Fibroblast Activation Protein Peptide Substrates Identified from Human Collagen I Derived Gelatin Cleavage Sites
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- 作者：Saurabh Aggarwal ; W. Nathaniel Brennen ; Thomas P. Kole ; Elizabeth Schneider ; Ozlem Topaloglu ; Melinda Yates ; Robert J. Cotter ; Samuel R. Denmeade
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：January 22, 2008
- 年：2008
- 卷：47
- 期：3
- 页码：1076 - 1086
- 全文大小：461K
- 年卷期：v.47,no.3(January 22, 2008)
- ISSN：1520-4995
文摘
A highly consistent trait of tumor stromal fibroblasts is the induction of the membrane-boundserine protease fibroblast activation protein-
(FAP), which is overexpressed on the surface of reactivestromal fibroblasts present within the stroma of the majority of human epithelial tumors. In contrast, FAPis not expressed by tumor epithelial cells or by fibroblasts or other cell types in normal tissues. Theproteolytic activity of FAP, therefore, represents a potential pan-tumor target that can be exploited for therelease of potent cytotoxins from inactive prodrugs consisting of an FAP peptide substrate coupled to acytotoxin. To identify FAP peptide substrates, we used liquid chromatography tandem mass spectroscopybased sequencing to generate a complete map of the FAP cleavage sites within human collagen I derivedgelatin. Positional analysis of the frequency of each amino acid at each position within the cleavage sitesrevealed FAP consensus sequences PPGP and (D/E)-(R/K)-G-(E/D)-(T/S)-G-P. These studies furtherdemonstrated that ranking cleavage sites based on the magnitude of the LC/MS/MS extracted ion currentpredicted FAP substrates that were cleaved with highest efficiency. Fluorescence-quenched peptides weresynthesized on the basis of the cleavage sites with the highest ion current rankings, and kinetic parametersfor FAP hydrolysis were determined. The substrate DRGETGP, which corresponded to the consensussequence, had the lowest Km of 21 
M. Overall the Km values were relatively similar for both high andlow ranked substrates, whereas the kcat values differed by up to 100-fold. On the basis of these results, theFAP consensus sequences are currently being evaluated as FAP-selective peptide carriers for incorporationinto FAP-activated prodrugs.
(FAP), which is overexpressed on the surface of reactivestromal fibroblasts present within the stroma of the majority of human epithelial tumors. In contrast, FAPis not expressed by tumor epithelial cells or by fibroblasts or other cell types in normal tissues. Theproteolytic activity of FAP, therefore, represents a potential pan-tumor target that can be exploited for therelease of potent cytotoxins from inactive prodrugs consisting of an FAP peptide substrate coupled to acytotoxin. To identify FAP peptide substrates, we used liquid chromatography tandem mass spectroscopybased sequencing to generate a complete map of the FAP cleavage sites within human collagen I derivedgelatin. Positional analysis of the frequency of each amino acid at each position within the cleavage sitesrevealed FAP consensus sequences PPGP and (D/E)-(R/K)-G-(E/D)-(T/S)-G-P. These studies furtherdemonstrated that ranking cleavage sites based on the magnitude of the LC/MS/MS extracted ion currentpredicted FAP substrates that were cleaved with highest efficiency. Fluorescence-quenched peptides weresynthesized on the basis of the cleavage sites with the highest ion current rankings, and kinetic parametersfor FAP hydrolysis were determined. The substrate DRGETGP, which corresponded to the consensussequence, had the lowest Km of 21 M. Overall the Km values were relatively similar for both high andlow ranked substrates, whereas the kcat values differed by up to 100-fold. On the basis of these results, theFAP consensus sequences are currently being evaluated as FAP-selective peptide carriers for incorporationinto FAP-activated prodrugs.