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Fibroblast Activation Protein α
Clan SC S9 | 750. Fibroblast Activation Protein α
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Chapter 750
Fibroblast Activation Protein α DATABANKS MEROPS name: fibroblast activation protein alpha subunit MEROPS classification: clan SC, family S9, subfamily S9B, peptidase S09.007 Tertiary structure: Available Species distribution: superclass Tetrapoda Reference sequence from: Homo sapiens (UniProt: Q12884)
Name and History The fibroblast activation protein α (FAP) was discovered with a monoclonal antibody, mAb F19, that was generated in the course of a serological survey of cell surface antigens expressed on cultured human fibroblasts, sarcomas and neuroectodermal tumor cells [1]. This antibody was used to characterize the plasma membrane-associated 95 kDa FAP glycoprotein [2,3], to isolate the FAP-encoding cDNA (Genbank U09278) [4], and to examine FAP expression in a broad range of normal and neoplastic human tissues [1,5]. The immunochemical studies also led to the detection of high molecular mass complexes, comprising FAP multimers, and noncovalently linked, heteromeric complexes between FAP and a distinct, FAPassociated protein of 105 kDa designated as FAPβ [2]. Subsequent investigations have shown that FAPβ is the CD26 cell surface glycoprotein, also called dipeptidyl peptidase IV (DPPIV) (Chapter 745). However, FAP/ CD26 heteromeric complexes are found only on the surface membrane of some cell types, notably cultured fibroblasts and melanocytes, whereas other cells express only FAP (e.g. some sarcoma cell lines) or CD26 (e.g. kidney epithelial cells, activated T lymphocytes) or neither molecule. Moreover, immunohistochemistry on human tissues has revealed distinct and generally nonoverlapping expression patterns for FAP and CD26 [48]. FAP has been independently studied under two other names; antiplasmin cleaving enzyme (APCE) [9] and seprase [10]. APCE was shown to be a soluble circulating form of FAP, lacking the transmembrane domain, after APCE was purified from plasma [9]. Seprase was
identified on melanoma cell lines including LOX and SKMEL28, then cloned (Genbank U76833) and shown to be identical to FAP [10,11]. The FAP homolog in Xenopus laevis was discovered independently in the course of a differential gene expression survey of the thyroid hormone-induced tail resorption program during tadpole metamorphosis [12]. The human and Xenopus FAP polypeptides show about 50% amino acid sequence identity.
Activity and Specificity FAP possesses both a dipeptidyl-peptidase activity, specific for N-terminal Xaa-(Pro/Ala) sequences, and an endopeptidase activity capable of degrading α2-antiplasmin, gelatin and denatured type I collagen [9,13], including MMP1-cleaved type I collagen [14]. Mutation of the catalytic Ser residue of FAP to Ala abolishes both dipeptidyl-peptidase and endopeptidase enzymatic activities [13]. FAP is assayed at 37 C in physiological buffers such as pH 7.6 Tris/EDTA [13,15]. As in prolyl endopeptidase (PEP) assays, 10 mM EDTA and a reducing agent such as 1 mM dithiothreitol are added to inhibit contaminating proteases; FAP is unaffected by such substances but all DPPIV family proteases are inhibited by metal ions [1618]. The solubility of some substrates can be improved by using 10% methanol or 1% DMF [15]. FAP enzymatic activity is detected in extracts of human cancerous tissues [13] and cirrhotic liver [19] but not in matched normal controls. The FAP cleavage sites within recombinant human collagen I include cleavage following Pro-Pro-Gly-Pro and a consensus sequence of (Asp/Glu)-(Arg/Lys)-Gly-(Glu/Asp)-(Thr/Ser)-Gly-Pro [20]. The FAP/APCE cleavage site in α2-antiplasmin similarly follows Gly11-Pro12 [21]. In contrast, the dipeptidyl-peptidase activity of FAP greatly prefers Ala-Pro-AFC over Gly-Pro-AFC [3]. However, the identification of several natural bioactive peptide substrates of FAP dipeptidylpeptidase activity, most notably neuropeptide Y, substance P and B-type natriuretic peptide with half-lives of less than 10 minutes, revealed that glycine is not dominant in P2 in natural substrates [15]. In contrast, endopeptidase
Clan SC S9 | 750. Fibroblast Activation Protein α
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cleavage of artificial substrates requires Gly-Pro preceded by a benzoyloxycarbonyl (Z) moiety, not a succinyl or acyl moiety [22]. FAP appears to prefer a positive charge at P10 [15,23]. Some DPPIV inhibitors also inhibit FAP, most notably PT-100 and PT-630. PT-630 inhibits DPPIV and FAP to similar extents [24,25]. More recently, substrate specificity has been used in FAP inhibitor design and some inhibitors are selective while others also inhibit prolyl endopeptidase (PEP/POP; Chapter 742) [21,23,26]. The incretin-based type 2 diabetes therapeutics, called gliptins, are selective DPPIV inhibitors that primarily act by raising circulating levels of bioactive GLP-1. The potent DPPIV inhibitor linagliptin is the only gliptin that displays some FAP inhibition, but linagliptin does not inhibit any other peptidase. Although FAP inhibition may be beneficial (see biology summary below), it is unlikely that the low level of FAP inhibition conferred by linagliptin, 90-fold less than its DPPIV inhibition [27], would have a clinical effect. The distinguishing features of linagliptin are its rapid absorption (Tmax 5 1.5 hr), in vivo longevity, non-renal (biliary) excretion, its low therapeutic dose of 5 mg day21, and excellent safety profile including cardiovascular safety [28].
Structural Chemistry FAP homodimers and FAP/DPPIV heterodimers as well as larger complexes of FAP have been identified in cell extracts of cultured human fibroblasts, melanocytes and sarcoma cells. When analyzed by immunoblotting of boiled extracts separated by SDS-PAGE, FAP (1760) appears as a single protein species of about 95 kDa. Digestion of this 95 kDa glycoprotein with neuraminidase or endoglycosidase H, but not O-glycanase, reduces the apparent molecular size on SDS gels, and complete removal of all five N-linked sugars (Figure 750.1) with N-glycanase generates polypeptides of 75 kDa [2]. FAP requires dimerization for activity. Each monomer consists of an αβ hydrolase domain and an 8-blade β-propeller domain (Protein Data Bank code 1Z68) [29]. The catalytic pocket is buried inside the protein between these two domains and contains essential residues contributed by both domains, including Ser624, Asp702 and His734 of the catalytic triad and Glu203 and Glu204, in the β-propeller domain, that contact the N-terminus of substrates when undergoing dipeptidyl-peptidase cleavage [7,13]. Arg123, Tyr656 and Asn704 also contribute to FAP catalysis [29,30]. Ala657 is essential for FAP endopeptidase activity because it is small and so makes space sufficient for endopeptidase substrates. In contrast, in DPP4 this three-dimensional location is occupied by the large negatively charged residue Asp663 that prevents
Hydrolase
Domains:
Propeller
Hydrolase
TransCytoplasmic membrane E203 E204
FAP (760 aa)
7
27
53
DPPIV (766 aa)
7
29
51
S624 D702 H734 499
E205 E206 L294 V341
S630 D708 H740 501
Key: N-glycosylation
Cysteine
Enzyme activity
ADA binding
FIGURE 750.1 Schematic of FAP and its nearest relative DPPIV. The arrangement of protein domains and some residues essential for function and positions of cysteines and potential N-linked glycosylation sites are depicted. Not to scale.
endopeptidase activity [29]. The transmembrane-bound form of FAP is a type II integral membrane protein with the transmembrane domain formed by residues 726 (Figure 750.1). The Mus musculus FAP cDNA (NM_007986) has been cloned on the basis of its sequence similarity to human FAP [31]. The predicted human and mouse FAP polypeptides share 89% amino acid sequence identity and contain identical catalytic domains. There is evidence from mouse FAP studies that differential mRNA splicing generates at least three distinct transcripts that differ in their extracellular portions adjacent to the transmembrane domain [31], but the corresponding protein isoforms have not yet been characterized. Disruption of the FAP locus by gene targeting results in homozygous FAP-deficient mice that develop normally, are fertile and show no overt phenotype [32]. Nonetheless, the function of FAP may still be elucidated in such mice using disease models of chronic inflammation, fibrosis or tumor induction.
Preparation Natural sources of human FAP include cultured fibroblasts, melanocytes and certain sarcoma cell lines as well as embryonic tissues, granulation tissue of healing wounds and certain tumor tissues [1,3,5]. Isolation of human FAP has been accomplished with a combination of lectin and antibody affinity chromatography [3]. Recombinant mouse and human FAP enzymes produced in mammalian or insect cell culture have been purified to homogeneity and possess the same catalytic activities as naturally isolated FAP enzyme [13,17,33]. Soluble FAP (27760) has been purified from bovine serum and human plasma [9].
Clan SC S9 | 750. Fibroblast Activation Protein α
(A)
(B)
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(C)
Malignant epithelial cells
Resting fibrocytes
Normal capillary
Tumor capillaries
Activated tumor stromal fibroblasts
FIGURE 750.2 Schematic of tumor stromal fibroblast activation, accompanied by induction of FAP cell surface expression, in epithelial cancers. (A) Early stage of epithelial cancer development with small deposits of malignant epithelial cells (nodules with a diameter less than approximately 2 mm) that do not require new blood vessel formation; (B) Larger nodules of malignant epithelial cells without new blood vessels and stroma formation, showing areas of central hypoxia and necrosis; (C) Tumor nodules (diameter greater than approximately 5 mm) with new blood vessels and accompanying, activated tumor stromal fibroblasts expressing cell surface FAP.
Biological Aspects Although FAP was discovered as a cell surface antigen of cultured normal fibroblasts, its expression pattern in vivo, as determined by immunohistochemistry with mAb F19 and other antibodies against distinct FAP epitopes [3], is highly restricted. In particular, resting fibroblasts in normal tissues lack detectable FAP expression, and FAP induction upon in vitro cell culture appears to be part of a more general activation program which fibroblasts assume when grown under artificial tissue culture conditions [34]. Corresponding activation phenotypes have been postulated for the reactive fibroblasts found in the granulation tissue of healing wounds, certain chronic inflammatory lesions, the supporting stroma of malignant epithelial neoplasms, and normal embryonic tissues, and all of these fibroblasts also express FAP [1,4,5,19,25,3538]. Notably, the activated tumor stromal fibroblasts found in carcinomas of the breast, colorectum, lung, stomach, pancreas and esophagus show prominent FAP expression (Figure 750.2). Moreover, these FAP1 cells appear to drive immune suppression [38] and epithelial-mesenchymal transition [37], and may be an excellent target for antitumor therapy [25,39]. Indeed, since very few cell types in normal organs express this cell surface antigen, it has been possible to use the 131I-labeled mAb F19 for selective immunological targeting in patients with metastatic colorectal cancers [40]. The few normal human cell types known to express FAP in vivo include fetal mesenchymal cells, placenta and a distinct subset of glucagon producing endocrine cells (A cells) in the pancreatic islets. The biological role of FAP expression in these
diverse tissue types and pathological processes is still unknown, but it is tempting to speculate about functions related to tissue remodeling and repair [5]. FAP is upregulated in liver fibrosis [6,19,41], pulmonary fibrosis [42], rheumatoid arthritis [35,43], osteoarthritis [36] and atherosclerosis [44]. The FAP substrate α2-antiplasmin, which is an inhibitor of fibrinolysis, is made by the liver and released into the circulation. Following cleavage by FAP, the activity of α2-antiplasmin is greatly increased [45]. Approaches to antitumor therapy exploiting FAP upregulation in tumors include inhibitors, antibodies and prodrugs. The FAP inhibitors talabostat (PT100) and PT630 have antitumor efficacy in mice [25] but talabostat is not effective in humans [46]. Antibody-toxin conjugate [47] and a protoxin that uses FAP-specific cleavage to activate the toxin [48] are efficacious in mouse models.
Distinguishing Features FAP is in the S9 or prolyl oligopeptidase family. FAP is the only S9 family member to possess both dipeptidylpeptidase and prolyl-endopeptidase activities. FAP has a restricted expression whereas the other S9 family proteases and non-proteases are ubiquitous. Unlike other gelatinases, primarily the MMPs (family M10) that have a precursor form, FAP is constitutively active. Specific antibodies identify FAP (Table 750.1); the most widely used and fully validated specific antibody is mouse monoclonal antibody F19 to human FAP. No specific substrate or selective inhibitor is available for FAP detection but
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TABLE 750.1 Antibodies recognizing FAP Supplier
Product ID
Antigen
Antibody
Reference
R&D Systems
3715-SE
Recombinant human FAP
sheep polyclonal
[38]
Abcam
ab53066
Peptide
rabbit polyclonal
[39,49]
Abcam
ab28244
Peptide of FAP stalk region near cell surface
rabbit polyclonal
[50]
Bender Medsystems
BMS168
Peptide
rabbit polyclonal
[51]
Santa Cruz
F11-24
Peptide
rabbit polyclonal
[37]
Abnova
1E5/H002191-M01
60 kDa carboxy fragment of FAP
mouse monoclonal
[6]
ATCC
F19
Human tumor cell line
mouse hybridoma
[1,7,19]
J. Cheng
6E1
Mouse FAP
rabbit monoclonal
[52]
FAP is the only peptidase known to hydrolyze both HAla-Pro-AFC and Z-Gly-Pro-AFC. Unlike PEP, FAP cannot hydrolyze succinyl-Ala-Pro- or succinyl-Gly-Proderived substrates [15,17,22]. Therefore, FAP can be identified by substrate combinations. FAP has a unique sequence and chromosomal location at 2q23 in human, 2:C1.3 in mouse and q21 in rat.
Related Peptidases Human FAP has 52% amino acid sequence identity with human DPPIV/CD26. Two cytosolic proteases with 28% sequence identity to FAP, DPP8 (Chapter 746) and DPP9 (Chapter 747), have been identified [16,53]. Like FAP, they cleave N-terminal Xaa-Pro peptides; however, unlike FAP, they have no endopeptidase activity and hydrolyze H-Ala-Pro- and H-Gly-Pro-derived substrates equally well [15,17,54]. FAP shares about 30% amino acid sequence identity with dipeptidyl-peptidase 6 (DPP6) cDNA and DPP10, which were isolated from brain [55,56]. The DPP6 and DPP10 proteins lack the characteristic Ser and Trp residues in the catalytic domain that are present in both FAP and the other three DPPIV family members [55,57]. The FAP gene is 72.8 kb. The DPPIV and FAP gene loci are adjacent, at human chromosome 2q24.2 and 2q24.3, respectively, suggesting that they may have arisen through gene duplication. By contrast, the other related genes of the DPPIV family are on different human chromosomes. All S9B family members, FAP, DPPIV, DPP8 and DPP9, hydrolyze H-Ala-Pro-derived substrates, so discrimination between these proteases can be difficult. Concordantly, some natural substrates overlap, including NPY, GLP-1, GLP-2 and some chemokines [15,58,59]. Several inhibitors of DPPIV also inhibit FAP, DPP8 and DPP9 [60], and some FAP inhibitors also inhibit PEP [26].
Further Reading For reviews on FAP in cancer and fibrosis, see Wang et al. [6], Pure [39], and Yu et al. [61].
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[34] Sappino, A.P., Masouye, I., Saurat, J.H., Gabbiani, G. (1990). Smooth muscle differentiation in scleroderma fibroblastic cells. Am. J. Pathol. 137(3), 585591. [35] Bauer, S., Jendro, M.C., Wadle, A., Kleber, S., Stenner, F., Dinser, R., Reich, A., Faccin, E., Godde, S., Dinges, H., MullerLadner, U., Renner, C. (2006). Fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes. Arthritis Res. Ther. 8(6), R171. [36] Milner, J.M., Kevorkian, L., Young, D.A., Jones, D., Wait, R., Donell, S.T., Barksby, E., Patterson, A.M., Middleton, J., Cravatt, B.F., Clark, I.M., Rowan, A.D., Cawston, T.E. (2006). Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis. Arthritis Res. Ther. 8(1), R23. [37] Gao, M.-Q., Kim, B.G., Kang, S., Choi, Y.P., Park, H., Kang, K.S., Cho, N.H. (2010). Stromal fibroblasts from the interface zone of human breast carcinomas induce an epithelial-mesenchymal transition-like state in breast cancer cells in vitro. J. Cell Sci. 123(20), 35073514. [38] Kraman, M., Bambrough, P.J., Arnold, J.N., Roberts, E.W., Magiera, L., Jones, J.O., Gopinathan, A., Tuveson, D.A., Fearon, D.T. (2010). Suppression of antitumor immunity by stromal cells expressing fibroblast activation protein-α. Science 330(6005), 827830. [39] Pure, E. (2009). The road to integrative cancer therapies: emergence of a tumor-associated fibroblast protease as a potential therapeutic target in cancer. Exp. Opin. Ther. Targets 13(8), 967973. [40] Welt, S., Divgi, C.R., Scott, A.M., Garin-Chesa, P., Finn, R.D., Graham, M., Carswell, E.A., Cohen, A., Larson, S.M., Old, L.J. (1994). Antibody targeting in metastatic colon cancer: a phase I study of monoclonal antibody F19 against a cell-surface protein of reactive tumor stromal fibroblasts. J. Clin. Oncol. 12(6), 11931203. [41] Levy, M.T., McCaughan, G.W., Marinos, G., Gorrell, M.D. (2002). Intrahepatic expression of the hepatic stellate cell marker fibroblast activation protein correlates with the degree of fibrosis in hepatitis C virus infection. Liver Int. 22(2), 93101. [42] Acharya, P.S., Zukas, A., Chandan, V., Katzenstein, A.L., Pure, E. (2006). Fibroblast activation protein: a serine protease expressed at the remodeling interface in idiopathic pulmonary fibrosis. Hum. Pathol. 37(3), 352360. [43] Ospelt, C., Mertens, J.C., Juengel, A., Brentano, F., MaciejewskaRodriguez, H., Huber, L.C., Hemmatazad, H., Wu¨est, T., Knuth, A., Gay, R.E., Michel, B.A., Gay, S., Renner, C., Bauer, S. (2010). Inhibition of fibroblast activation protein and dipeptidyl peptidase IV increases cartilage invasion by rheumatoid arthritis synovial fibroblasts. Arthritis Rheum. 62(5), 12241235. [44] Brokopp, C.E., Schoenauer, R., Richards, P., Bauer, S., Lohmann, C., Emmert, M.Y., Weber, B., Winnik, S., Aikawa, E., Graves, K., Genoni, M., Vogt, P., Lu¨scher, T.F., Renner, C., Hoerstrup, S.P., Matter, C.M. (2011). Fibroblast activation protein is induced by inflammation and degrades type I collagen in thincap fibroatheromata. Eur. Heart J. 32(21), 27132722. [45] Lee, K.N., Jackson, K.W., Christiansen, V.J., Chung, K.H., McKee, P.A. (2004). A novel plasma proteinase potentiates alpha2-antiplasmin inhibition of fibrin digestion. Blood 103(10), 37833788. [46] Henry, L.R., Lee, H.O., Lee, J.S., Klein-Szanto, A., Watts, P., Ross, E.A., Chen, W.T., Cheng, J.D. (2007). Clinical implications
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
of fibroblast activation protein in patients with colon cancer. Clin. Cancer Res. 13(6), 17361741. Ostermann, E., Garin-Chesa, P., Heider, K.H., Kalat, M., Lamche, H., Puri, C., Kerjaschki, D., Rettig, W.J., Adolf, G.R. (2008). Effective immunoconjugate therapy in cancer models targeting a serine protease of tumor fibroblasts. Clin. Cancer Res. 14(14), 45844592. LeBeau, A.M., Brennen, W.N., Aggarwal, S., Denmeade, S.R. (2009). Targeting cancer stroma with a fibroblast activation protein-activated promelittin protoxin. Mol. Cancer Ther. 8(5), 13781386. Goscinski, M.A., Suo, Z., Florenes, V.A., Vlatkovic, L., Nesland, J.M., Giercksky, K.-E. (2008). FAP-alpha and uPA show different expression patterns in premalignant and malignant esophageal lesions. Ultrastruct. Pathol. 32(3), 8996. Bhati, R., Patterson, C., Livasy, C.A., Fan, C., Ketelsen, D., Hu, Z., Reynolds, E., Tanner, C., Moore, D.T., Gabrielli, F., Perou, C.M., Klauber-DeMore, N. (2008). Molecular characterization of human breast tumor vascular cells. Am. J. Pathol. 172(5), 13811390. Wen, Y., Wang, C., Ma, T., Li, Z., Zhou, L., Mu, B., Leng, F., Shi, H., Li, Y., Wei, Y. (2010). Immunotherapy targeting fibroblast activation protein inhibits tumor growth and increases survival in a murine colon cancer model. Cancer Sci. 101(11), 23252332. Cheng, J.D., Dunbrack, R.L., Valianou, M., Rogatko, A., Alpaugh, R.K., Weiner, L.M. (2002). Promotion of tumor growth by murine fibroblast activation protein, a serine protease, in an animal model. Cancer Res. 62(16), 47674772. Abbott, C.A., Yu, D.M.T., Woollatt, E., Sutherland, G.R., McCaughan, G.W., Gorrell, M.D. (2000). Cloning, expression and chromosomal localization of a novel human dipeptidyl peptidase (DPP) IV homolog, DPP8. Eur. J. Biochem. 267(20), 61406150. Yu, D.M.T., Ajami, K., Gall, M.G., Park, J., Lee, C.S., Evans, K., McLaughlin, E.A., Pitman, M.R., Abbott, C.A., McCaughan, G.W., Gorrell, M.D. (2009). The in vivo expression of dipeptidyl peptidases 8 and 9. J. Histochem. Cytochem. 57(11), 10251040. Chen, T., Ajami, K., McCaughan, G.W., Gai, W.-P., Gorrell, M.D., Abbott, C.A. (2005). Molecular characterization of a novel dipeptidyl peptidase like 2 short form (DPL2-s) that is highly expressed in the brain and lacks dipeptidyl peptidase activity. Biochim. Biophys. Acta 1764(1), 3343. McNicholas, K., Chen, T., Abbott, C. (2009). Dipeptidyl peptidase (DP) 6 and DP10: novel brain proteins implicated in human health and disease. Clin. Chem. Lab. Med. 47(3), 262. Yokotani, N., Doi, K., Wenthold, R.J., Wada, K. (1993). Nonconservation of a catalytic residue in a dipeptidyl aminopeptidase IV-related protein encoded by a gene on human chromosome 7. Hum. Mol. Gen. 2(7), 10371039. Ajami, K., Pitman, M.R., Wilson, C.H., Park, J., Menz, R.I., Starr, A.E., Cox, J.H., Abbott, C.A., Overall, C.M., Gorrell, M.D. (2008). Stromal cell-derived factors 1 alpha and 1 beta, inflammatory protein-10 and interferon-inducible T cell chemo-attractant are novel substrates of dipeptidyl peptidase 8. FEBS Lett. 582(5), 819825. Bjelke, J.R., Christensen, J., Nielsen, P.F., Branner, S., Kanstrup, A.B., Wagtmann, N., Rasmussen, H.B. (2006). Dipeptidyl peptidase 8 and 9 specificity and molecular
Clan SC S9 | 750. Fibroblast Activation Protein α
characterization compared to dipeptidyl peptidase IV. Biochem. J. 396(2), 391399. [60] Lankas, G., Leiting, B., Roy, R., Eiermann, G., Beconi, M., Biftu, T., Chan, C., Edmondson, S., Feeney, W., He, H., Ippolito, D., Kim, D., Lyons, K., Ok, H., Patel, R., Petrov, A., Pryor, K., Qian, X., Reigle, L., Woods, A., Wu, J., Zaller, D., Zhang, X., Zhu, L., Weber, A., Thornberry, N. (2005). Dipeptidyl
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peptidase IV inhibition for the treatment of type 2 diabetes Potential importance of selectivity over dipeptidyl peptidases 8 and 9. Diabetes 54(10), 29882994. [61] Yu, D.M.T., Yao, T.-W., Chowdhury, S., Nadvi, N.A., Osborne, B., Church, W.B., McCaughan, G.W., Gorrell, M.D. (2010). The dipeptidyl peptidase IV family in cancer and cell biology. FEBS J. 277, 11261144.
Mark D. Gorrell Centenary Institute, Sydney Medical School, University of Sydney, NSW 2006, Australia. Email: [email protected]
John E. Park NBE Discovery, Boehringer Ingelheim Pharma KG, Birkendorferstr. 65, 88397 Biberach, Germany. Email: [email protected] Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00750-X
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Chapter 750
Fibroblast Activation Protein α DATABANKS MEROPS name: fibroblast activation protein alpha subunit MEROPS classification: clan SC, family S9, subfamily S9B, peptidase S09.007 Tertiary structure: Available Species distribution: superclass Tetrapoda Reference sequence from: Homo sapiens (UniProt: Q12884)
Name and History The fibroblast activation protein α (FAP) was discovered with a monoclonal antibody, mAb F19, that was generated in the course of a serological survey of cell surface antigens expressed on cultured human fibroblasts, sarcomas and neuroectodermal tumor cells [1]. This antibody was used to characterize the plasma membrane-associated 95 kDa FAP glycoprotein [2,3], to isolate the FAP-encoding cDNA (Genbank U09278) [4], and to examine FAP expression in a broad range of normal and neoplastic human tissues [1,5]. The immunochemical studies also led to the detection of high molecular mass complexes, comprising FAP multimers, and noncovalently linked, heteromeric complexes between FAP and a distinct, FAPassociated protein of 105 kDa designated as FAPβ [2]. Subsequent investigations have shown that FAPβ is the CD26 cell surface glycoprotein, also called dipeptidyl peptidase IV (DPPIV) (Chapter 745). However, FAP/ CD26 heteromeric complexes are found only on the surface membrane of some cell types, notably cultured fibroblasts and melanocytes, whereas other cells express only FAP (e.g. some sarcoma cell lines) or CD26 (e.g. kidney epithelial cells, activated T lymphocytes) or neither molecule. Moreover, immunohistochemistry on human tissues has revealed distinct and generally nonoverlapping expression patterns for FAP and CD26 [48]. FAP has been independently studied under two other names; antiplasmin cleaving enzyme (APCE) [9] and seprase [10]. APCE was shown to be a soluble circulating form of FAP, lacking the transmembrane domain, after APCE was purified from plasma [9]. Seprase was
identified on melanoma cell lines including LOX and SKMEL28, then cloned (Genbank U76833) and shown to be identical to FAP [10,11]. The FAP homolog in Xenopus laevis was discovered independently in the course of a differential gene expression survey of the thyroid hormone-induced tail resorption program during tadpole metamorphosis [12]. The human and Xenopus FAP polypeptides show about 50% amino acid sequence identity.
Activity and Specificity FAP possesses both a dipeptidyl-peptidase activity, specific for N-terminal Xaa-(Pro/Ala) sequences, and an endopeptidase activity capable of degrading α2-antiplasmin, gelatin and denatured type I collagen [9,13], including MMP1-cleaved type I collagen [14]. Mutation of the catalytic Ser residue of FAP to Ala abolishes both dipeptidyl-peptidase and endopeptidase enzymatic activities [13]. FAP is assayed at 37 C in physiological buffers such as pH 7.6 Tris/EDTA [13,15]. As in prolyl endopeptidase (PEP) assays, 10 mM EDTA and a reducing agent such as 1 mM dithiothreitol are added to inhibit contaminating proteases; FAP is unaffected by such substances but all DPPIV family proteases are inhibited by metal ions [1618]. The solubility of some substrates can be improved by using 10% methanol or 1% DMF [15]. FAP enzymatic activity is detected in extracts of human cancerous tissues [13] and cirrhotic liver [19] but not in matched normal controls. The FAP cleavage sites within recombinant human collagen I include cleavage following Pro-Pro-Gly-Pro and a consensus sequence of (Asp/Glu)-(Arg/Lys)-Gly-(Glu/Asp)-(Thr/Ser)-Gly-Pro [20]. The FAP/APCE cleavage site in α2-antiplasmin similarly follows Gly11-Pro12 [21]. In contrast, the dipeptidyl-peptidase activity of FAP greatly prefers Ala-Pro-AFC over Gly-Pro-AFC [3]. However, the identification of several natural bioactive peptide substrates of FAP dipeptidylpeptidase activity, most notably neuropeptide Y, substance P and B-type natriuretic peptide with half-lives of less than 10 minutes, revealed that glycine is not dominant in P2 in natural substrates [15]. In contrast, endopeptidase
Clan SC S9 | 750. Fibroblast Activation Protein α
3396
cleavage of artificial substrates requires Gly-Pro preceded by a benzoyloxycarbonyl (Z) moiety, not a succinyl or acyl moiety [22]. FAP appears to prefer a positive charge at P10 [15,23]. Some DPPIV inhibitors also inhibit FAP, most notably PT-100 and PT-630. PT-630 inhibits DPPIV and FAP to similar extents [24,25]. More recently, substrate specificity has been used in FAP inhibitor design and some inhibitors are selective while others also inhibit prolyl endopeptidase (PEP/POP; Chapter 742) [21,23,26]. The incretin-based type 2 diabetes therapeutics, called gliptins, are selective DPPIV inhibitors that primarily act by raising circulating levels of bioactive GLP-1. The potent DPPIV inhibitor linagliptin is the only gliptin that displays some FAP inhibition, but linagliptin does not inhibit any other peptidase. Although FAP inhibition may be beneficial (see biology summary below), it is unlikely that the low level of FAP inhibition conferred by linagliptin, 90-fold less than its DPPIV inhibition [27], would have a clinical effect. The distinguishing features of linagliptin are its rapid absorption (Tmax 5 1.5 hr), in vivo longevity, non-renal (biliary) excretion, its low therapeutic dose of 5 mg day21, and excellent safety profile including cardiovascular safety [28].
Structural Chemistry FAP homodimers and FAP/DPPIV heterodimers as well as larger complexes of FAP have been identified in cell extracts of cultured human fibroblasts, melanocytes and sarcoma cells. When analyzed by immunoblotting of boiled extracts separated by SDS-PAGE, FAP (1760) appears as a single protein species of about 95 kDa. Digestion of this 95 kDa glycoprotein with neuraminidase or endoglycosidase H, but not O-glycanase, reduces the apparent molecular size on SDS gels, and complete removal of all five N-linked sugars (Figure 750.1) with N-glycanase generates polypeptides of 75 kDa [2]. FAP requires dimerization for activity. Each monomer consists of an αβ hydrolase domain and an 8-blade β-propeller domain (Protein Data Bank code 1Z68) [29]. The catalytic pocket is buried inside the protein between these two domains and contains essential residues contributed by both domains, including Ser624, Asp702 and His734 of the catalytic triad and Glu203 and Glu204, in the β-propeller domain, that contact the N-terminus of substrates when undergoing dipeptidyl-peptidase cleavage [7,13]. Arg123, Tyr656 and Asn704 also contribute to FAP catalysis [29,30]. Ala657 is essential for FAP endopeptidase activity because it is small and so makes space sufficient for endopeptidase substrates. In contrast, in DPP4 this three-dimensional location is occupied by the large negatively charged residue Asp663 that prevents
Hydrolase
Domains:
Propeller
Hydrolase
TransCytoplasmic membrane E203 E204
FAP (760 aa)
7
27
53
DPPIV (766 aa)
7
29
51
S624 D702 H734 499
E205 E206 L294 V341
S630 D708 H740 501
Key: N-glycosylation
Cysteine
Enzyme activity
ADA binding
FIGURE 750.1 Schematic of FAP and its nearest relative DPPIV. The arrangement of protein domains and some residues essential for function and positions of cysteines and potential N-linked glycosylation sites are depicted. Not to scale.
endopeptidase activity [29]. The transmembrane-bound form of FAP is a type II integral membrane protein with the transmembrane domain formed by residues 726 (Figure 750.1). The Mus musculus FAP cDNA (NM_007986) has been cloned on the basis of its sequence similarity to human FAP [31]. The predicted human and mouse FAP polypeptides share 89% amino acid sequence identity and contain identical catalytic domains. There is evidence from mouse FAP studies that differential mRNA splicing generates at least three distinct transcripts that differ in their extracellular portions adjacent to the transmembrane domain [31], but the corresponding protein isoforms have not yet been characterized. Disruption of the FAP locus by gene targeting results in homozygous FAP-deficient mice that develop normally, are fertile and show no overt phenotype [32]. Nonetheless, the function of FAP may still be elucidated in such mice using disease models of chronic inflammation, fibrosis or tumor induction.
Preparation Natural sources of human FAP include cultured fibroblasts, melanocytes and certain sarcoma cell lines as well as embryonic tissues, granulation tissue of healing wounds and certain tumor tissues [1,3,5]. Isolation of human FAP has been accomplished with a combination of lectin and antibody affinity chromatography [3]. Recombinant mouse and human FAP enzymes produced in mammalian or insect cell culture have been purified to homogeneity and possess the same catalytic activities as naturally isolated FAP enzyme [13,17,33]. Soluble FAP (27760) has been purified from bovine serum and human plasma [9].
Clan SC S9 | 750. Fibroblast Activation Protein α
(A)
(B)
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(C)
Malignant epithelial cells
Resting fibrocytes
Normal capillary
Tumor capillaries
Activated tumor stromal fibroblasts
FIGURE 750.2 Schematic of tumor stromal fibroblast activation, accompanied by induction of FAP cell surface expression, in epithelial cancers. (A) Early stage of epithelial cancer development with small deposits of malignant epithelial cells (nodules with a diameter less than approximately 2 mm) that do not require new blood vessel formation; (B) Larger nodules of malignant epithelial cells without new blood vessels and stroma formation, showing areas of central hypoxia and necrosis; (C) Tumor nodules (diameter greater than approximately 5 mm) with new blood vessels and accompanying, activated tumor stromal fibroblasts expressing cell surface FAP.
Biological Aspects Although FAP was discovered as a cell surface antigen of cultured normal fibroblasts, its expression pattern in vivo, as determined by immunohistochemistry with mAb F19 and other antibodies against distinct FAP epitopes [3], is highly restricted. In particular, resting fibroblasts in normal tissues lack detectable FAP expression, and FAP induction upon in vitro cell culture appears to be part of a more general activation program which fibroblasts assume when grown under artificial tissue culture conditions [34]. Corresponding activation phenotypes have been postulated for the reactive fibroblasts found in the granulation tissue of healing wounds, certain chronic inflammatory lesions, the supporting stroma of malignant epithelial neoplasms, and normal embryonic tissues, and all of these fibroblasts also express FAP [1,4,5,19,25,3538]. Notably, the activated tumor stromal fibroblasts found in carcinomas of the breast, colorectum, lung, stomach, pancreas and esophagus show prominent FAP expression (Figure 750.2). Moreover, these FAP1 cells appear to drive immune suppression [38] and epithelial-mesenchymal transition [37], and may be an excellent target for antitumor therapy [25,39]. Indeed, since very few cell types in normal organs express this cell surface antigen, it has been possible to use the 131I-labeled mAb F19 for selective immunological targeting in patients with metastatic colorectal cancers [40]. The few normal human cell types known to express FAP in vivo include fetal mesenchymal cells, placenta and a distinct subset of glucagon producing endocrine cells (A cells) in the pancreatic islets. The biological role of FAP expression in these
diverse tissue types and pathological processes is still unknown, but it is tempting to speculate about functions related to tissue remodeling and repair [5]. FAP is upregulated in liver fibrosis [6,19,41], pulmonary fibrosis [42], rheumatoid arthritis [35,43], osteoarthritis [36] and atherosclerosis [44]. The FAP substrate α2-antiplasmin, which is an inhibitor of fibrinolysis, is made by the liver and released into the circulation. Following cleavage by FAP, the activity of α2-antiplasmin is greatly increased [45]. Approaches to antitumor therapy exploiting FAP upregulation in tumors include inhibitors, antibodies and prodrugs. The FAP inhibitors talabostat (PT100) and PT630 have antitumor efficacy in mice [25] but talabostat is not effective in humans [46]. Antibody-toxin conjugate [47] and a protoxin that uses FAP-specific cleavage to activate the toxin [48] are efficacious in mouse models.
Distinguishing Features FAP is in the S9 or prolyl oligopeptidase family. FAP is the only S9 family member to possess both dipeptidylpeptidase and prolyl-endopeptidase activities. FAP has a restricted expression whereas the other S9 family proteases and non-proteases are ubiquitous. Unlike other gelatinases, primarily the MMPs (family M10) that have a precursor form, FAP is constitutively active. Specific antibodies identify FAP (Table 750.1); the most widely used and fully validated specific antibody is mouse monoclonal antibody F19 to human FAP. No specific substrate or selective inhibitor is available for FAP detection but
Clan SC S9 | 750. Fibroblast Activation Protein α
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TABLE 750.1 Antibodies recognizing FAP Supplier
Product ID
Antigen
Antibody
Reference
R&D Systems
3715-SE
Recombinant human FAP
sheep polyclonal
[38]
Abcam
ab53066
Peptide
rabbit polyclonal
[39,49]
Abcam
ab28244
Peptide of FAP stalk region near cell surface
rabbit polyclonal
[50]
Bender Medsystems
BMS168
Peptide
rabbit polyclonal
[51]
Santa Cruz
F11-24
Peptide
rabbit polyclonal
[37]
Abnova
1E5/H002191-M01
60 kDa carboxy fragment of FAP
mouse monoclonal
[6]
ATCC
F19
Human tumor cell line
mouse hybridoma
[1,7,19]
J. Cheng
6E1
Mouse FAP
rabbit monoclonal
[52]
FAP is the only peptidase known to hydrolyze both HAla-Pro-AFC and Z-Gly-Pro-AFC. Unlike PEP, FAP cannot hydrolyze succinyl-Ala-Pro- or succinyl-Gly-Proderived substrates [15,17,22]. Therefore, FAP can be identified by substrate combinations. FAP has a unique sequence and chromosomal location at 2q23 in human, 2:C1.3 in mouse and q21 in rat.
Related Peptidases Human FAP has 52% amino acid sequence identity with human DPPIV/CD26. Two cytosolic proteases with 28% sequence identity to FAP, DPP8 (Chapter 746) and DPP9 (Chapter 747), have been identified [16,53]. Like FAP, they cleave N-terminal Xaa-Pro peptides; however, unlike FAP, they have no endopeptidase activity and hydrolyze H-Ala-Pro- and H-Gly-Pro-derived substrates equally well [15,17,54]. FAP shares about 30% amino acid sequence identity with dipeptidyl-peptidase 6 (DPP6) cDNA and DPP10, which were isolated from brain [55,56]. The DPP6 and DPP10 proteins lack the characteristic Ser and Trp residues in the catalytic domain that are present in both FAP and the other three DPPIV family members [55,57]. The FAP gene is 72.8 kb. The DPPIV and FAP gene loci are adjacent, at human chromosome 2q24.2 and 2q24.3, respectively, suggesting that they may have arisen through gene duplication. By contrast, the other related genes of the DPPIV family are on different human chromosomes. All S9B family members, FAP, DPPIV, DPP8 and DPP9, hydrolyze H-Ala-Pro-derived substrates, so discrimination between these proteases can be difficult. Concordantly, some natural substrates overlap, including NPY, GLP-1, GLP-2 and some chemokines [15,58,59]. Several inhibitors of DPPIV also inhibit FAP, DPP8 and DPP9 [60], and some FAP inhibitors also inhibit PEP [26].
Further Reading For reviews on FAP in cancer and fibrosis, see Wang et al. [6], Pure [39], and Yu et al. [61].
References [1] Rettig, W.J., Garin-Chesa, P., Beresford, H.R., Oettgen, H.F., Melamed, M.R., Old, L.J. (1988). Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells. Proc. Natl. Acad. Sci. USA 85(9), 31103114. [2] Rettig, W.J., Garin-Chesa, P., Healey, J.H., Su, S.L., Ozer, H.L., Schwab, M., Albino, A.P., Old, L.J. (1993). Regulation and heteromeric structure of the fibroblast activation protein in normal and transformed cells of mesenchymal and neuroectodermal origin. Cancer Res. 53(14), 33273335. [3] Rettig, W.J., Su, S.L., Fortunato, S.R., Scanlan, M.J., Raj, B.K., Garin-Chesa, P., Healey, J.H., Old, L.J. (1994). Fibroblast activation protein: purification, epitope mapping and induction by growth factors. Int. J. Cancer 58(3), 385392. [4] Scanlan, M.J., Raj, B.K., Calvo, B., Garin-Chesa, P., SanzMoncasi, M.P., Healey, J.H., Old, L.J., Rettig, W.J. (1994). Molecular cloning of fibroblast activation protein alpha, a member of the serine protease family selectively expressed in stromal fibroblasts of epithelial cancers. Proc. Natl. Acad. Sci. USA 91(12), 56575661. [5] Garin-Chesa, P., Old, L.J., Rettig, W.J. (1990). Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers. Proc. Natl. Acad. Sci. USA 87(18), 72357239. [6] Wang, X.M., Yao, T.-W., Nadvi, N.A., Osborne, B., McCaughan, G.W., Gorrell, M.D. (2008). Fibroblast activation protein and chronic liver disease. Front. Biosci. 13, 31683180. [7] Wang, X.M., Yu, D.M.T., McCaughan, G.W., Gorrell, M.D. (2005). Fibroblast activation protein increases apoptosis, cell adhesion and migration by the LX-2 human stellate cell line. Hepatology 42(4), 935945.
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characterization compared to dipeptidyl peptidase IV. Biochem. J. 396(2), 391399. [60] Lankas, G., Leiting, B., Roy, R., Eiermann, G., Beconi, M., Biftu, T., Chan, C., Edmondson, S., Feeney, W., He, H., Ippolito, D., Kim, D., Lyons, K., Ok, H., Patel, R., Petrov, A., Pryor, K., Qian, X., Reigle, L., Woods, A., Wu, J., Zaller, D., Zhang, X., Zhu, L., Weber, A., Thornberry, N. (2005). Dipeptidyl
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peptidase IV inhibition for the treatment of type 2 diabetes Potential importance of selectivity over dipeptidyl peptidases 8 and 9. Diabetes 54(10), 29882994. [61] Yu, D.M.T., Yao, T.-W., Chowdhury, S., Nadvi, N.A., Osborne, B., Church, W.B., McCaughan, G.W., Gorrell, M.D. (2010). The dipeptidyl peptidase IV family in cancer and cell biology. FEBS J. 277, 11261144.
Mark D. Gorrell Centenary Institute, Sydney Medical School, University of Sydney, NSW 2006, Australia. Email: [email protected]
John E. Park NBE Discovery, Boehringer Ingelheim Pharma KG, Birkendorferstr. 65, 88397 Biberach, Germany. Email: [email protected] Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00750-X