In the present study we identified the epitopes of antibodies against amyloid β-(1–42)-peptide (Aβ1–42): 4G8 reacted with peptides corresponding to residues 17–21, 6F/3D reacted with peptides corresponding to residues 9–14, and anti 5-10 reacted with peptides corresponding to residues 5–10. The study also yielded some insight into the Aβ1–42 structures resulting from differences in pH. An ELISA study using monoclonal antibodies showed that pH-dependent conformational changes occur in the 6F/3D and 4G8 epitopes modified at pH 4.6, but not in the sequences recognized by anti 1-7 and anti 5-10. This was unique to Aβ1–40 and Aβ1–42 and did not occur with Aβ1–16 or Aβ17–42. The reactivity profile of 4G8 was not affected by blockage of histidine residues of pH-modified Aβ1–40 and Aβ1–42 with diethyl pyrocarbonate; however, the mutant [Gln11]Aβ1–40 abrogated the unique pH-dependence towards 4G8 observed with Aβ1–40. These findings suggest that these epitopes are cryptic at pH4.6, and that Glu11 is responsible for the changes. We suggest that the abnormal folding of 6F/3D epitope affected by pH masked the 4G8 epitope. A study of the binding of metal ions to Aβ1–42 suggested that Cu2+ and Zn2+ induced a conformational transition around the 6F/3D region at pH7.4, but did not affect the region when it was modified at pH4.6. However, Fe2+ had no effect, irrespective of pH. Aβ modified at pH 4.6 appeared to be relatively resistant to proteinase K compared with Aβs modified at pH7.4, and the former might be preferentially internalized and accumulated in a human glial cell. Our findings suggest the importance of microenvironmental changes, such as pH, in the early stage of formation of Aβ aggregates in the glial cell.
Structural insight into a partially unfolded state preceding aggregation in an intracellular lipid-binding protein
