title: 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.
authors.affiliation: Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China.
authors.forename: Chih-Hsiang
authors.initials: CH
authors.lastname: Huang
authors.forename: Ching-Shu
authors.initials: CS
authors.lastname: Suen
authors.forename: Ching-Ting
authors.initials: CT
authors.lastname: Lin
authors.forename: Chia-Hui
authors.initials: CH
authors.lastname: Chien
authors.forename: Hsin-Ying
authors.initials: HY
authors.lastname: Lee
authors.forename: Kuei-Min
authors.initials: KM
authors.lastname: Chung
authors.forename: Ting-Yueh
authors.initials: TY
authors.lastname: Tsai
authors.forename: Weir-Tong
authors.initials: WT
authors.lastname: Jiaang
authors.forename: Ming-Jing
authors.initials: MJ
authors.lastname: Hwang
authors.forename: Xin
authors.initials: X
authors.lastname: Chen
mesh_terms: D002478:Cells, Cultured; D010450:Endopeptidases; D018093:Gelatinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D000099167:Fibroblast Activation Protein Alpha
chemical_list: D010450:Endopeptidases; D018093:Gelatinases; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D000099167:Fibroblast Activation Protein Alpha; C485561:SPRY2 protein, human