Cleavage-site specificity of prolyl endopeptidase FAP investigated with a full-length protein substrate

Abstract

Fibroblast activation protein (FAP) is a prolyl-cleaving endopeptidase proposed as an anti-cancer drug target. It is necessary to define its cleavage-site specificity to facilitate the identification of its in vivo substrates and to understand its biological functions. We found that the previously identified substrate of FAP, α(2)-anti-plasmin, is not a robust substrate in vitro. Instead, an intracellular protein, SPRY2, is cleavable by FAP and more suitable for investigation of its substrate specificity in the context of the full-length globular protein. FAP prefers uncharged residues, including small or bulky hydrophobic amino acids, but not charged amino acids, especially acidic residue at P1', P3 and P4 sites. Molecular modelling analysis shows that the substrate-binding site of FAP is surrounded by multiple tyrosine residues and some negatively charged residues, which may exert least preference for substrates with acidic residues. This provides an explanation why FAP cannot cleave interleukins, which have a glutamate at either P4 or P2', despite their P3-P2-P1 sites being identical to SPRY2 or α-AP. Our study provided new information on FAP cleavage-site specificity, which differs from the data obtained by profiling with a peptide library or with the denatured protein, gelatin, as the substrate. Furthermore, our study suggests that negatively charged residues should be avoided when designing FAP inhibitors.

Fibroblast Activation Protein Cleaves and Inactivates Fibroblast Growth Factor 21

“…Therefore, selective inhibition of FAP may have minimum effects on blood clotting. In addition, ␣(2)-antiplasmin may not be cleaved by FAP in vivo because of posttranslational modification (52). Another class of substrate is gelatin, after initial denaturation of collagen by matrix metalloproteinases.…”

“…Another class of substrate is gelatin, after initial denaturation of collagen by matrix metalloproteinases. Again, the physiological importance of the gelatinase activity of FAP has not been demonstrated (52).…”

Fibroblast Activation Protein-α

“…FAP endopeptidase activity was shown to cleave the SPRY2 protein (also called SPROUTY2), a natural inhibitor of receptor tyrosine kinase (Huang et al, 2011a). Although SPRY2 is readily cleaved by FAP, it likely is not an in vivo substrate of FAP because it is localized inside the cells.…”

Over-expression of fibroblast activation protein alpha increases tumor growth in xenografts of ovarian cancer cells

“…It is a 95-kDa cell surface glycoprotein and its full-length shares 52% sequence homology with dipeptidyl peptidase IV (DPPIV, CD26). FAPa has been shown to have both DPP and collagenase activity, and is capable of degrading gelatin and type I collagen [7,8].…”