Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells.

Abstract

Abstract. We have studied the posttranslational modifications of the 52-kD protein, an estrogenl~gulated 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-13-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.

Cathepsin D is partly endocytosed by the LRP1 receptor and inhibits LRP1-regulated intramembrane proteolysis

“…We first analyzed the endocytosis of S 35 -radiolabelled pro-cath-D secreted by cancer cells in human mammary fibroblasts (HMF) in which endogenous LRP1 synthesis had been blocked by RNA interference ( Figure 1A). Internalized labelled 52-kDa pro-cath-D was first transformed into a 48-kDa endosomal intermediate and then into the 34-kDa lysosomal mature enzyme ( Figure 1A, panel a, lane 1) (Capony et al, 1987). Excess M6P prevented pro-cath-D from binding to its M6P receptors and inhibited the internalization of pro-cath-D by 82% ( Figure 1A, panel a, compare lanes 1 and 3).…”

Elevations in Cathepsin B Protein Content and Enzyme Activity Occur Independently of Glycosylation during Colorectal Tumor Progression

“…In breast carcinomas, cathepsin D was also found to have a more acidic isoelectric point compared with normal breast tissue, due to modifications of the oligosaccharides expressed on the cathepsin D protein (45). Thus, our data may suggest a similar occurrence to that observed by others in that both nonglycosylated cathepsin B protein in adenomas and glycosylated cathepsin B protein in carcinomas have similar subcellular localizations, but in carcinomas the altered processing of cathepsin B protein, detected as increased amounts of glycosylation, may act to modify the endogenous rate of inhibition at a particular intracellular site or the ability to cleave subcellular or extracellular substrates.…”

Cathepsin D: newly discovered functions of a long-standing aspartic protease in cancer and apoptosis

“…However, cath-D can cleave its substrates up to a pH of 6.2 in vitro, but not above that level [123]. Hence, it is predictable that the proteolytic activity of cytosolic cath-D would be drastically impaired under adverse pH conditions, unfavourable for its catalytic function.…”