HUMAN BLEOMYCIN HYDROLASE - MOLECULAR-CLONING, SEQUENCING, FUNCTIONALEXPRESSION, AND ENZYMATIC CHARACTERIZATION

We have cloned the cDNA of human bleomycin hydrolase (hBH), a protease which is thought to be involved in the metabolic inactivation of-the antineoplastic drug bleomycin. The open reading frame consists of 1365 base pairs and is predicted to encode a 52 kDa protein. The protein s hares 40% identity with yeast bleomycin hydrolase and contains the con served active site residues (Cys, His, Asn) characteristic for cystein e proteases of-the papain superfamily. Human bleomycin hydrolase has b een functionally expressed in Spodoptera frugiperda Sf9 cells using th e Autographa californica nuclear polyhedrosis virus. The 52 kDa recomb inant protein forms a hexamer of 310 kDa and acts strictly as an amino peptidase with a broad substrate specificity. The lack of a leader seq uence and its pH optimum at 7.2 suggest a cytosolic/nuclear localizati on. Human bleomycin hydrolase was detected at low to moderate expressi on levels in most of the human organs tested. Significantly higher RNA levels have been observed in a variety of tumor cell lines. The human enzyme effectively degrades both farms of bleomycin (A2 and B2) in vi tro and could indeed be responsible for the resistance of various tumo rs to this widely used anticancer drug.