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Purification, identification and characterisation of the active site of a Seprase-like activity from bovine serum

Collins, Patrick and McMahon, Gillian and O'Brien, Pamela and O'Connor, Brendan (2004) Purification, identification and characterisation of the active site of a Seprase-like activity from bovine serum. International Journal of Biochemistry & Cell Biology, 36 (11). pp. 2320-2333. ISSN 1878-5875

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Abstract

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 IC50 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 S1. This analysis revealed at least five subsites to be involved in enzyme-substrate binding, 1 with the smallest peptide cleaved being a tetrapeptide. A proline residue in position P1 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 (P1ʹ′) 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.

Item Type:Article (Published)
Refereed:Yes
Subjects:Biological Sciences > Biotechnology
Biological Sciences > Biochemistry
Biological Sciences > Enzymology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Chemical Sciences
DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Research Initiatives and Centres > Irish Separation Science Cluster (ISSC)
Publisher:Elsevier
Official URL:http://dx.doi.org/10.1016/j.biocel.2004.05.006
Copyright Information:© 2004 Elsevier
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 3.0 License. View License
Funders:Enterprise Ireland, Health Research Board
ID Code:17814
Deposited On:28 Feb 2013 14:37 by Brendan O'Connor. Last Modified 27 Apr 2017 13:54

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