Peptide substrate profiling defines fibroblast activation protein as an endopeptidase of strict Gly2‐Pro1‐cleaving specificity

Abstract

Fibroblast activation protein (FAP) is a serine protease of undefined endopeptidase specificity implicated in tumorigenesis. To characterize FAP's specificity, we synthesized intramolecularly quenched fluorescent substrate sets based on the FAP cleavage site in α₂‐antiplasmin (TSGP‐NQ). FAP required substrates with Pro at P₁ and Gly or d‐amino acids at P₂ and preferred small, uncharged amino acids at P₃, but tolerated most amino acids at P₄, and . These substrate preferences allowed design of peptidyl‐chloromethyl ketones that inhibited FAP, but not the related protease, dipeptidyl peptidase‐4. Thus, FAP is a narrow specificity endopeptidase and this can be exploited for inhibitor design.