CHARACTERIZATION BY RADIOSEQUENCING OF THE CARBOXYL-TERMINAL DERIVATIVES PRODUCED FROM NORMAL AND MUTANT AMYLOID-BETA PROTEIN PRECURSORS

The 39-43 amino acid (similar to 4 kD) amyloid beta-protein (A beta) d eposited as amyloid Alzheimer's disease is an infernal peptide beginni ng 99 residues from the COOH end of a much larger amyloid beta-protein precursor (beta APP). In cultured cells, normal processing of the bet a APP produces similar to 8.7, similar to 9.6, similar to 10.9, and si milar to 11.4 kD COOH-terminal derivatives that appear to contain all or part of the A beta domain. In this study, we metabolically labeled transfected human neuroblastoma (M17) cells with [H-3]phenylalanine pl us [S-35]methionine and then radiosequenced the immunoprecipitated COO H-terminal beta APP derivatives faking advantage of the fact that the A beta has phenylalanines at positions 4, 19, and 20, and a single met hionine at position 35. Our analysis of the derivatives produced by tr ansfected M17 cells expressing beta APP(695) confirms that the similar to 8.7 kD COOH-terminal derivative begins at A beta(17) and indicates that the similar to 9.6 and similar to 10.9 kD derivatives begin at A beta(10) and A beta(4) respectively. Significantly we find that the 1 1.4 kD derivative begins at A beta(1). Thus normal beta APP processing produces a potentially amyloidogenic COOH-terminal derivative that ha s the AP domain intact at its amino terminus. We have previously shown that cells expressing beta APP(Delta NL) a mutant linked to familial Alzheimer's disease, produce an increased amount of the 11.4 kD COOH-t erminal derivative and secrete more A beta. Radiosequencing of these d erivatives showed that the Delta NL mutant is cleaved at the same loca tion as wild type beta APP producing an 11.4 kD COOH-terminal derivati ve and A beta that both begin at A beta(1). Thus the Delta NL mutation appears to accelerate a cleavage that releases an 11.4 kD COOH-termin al derivative identical to that normally produced from wild type beta APP, and it appears that this 11.4 kD derivative is further processed to release excess A beta.