Title: The protease complex consisting of dipeptidyl peptidase IV and seprase plays a role in the migration and invasion of human endothelial cells in collagenous matrices Ghersi G, Zhao Q, Salamone M, Yeh Y, Zucker S, Chen WT Ref: Cancer Research, 66:4652, 2006 : PubMed
Dipeptidyl peptidase IV (DPP4/CD26) and seprase/fibroblast activation protein alpha are homologous type II transmembrane, homodimeric glycoproteins that exhibit unique prolyl peptidase activities. Human DPP4 is ubiquitously expressed in epithelial and endothelial cells and serves multiple functions in cleaving the penultimate positioned prolyl bonds at the NH(2) terminus of a variety of physiologically important peptides in the circulation. Recent studies showed a linkage between DPP4 and down-regulation of certain chemokines and mitogenic growth factors, and degradation of denatured collagens (gelatin), suggesting a role of DPP4 in the cell invasive phenotype. Here, we found the existence of a novel protease complex consisting of DPP4 and seprase in human endothelial cells that were activated to migrate and invade in the extracellular matrix in vitro. DPP4 and seprase were coexpressed with the three major protease systems (matrix metalloproteinase, plasminogen activator, and type II transmembrane serine protease) at the cell surface and organize as a complex at invadopodia-like protrusions. Both proteases were colocalized at the endothelial cells of capillaries, but not large blood vessels, in invasive breast ductal carcinoma in vivo. Importantly, monoclonal antibodies against the gelatin-binding domain of DPP4 blocked the local gelatin degradation by endothelial cells in the presence of the major metallo- and serine protease systems that modified pericellular collagenous matrices and subsequent cell migration and invasion. Thus, we have identified a novel mechanism involving the DPP4 gelatin-binding domain of the DPP4-seprase complex that facilitates the local degradation of the extracellular matrix and the invasion of the endothelial cells into collagenous matrices.
Seprase is a homodimeric 170 kDa integral membrane gelatinase whose expression correlates with the invasiveness of the human melanoma cell line LOX. Here, we report the molecular cloning of a cDNA that encodes the 97 kDa subunit of seprase. Its deduced amino acid sequence predicts a type II integral membrane protein with a cytoplasmic tail of 6 amino acids, followed by a transmembrane domain of 20 amino acids and an extracellular domain of 734 amino acids. The carboxyl terminus contains a putative catalytic region (approximately 200 amino acids) which is homologous (68% identity) to that of the nonclassical serine protease dipeptidyl peptidase IV (DPPIV). The conserved serine protease motif G-X-S-X-G is present as G-W-S-Y-G. However, sequence analysis of seprase cDNA from LOX and other cell lines strongly suggests that seprase and human fibroblast activation protein alpha (FAP alpha) are products of the same gene. We propose that seprase/FAP alpha and DPPIV represent a new subfamily of serine integral membrane proteases (SIMP).
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 alpha (FAPalpha), 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 FAPalpha 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 affinity-labeled by [3H]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, FAPalpha, 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.