Characterization of a novel, membrane-bound, 80-kDa matrix-degrading protease from human breast cancer cells. Monoclonal antibody production, isolation, and localization.
A major, apparently novel extracellular matrix-degrading protease was previously identified and partially isolated from hormone-dependent but not from hormone-independent human breast cancer cells (Shi, Y. E., Torri, J., Yieh, L., Wellstein, A., Lippman, M. E., and Dickson, R. B. (1993) Cancer Res. 53, 1409-1415). Although initially the 80-kDa protease was identified from breast cancer cell-conditioned medium, immunofluorescence staining of breast cancer cells with anti-80-kDa protease monoclonal antibody 21-9 showed that in addition to its detection in intracellular compartments, the protease was uniformly localized around periphery of the cells with more intensive staining on the pseudopodia and membrane ruffles. A surface biotinylation technique confirmed the plasma membrane localization of the protease. In addition, the 80-kDa protease could not be washed from the membrane fraction of homogenized breast cancer cells with high concentrations of salts or with EDTA. The 80-kDa protease may noncovalently associate with other protein(s) to form complexes, the 95- and 110-kDa proteases. Both complexes showed gelatinolytic activity and bore the epitopes recognized by monoclonal antibody 21-9. Furthermore, both complexes could be converted to 80-kDa forms by boiling in SDS in the absence of reducing agents. Expression of this novel, integral membrane gelatinase could allow breast cancer cells an alternative to other previously described matrix-degrading enzymes for degradation of the extracellular matrix in close proximity to their surfaces.
Lin
C Y
CY
Lombardi Cancer Center, Georgetown University Medical Center, Washington, D. C. 20007, USA.
Wang
J K
JK
Torri
J
J
Dou
L
L
Sang
Q A
QA
Dickson
R B
RB
D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000378:metabolism*; D001943:Breast Neoplasms / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000032:analysis / Q000276:immunology*; D005109:Extracellular Matrix / Q000378:metabolism*; D005260:Female; D019085:Fluorescent Antibody Technique, Direct; D018093:Gelatinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D010450:Endopeptidases; D018093:Gelatinases