Abstract
Fibroblast Activation Protein (FAP) is a membrane-bound serine protease whose expression is often elevated in activated fibroblasts associated with tissue remodeling in various common diseases such as cancer, arthritis and fibrosis. Like the closely related dipeptidyl peptidase DPPIV, the extracellular domain of FAP can be released into circulation as a functional enzyme, and limited studies suggest that the circulating level of FAP correlates with the degree of tissue fibrosis. Here we describe a novel homogeneous fluorescence intensity assay for circulating FAP activity based on a recently identified natural substrate, FGF21. This assay is unique in that it can effectively distinguish endopeptidase activity of FAP from that of other related enzymes such as prolyl endopeptidase (PREP) and was validated using Fap-deficient mice. Structural modeling was used to elucidate the mechanistic basis for the observed specificity in substrate recognition by FAP, but not by DPPIV or PREP. Finally, the assay was used to detect elevated FAP activity in human patients diagnosed with liver cirrhosis and to determine the effectiveness of a chemical inhibitor for FAP in mice. We propose that the assay presented here could thus be utilized for diagnosis of FAP-related pathologies and for the therapeutic development of FAP inhibitors.
Highlights
Fibroblast activation protein, (FAP, known as FAPα, seprase, or circulating antiplasmin-cleaving enzyme, (EC 3.4.21.B28) is a type II transmembrane serine protease and a member of the S9 family of proline-specific proteases which includes dipeptidyl peptidase IV (DPPIV), DPP8, DPP9, prolyl endopeptidase (PREP, known as POP) (1–4)
Fibroblast Activation Protein (FAP) is produced as a membrane-bound protein, the extracellular domain encoding the active enzyme can be shed from the cell surface, and soluble FAP protein is readily detectable in serum and plasma by a standard sandwich ELISA
We have previously demonstrated by immunoblot analysis that FAP is present in the plasma from wild type (WT) mice, to a lesser degree in heterozygotes and undetectable in KO mice[29]
Summary
Fibroblast activation protein, (FAP, known as FAPα, seprase, or circulating antiplasmin-cleaving enzyme, (EC 3.4.21.B28) is a type II transmembrane serine protease and a member of the S9 family of proline-specific proteases which includes dipeptidyl peptidase IV (DPPIV), DPP8, DPP9, prolyl endopeptidase (PREP, known as POP) (1–4). We and others have recently found that FAP cleaves FGF21 at a specific site proximal to the C-terminus, leading to its inactivation, as this region of the molecule is crucial for binding βKlotho[29,30,31] In both α2AP and FGF21, the specific cleavage site targeted by FAP possesses the consensus Gly-Pro sequence at P2-P1 position, and these amino acid residues are essential for cleavage by FAP32. FAP protein can be isolated from tissue or blood samples by immunocapture with an FAP-specific antibody, followed by a general fluorescence intensity assay for dipeptidyl-peptidases using a peptide substrate attached to a chemically quenched dye, such as Z-Gly-Pro-7-amido-4-methylcoumarin (AMC) or Ala-Pro-7-amino-4-trifluoromethyl-coumarin (AFC)[13,21,35,36]. This assay utilizes a modified peptide substrate based on the endopeptidase cleavage site of FGF21, a newly identified natural substrate for FAP, in a quenched dye format and is selective for FAP
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