Fibroblast activation protein peptide substrates identified from human collagen I derived gelatin cleavage sites
Fibroblast activation protein peptide substrates identified from human collagen I derived gelatin cleavage sites
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DOI：
10.1021/bi701921b
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发表时间：
2008-01-22
期刊：
影响因子：
2.9
通讯作者：
Denmeade, Samuel R.
中科院分区：
文献类型：
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作者：
Aggarwal, Saurabh;Brennen, W. Nathaniel;Denmeade, Samuel R.
肿瘤基质成纤维细胞的一个高度一致的特征是诱导膜结合丝氨酸蛋白酶成纤维细胞激活蛋白-α（FAP），该蛋白在大多数人类上皮肿瘤基质内存在的反应性基质成纤维细胞的表面过度表达。相反，FAP 不由肿瘤上皮细胞或正常组织中的成纤维细胞或其他细胞类型表达。因此，FAP 的蛋白水解活性代表了潜在的泛肿瘤靶点，可用于从由与细胞毒素偶联的 FAP 肽底物组成的无活性前药中释放有效的细胞毒素。为了鉴定 FAP 肽底物，我们使用基于液相色谱串联质谱的测序来生成人 I 型胶原蛋白衍生明胶中 FAP 裂解位点的完整图谱。对切割位点内每个位置处每个氨基酸的频率进行位置分析揭示了FAP共有序列PPGP和(D/E)-(R/K)-G-(E/D)-(T/S)-G-P。这些研究进一步证明，根据 LC/MS/MS 提取的离子电流大小对裂解位点进行排序，可以预测裂解效率最高的 FAP 底物。根据离子流排名最高的裂解位点合成荧光淬灭的肽，并确定 FAP 水解的动力学参数。对应于共有序列的底物 DRGETGP 具有最低的 K-m，为 21 μM。总体而言，高排名底物和低排名底物的 K-m 值相对相似，而 k(cat) 值相差高达 100 倍。基于这些结果，目前正在评估 FAP 共有序列作为 FAP 选择性肽载体，用于掺入 FAP 激活的前药中。
A highly consistent trait of tumor stromal fibroblasts is the induction of the membrane-bound serine protease fibroblast activation protein-alpha (FAP), which is overexpressed on the surface of reactive stromal fibroblasts present within the stroma of the majority of human epithelial tumors. In contrast, FAP is not expressed by tumor epithelial cells or by fibroblasts or other cell types in normal tissues. The proteolytic activity of FAP, therefore, represents a potential pan-tumor target that can be exploited for the release of potent cytotoxins from inactive prodrugs consisting of an FAP peptide substrate coupled to a cytotoxin. To identify FAP peptide substrates, we used liquid chromatography tandem mass spectroscopy based sequencing to generate a complete map of the FAP cleavage sites within human collagen I derived gelatin. Positional analysis of the frequency of each amino acid at each position within the cleavage sites revealed FAP consensus sequences PPGP and (D/E)-(R/K)-G-(E/D)-(T/S)-G-P. These studies further demonstrated that ranking cleavage sites based on the magnitude of the LC/MS/MS extracted ion current predicted FAP substrates that were cleaved with highest efficiency. Fluorescence-quenched peptides were synthesized on the basis of the cleavage sites with the highest ion current rankings, and kinetic parameters for FAP hydrolysis were determined. The substrate DRGETGP, which corresponded to the consensus sequence, had the lowest K-m of 21 mu M. Overall the K-m values were relatively similar for both high and low ranked substrates, whereas the k(cat) values differed by up to 100-fold. On the basis of these results, the FAP consensus sequences are currently being evaluated as FAP-selective peptide carriers for incorporation into FAP-activated prodrugs.