Oligopeptides Generated by Neprilysin Degradation of ß-Amyloid Have the Highest Cu(II) Affinity in the Whole Aß Family.

The catabolism of ß-amyloid (Aß) is carried out by numerous endopeptidases including neprilysin, which hydrolyzes peptide bonds preceding positions 4, 10, and 12 to yield Aß4-9 and a minor Aß12- x species. Alternative processing of the amyloid precursor protein by ß-secretase also generates the Aß11- x species. All these peptides contain a Xxx-Yyy-His sequence, also known as an ATCUN or NTS motif, making them strong chelators of Cu(II) ions. We synthesized the corresponding peptides, Phe-Arg-His-Asp-Ser-Gly-OH (Aß4-9), Glu-Val-His-His-Gln-Lys-am (Aß11-16), Val-His-His-Gln-Lys-am (Aß12-16), and pGlu-Val-His-His-Gln-Lys-am (pAß11-16), and investigated their Cu(II) binding properties using potentiometry, and UV-vis, circular dichroism, and electron paramagnetic resonance spectroscopies. We found that the three peptides with unmodified N-termini formed square-planar Cu(II) complexes at pH 7.4 with analogous geometries but significantly varied Kd values of 6.6 fM (Aß4-9), 9.5 fM (Aß12-16), and 1.8 pM (Aß11-16). Cyclization of the N-terminal Glu11 residue to the pyroglutamate species pAß11-16 dramatically reduced the affinity (5.8 nM). The Cu(II) affinities of Aß4-9 and Aß12-16 are the highest among the Cu(II) complexes of Aß peptides. Using fluorescence spectroscopy, we demonstrated that the Cu(II) exchange between the Phe-Arg-His and Val-His-His motifs is very slow, on the order of days. These results are discussed in terms of the relevance of Aß4-9, a major Cu(II) binding Aß fragment generated by neprilysin, as a possible Cu(II) carrier in the brain.