STABLY TRANSFECTED HUMAN-CELLS OVEREXPRESSING RAT-BRAIN ENDOPEPTIDASE-3.4.24.16 - BIOCHEMICAL-CHARACTERIZATION OF THE ACTIVITY AND EXPRESSION OF SOLUBLE AND MEMBRANE-ASSOCIATED COUNTERPARTS

We recently cloned endopeptidase-24.16 (neurolysin; EC 3.4.24.16), a n eurotensin-degrading peptidase likely involved in the physiological te rmination of the neurotensinergic signal in the central nervous system and in the gastrointestinal tract, We stably transfected human kidney cells with the pcDNA3-lambda 7aB1 construction bearing the whole open reading frame encoding the rat brain peptidase. Transfectants display ed endopeptidase-24.16 immunoreactivity and exhibited QFS- and neurote nsin-hydrolyzing activities, the biochemical and specificity propertie s of which fully matched those observed with the purified murine enzym e. Cryoprotection experiments and substrate degradation by intact plat ed cells indicated that transfectants exhibited a membrane-associated form of endopeptidase-24.16, the catalytic site of which clearly faced the extracellular domain. Transfected cells were unable to secrete th e enzyme. Overall, our experiments indicate that we have obtained stab ly transfectant cells that overexpress an enzymatic activity displayin g biochemical properties identical to those of purified endopeptidase- 24.16, The membrane-associated counterpart and lack of secretion of th e enzyme were clearly reminiscent of what was observed with pure cultu red neurons, but not with astrocytes. Therefore, the transfected cell model described here could prove useful for establishing, by a mutagen esis approach, the structural elements responsible for the ''neuronal' ' phenotype exhibited by the enzyme in transfected cells.