Fibroblast Activation Protein (FAP) is a cell-surface anchored dimeric protease, closely related to Dipeptidyl peptidase 4 (DPP4).  Both enzymes are also found in a soluble, secreted form.  FAP expression is predominantly associated with disease states, such as cancer and liver fibrosis but few substrates of FAP are known.  The aim of this study is to identify novel FAP substrates and downstream effects of knocking out FAP enzyme activity.

Primary mouse embryonic fibroblasts (MEFs) were isolated from FAP gene knockout mice.  These cells were immortalised by transduction with the SV40 Large T antigen.  FAP and GFP were then cloned into an inducible expression system which produced both proteins in stoichiometric amounts.  A second, FAP inactive, construct contained a serine to alanine substitution at the catalytic site.

To identify substrates, Terminal Amine Isotopic Labelling of Substrates (TAILS) was employed to examine the differences between cell lines expressing functional enzyme (FAP e+) vs inactive FAP (FAP e-).  Several cleavage sites in type 1 collagen depend on active FAP and typically adhere to biochemical in vitro FAP specificity. These findings are consistent with previous reports that suggested type 1 collagen as a FAP substrate. In addition, the impact of FAP activity on secretome and proteome composition was investigated using a quantitative proteomic strategy.

These data will provide insights into FAP functions by elucidating its substrates and the global cellular effects of knocking out its enzyme activity.