Selective Homogeneous Assay for Circulating Endopeptidase Fibroblast Activation Protein (FAP)

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.

Conflict of interest statement

This work was funded by Genentech, Inc. All the authors are present or former paid employees of Genentech/Roche. Genentech has filed patent applications related to this work.

Figures

Figure 1

Design of fluorescence-quenched peptides. Peptides contain an N-terminal fluorescent donor, followed by six amino acid residues of the region flanking the dominant FAP endopeptidase cleavage site of human FGF21, ending with an additional C-terminal lysine conjugated to a dark quencher. Variants include substitution of the P1 proline with glycine, the P2 glycine with D-alanine or the entire homologous region of murine FGF21.

Figure 2

Plasma cleavage of fluorescence-quenched peptides. (A) Cleavage rate of human WT (GP), human mutant (GG) and murine (EP) FGF21-based peptides in plasma samples isolated from Fap+/+, Fap+/−, and Fap−/− mice, as indicated (N = 3 mice per genotype). As a control, the cleavage reaction was run with 1 nM recombinant mouse FAP. Concentration of peptide substrates in this experiment was 3 µM. (B) Cleavage rate of the GP peptide by recombinant human FAP or PREP proteins at 1 nM enzyme concentration. (C) Cleavage rate of the GP peptide by recombinant human FAP (left, blue) or PREP (right, red) in the presence of an FAP-specific inhibitor cpd60 and/or PREP-specific inhibitor KYP-2047 (KYP). (D) Cleavage rate of the GP peptide in plasma from Fap+/+ (blue), Fap+/− (red), and Fap−/− (black) mice in the presence of cpd60 and/or KYP (N = 3 per group).

Figure 3

Cleavage kinetics of GP and aP substrates by recombinant FAP. (A and B) Michaelis-Menten saturation curves for human (black) or mouse (blue) recombinant FAP cleavage of (A) the GP or (B) aP peptides. (C) Plot of fractional velocity as a function of cpd60 concentration, for recombinant mouse or human FAP with cpd60, using GP peptide. Mouse and human FAP preparations were found to be 74% and 57% active, respectively. (D) Human and mouse FAP enzyme kinetics constants with the GP and aP substrates, corrected for experimentally determined active enzyme concentrations determined in (C). Error is SEM, determined from three experimental replicates. FAP molar concentrations are based on monomer for active site titration and kinetics calculations.

Figure 4

Specificity of aP peptide cleavage. (A) Cleavage rate of the aP or GG peptide by 1 nM recombinant, human FAP or PREP. (B) Cleavage rate of the aP peptide by a panel of recombinant, human prolyl peptidases. Each recombinant peptidase was used at 2.5 µg/ml. (C and D) Cleavage rate of ANPFAP (C) or aP NIRF (D) by 10 nM recombinant, human FAP or PREP. (E) Cleavage rate of the aP peptide in plasma from Fap+/+, Fap+/−, and Fap−/− mice. Plasma was diluted 10-fold. (F) Cleavage rate of the aP peptide in anti-FAP or control IgG-immunodepleted human serum. Serum was diluted 2.5-fold. Complete FAP removal following anti-FAP immunodepletion was shown previously by immunoblotting.

Figure 5

Binding mode of peptide substrates to FAP, DPPIV and PREP. (A) FAP active site (grey surface and pale green sticks) bound to modeled IPI substrate tripeptide (green sticks). (B) DPPIV active site (grey surface and pale cyan sticks) bound to IPI substrate tripeptide (cyan sticks). (C) FAP active site (grey surface and pale blue sticks) bound to modeled acetyl-VaPSQ substrate peptide (blue sticks). (D) Representative induced-fit docking model of PREP (grey surface and pale pink sticks) bound to acetyl-VAPSQ-amide substrate peptide (pink sticks). In all panels, red dashed lines indicate intermolecular and intra-substrate hydrogen bonds. (E) Comparison of cleavage rates by human FAP on VxPSQG peptide substrates containing various D-enantiomer residues at the P2 position (xP).

Figure 6

FAP activity in serum correlates with liver disease and FAP levels. (A) Scatter plot showing FAP activity levels determined by using the aP peptide (x axis) and FAP protein levels determined by ELISA in serum samples (y axis) from healthy individuals (N = 16, black circles) or individuals previously diagnosed with liver cirrhosis (N = 17, red circles). R2 value is indicated in the graph. (B and C) The same data as (A), with FRET and ELISA data presented separately, including mean ± SEM. P values indicated were calculated by student t-test.

Figure 7

Plasma FAP activity is completely suppressed in mice fed chow with cpd60. Ten to eighteen-week old C57/BL6 background mice were fed ad libitum control chow (blue circles), diet containing 20 ppm of cpd60 (green squares), or diet containing 100 ppm of cpd60 (red triangles). Plasma was isolated from blood retrieved at the indicated time points and FAP activity levels determined using the aP peptide. N = 5 animals per group. P values indicated were calculated by student t-test (****P < 0.0001 for 20 ppm cpd60 vs control diet; ####P < 0.0001 for 100 ppm cpd60 vs control diet).