Fibroblast activation protein peptide substrates identified from human collagen I derived gelatin cleavage sites

Abstract

A highly consistent trait of tumor stromal fibroblasts is the induction of the membrane-bound serine protease fibroblast activation protein-alpha (FAP), which is overexpressed on the surface of reactive stromal fibroblasts present within the stroma of the majority of human epithelial tumors. In contrast, FAP is not expressed by tumor epithelial cells or by fibroblasts or other cell types in normal tissues. The proteolytic activity of FAP, therefore, represents a potential pan-tumor target that can be exploited for the release of potent cytotoxins from inactive prodrugs consisting of an FAP peptide substrate coupled to a cytotoxin. To identify FAP peptide substrates, we used liquid chromatography tandem mass spectroscopy based sequencing to generate a complete map of the FAP cleavage sites within human collagen I derived gelatin. Positional analysis of the frequency of each amino acid at each position within the cleavage sites revealed FAP consensus sequences PPGP and (D/E)-(R/K)-G-(E/D)-(T/S)-G-P. These studies further demonstrated that ranking cleavage sites based on the magnitude of the LC/MS/MS extracted ion current predicted FAP substrates that were cleaved with highest efficiency. Fluorescence-quenched peptides were synthesized on the basis of the cleavage sites with the highest ion current rankings, and kinetic parameters for FAP hydrolysis were determined. The substrate DRGETGP, which corresponded to the consensus sequence, had the lowest Km of 21 microM. Overall the Km values were relatively similar for both high and low ranked substrates, whereas the kcat values differed by up to 100-fold. On the basis of these results, the FAP consensus sequences are currently being evaluated as FAP-selective peptide carriers for incorporation into FAP-activated prodrugs.

Figure 1

Characterization of recombinant His-tagged FAP. (A) Hydrolysis of the dipeptide substrate Ala-Pro-AFC (500 μM) by FAP (5 μg/mL) compared to DPPIV. The inset shows Western blot analysis of purified His–FAP following denaturing SDS–PAGE. (B) Digestion of indicated FITC-labeled proteins (100 μg/mL) by FAP (5 μg/mL).

Figure 2

(A) MALDI-TOF mass spectra demonstrating FAP digestion of human collagen I. (B) Example of cleavage site sequence(s) determination using the FindPept tool for a selected mass of 2114.5 from MALDI-TOF mass spectra. Multiple potential cleavage site sequences within ±1 mass unit are identified by this analysis.

Figure 3

(A) FAP cleavage sites within the 8.5 kDa fragment of recombinant human collagen I derived gelatin. Cleavage sites are bracketed by “/”, and [M + H]⁺ denotes the mass of the cleavage fragment. Amino acids on the N-terminal side of the cleavage site (i.e., P7–P1) are in bold font. (B) Amino acid sequence of the 8.5 kDa fragment with FAP cleavage sites indicated by arrows.

Figure 4

FAP cleavage sites within the 100 kDa recombinant human collagen I derived gelatin. Cleavage sites are bracketed by “/”. The mass of the cleavage fragment, [M + H]⁺, and extracted ion current are included for each cleavage site. Underlined sequences indicate cleavage sites which do not contain Pro (P) in the P1 position. The consensus sequences PPGP and D-(R/K)-G-E-(T/S)-G-P are indicated by bold text.

Figure 5

Frequency (%) of each amino acid in positions P7–P′1 within the FAP cleavage sites within the 100 kDa recombinant human collagen I derived gelatin. Gray bars indicate the frequency for all 101 cleavage sites. Black bars indicate the frequency in only those cleavage sites containing Pro (P) at the P1 position. The amino acids C, H, W, and Y are not present in human collagen I and, therefore, are not included.