Identification of a membrane-bound pteroyl poly gamma-glutamyl carboxypeptidase (folate hydrolase) that is highly expressed in human prostatic carcinoma cells

We investigated using capillary electrophoresis pteroyl poly-gamma-glutamate carboxypeptidase (hydrolase) activity in membrane preparations from androgen-sensitive human prostatic carcinoma cells (LNCaP). The enzyme immunologically cross-reacts with a derivative of an anti-prostate monoclonal antibody (7E11-CS) that recognizes prostate specific membrane (PSM) antigen. The PSM enzyme hydrolyzes gamma-glutamyl linkages and is an exopeptidase as it liberates progressively glutamates from methotrexate triglutamate (MTXGlu₃) and folate pentaglutamate (PteGlu₅) with accumulation of MTX and PteGlu₃ respectively. The semi-purified membrane-bound enzyme has a broad activity from pH 2 to 10 and is maximally active at pH 4.0. Enzymatic activity was weakly inhibited by dithiothreitol (≥ 0.2 mM) but not by reduced glutathione, homocysteine, or phydroxymercuribenzoate (0.05-0.5 mM). By contrast to LNCaP cell membranes, membranes isolated from androgen-insensitive human prostate (TSU-Prl, Duke-145, PC-3) and estrogen-sensitive mammary adenocarcinoma (MCF-7) cells do not exhibit comparable hydrolase activity nor do they react with 7E11-C5. Thus, we have identified in LNCaP cells a folate hydrolase that exhibits exopeptidase activity and is strongly expressed by these cells. (Supported by NIH grants CA39203, -29502, -08748-29 & DK/CA 47650).