We have studied the posttranslational modifications of the 52-kD
protein, an
estrogen-regulated autocrine
mitogen secreted by several human
breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD
protein was found to be phosphorylated mostly (94%) on high-
mannose N-linked
oligosaccharide chains, and
mannose-6-phosphate signals were identified. The
phosphate signal was totally removed by
alkaline phosphatase hydrolysis. The secreted 52-kD
protein was partly taken up by MCF7 cells via
mannose-6-phosphate receptors and processed into 48- and 34-kD
protein moieties as with lysosomal
hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive
proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD
protein and its processed cellular forms as aspartic and acidic
proteinases specifically inhibited by
pepstatin. The 52-kD
protease is secreted in
breast cancer cells under its inactive
proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The
enzyme of
breast cancer cells, when compared with
cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD
protein and its cellular processed forms of
breast cancer cells were totally sensitive to
endo-beta-N-acetylglucosaminidase H (Endo H), whereas several cellular
cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD
cathepsin D-like
enzyme of
breast cancer cells is different from previously described
cathepsin D(s). The 52-kD
estrogen-induced lysosomal
proteinase may have important functions in facilitating the
mammary cancer cells to proliferate, migrate, and metastasize.