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 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.
Piñeiro-Sánchez
M L
ML
Lombardi Cancer Center and Department of Cell Biology, Georgetown University Medical Center, Washington, D. C. 20007, USA.
Goldstein
L A
LA
Dodt
J
J
Howard
L
L
Yeh
Y
Y
Tran
H
H
Argraves
W S
WS
Chen
W T
WT
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D018093:Gelatinases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008545:Melanoma / Q000201:enzymology*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D008565:Membrane Proteins; D012697:Serine Endopeptidases; D018093:Gelatinases