Examination of the role of endopeptidase 3.4.24.15 in A β secretion by human transfected cells

We have taken advantage of our recent development of highly potent and specific phosphinic inhibitors of endopeptidase 3.4.24.15 to examine the putative contribution of the enzyme in the secretion of Aβ by HK293 transfected cells overexpressing the wild type and the Swedish (Sw) double mutated form of βAPP 751 . First, we showed that HK293 cells contain a peptidase activity, the inhibition profile of which fully matches that of purified endopeptidase 3.4.24.15. Second, we established that the treatment of HK293 cells with specific phosphinic inhibitors leads to about 80% inhibition of intracellular endopeptidase 3.4.24.15 activity, indicating that these inhibitors penetrate the cells. Metabolic labelling of wild type and SwβAPP 751 ‐expressing cells, followed by immunoprecipitation of Aβ‐containing peptides, revealed the secretion of Aβ and the intracellular formation of an Aβ‐containing 12 kDa product. Aβ secretion by SwβAPP 751 transfected cells was drastically enhanced when compared to cells expressing wild type βAPP 751 . This production was not affected by endopeptidase 3.4.24.15 inhibitors in either cell type. This correlates well with the observation that endopeptidase 3.4.24.15 does not cleave recombinant baculoviral SwβAPP 751 , in vitro . Our previous data indicated that endopeptidase 3.4.24.15 activity was reduced in the parietal cortex of Alzheimer's disease affected brains and that the enzyme probably participated, in this brain area, to the catabolism of somatostatin 1‐14. However, the present work indicates that endopeptidase 3.4.24.15 does not seem to behave as a β‐secretase in HK293 transfected cells. Therefore, it is suggested that endopeptidase 3.4.24.15 could participate in the symptomatology, but probably not in the aetiology of Alzheimer's disease.