IDENTIFICATION OF THE 170-KDA MELANOMA MEMBRANE-BOUND GELATINASE (SEPRASE) AS A SERINE INTEGRAL MEMBRANE PROTEASE

The 170-kDa membrane-bound gelatinase, seprase, is a cell surface prot ease, the expression of which correlates with the invasive phenotype o f human melanoma and carcinoma cells, We have isolated seprase from ce ll membranes and shed vesicles of LOX human melanoma cells, The active enzyme is a dimer of N-glycosylated 97-kDa subunits, Sequence analysi s of three internal proteolytic fragments of the 97-kDa polypeptide re vealed up to 87.5% identity to the 95-kDa fibroblast activation protei n alpha (FAP alpha), the function of which is unknown, Thus, we used r everse transcription-polymerase chain reaction to generate a 2.4-kilob ase cDNA from LOX mRNA with FAP alpha primers, COS-7 cells transfected with this cDNA expressed a 170 kDa gelatinase that is recognized by m onoclonal 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 fluoroph osphate, Seprase could be affinity-labeled by [H-3]diisopropyl fluorop hosphate, but the proteolytically inactive 97-kDa subunit could not, c onfirming the existence of a serine protease active site on the dimeri c form, Proteolytic activity is lost upon dissociation into its 97-kDa subunit following treatment with acid, heat, or cysteine and histidin e-modifying agents, We conclude that seprase, FAP alpha, and DPPIV are related serine integral membrane proteases and that seprase is simila r to DPPIV, the proteolytic activities of which are dependent upon sub unit association.