Morphology and antibody recognition of synthetic β‐amyloid peptides

Abstract

To elucidate the relationship between amyloid fibril formation in Alzheimer disease (AD) and the primary structure of the beta-amyloid protein (beta-AP), we investigated the ability of peptides sharing sequences with beta-AP to form fibrils in vitro and to recognize anti-beta-amyloid antisera. The peptides, which were synthesized using a FMOC solid phase procedure and purified by HPLC, consisted of residues 6-25 from the putative aqueous domain, residues 22-35, which overlaps the putative aqueous and transmembrane domains, and residues 1-38 and 1-40 representing nearly the full length of beta-AP. Electron microscopy of negative-stained or thin-sectioned preparations revealed that the peptides assembled into fibrils having different morphologies, some of which resembled in situ AD amyloid. Peptide 6-25 fibrils had diameters of 50-80 A and occasionally showed a central groove suggestive of constituent filaments. Cross sections of the fibril showed a penta- or hexameric arrangement of globular subunits with diameters of 25-30 A. Peptide 22-35 fibrils were helical, with a pitch of 1,100 A and a width of 120 A at its greatest and 50-60 A at its narrowest. The fibrils formed by peptides 1-38 and 1-40 were 70-90 A in diameter. When the peptide assemblies were singly oriented by sedimentation or doubly oriented in a magnetic field, their X-ray diffraction patterns all showed reflections typical of a cross-beta pleated sheet conformation. The patterns differed mainly in their small-angle equatorial intensity, which arises from the packing of fibrils having different widths. Antiserum raised to either native amyloid or to synthetic peptide beta-(1-28) was highly reactive in an inhibition-ELISA assay to beta-(6-25) and beta-(1-38), but not to beta-(22-35), and immunostained beta-(1-40) on Western blots. These studies show that the beta-(6-25), beta-(1-38) and beta-(1-40) peptides can assemble into cross-beta fibrils that retain epitopes characteristic of AD amyloid.

Propagating structure of Alzheimer’s β-amyloid (10–35) is parallel β-sheet with residues in exact register

“…2 and illustrated in Fig. 4, distance constraints of 5 Å for both Gln 15 -Gln 15 and Lys 16 -Lys 16 could fit either a parallel or an antiparallel ␤-strand structure. In the parallel structure, every 13 C carbonyl interacts with two, equidistant 13 C carbonyls (''multiple spins''; Fig.…”

“…This enzyme specifically crosslinks Gln 15 and Lys 16 of A␤ but never involves Lys 28 (70), suggesting that Gln 15 and Lys 16 are proximate in an aggregated form of A␤. For our experiments, we synthesized a 26-aa peptide, comprising residues 10-35 of the A␤ peptide (A␤ (10-35) , Fig.…”

“…Our selection of A␤ was based on the following considerations: (i) Model peptide studies have demonstrated that truncated peptides form fibrils (12)(13)(14)(15)(16)(17)(18)(19)(20); (ii) neurotoxicity is linked with a peptide's ability to self-associate into filamentous ␤-strand aggregates (71-76); (iii) A␤ incorporates the core region, point mutations of which significantly obstruct fibril formation and have been used to generate inhibitors of fibrillogenesis (33)(34)(35)(36)(37)(38)(39); (iv) A␤ retains the ability to add to bona fide Alzheimer's plaques, in contrast to other truncated peptides (77)(78)(79)(80)(81), and forms fibrils morphologically similar to those of the full length peptide; (v) and of most importance, the full length peptide, A␤ , is intractable for the controlled formation of fibrils from aqueous media because at the earliest time points, some of the peptide exists as an amorphous precipitate. In contrast, the use of A␤ allowed the reproducible and controlled formation of fibrils from aqueous solutions, under defined conditions of pH, ionic strength, and peptide concentration and thus yielded the required homogeneous fibrils.…”

Thioflavine T interaction with synthetic Alzheimer's disease β‐amyloid peptides: Detection of amyloid aggregation in solution

“…The fluorescence change is related to peptide concentration and is dependent upon the total dye concentration, shown in the aggregated state of the amyloid peptides. A number of studies (Halverson et al, 1990;Fraser et al, 1991;Burdick et al, 1992) have correlated the sedimentability and electron microscopic appearance of amyloid fibrils for several synthetic P/A4 peptides. A more limited series of experiments confirms the sedimentation characteristics for P(1-28) and extends them to P(1-40).…”

Pauling and Corey's α-pleated sheet structure may define the prefibrillar amyloidogenic intermediate in amyloid disease

“…As described by Worcester (63) and Pauling (64), the diamagnetic anisotropy of the planar peptide groups in an ␣-helix contributes to a greater total magnetic anisotropy for ␣-helices compared with ␤-sheets. The diamagnetic anisotropy (63,64) of the peptide groups in the ␣-sheet structure may explain why amyloid fibrils grow more efficiently and in an ordered orientation in the presence of a magnetic field (65,66). Both amyloid fibrils (65,66) and ␣-helices (63, 64) orient with the helical axis parallel to the magnetic field.…”