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 neurotensin-degrading peptidase likely involved in the physiological termination 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 displayed endopeptidase-24.16 immunoreactivity and exhibited QFS- and neurotensin-hydrolyzing activities, the biochemical and specificity properties of which fully matched those observed with the purified murine enzyme. Cryoprotection experiments and substrate degradation by intact plated 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 the enzyme. Overall, our experiments indicate that we have obtained stably transfectant cells that overexpress an enzymatic activity displaying biochemical properties identical to those of purified endopeptidase-24.16. The membrane-associated counterpart and lack of secretion of the enzyme were clearly reminiscent of what was observed with pure cultured neurons, but not with astrocytes. Therefore, the transfected cell model described here could prove useful for establishing, by a mutagenesis approach, the structural elements responsible for the "neuronal" phenotype exhibited by the enzyme in transfected cells.
Vincent
B
B
IPMC du CNRS, UPR 411, Valbonne, France.
Dauch
P
P
Vincent
J P
JP
Checler
F
F
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002460:Cell Line / Q000502:physiology; D004072:Digitonin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry; D007202:Indicators and Reagents / Q000494:pharmacology; D007668:Kidney / Q000166:cytology; D008565:Membrane Proteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D017693:Sodium Bicarbonate / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D012995:Solubility; D014162:Transfection
D007202:Indicators and Reagents; D008565:Membrane Proteins; D012965:Sodium Chloride; D017693:Sodium Bicarbonate; D008666:Metalloendopeptidases; C062316:neurolysin; D004072:Digitonin