Purification, identification and characterisation of seprase from bovine serum.
The study and identification for the first time of a soluble form of a seprase activity from bovine serum is presented. To date, this activity has only been reported to be an integral membrane protease but has been known to shed from its membrane. The activity was purified 30,197-fold to homogeneity, using a combination of column chromatographies, from bovine serum. Inhibition by DFP, resulting in an IC(50) of 100:nM confirms classification as a serine protease. The protease after separation and visualisation by native PAGE was subjected to tryptic digestion and the subsequent peptides sequenced. Each peptide sequenced was found to be present in the primary structure of seprase/fibroblast activation protein (FAP), a serine gelatinase specific for proline-containing peptides and macromolecules. Substrate specificity studies using kinetic, RP-HPLC and LC-MS analysis of synthetic peptides suggest that this peptidase has an extended substrate-binding region in addition to the primary specificity site S(1). This analysis revealed at least five subsites to be involved in enzyme-substrate binding, with the smallest peptide cleaved being a tetrapeptide. A proline residue in position P(1) was absolutely necessary therefore showing high primary substrate specificity for the Pro-X bond, while a preference for a hydrophobic residue at the C-terminal end of the scissile bond (P'(1)) was evident. The enzyme also showed complete insensitivity to the prolyl oligopeptidase specific inhibitors, JTP-4819, Fmoc-Ala-pyrrCN and Z-Phe-Pro-BT. To date, no physiological substrate has clearly been defined for this protease but its ability to effectively degrade gelatin suggests a candidate protein substrate in vivo and a possible role in extracellular matrix protein degradation.
School of Biotechnology and National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland.
Patrick J
PJ
Collins
Gillian
G
McMahon
Pamela
P
O'Brien
Brendan
B
O'Connor
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D005109:Extracellular Matrix / Q000201:enzymology*; D005780:Gelatin / Q000378:metabolism; D018093:Gelatinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology*; D012697:Serine Endopeptidases / Q000378:metabolism*; D044967:Serum / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000099167:Fibroblast Activation Protein Alpha
D010450:Endopeptidases; D005780:Gelatin; D018093:Gelatinases; D008565:Membrane Proteins; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D000099167:Fibroblast Activation Protein Alpha