PubMed Scopus (1753) Google Scholar, 8Jarrett J.T. Berger E.P. Lansbury Jr., P.T. Ann. N. Y. Acad. Sci. 1993; 695: 144-148Crossref PubMed Scopus (226) Google Scholar, 9Pike C.J. Burdick D. Walencewicz A.J. Glabe C.G. Cotman C.W. J. Neurosci. 1993; 13: 1676-1687Crossref PubMed Google Scholar). Certain mutations associated with familial AD lead to an increased secretion of the 42-amino acid form (10Suzuki N. Cheung T.T. Cai X.D. Odaka A. Otvos Jr., L. Eckman C. Golde T.E. Younkin S.G. Science. 1994; 264: 1336-1340Crossref PubMed Scopus (1351) Google Scholar) and an enhanced accumulation of amyloid. β-Amyloid displays several important features that distinguish it from other types of amyloid. (i) The peptide forming the amyloid deposits is present at very low concentrations in the circulation. This is in contrast to peripheral amyloid disorders in which the amyloid proteins are present at high concentrations. Examples of such non-central nervous system amyloid proteins include serum amyloid A, myeloma protein, and transthyretin (11Kisilevsky R. Rubin E. Farber J.L. Pathology. J. B. Lippincott Company, London1988: 1178-1193Google Scholar). (ii) The levels of Aβ are not higher and the peptide is not structurally different (except in extremely rare cases of familial AD) in individuals with the disease than in healthy controls (for a review, see Ref. 3Selkoe D.J. J. Biol. Chem. 1996; 271: 18295-18298Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar). (iii) It is well known that most and possibly all nucleated cells in the body produce the Aβ (12Haass C. Selkoe D.J. Cell. 1993; 75: 1039-1042Abstract Full Text PDF PubMed Scopus (739) Google Scholar, 13Haass C. Hung A.Y. Schlossmacher M.G. Oltersdorf T. Teplow D.B. Selkoe D.J. Ann. N. Y. Acad. Sci. 1993; 695: 109-116Crossref PubMed Scopus (104) Google Scholar); however, for unknown reasons, β-amyloid is only deposited in the central nervous system. In the present study, we aimed at investigating why β-amyloid exclusively is formed in the central nervous system. Previous work has demonstrated that some plasma proteins and lipoproteins bind Aβ and serve as carrier proteins (14Biere A.L. Ostaszewski B. Stimson E.R. Hyman B.T. Maggio J.E. Selkoe D.J. J. Biol. Chem. 1996; 271: 32916-32922Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 15Koudinov Matsubara Frangione B. Ghiso J. Biochem. Biophys. Res. Commun. 1994; 205: 1164-1171Crossref PubMed Scopus (132) Google Scholar). Protein binding is a general mechanisms for the transport of endogenous substances such as hormones and lipids as well as clinically used drugs (16Benet L.Z. Kroetz D.L. Sheiner L.B. Hardman J.G. Limbird L.E. Molinoff P.B. Ruddon R.W. Gilman A.G. Goodman & Gilman's The Pharmacological Basis of Therapeutics. McGraw-Hill, New York1996: 3-28Google Scholar). Generally, it is only the non-protein-bound fraction of the substances that is biologically active. We therefore hypothesized that only the free fraction of Aβ can take part in the polymerization process generating amyloid fibrils. Hence, Aβ-carrier proteins may have an important role in preventing amyloid formation by increasing the bound fraction. The bulk of large proteins do not penetrate the blood-brain barrier efficiently. Thus, the levels of soluble proteins in the central nervous system are much lower than those in peripheral tissues. It has been estimated that the ratio between protein content in the CSF and plasma is approximately 0.004 (17Hardman J.G. Limbird L.E. Molinoff P.B. Ruddon R.W. Gilman A.G. Goodman & Gilman's The Pharmacological Basis of Therapeutics. 9th Ed. McGraw-Hill, New York1996Google Scholar). However, in contrast to large proteins, the Aβ levels are higher in CSF than in plasma (18Lannfelt L. Basun H. Vigo-Pelfrey C. Wahlund L.O. Winblad B. Lieberburg I. Schenk D. Neurosci. Lett. 1995; 199: 203-206Crossref PubMed Scopus (34) Google Scholar, 19Lannfelt L. Basun H. Wahlund L.O. Rowe B.A. Wagner S.L. Nat. Med. 1995; 1: 829-832Crossref PubMed Scopus (143) Google Scholar), which probably reflects a higher rate of secretion from neuronal cells than from other cell types. Overall, this suggests that a smaller fraction of the Aβ is protein-bound in the central nervous system than in the periphery. With this background, we decided to investigate whether plasma and CSF proteins can indeed inhibit β-amyloidogenesis. We establishedin vitro assays allowing quantitative and qualitative studies of amyloid formation in the presence of several different proteins. The proteins studied here represent more than 90% of the protein content in plasma and CSF (20Hawkins P.N. Rossor M.N. Gallimore J.R. Miller B. Moore E.G. Pepys M.B. Biochem. Biophys. Res. Commun. 1994; 201: 722-726Crossref PubMed Scopus (41) Google Scholar, 21Doolittle D.F. Stamatoyannopoulos G.N. Majerus A.W.P.W. Varmus H. The Molecular Basis of Blood Diseases. W. B. Saunders Company, Philadelphia1994: 701-723Google Scholar, 22Smith M.D. J. Immunol. Methods. 1976; 9: 373-380Crossref PubMed Scopus (1) Google Scholar, 23Wallum B.J. Taborsky Jr., G.J. Porte J. Figlewicz D.P. Beard J.C. Ward W.K. Dorsa D. J. Clin. Endocrinol. Metab. 1987; 64: 190-194Crossref PubMed Scopus (217) Google Scholar, 24Vatassery G.T. Quach H.T. Smith W.E. Benson B.A. Eckfeldt J.H. Clin. Chim. Acta. 1991; 197: 19-25Crossref PubMed Scopus (31) Google Scholar, 25Tietz N.W. Burtis C.A. Ashwood E.R. Tietz Textbook of Clinical Chemistry. 2nd Ed. W. B. Saunders Company, Philadelphia1994Google Scholar). Many drugs bind to plasma proteins, which can lead to interactions with severe consequences (16Benet L.Z. Kroetz D.L. Sheiner L.B. Hardman J.G. Limbird L.E. Molinoff P.B. Ruddon R.W. Gilman A.G. Goodman & Gilman's The Pharmacological Basis of Therapeutics. McGraw-Hill, New York1996: 3-28Google Scholar). If some drugs bind to the same site on plasma protein molecules as Aβ, it may lead to increased levels of free Aβ and enhanced amyloid formation. Therefore, we decided to also address this possibility experimentally. Synthetic Aβ1–40, Aβ1–40, and PrP106–126 biotinylated at the N terminus were obtained from ANAWA (Wangen, Switzerland). Nonlabeled Aβ1–40, Aβ1–42, and PrP106–126 were obtained from Bachem (Bubendorff, Switzerland). The peptides were stored in Me2SO at −20 °C. Human serum albumin, (fatty acid-free; 99% purity) was from Sigma. All other proteins were from Calbiochem. Streptavidin-peroxidase was bought from Roche Molecular Biochemicals. All other reagents were from Sigma. Iodinated Aβ1–42 was obtained from Amersham Pharmacia Biotech. 96-well plates (Maxisorp; Nunc) were coated with peptide by incubating them with a solution of Aβ1–42 or Aβ1–40 (2.5 μm) in Tris-buffered saline (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, and NaN3). Solution (100 μl) was added to each well, and the plates were incubated at 37 °C with shaking for 48 h. The peptide solution was then flicked off. Staining with a solution of Congo red (20 μm) in Tris-buffered saline showed that the polymeric peptide had bound to the wells (data not shown). After removal of the peptide solution, the plates were placed upside down on absorbing paper and allowed to dry. Coated plates were stored at −20 °C in a desiccator. On the day of experiment, the plates were blocked by the addition of 300 μl of PBS containing 0.05% Tween 20 (PBS-T) and 1% bovine serum albumin/well for 2 h at room temperature. The plates were then washed with PBS-Tween (0.05% Tween 20), and the fluid was flicked off. Biotin-Aβ1–40 or biotin-Aβ1–42 was dissolved in Me2SO and diluted with Tris-buffered saline with NaN3 (0.05%). Unless stated otherwise, the final concentration of the labeled peptide was 20 nm. The plates were incubated overnight at 37 °C with agitation. Nonbound peptide was removed by washing the plates three times with PBS-T (300 μl/well). Streptavidin-peroxidase was diluted with PBS-T and 1% BSA and added to the plates (150 μl/well). After incubation (2 h at room temperature), the solution was flicked off, and the plates were washed four times with PBS-T. Tetramethyl-benzidin was used as chromogenic substrate for the peroxidase. After termination of the reaction with sulfuric acid (0.33 m, final concentration), absorbance was measured at 455 nm with a SpectraMAX 250 96-well plate reader. Nonspecific binding was defined as the binding of biotin-Aβ to wells that had not been coated with Aβ. There was a linear relationship between peroxidase activity and the amount of peptide bound (data not shown). Nonspecific binding was, on average, approximately 15% of total binding (data not shown). We also studied the incorporation of125I-Aβ into tissue sections of human AD brain using the method of Maggio et al. (29Maggio J.E. Stimson E.R. Ghilardi J.R. Allen C.J. Dahl C.E. Whitcomb D.C. Vigna S.R. Vinters H.V. Labenski M.E. Mantyh P.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5462-5466Crossref PubMed Scopus (204) Google Scholar). In experiments with the prion protein-derived peptide PrP106–126 (26Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5131) Google Scholar), similar methodology was used, but with two exceptions. First, the Maxisorp plates were coated with a solution of 10 μm peptide for 14 days. Second, incubation with N-terminally biotinylated PrP106–126 was performed for 4 h. The method used was validated by several means. (i) biotinylated-Aβ1–42 or Aβ1–40 was incubated at a high concentration (10 μm) for 72 h at 37 °C and then examined by electron microscopy. Both peptides were capable of forming fibrils that were indistinguishable from nonbiotinylated controls. (ii) Both biotinylated peptides required the Maxisorp plates to be coated with amyloid fibrils in order for them to bind. When the plates were coated with truncated variants of Aβ (Aβ12–28, Aβ35–42, Aβ10–20, Aβ1–16, or Aβ25–35), no significant binding over that obtained with noncoated control wells was observed. (iii) We also studied the incorporation of biotin-Aβ1–40 into preformed Aβ1–42 fibrils. A low concentration of fibrils (corresponding to 20 nm monomeric peptide) was incubated with equimolar amounts of biotin Aβ1–40. After overnight incubation and centrifugation, the material was stained with anti-biotin Ig labeled with colloidal gold and negatively stained with uranyl acetate. Using this protocol, gold-labeled amyloid fibrils were observed, demonstrating that biotin-Aβ1–40 could bind to the preformed fibers. The controls used were biotin-Aβ1–40 or preformed Aβ1–42 fibrils alone. In these control experiments, biotin-Aβ1–40 did not produce any detectable fibrils, whereas the Aβ1–42 fibrils were not labeled by gold. Negative staining was performed by adsorption of a 5-μl aliquot of the sample to a carbon-coated 200-mesh copper grid for 60 s. Staining was done by adding 10 μl of 2% uranyl acetate directly to the adsorbed sample droplet for 2 min and air drying after the removal of excess liquid with filter paper. Specimens were examined in a JEOL 1210 electron microscope operated at 100 kV. Digitized micrographs were recorded with a slow scan charge-coupled device camera (Gatan; model 679). Data acquisition with the slow scan charge-coupled device camera and processing of the digitized images were controlled by a Macintosh PowerPC 8500 using DigitalMicrograph software from Gatan. Images were printed on a Thermoprinter Phaser 440 (Tektronix). Magnification calibration was performed as described previously (27Wrigley N.G. J. Ultrastruc. Res. 1968; 24: 454-464Crossref PubMed Scopus (324) Google Scholar) using negatively stained catalase crystals. Interactions between albumin and monomeric/polymeric Aβ were measured using a BiaCore 2000 instrument (BiaCore AB, Uppsala, Sweden) essentially as described previously (28Karlsson R. Michaelsson A. Mattsson M. J. Immunol. Methods. 1991; 145: 229-240Crossref PubMed Scopus (1011) Google Scholar). Briefly, monomeric biotin-Aβ1–40 at a concentration of 115 nm was attached to a sensor cell to which streptavidin had been coupled. The peptide was stored in Me2SO and diluted in running buffer (see below) immediately before it was coupled to the chip. Under these conditions, no evidence suggesting that the peptide polymerized could be obtained (data not shown). Polymeric peptide was attached via a monoclonal antibody against amino acid residues 2–8 of Aβ (BAP-1A). Unless otherwise stated, the running buffer used contained 10 mm Hepes, pH 7.5, 150 mm NaCl, and 0.05% P20 detergent. After each experiment, the cells were washed with ethanolamine (1 m, pH 8.5) until the sensor signal remained stable in contact with running buffer. In the first set of experiments, we investigated whether albumin, which is quantitatively the most important Aβ-binding protein (14Biere A.L. Ostaszewski B. Stimson E.R. Hyman B.T. Maggio J.E. Selkoe D.J. J. Biol. Chem. 1996; 271: 32916-32922Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar) and also the most abundant protein in plasma and CSF, could interfere with the incorporation of biotin-Aβ1–40 into amyloid fibrils. The test system used is based on the finding that Aβ monomers bind with high affinity to preformed polymers of Aβ (29Maggio J.E. Stimson E.R. Ghilardi J.R. Allen C.J. Dahl C.E. Whitcomb D.C. Vigna S.R. Vinters H.V. Labenski M.E. Mantyh P.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5462-5466Crossref PubMed Scopus (204) Google Scholar). We immobilized Aβ1–42 polymers as seeds (7Jarrett J.T. Berger E.P. Lansbury Jr., P.T. Biochemistry. 1993; 32: 4693-4697Crossref PubMed Scopus (1753) Google Scholar) in 96-well plates and measured the incorporation of soluble biotin-Aβ1–40 in the presence of human serum albumin (HSA) or BSA. In Fig.1 A, the effects of different concentrations of HSA and BSA on biotin-Aβ1–40incorporation are shown. The highest concentrations of albumin used corresponded approximately to the plasma levels of a healthy human adult (21Doolittle D.F. Stamatoyannopoulos G.N. Majerus A.W.P.W. Varmus H. The Molecular Basis of Blood Diseases. W. B. Saunders Company, Philadelphia1994: 701-723Google Scholar). Both HSA and BSA had the capacity to completely inhibit the incorporation of biotin-Aβ1–40 into immobilized Aβ polymers with apparent IC50 values of 10 and 12 μm, respectively. The effects of HSA on the polymerization of Aβ1–42 in this system were also studied. Here, nonlabeled Aβ1–40 or Aβ1–42 was immobilized in the Maxisorp plates as described under "Experimental Procedures." Biotinylated Aβ1–40 or Aβ1–42 was then allowed to bind the immobilized peptide in the presence of various concentrations of HSA. The IC50 values of HSA on the inhibition of Aβ1–40 or Aβ1–42 binding were essentially identical (data not shown), suggesting that HSA is indeed capable of inhibiting polymerization of the two major forms of the Aβ. These findings were confirmed in a second set of experiments in which the incorporation of 125I-Aβ1–42 into brain tissue sections from an individual with AD was measured. As seen in Fig. 1 B, the radiolabeled peptide bound to the amyloid deposits in the tissue, as demonstrated previously (29Maggio J.E. Stimson E.R. Ghilardi J.R. Allen C.J. Dahl C.E. Whitcomb D.C. Vigna S.R. Vinters H.V. Labenski M.E. Mantyh P.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5462-5466Crossref PubMed Scopus (204) Google Scholar). In the presence of 227 μm HSA, binding was heavily reduced (Fig.1 C). Measurement of the incorporated radioactivity using a phosphorimager showed that overall binding (binding to amyloid deposits in the tissue and background together) had been reduced with 55% by the addition of HSA. Aβ incubated at high concentrations also rapidly polymerizes in the absence of preformed polymers, but through primary nucleation (30Jarrett J.T. Lansbury Jr., P.T. Cell. 1993; 73: 1055-1058Abstract Full Text PDF PubMed Scopus (1918) Google Scholar, 31Orgel L.E. Chem. Biol. 1996; 3: 413-414Abstract Full Text PDF PubMed Scopus (32) Google Scholar). Therefore, in other experiments, we studied the effects of HSA on soluble Aβ1–40 and Aβ1–42 in the absence of seeds. As seen in Fig. 2, Aand C, both peptides formed fibrils when incubated for 24 h at 37 °C at a concentration of 20 μm. This concentration is approximately 6,000 times higher than that in CSF (17Hardman J.G. Limbird L.E. Molinoff P.B. Ruddon R.W. Gilman A.G. Goodman & Gilman's The Pharmacological Basis of Therapeutics. 9th Ed. McGraw-Hill, New York1996Google Scholar,29Maggio J.E. Stimson E.R. Ghilardi J.R. Allen C.J. Dahl C.E. Whitcomb D.C. Vigna S.R. Vinters H.V. Labenski M.E. Mantyh P.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5462-5466Crossref PubMed Scopus (204) Google Scholar, 39Strittmatter W.J. Weisgraber K.H. Huang D.Y. Dong L.M. Salvesen G.S. Pericak-Vance M. Schmechel D. Saunders A.M. Goldgaber D. Roses A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8098-8102Crossref PubMed Scopus (1238) Google Scholar). When incubated in the presence of 227 μm HSA, the polymerization of Aβ1–40 into amyloid fibrils was completely inhibited (Fig. 2 B). Under the same conditions, Aβ1–42 only formed occasional fibrils (Fig.2 D). Moreover, spherical structures of 10–30 nm in diameter were also detected frequently. PrP106–126 represents the central core of the prion protein (for a recent review, see Ref. 26Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5131) Google Scholar) and spontaneously forms amyloid-like fibrils. Similar to the Aβ, prion protein can form amyloid deposits in the CNS and cause neurodegeneration. We therefore decided to study whether albumin can also prevent polymerization of this peptide. As seen in Fig. 3, HSA dose-dependently inhibited the binding of biotinylated PrP106–126 to immobilized homologous peptide. The IC50 value for HSA in this system was approximately 100 μm, 10 times higher than that for Aβ. This concentration represents less then one-sixth of the albumin concentration in blood but is more than 30 times higher than the albumin concentration in CSF. Hence, these findings demonstrate that HSA displays a certain degree of specificity for β-amyloid. Surface plasmon resonance spectroscopy allows protein-protein interaction studies in real time (28Karlsson R. Michaelsson A. Mattsson M. J. Immunol. Methods. 1991; 145: 229-240Crossref PubMed Scopus (1011) Google Scholar), and this methodology was therefore used to study how BSA and HSA interact with monomeric and polymeric Aβ. Preformed Aβ1–42 fibrils were immobilized to the sensor chip as described under "Experimental Procedures." A solution (25 μm) of BSA (Fig.4 A) or HSA (Fig.4 B) was then allowed to flow through the cell. The protein bound avidly to the polymers, indicating that albumin indeed has an affinity for the polymeric peptide. In parallel flow cells, monomeric biotin-Aβ1–40 was immobilized using streptavidin. In this case, no binding was observed with either BSA (Fig. 4 A) or HSA (Fig. 4 B). When the experiment was repeated using nonbiotinylated Aβ1–40 that was immobilized with a monoclonal antibody, essentially identical results were obtained (data not shown). It is well known that several clinically important drugs bind to albumin with various affinities. We speculated that some of these substances may bind to the same site(s) on the albumin molecule as Aβ and may therefore be able to displace the peptide from its binding site(s). This may lead to increased levels of free Aβ and the enhancement of amyloid formation. We therefore screened a number of albumin ligands with regard to their effects on biotin-Aβ1–40 incorporation into preformed amyloid polymers in the presence or absence of 100 μmHSA. It was found that tolbutamide, at concentrations corresponding to therapeutic levels (17Hardman J.G. Limbird L.E. Molinoff P.B. Ruddon R.W. Gilman A.G. Goodman & Gilman's The Pharmacological Basis of Therapeutics. 9th Ed. McGraw-Hill, New York1996Google Scholar), enhanced biotin-Aβ1–40incorporation in the presence but not in the absence of HSA (Fig.5). This strongly suggests that tolbutamide is capable of interfering with Aβ-albumin binding and indirectly stimulating amyloid fibril formation. In these experiments, we investigated the effects of various plasma/CSF proteins on Aβ polymerization (Table I). The proteins listed in Table I represent more than 90% of the protein content in plasma and CSF. The concentrations tested covered the levels in plasma and CSF for all but two proteins, IgM and α1-antichymotrypsin. The highest concentrations used were 0.55 and 1.0 μm, respectively, which were lower than their plasma concentrations but higher than their CSF concentrations (see Table I). Seven of the 13 tested proteins had very little or no effect (i.e. the IC50 was higher than the plasma concentrations). Of the remaining six proteins, three had IC50 values in the range of 10–30 μm, and three had IC50 values below 10 μm. Albumin, α1-antitrypsin, IgG, and IgA had IC50 values that were substantially below their plasma concentrations, which strongly suggests that these proteins may be potent inhibitors of β-amyloidogenesis in vivo.Table IIC50 values for the inhibition of biotin-Aβ1–40incorporation into immobilized Aβ1–42 polymersProteinIC50*nPlasma concentrationCSF concentration(μm)(μm)(μm)Albumin10106443.0IgG104750.3IgA1.34130.007IgM>0.5541.90.0002α1-Antitrypsin1.24250.12Transferrin304380.17α2-Macroglobulin2.543.40.0024Insulin>10040.0001aHighly variable.0.000008aHighly variable.α1-Antichymotrypsin>1.048.60.038Antithrombin III>7.744.0N.I.bN.I., no information available.Serum amyloid P>0.240.20.000033Transthyretin>5.545.50.27Fibrinogen>8.848.8N.I.The two columns to the right indicate plasma and CSF concentrations of the studied proteins in healthy adults (21Doolittle D.F. Stamatoyannopoulos G.N. Majerus A.W.P.W. Varmus H. The Molecular Basis of Blood Diseases. W. B. Saunders Company, Philadelphia1994: 701-723Google Scholar, 22Smith M.D. J. Immunol. Methods. 1976; 9: 373-380Crossref PubMed Scopus (1) Google Scholar, 23Wallum B.J. Taborsky Jr., G.J. Porte J. Figlewicz D.P. Beard J.C. Ward W.K. Dorsa D. J. Clin. Endocrinol. Metab. 1987; 64: 190-194Crossref PubMed Scopus (217) Google Scholar, 24Vatassery G.T. Quach H.T. Smith W.E. Benson B.A. Eckfeldt J.H. Clin. Chim. Acta. 1991; 197: 19-25Crossref PubMed Scopus (31) Google Scholar, 41Vasileva T.G. Dobrogorskaia L.N. Kraeva L.N. Goncharova V.P. Vopr. Med. Khim. 1989; 35: 48-51Google Scholar).a Highly variable.b N.I., no information available. Open table in a new tab The two columns to the right indicate plasma and CSF concentrations of the studied proteins in healthy adults (21Doolittle D.F. Stamatoyannopoulos G.N. Majerus A.W.P.W. Varmus H. The Molecular Basis of Blood Diseases. W. B. Saunders Company, Philadelphia1994: 701-723Google Scholar, 22Smith M.D. J. Immunol. Methods. 1976; 9: 373-380Crossref PubMed Scopus (1) Google Scholar, 23Wallum B.J. Taborsky Jr., G.J. Porte J. Figlewicz D.P. Beard J.C. Ward W.K. Dorsa D. J. Clin. Endocrinol. Metab. 1987; 64: 190-194Crossref PubMed Scopus (217) Google Scholar, 24Vatassery G.T. Quach H.T. Smith W.E. Benson B.A. Eckfeldt J.H. Clin. Chim. Acta. 1991; 197: 19-25Crossref PubMed Scopus (31) Google Scholar, 41Vasileva T.G. Dobrogorskaia L.N. Kraeva L.N. Goncharova V.P. Vopr. Med. Khim. 1989; 35: 48-51Google Scholar). 1 unit of inhibitory activity was defined as the number of μmol/liter of protein required to inhibit polymerization by 50% under the conditions specified under "Experimental Procedures." When taking the plasma concentration of the studied proteins into consideration, it is possible to estimate how much inhibitory activity each protein contributes (Fig. 6). Albumin is probably the most important regulator of β-amyloidogenesis in plasma. Although α1-antitrypsin has an IC50 value eight times lower than that of albumin (1.25 and 10 μm, respectively), the concentration of the former is substantially lower (25.3 and 644 μm, respectively). Therefore, despite its higher efficacy, it probably plays a less important role in the regulation of Aβ polymerization. Cerebrospinal fluid contains essentially the same proteins as plasma, but the concentrations are considerably lower (see Table I). None of the tested proteins are present in the CSF in a concentration equal to or higher than its IC50 value, which was obtained in the amyloid formation assay (see Table I). When comparing the total amount of inhibitory activity in plasma and CSF, we found that CSF contains only about 0.3% of that seen in plasma (Fig. 6). Plasma and CSF proteins with affinity to Aβ serve as carriers for the peptide (14Biere A.L. Ostaszewski B. Stimson E.R. Hyman B.T. Maggio J.E. Selkoe D.J. J. Biol. 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Albumin is the most abundant protein in CSF, but it is present at a concentration below its IC50value at which only a partial inhibition of Aβ polymerization is obtained (see Fig. 1). Plasma also contains significant levels of other proteins, such as α1-antitrypsin, IgG, and IgA, that are capable of inhibiting Aβ polymerization. The other studied proteins capable of inhibiting polymerization are also present in CSF in concentrations substantially below their IC50 values (see Table I). These results point to a dramatic difference between plasma and CSF: the former contains large quantities of inhibitory proteins, whereas the latter contains small quantities of inhibitory proteins. For unknown reasons, β-amyloid deposits are not formed outside the central nervous system (34Selkoe D.J. Ann. Med. 1989; 21: 73-76Crossref PubMed Scopus (52) Google Scholar). The present results suggest that the high concentrations of inhibitory proteins in plasma prevent the formation of β-amyloid in peripheral tissues, but the low levels in CSF do not block β-amyloid formation in the central nervous system. This conclusion is also supported by previous experimental data (35Wisniewski T. Castano E. Ghiso J. Frangione B. Ann. Neurol. 1993; 34: 631-633Crossref PubMed Scopus (52) Google Scholar), showing that CSF only partially inhibits the formation of thioflavin-binding amyloid from synthetic Aβ1–40. Pathologically reduced levels of albumin might promote β-amyloidosis and possibly also AD. In clinical studies, it was observed that anti-inflammatory drugs may have beneficial effects on AD (36Breitner J.C. Neurobiol. Aging. 1996; 17: 789-794Crossref PubMed Scopus (173) Google Scholar). Levels of albumin are often reduced in association with inflammation (25Tietz N.W. Burtis C.A. Ashwood E.R. Tietz Textbook of Clinical Chemistry. 2nd Ed. W. B. Saunders Company, Philadelphia1994Google Scholar) and, hence, the antiamyloidogenic activity in plasma and CSF is also reduced. However, even heavily reduced plasma levels of albumin are probably still sufficiently high to prevent amyloid formation in peripheral tissues. It may be different in the central nervous system. Because albumin (and other inhibitory proteins) is present in low concentrations having limited effects on amyloid formation (35Wisniewski T. Castano E. Ghiso J. Frangione B. Ann. Neurol. 1993; 34: 631-633Crossref PubMed Scopus (52) Google Scholar), even small reductions in albumin levels in association with inflammation may lead to increased amyloid formation. The structural background as to why Aβ binds albumin and other proteins is not known. However, it is reasonable to assume that hydrophobic interactions are involved. It was surprising that monomeric Aβ did not display binding to albumin when studied by surface plasmon resonance spectroscopy, considering the findings of Biere et al. (14Biere A.L. Ostaszewski B. Stimson E.R. Hyman B.T. Maggio J.E. Selkoe D.J. J. Biol. Chem. 1996; 271: 32916-32922Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar) showing that soluble Aβ binds albumin and lipoproteins. One explanation may be that Aβ molecules rapidly form small, soluble, oligomers with an affinity to albumin (37Podlisny M.B. Walsh D.M. Amarante P. Ostaszewski B.L. Stimson E.R. Maggio J.E. Teplow D.B. Selkoe D.J. Biochemistry. 1998; 37: 3602-3611Crossref PubMed Scopus (189) Google Scholar, 38Pitschke M. Prior R. Haupt M. Riesner D. Nat. Med. 1998; 4: 832-834Crossref PubMed Scopus (297) Google Scholar). Tolbutamide is a drug used to regulate blood glucose levels in diabetes mellitus. It also displays a high affinity for albumin. As a result, its clinical use is often associated with interactions with other drugs when the compounds compete for the same binding site on the albumin molecule (17Hardman J.G. Limbird L.E. Molinoff P.B. Ruddon R.W. Gilman A.G. Goodman & Gilman's The Pharmacological Basis of Therapeutics. 9th Ed. McGraw-Hill, New York1996Google Scholar). Here, we found that tolbutamide, at concentrations corresponding to therapeutic levels, enhanced amyloid formation in the presence but not in the absence of HSA. A reasonable explanation is that tolbutamide and Aβ bind to the same site on albumin. Tolbutamide may therefore displace Aβ from albumin and generate higher free Aβ fractions that can participate in amyloid formation. Drugs that can penetrate into the central nervous system, bind to the Aβ site(s) on albumin, and increase the free fraction of the peptide may thus be capable of enhancing amyloid formation in vivo. Mutations affecting proteins capable of binding Aβ may promote the development of AD (39Strittmatter W.J. Weisgraber K.H. Huang D.Y. Dong L.M. Salvesen G.S. Pericak-Vance M. Schmechel D. Saunders A.M. Goldgaber D. Roses A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8098-8102Crossref PubMed Scopus (1238) Google Scholar, 40Blacker D. Wilcox M.A. Laird N.M. Rodes B. Horvath S.M. Go R.C. Bassett S.S. McInnis M.G. Albert M.S. Hyman B.T. Tanzi R.E. Nat. Genet. 1998; 19: 357-360Crossref PubMed Scopus (582) Google Scholar). It is therefore possible that mutations affecting the proteins studied here may also have an impact on the development of AD through a similar mechanism. In conclusion, the present data suggest a novel and possibly important physiological role for albumin and other plasma/CSF proteins in controlling amyloidogenesis in the central nervous system and possibly also in peripheral tissues. The data also suggest that drugs with certain pharmacokinetic properties may be capable of enhancing amyloidogenesis. Moreover, the reduced levels of albumin seen in association with inflammatory reactions may provide an opportunity for the Aβ to polymerize and thereby more easily form amyloid in the central nervous system. Antibody BAP-1A was a generous gift from Dr. Manfred Brockhaus (Hoffmann-La Roche AG). We thank Dr. John Kemp for valuable suggestions during the preparation of the manuscript.
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