Monsky W L, Lin C Y, Aoyama A, Kelly T, Akiyama S K, Mueller S C, Chen W T
Department of Cell Biology, Georgetown University School of Medicine, Washington, DC 20007.
Cancer Res. 1994 Nov 1;54(21):5702-10.
Seprase, a large, gelatin-degrading membrane-protease complex, is expressed at the invasive front of malignant melanoma cells on invadopodia, and its surface expression contributes to the invasive phenotype. An in 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 > RPM17951 > A375 > SKMEL28. The seprase and Gelatinase A activities of these cell lines were also investigated. Seprase and active gelatinase A are found in cell membranes of LOX and RPM17951 cells but not those of SKMEL28 cells. Experiments using anti-seprase monoclonal antibodies in conjunction with a cell fractionation technique indicate that seprase consists of M(r) 97,000 polypeptides and is enriched on the ventral membrane of LOX in contact with planar extracellular matrix substratum. Confocal microscopy further substantiates our biochemical findings that seprase, as well as Gelatinase A, is localized on invadopodia membranes with a 6-fold increase of seprase and 4-fold increase of Gelatinase A intensity over the level expressed on dorsal membranes. In addition, LOX cells expressing higher levels of seprase at the cell surface, as selected by fluorescence-activated cell sorting, are significantly more degradative than LOX cells with lower seprase expression. Taken together, our data show a concordance between seprase and Gelatinase A expression on the cell surface at invadopodia and the matrix-degrading activity of human malignant melanoma cells. Seprase and major secreted proteases may act in concert to degrade components of the extracellular matrix during invasion.
分离酶是一种大型的、能降解明胶的膜蛋白酶复合物，在恶性黑色素瘤细胞侵袭前沿的侵袭伪足上表达，其表面表达有助于形成侵袭表型。采用体外试验来测定四种人恶性黑色素瘤细胞系的基质降解活性。这些细胞系在基质降解活性上存在差异，LOX > RPM17951 > A375 > SKMEL28。还对这些细胞系的分离酶和明胶酶A活性进行了研究。在LOX和RPM17951细胞的细胞膜中发现了分离酶和活性明胶酶A，而在SKMEL28细胞中未发现。使用抗分离酶单克隆抗体结合细胞分级分离技术进行的实验表明，分离酶由分子量为97,000的多肽组成，并且在与平面细胞外基质底物接触的LOX细胞腹侧膜上富集。共聚焦显微镜进一步证实了我们的生化研究结果，即分离酶以及明胶酶A定位于侵袭伪足膜上，其强度比背侧膜上表达的水平分别增加了6倍和4倍。此外，通过荧光激活细胞分选选择的在细胞表面表达较高水平分离酶的LOX细胞，其降解能力明显高于分离酶表达较低的LOX细胞。综上所述，我们的数据表明，在侵袭伪足处细胞表面的分离酶和明胶酶A表达与人类恶性黑色素瘤细胞的基质降解活性之间存在一致性。分离酶和主要分泌蛋白酶可能协同作用，在侵袭过程中降解细胞外基质成分。
