CHARACTERIZATION OF HUMAN CELLULAR GAMMA-GLUTAMYL HYDROLASE

A previously identified cDNA encoding a human gamma-glutamyl hydrolase was expressed in a baculovirus system. The expressed protein had mole cular mass of 37 kDa. Treatment of the protein with PNGase F produced a protein of molecular mass of 30 kDa, indicating that the protein con tained asparagine-linked glycosylation. Sequence analysis of the expre ssed protein indicated that a 24-amino-acid signal peptide had been re moved. A polyclonal antibody to the expressed enzyme was used in Weste rn blot analysis of partially purified lysates of HL-60 promyeloid leu kemia cells and MCF-7 breast cancer cells. The HL-60 and MCF-7 enzymes appeared as two closely spaced bands with a molecular mass of 37 kDa. Treatment of the HL-60 enzyme with PNGase F produced a protein with a molecular mass of 30 kDa. The activities of the expressed enzyme and the enzyme from HL-60 cells were similar on methotrexate polyglutamate s. Methotrexate-gamma-Glu is a poor substrate for the human enzyme rel ative to methotrexate gamma-Glu(2-5). During hydrolysis of methotrexat e-gamma-Glu(4), all possible pterin-containing cleavage products (meth otrexate and methotrexate-gamma-Glu(1-3)) appear. The results demonstr ated that the human enzyme cleaves both the ultimate and penultimate g amma-linkages of methotrexate polyglutamates. Glutamate was released a s either glutamic acid or gamma-Glu(2). Longer chain species of gamma- Glu(n>2) were not observed. Inhibition by iodoacetic acid suggested th at both the expressed enzyme and the HL-60 enzyme may contain a cataly tically essential cysteine. These results indicate that the identified cDNA encodes the intracellular gamma-glutamyl hydrolase found in a va riety of human tumor cells and that the baculovirus-expressed enzyme i s a suitable model for further structural and enzymatic studies.