Quantitation of fibroblast activation protein (FAP)-specific protease activity in mouse, baboon and human fluids and organs

• Published in: FEBS open bio Vol. 4; no. 1; pp. 43 - 54
• Main Authors: Keane, Fiona M., Yao, Tsun-Wen, Seelk, Stefanie, Gall, Margaret G., Chowdhury, Sumaiya, Poplawski, Sarah E., Lai, Jack H., Li, Youhua, Wu, Wengen, Farrell, Penny, Vieira de Ribeiro, Ana Julia, Osborne, Brenna, Yu, Denise M.T., Seth, Devanshi, Rahman, Khairunnessa, Haber, Paul, Topaloglu, A. Kemal, Wang, Chuanmin, Thomson, Sally, Hennessy, Annemarie, Prins, John, Twigg, Stephen M., McLennan, Susan V., McCaughan, Geoffrey W., Bachovchin, William W., Gorrell, Mark D.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 01-01-2014; John Wiley & Sons, Inc; Elsevier; Wiley
• Subjects: Adipose tissue; Bile; Biomarker; Biomarkers; Cirrhosis; Colon; Diabetes; Dipeptidyl peptidase; Enzymatic activity; Enzymes; Epididymis; Fibroblast; Fibroblast activation protein; Fibroblasts; Fibrosis; Fluorides; Genomes; Hepatitis; Leukocytes; Liver; Liver cirrhosis; Liver disease; Liver diseases; Lymph nodes; Lymphatic system; Medical research; Mesenchyme; Pancreas; Physiology; Proline; Prostate; Protease activity; Protease substrates; Proteinase; Proteins; Quantitation; Skin; Stroma; Submaxillary gland; Tumors; Uterus; Germany; Biomarker; LN; DMSO; gko; STLV; DPP4; PBC; het; AMC; LDS; wt; PBS; Protease activity; Liver disease; EDTA; PBMC; PVDF; Dipeptidyl peptidase; mAb; FAP; ND; Fibrosis; ALD; HCV; PEP; yrs; Fibroblast; Protease substrates; HCV, hepatitis C virus; PBMC, peripheral blood mononuclear cells; STLV, simian T-cell lymphotrophic virus; mAb, monoclonal antibody; LDS, lithium dodecyl sulphate; FAP, fibroblast activation protein-α; PVDF, polyvinylidene fluoride; wt, wild type; PBS, phosphate-buffered saline; gko, gene knock out; LN, lymph node; ALD, alcoholic liver disease; PBC, primary biliary cirrhosis; EDTA, ethylene diamine tetra acetic acid; AMC, amino-4-methylcoumarin; DPP4, dipeptidyl peptidase 4; het, heterozygous; yrs, years; DMSO, dimethyl sulfoxide; PEP, prolyl endopeptidase; ND, non-diseased
• ISSN: 2211-5463; 2211-5463
• DOI: 10.1016/j.fob.2013.12.001

The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.
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•A novel synthetic fluorogenic substrate is proven to be FAP-specific.•Mice have higher levels of circulating FAP activity compared to baboons or humans.•No FAP activity was detected in urine or bile but bile contained high DPP4 activity.•FAP activity is greatest in pancreas, uterus, salivary gland, skin and lymph node.•FAP activity and protein is elevated in both serum and liver in human liver disease.