Fibroblast activation protein: Purification, epitope mapping and induction by growth factors

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Abstract
The human fibroblast activation protein (FAP) defined by monoclonal antibody (MAb) F19 is a cell surface antigen expressed in reactive stromal fibroblasts of breast, colorectal, lung and other epithelial cancers. In contrast to its stroma-specific localization in epithelial neoplasms, FAP is expressed in the malignant mesenchymal cells of bone and soft-tissue sarcomas. FAP is transiently expressed in some fetal mesenchymal tissues but is absent or expressed at low levels in most adult tissues. FAP is induced in cultured fibroblasts and, in these cells, consists of a Mr 95,000 subunit (FAPα) carrying the F19 epitope and a non-covalently bound Mr 105,000 subunit (FAPβ) lacking the F19 epitope. Using MAb F19 and 5 newly derived MAbs, we identify 3 distinct epitopes on FAPα and tentatively assign one epitope to FAPβ. Analysis of detergent extracts of a FAPαhighβ-sarcoma cell line by size exclusion–high performance liquid chromatography (HPLC) revealed that FAPα does not elute as a Mr, 95,000 species but as part of a high-molecular weight complex (Mr > 400,000) that dissociates into Mr 95,000 subunits in SDS gels. Immunoaffinity purification of FAPα followed by tryptic digestion, reversed-phase HPLC and microsequencing identified 3 unique FAPα peptides, with 2 showing sequence similarity (23/38 identical amino acids) to segments of CD26, a T-cell activation antigen. CD26 is a membrane-bound enzyme (dipeptidyl aminopeptidase IV), but immunopurified FAPα has little if any dipeptidase activity with typical CD26 substrates. Finally, studies with FAPlow leptomeningeal fibroblasts revealed that transforming growth factor-β, 12-O-tetradecanoyl phorbol-13-acetate and retinoids can upregulate FAP expression, whereas serum and several other factors had no or little effect on FAP levels. FAP and CD26 may belong to a family of structurally related but functionally distinct activation proteins that are expressed on different cell types and show unique modes of regulation in normal and malignant cells.

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