Abstract
Proteolytic degradation of extracellular matrix (ECM) components during tissue remodeling plays a pivotal role in normal and pathological processes including wound healing, inflammation, tumor invasion, and metastasis. Proteolytic enzymes in tumors may activate or release growth factors from the ECM or act directly on the ECM itself, thereby facilitating angiogenesis or tumor cell migration. Fibroblast activation protein (FAP) is a cell surface antigen of reactive tumor stromal fibroblasts found in epithelial cancers and in granulation tissue during wound healing. It is absent from most normal adult human tissues. FAP is conserved throughout chordate evolution, with homologues in mouse and Xenopus laevis, whose expression correlates with tissue remodeling events. Using recombinant and purified natural FAP, we show that FAP has both dipeptidyl peptidase activity and a collagenolytic activity capable of degrading gelatin and type I collagen; by sequence, FAP belongs to the serine protease family rather than the matrix metalloprotease family. Mutation of the putative catalytic serine residue of FAP to alanine abolishes both enzymatic activities. Consistent with its in vivo expression pattern determined by immunohistochemistry, FAP enzyme activity was detected by an immunocapture assay in human cancerous tissues but not in matched normal tissues. This study demonstrates that FAP is present as an active cell surface-bound collagenase in epithelial tumor stroma and opens up investigation into physiological substrates of its novel, tumor-associated dipeptidyl peptidase activity.
Highlights
Tumor capillaries, permitting malignant tumors to spread as well as acquire nutrients and discard wastes
One recently identified marker of reactive tumor stromal fibroblasts, the so-called fibroblast activation protein (FAP), is expressed in over 90% of common human epithelial cancers, and its expression in tumors is restricted to the tumor stromal fibroblast, as detected by immunohistochemistry with the prototype monoclonal antibody F19 [3] and other FAPspecific antibodies [4]
This study was designed to link the known expression pattern of human FAP in tumor stromal fibroblasts with a biochemical function that may help to explain its role in tumorigenesis and other tissue remodeling events
Summary
Materials—Geneticin, Lipofectin, DMEM, and DMEM/F12 media were obtained from Life Technologies, Inc. Fetal bovine serum was supplied by Biological Industries (Kibbutz Beit Hemeetz, Israel). All restriction enzymes were from Roche Molecular Biochemicals. Pfu polymerase was from Stratagene GmbH (Heidelberg Germany). All chromatography columns and matrices were purchased from Amersham Pharmacia Biotech. Batimastat® was obtained from British Biotech (Oxford, UK). High Five cells, pVL1393 vector, and Sf9 cells were obtained from Invitrogen (NV Leek, Netherlands). 2,2Ј-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid, diisopropylfluorophosphate (DFP), bovine trypsin, and DMEM lacking methionine and cysteine were from Sigma. Radiolabeled diisopropylfluorophosphate as 14C-DFP or 3H-DFP and [35S]methionine/cysteine mix were from NEN Life Science Products. Primers for site-directed mutagenesis were obtained from MWG Biotech (Ebersberg, Germany). Dipeptidic 4-methoxy--napthylamide and 7-amino-trifluoromethyl-coumarin (AFC) conjugates were obtained from Bachem (Heidelberg, Germany).
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