Major Carboxyl Terminal Fragments Generated by [gamma]-Secretase Processing of the Alzheimer Amyloid Precursor Are 50 and 51 Amino Acids Long

To understand the cleavage of the amyloid [beta] protein (A[beta]) precursor (APP) by γ-secretase and to determine its changes in a representative familial Alzheimer disease (FAD) mutation. Transfected cells expressing wild-type and FAD mutant APP were analyzed for changes in the levels of the major secreted A[beta] species and of the corresponding intracellular C-terminal APP fragments (APP intracellular domain, AICD) generated by γ-secretase, whereas radio-sequencing was used to precisely identify the resulting cleavage site(s). The AICD fragment(s) generated by γ-secretase cleavage comigrated in gels with a 50-residue synthetic peptide used as control, which is smaller than the 59 and 57 residues predicted from A[beta] ending at positions 40 (A[beta]40) and 42 (A[beta]42), respectively. In agreement with previous findings, an FAD mutant form of presenilin 1 (PS1-M139V) significantly increased the longer A[beta]42 while showing trends toward reducing A[beta]40. AICD levels were reduced by the mutation, suggesting that γ-secretase activity may be actually impaired by the mutation. Radiosequence analysis in cells expressing wild-type PS1 detected γ-secretase cleavage sites at the A[beta] peptide bond L(49)-V(50) to generate a 50-amino acid (aa) AICD fragment (AICD50) and the A[beta] peptide bond T(48)-L(49), generating an AICD of 51 aa (AICD51). No other cleavage sites were reliably detected. Based on findings that the FAD mutation that increases A[beta]42 also reduces AICD, we propose that γ-secretase activity is impaired by FAD mutations and predict that physiologic and environmental agents that inhibit γ-secretase will actually induce AD pathogenesis rather that prevent it. Furthermore, we propose that the cleavage site to generate AICD is naturally ragged and occurs predominantly at two sites 48 and 49 aa from the start of the A[beta] sequence. Thus, end specific antibodies to these two sites will need to be generated to study the quantitative relationships between these two cleavages in sporadic AD and FAD.