Characterization and properties of two monoclonal antibodies specific for the Mr 52,000 precursor of cathepsin D in human breast cancer cells.
The Mr 52,000 estrogen-induced protein secreted by MCF7 cells has been identified as a procathepsin D (procath D), which increases cell growth in vitro, may stimulate invasiveness by digesting extracellular matrix and appears to be a tissue marker for predicting relapses in breast cancer patients. The protease is also present within mammary cells as Mr 48,000, 34,000, 14,000 mature forms that were also recognized by the previously described antibodies to the Mr 52,000 protein (M. Garcia, F. Capony, D. Derocq, D. Simon, B. Pau, and H. Rochefort, Cancer Res., 45: 709-716, 1985). Using selective screening with a [35S]methionine-labeled MCF7 cell lysate, we have now isolated two new monoclonal antibodies interacting exclusively with the Mr 52,000 procath D. The two monoclonal antibodies, M2E8* and D9H8*, purified from ascitic fluids are IgG1. Their respective Kds for Mr 52,000 procath D are 0.96 and 0.18 nM. They are directed against two separate domains of the proenzyme that differ from the three domains of previously described antibodies. They both interact with the deglycosylated protein and recognize the autoactivated secreted proenzyme (Mr 51,000), which is devoid of the first part of the NH2-terminal end. By immunodetection of cathepsin D proteolytic activity in plasma, these two antibodies were found to recognize selectively human cathepsin D but not the cathepsin D of other species (rat, mouse, rabbit, goat, and horse) whereas antibodies to mature cathepsin D were less species specific. Using sequential passages on concanavalin A-Sepharose and Sepharose matrices coupled to antibodies to the precursor and antibodies to the mature cathepsin D, we separately purified to homogeneity the Mr 52,000 procath D form and its processed cellular forms, whose biological activities can now be assessed independently. The two monoclonal antibodies were also shown to inhibit the uptake and processing of the Mr 52,000 cathepsin D in MCF7 cells (mostly D9H8*) and to decrease its proteolytic activity, mostly on extracellular matrix (M2E8*). These two monoclonal antibodies are therefore new tools for studying the function, regulation, cellular processing, and localization of procath D as well as the clinical significance of its concentration in normal and tumoral mammary epithelial cells.
Freiss
G
G
INSERM U 148, Unité Hormones et Cancer, Montpellier, France.
Vignon
F
F
Rochefort
H
H
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001692:Biological Transport; D001943:Breast Neoplasms / Q000201:enzymology / Q000276:immunology*; D002402:Cathepsin D / Q000276:immunology* / Q000302:isolation & purification; D004792:Enzyme Precursors / Q000276:immunology* / Q000302:isolation & purification; D000939:Epitopes; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D013045:Species Specificity; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D000939:Epitopes; D006023:Glycoproteins; D002402:Cathepsin D