Characterization of purified cathepsin D from malignant human breast tissue

The aspartyl protease cathepsin D (EC 3.4.23.5) appears to be found in incr eased amounts and/or abnormally secreted in breast cancer cells, and may co ntribute to the metastatic spread of malignancy. In the present study, cath epsin D was purified 4800-fold in 20% yield from malignant human breast tis sue using affinity chromatography on pepstatin-agarose and DEAE-Sephadex ch romatography. Slab gel SDS-PAGE of the purified cathepsin D indicated the p resence of three major protein bands (31, 13, 12, kDa) and two minor protei n bands (47, 29 kDa). Western blotting indicated that the 31 kDa band was t he major immunoreactive species. Isoelectric focusing indicated that the pu rified cathepsin D consisted of three major isoforms at approximate pIs of 7.4, 7.0 and 6.6, and a possible isoform of lower activity centered around pI 3.2. The pH curve of purified cathepsin D indicated a broad optimum cent ered around pH 3.4. Lectin blotting suggested the presence of mannose resid ues but no evidence was found for lectin-available sialic acid, fucose, N-a cetylglucosamine and galactose residues. The investigated properties of pur ified cathepsin D from malignant breast tissue are very similar, if not ide ntical, to the properties of cathepsin D previously purified from normal hu man breast tissue. Our findings suggest that the elevated activity and anti genic levels of cathepsin D in malignant breast tissue are due to increased amounts of apparently normal enzyme.