Characterization by radiosequencing of the carboxyl-terminal derivatives produced from normal and mutant amyloid β protein precursors

Abstract

The 39–43 amino acid (∼ kD) amyloid β protein (Aβ) deposited as amyloid Alzheimer's disease is an internal peptide beginning 99 residues from the COOH end of a much larger amyloid fiprotein precursor (βAPP). In cultured cells, normal processing of the PAPP produces ∼8.7, ∼9.6, ∼10.9, and ∼ 11.4 kD COOH-terminal derivatives that appear to contain all or part of the Aβ domain. In this study, we metabolically labeled transfected human neuroblastoma (Ml 7) cells with PH] phenylalanine plus (35SJmethionine and then radiosequenced the immunopre-cipitated COOH-terminal βAPP derivatives taking advantage of the fact that the Aβ has phenylalanines at positions 4, 19, and 20, and a single methionine at position 35. Our analysis of the derivatives produced by transfected Ml 7 cells expressing βAPP695 confirms that the ∼8.7 kD COOH-terminal derivative begins at Aβ17 and indicates that the ∼9.6 and ∼10.9 kD derivatives begin at Aβ10 and Aβ4 respectively. Significantly, we find that the 11.4 kD derivative begins at APr Thus normal PAPP processing produces a potentially amyloidogenic COOH-terminal derivative that has the Aβ domain intact at its amino terminus. We have previously shown that cells expressing βAPPWL, a mutant linked to familial Alzheimer's disease, produce an increased amount of the 11.4 kD COOH-terminal derivative and secrete more Aβ. Radiosequencing of these derivatives showed that the ANL mutant is cleaved at the same location as wild type βAPP producing an 11.4 kD COOH-terminal derivative and Aβ that both begin at Aβ Thus the ANL mutation appears to accelerate a cleavage that releases an 11.4 kD COOH-terminal derivative identical to that normally produced from wild type PAPP, and it appears that this 11.4 kD derivative is further processed to release excess Aβ.