Genetic forms of disease provide the opportunity to identify the causative mutation(s), and in the case of a mutation within a protein coding region, to study the properties of the resultant mutant protein and attempt to relate these properties to disease mechanism. In AD, familial forms of the disease may arise from mutations affecting the absolute amounts of Aβ produced, the relative amounts of Aβ40, Aβ42, or Aβ peptides of other lengths, or the Aβ peptide sequence. A recently discovered FAD mutation in a family in Osaka, Japan, results in the production of Aβ peptides lacking Glu22 (ΔE22), [18
]. Initial studies seeking to determine the effect of this deletion on the biophysical behavior of the peptide suggested that the Osaka form of Aβ did not form fibrils in vitro
, although it did form oligomers, and that patients from the Osaka family did not have amyloid plaques [18
]. [ΔE22]Aβ inhibited hippocampal long-term potentiation (LTP) in rats and caused synapse loss in mouse hippocampal slices, although little effect was observed in MTT assays [18
]. Subsequent studies reported the identification of ΔE22Aβ oligomers in transfected cells, based on immunoreactivity with the ADDL-specific antibody NU1 [36
Our interest in the Osaka peptide was stimulated by the fact that Glu22 plays an important role in controlling Aβ monomer folding and subsequent peptide self-association [19
], and thus its elimination would be expected to have significant effects on these processes. Such effects have been observed in studies of Aβ mutants containing amino acid substitutions for Glu22 [20
]. The results of our studies of the ΔE22 forms of Aβ40 and Aβ42 show, contrary to prior published work, that elimination of Glu22 causes an extraordinary increase in the propensity of Aβ to form fibrils. We use the term “extraordinary” because the magnitude of the kinetic alteration, especially in the case of [ΔE22]Aβ42, was so great that fibril formation occurred during the initial solvation of the peptide.
It is possible that the prior failure to observe significant ThT fluorescence in [ΔE22]Aβ40 or [ΔE22]Aβ42 peptides [18
] may have been due to loss of protein by simple precipitation, as these experiments were done using a relatively high Aβ concentration (100 μM) and in a solvent, PBS, in which relatively rapid fibril formation occurs with even WT peptides. In our initial experiments, we encountered this phenomenon (data not shown) and were able to recognize it only by careful monitoring of protein concentration at each step of the experiments. Differences in peptide preparation methods also may have contributed to the different experimental results. Tomiyama et al.
] prepared their peptides using hexafluoroisopropanol solvation, evaporation, and ammonium hydroxide solubilization. A 1:9 (v/v; peptide:PBS) dilution then was made prior to certain experimental studies. Hexafluroisopropanol solvation, evaporation, DMSO solubilization, and PBS dilution was employed in other experiments. In addition to the distinct preparation procedures, the system of Tomiyama et al.
differs from ours in the presence of the ammonium cation or DMSO in the final PBS solution used for the experiments.
We began our studies by examining the time evolution of secondary structure. Consistent with prior studies [42
], Aβ40, Aβ42 and [ΔE22]Aβ40 existed predominately as statistical coils immediately after solvation, but all then displayed SC→β-sheet transitions. Remarkably, [ΔE22]Aβ40 underwent this transition ≈400-fold faster than did its WT homologue. [ΔE22]Aβ42 did not display such a transition at all, but rather possessed a level of β-sheet equivalent to that observed after fibril formation. The relative kinetics of secondary structure changes among the peptides was maintained in ThT fluorescence experiments. WT Aβ40 displayed lag and growth phases, whereas WT Aβ42 and [ΔE22]Aβ40 displayed no lag phases. [ΔE22]Aβ42 displayed no substantial transition in ThT fluorescence intensity, but rather exhibited high ThT fluorescence when the initial measurement was made.
The most significant factor contributing to the acceleration of Aβ40 assembly was diminution of the lag phase, which suggests that the Glu22 deletion facilitates the folding of the Aβ monomer into a conformer with a high propensity to self-associate into fibril nuclei. The effect of the Glu22 deletion in the Aβ42 system was primarily on overall fibril formation kinetics because no lag phases were observed. This observation is consistent with experimental and computational studies that have demonstrated that Aβ42 possesses greater initial structural order than does Aβ40 [19
We reported previously that Aβ40, Aβ42, and all reported mutant or truncated forms of these two peptides undergo a SC→α-helix→β-sheet conformational transition during assembly from nascent monomer to fibril [44
]. Fluorinated alcohols (TFE or HFIP) affected the kinetics of these conformational transitions, and thus the kinetics of peptide assembly, through their ability to stabilize helical forms of the peptides [48
]. The stabilization of α-helices depended not only on the intrinsic propensity of the peptide backbone to adopt helical states, due to its solvophobicity in fluorinated alcohols, but also on the primary structure-dependence of α-helix stability [30
]. Here, we probed the effect of the ΔE22 primary structure change on peptide conformational stability by determining the secondary structure content of Aβ40, Aβ42, and their ΔE22 homologues in different concentrations of fluorinated alcohols. In the Aβ40 system, increasing concentrations of TFE produced a SC→α-helix transition in the WT peptide. In contrast, a SC→β-sheet→α-helix transition occurred with the ΔE22 peptide. In kinetic studies of WT Aβ fibril formation, a SC→α-helix transition occurs obligatorily before
β-sheet formation [44
]. For this reason, low concentrations of TFE accelerate fibril formation by accelerating this initial conformational transition. The lack of an observed α-helix state in the [ΔE22]Aβ40 peptide at low TFE concentrations suggests that the ΔE22 mutation increases the peptide's propensity for β-sheet formation so significantly that the SC→α-helix transition occurs too quickly to be monitored in the experimental system employed. Higher concentrations of TFE block the α-helix→β-sheet transition, and hence fibril formation, by increasing the activation energy for the transition or by decreasing the free energy of the α-helix state [48
]. Consistent with this observation, higher TFE concentrations did result in α-helix formation in the [ΔE22]Aβ40 peptide. The effect of the ΔE22 mutation on Aβ40 was so large that the behavior of the mutant peptide was similar to that of WT Aβ42. Not surprisingly, the propensity of the ΔE22 variant of Aβ42 for β-sheet formation was so high that no α-helix conformer could be observed at low TFE concentration and no such conformer was formed up to 60% TFE. An α-helix state was
observed when 100% TFE was used. Qualitatively similar data were obtained using HFIP, with the one exception that α-helix was observed in [ΔE22]Aβ42 when the HFIP concentration reached 20%. The rank order of β-sheet propensity, [ΔE22]Aβ42 >> Aβ42 > [ΔE22]Aβ40 > Aβ40, also was observed in studies of the effect of pH 10 and pH 12 on conformation. In these experiments, the latter two peptides largely existed in SC form. [ΔE22]Aβ40 displayed a β-sheet element, whereas [ΔE22]Aβ42 was largely β-sheet. Taken together, the results of these three experiments support a conclusion that the ΔE22 mutation substantially increases peptide β-sheet propensity in Aβ40 and produces β-sheet structure of extraordinary stability in [ΔE22]Aβ42.
Conformational and kinetics differences among the peptides were mirrored by their oligomerization states. [ΔE22]Aβ40 had a more restricted distribution than did WT Aβ40, one in which the largest predominant oligomer was trimer instead of tetramer. The molecular basis for this observation is unclear. In addition, the [ΔE22]Aβ40 oligomers all displayed electrophoretic mobilities greater than those of their WT homologues. The simplest explanation for this difference is the 128 molecular weight decrement in the mutant peptides. However, it also is possible that the mutant oligomers have more compact structures or bind different amounts of SDS.
The largest differences in oligomerization were seen in the [ΔE22]Aβ42 peptides, which showed higher propensities for oligomerization. Increased oligomer frequencies were observed in the region corresponding to paranuclei [34
]. In addition, higher-order oligomers (dodecamer and octadecamer regions) were observed in the [ΔE22]Aβ42 samples that were not seen at all in the WT samples. The accelerated conformational and assembly kinetics displayed by the mutant peptides thus correlates with an increased oligomerization propensity. Not surprisingly, the rapid early kinetics also was reflected in rapid fibril formation. Whereas both WT Aβ40 and Aβ42 peptides possessed nebulous globular and very short irregular string-like morphologies immediately upon solvation from lyophilizates, short protofibrillar and fibrillar structures were evident immediately in the ΔE22 samples.
A kinetic effect of the ΔE22 mutation also was supported by the results of measurement of the Cr
for each of the four peptides. These values ranged from 0.32–1.24 μM, consistent with values reported before for Aβ40 in PBS [49
]. To the nearest 0.5 μM, the Cr
values of the ΔE22 peptides were ≈1/2 those of the respective WT peptides. Using these Cr
calculations revealed that the stability of the fibrils formed by each ΔE22 peptide was ≈0.5 kcal/mol larger than its respective WT homologue. This small stability increase, less than a single H-bond, is too low to account for the observed rate differences in CD spectral changes, increases in ThT fluorescence, and fibril evolution. We conclude that the primary effect of the ΔE22 mutation is to stabilize β-structure within the Aβ monomer or within low-order oligomers, which results in an extraordinary change in the kinetics of fibril formation without producing a change of equivalent magnitude in system thermodynamics (i.e., fibril stability). This mechanism is consistent with prior experimental and computational studies of the kinetics and thermodynamics of Aβ fibril formation that suggest that monomer conformational rearrangement is a rate-limiting step in fibril elongation [51