Purification, identification and characterisation of seprase from bovine serum

Abstract

ABBREVIATIONSAMC, 7-amino-4-methylcoumarin; BCA, bicinchoninic acid; CPC, calcium phosphate cellulose; DFP, diisopropylfluorophosphate; FPLC, fast protein liquid chromatography; HIC, hydrophobic interaction chromatography; PO, prolyl oligopeptidase; TFA, trifluoroacetic acid; Z, N-benzyloxycarbonyl; ZIP, Z-Pro-prolinal-insensitive Z-Gly-Pro-AMC-hydrolysing peptidase. ABSTRACTThe 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 100nM 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, 2 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 ʹ′) 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.

Antiplasmin-cleaving enzyme is a soluble form of fibroblast activation protein

“…Interestingly, Collins et al 25 have found FAP-like activity in bovine serum, which is in accord with our report of APCE in human plasma. APCE may be a soluble, circulating derivative of FAP, resulting from cleavage of the Cys23-Ile24 bond in the transmembrane or extracellular domain of FAP, and if so, its presence in plasma could have an impact on the use of therapeutic agents designed to target FAP at the tissue level.…”

“…These values are about 2-fold smaller when compared to a K m of 0.270 mM for bovine FAP toward Z-Gly-Pro-AMC. 25 Using Z-Gly-Pro-AMC, we determined k cat values for human rFAP and APCE to be 30.5 minute Ϫ1 and 32.2 minute Ϫ1 ; k cat for bovine FAP has not been reported. For Ala-Pro-AFC substrate, human rFAP and APCE have K m and k cat values of 0.323 mM and 64.7 minute Ϫ1 and 0.272 mM and 59.7 minute Ϫ1 , respectively, in contrast to 0.20 mM and 120 minutes Ϫ1 , which Sun et al 32 reported for full-length human rFAP.…”

Emerging roles of serine proteinases in tissue turnover in arthritis

“…The active enzyme is a homodimer of two 97-kd subunits, and a soluble form of FAP␣ has also been reported (131,132). In contrast to DPP-4, FAP␣ typically is not expressed in normal tissues.…”

Seprase: An overview of an important matrix serine protease

“…Reports show that the gelatinase activity of Seprase was completely blocked by serine-protease inhibitors, including DFP and PMSF [11,25] [40]. Table 1 summarises some of the biochemical properties of Seprase.…”