A POTENTIAL MARKER PROTEASE OF INVASIVENESS, SEPRASE, IS LOCALIZED ONINVADOPODIA OF HUMAN-MALIGNANT MELANOMA-CELLS

Seprase, a large, gelatin-degrading membrane-protease complex, is expr essed at the invasive front of malignant melanoma cells on invadopodia , and its surface expression contributes to the invasive phenotype. An ia vitro assay was used to determine the matrix-degrading activity of four malignant human melanoma cell lines. The lines differ in matrix- degrading activity with LOX > RPMI7951 > A375 > SKMEL28. The seprase a nd Gelatinase A activities of these cell lines were also investigated. Seprase and active gelatinase A are found in cell membranes of LOX an d RPMI7951 cells but not those of SKMEL28 cells. Experiments using ant i-seprase monoclonal antibodies in conjunction with a cell fractionati on technique indicate that seprase consists of M(r) 97,000 polypeptide s and is enriched on the ventral membrane of LOX in contact with plana r extracellular matrix substratum. Confocal microscopy further substan tiates our biochemical findings that seprase, as well as Gelatinase A, is localized on invadopodia membranes with a 6-fold increase of sepra se and 4-fold increase of Gelatinase A intensity over the level expres sed on dorsal membranes. In addition, LOX cells expressing higher leve ls of seprase at the cell surface, as selected by fluorescence-activat ed cell sorting, are significantly more degradative than LOX Fells wit h lower seprase expression. Taken together, our data show a concordanc e between seprase and Gelatinase A expression on the cell surface at i nvadopodia and the matrix-degrading activity of human malignant melano ma cells. Seprase and major secreted proteases may act in concert to d egrade components of the extracellular matrix during invasion.