Identification of novel natural substrates of fibroblast activation protein by differential degradomics and proteomics

ABSTRACT: Fibroblast Activation Protein (FAP) is a cell-surface transmembrane-anchored anchored dimeric protease. This unique constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities, and can hydrolyse post-proline bonds. FAP expression is very low in adult organs, but is greatly up regulated by activated fibroblasts in sites of tissue remodelling, including fibrosis, atherosclerosis, arthritis and tumours. Primary mouse embryonic fibroblasts (MEF) were isolated from FAP gene knockout (GKO) embryos, immortalised and then transduced to express either enzyme active or inactive FAP. The cell secretomes were analysed using degradomic and proteomic techniques. Terminal Amine Isotopic Labelling of Substrates based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CCL2, IL13, C1qT6 and CSF-1 that were confirmed in vitro. In addition, differential metabolic labelling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation and wound healing associated proteins. FAP GKO plasma exhibited slower that wildtype clotting times. This study greatly enhances the understanding of the roles of FAP through a significant expansion of the substrate repertoire of this protease and identifying downstream effects of its activity that support roles in pathological processes.

INSTRUMENT(S): QSTAR

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Fibroblast

SUBMITTER: Oliver Schilling  

LAB HEAD: Oliver Schilling

PROVIDER: PXD004754 | Pride | 2018-12-19

REPOSITORIES: Pride