Identification of the 170-kDa Melanoma Membrane-bound Gelatinase (Seprase) as a Serine Integral Membrane Protease

Abstract

The 170-kDa membrane-bound gelatinase, seprase, is a cell surface protease, the expression of which correlates with the invasive phenotype of human melanoma and carcinoma cells. We have isolated seprase from cell membranes and shed vesicles of LOX human melanoma cells. The active enzyme is a dimer of N-glycosylated 97-kDa subunits. Sequence analysis of three internal proteolytic fragments of the 97-kDa polypeptide revealed up to 87.5% identity to the 95-kDa fibroblast activation protein ␣ (FAP␣), the function of which is unknown. Thus, we used reverse transcription-polymerase chain reaction to generate a 2.4-kilobase cDNA from LOX mRNA with FAP␣ primers. COS-7 cells transfected with this cDNA expressed a 170-kDa gelatinase that is recognized by monoclonal antibodies directed against seprase. Sequence analysis also showed similarities to the 110-kDa subunit of dipeptidyl peptidase IV (DPPIV). Like DPPIV, the gelatinase activity of seprase was completely blocked by serine-protease inhibitors, including diisopropyl fluorophosphate. Seprase could be affinitylabeled by [ 3 H]diisopropyl fluorophosphate, but the proteolytically inactive 97-kDa subunit could not, confirming the existence of a serine protease active site on the dimeric form. Proteolytic activity is lost upon dissociation into its 97-kDa subunit following treatment with acid, heat, or cysteine and histidine-modifying agents. We conclude that seprase, FAP␣, and DPPIV are related serine integral membrane proteases and that seprase is similar to DPPIV, the proteolytic activities of which are dependent upon subunit association.Seprase was originally identified from a human malignant melanoma cell line LOX, which exhibited aggressive behavior in experimental metastasis (1, 2). It is a membrane glycoprotein with gelatinase activity that is expressed and localized at the invasion front during invasion into the ECM 1 by human melanoma, breast carcinoma cells, and chicken embryo fibroblasts transformed by Rous sarcoma virus (3-6). The protease is a hydrophobic glycoprotein soluble in Triton X-100 and in SDS (3). Seprase shows gelatinolytic activity as demonstrated by gelatin zymography (3,4). The enzyme maintains maximal activity at neutral pH, which can be further enhanced by SDS buffer, EDTA, the cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane and dithiothreitol. However, seprase has a peculiar protease inhibitor profile; it is inhibited by the protease inhibitors PMSF and NEM (3). A major problem in purifying seprase was the apparent low level of this protein in cultured cell lines. Because LOX cells produced a large quantity of membrane vesicles that were enriched in seprase, we isolated seprase from the cell membranes and shed vesicles in LOX conditioned medium and characterized its subunit composition and its enzymatic activity. We found that the protease had an apparent molecular mass of 170 kDa composed of proteolytically inactive 97-kDa subunits. Amino acid analysis of internal proteolytic fragments of the 97-kDa polypep...

Purification, identification and characterisation of seprase from bovine serum

“…Even though conflicting reports exist on possible prolyl dipeptidyl peptidase activity by seprase (Chen, Kelly & Ghersi 2003;Ghersi et al 2002;Pineiro-Sanchez et al 1997) no such activity was detected by purified ZIP/Seprase using Gly-Pro-AMC. Further substrate specificity studies suggest ZIP/Seprase has an extended substrate-binding region in addition to the primary specificity site, S 1 .…”

“…Seprase has also been shown to be one of a number of serine proteases required for microvascular endothelial cell reorganization and capillary morphogenesis in vitro (Aimes et al 2003). It is overexpressed by invasive tumour cells (Pineiro-Sanchez et al 1997;Monsky et al 1994) in particular it has been shown to be expressed by activated fibroblasts in the stroma of various epithelial cancers, mesenchymal tumours and breast-cancer cells (Ariga et al 2001). Clinicopathologic analysis has revealed increased Seprase expression in cases of invasive ductal carcinoma is associated with longer overall and disease-free survival (Ariga et al 2001;Kelly et al 1998).…”

Phase II trial of single agent Val-boroPro (talabostat) inhibiting fibroblast activation protein in patients with metastatic colorectal cancer

“…An indirect sandwich ELISA-based assay based on previously published methods 24,25 was performed using patient plasma samples to determine circulating FAP concentrations for patients on Val-boroPro treatment. 96 well polystyrene plates with maxi-sorp treated surfaces were coated with 50μg/ml monoclonal antibody D8, 26 obtained from rat hybridoma serum-free supernatant (kind gift from Wen-Tien Chen, SUNY, Stony Brook, NY) for 2 h. PBS with 0.05% Tween 20 and 3% BSA was used to block non-specific binding. Plates were blocked overnight and washed, and then known concentrations of recombinant human FAP (0-6 μg/ml) were added to the standard wells to develop a standard curve for each assay.…”

A Novel Protease-docking Function of Integrin at Invadopodia

“…Antibodies and Immunofluorescence Labeling-Anti-seprase monoclonal antibodies (mAbs) D28 and D8 have previously been described (14,15). Anti-␤ 1 polyclonal (number 3847) and mAb 13 antibodies were used to detect ␤ 1 integrins (17), and anti-vitronectin receptor antibodies was used to detect the ␤ 3 subunit of the vitronectin receptor (Life Technologies, Inc.).…”

“…Moreover, both adhesive and signaling activities of integrins can be regulated by the interaction between integrins and the urokinase plasminogen activator/receptor (12). We have shown that in LOX melanoma cells, a 170-kDa membrane gelatinase, seprase, was localized to invadopodia and associated with the invasive phenotype (13)(14)(15)(16). Sequencing data on the 97-kDa protein subunit of seprase indicates only a short (six) amino acid sequence at the cytoplasmic amino terminus (14), suggesting that seprase localization at invadopodia may be dependent upon other membrane proteins such as integrins.…”