Identification of a ligand binding site in the human neutrophil formyl peptide receptor using a site-specific fluorescent photoaffinity label and mass spectrometry
Identification of a ligand binding site in the human neutrophil formyl peptide receptor using a site-specific fluorescent photoaffinity label and mass spectrometry
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DOI：
10.1074/jbc.273.17.10428
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发表时间：
1998-04-24
影响因子：
4.8
通讯作者：
Jesaitis, AJ
中科院分区：
文献类型：
--
作者：
Mills, JS;Miettinen, HM;Jesaitis, AJ
合成了一种新型荧光光亲和交联探针——甲酰基-甲基-对苯甲酰-l -苯丙氨酸- h - tyr - lys -epsilon- n-荧光素（fMBpaFYK-fl），用于鉴定重组人吞噬细胞化学引诱剂甲酰基肽受体（FPR）的结合位点残基。光激活后，将荧光素标记的中国鼠卵巢细胞膜溶解于辛基葡萄糖苷中，通过阴离子交换和凝胶过滤层析分离。在表达fpr细胞的提取物中观察到单峰荧光，而野生型对照提取物中没有。用CNBr切割光标记的中国仓鼠卵巢膜，并在抗荧光素免疫亲和基质上分离荧光片段。基质辅助激光解吸电离质谱鉴定出一个质量为1754的主要物种，与fMBpaFYK-fl交联val - arg - lys - al- hse的CNBr片段（FPR的预期CNBr片段，残基83-87）一致，该肽进一步用胰蛋白酶裂解，用抗荧光素免疫亲和再纯化，并进行基质辅助激光解吸电离质谱分析，观察到质量为1582的胰蛋白酶片段。这是与Val-Arg-Lys （FPR残基83-85）交联的fMBpaFYK-fl的质量，Val-Arg-Lys- ala - hse的预期胰蛋白酶裂解产物。残基83-85位于靠近细胞外表面的FPR的假设的第二跨膜区域内，提出了FPR的3D模型，该模型考虑了膜内，位点定向诱变结果（Miettinen， H. M., Mills， J., Gripentrog， J., Dratz， E. A., Granger， B. L.和Jesaitis， A. J. (1997) J. Immunol. 159, 4045-4054）和光化学交联数据。
A novel fluorescent photoaffinity cross-linking probe, formyl-Met-p-benzoyl-L-phenylalanine-Phe-Tyr-Lys-epsilon-N-fluorescein (fMBpaFYK-fl), was synthesized and used to identify binding site residues in recombinant human phagocyte chemoattractant formyl peptide receptor (FPR), After photoactivation, fluorescein-labeled membranes from Chinese hamster ovary cells were solubilized in octylglucoside and separated by tandem anion exchange and gel filtration chromatography, A single peak of fluorescence was observed in extracts of FPR-expressing cells that was absent in extracts from wild type controls. Photolabeled Chinese hamster ovary membranes were cleaved with CNBr, and the fluorescent fragments were isolated on an antifluorescein immunoaffinity matrix. Matrix-assisted laser desorption ionization mass spectrometry identified a major species with mass = 1754, consistent with the CNBr fragment of fMBpaFYK-fl cross-linked to Val-Arg-Lys-Ala-Hse (an expected CNBr fragment of FPR, residues 83-87), This peptide was further cleaved with trypsin, repurified by antifluorescein immunoaffinity, and subjected to matrix-assisted laser desorption ionization mass spectrometry, A tryptic fragment with mass = 1582 was observed, which is the mass of fMBpaFYK-fl cross-linked to Val-Arg-Lys (FPR residues 83-85), an expected trypsin cleavage product of Val-Arg-Lys-Ala-Hse. Residues 83-85 lie within the putative second transmembrane-spanning region of FPR near the extracellular surface, A 3D model of FPR is presented, which accounts for intramembrane, site-directed mutagenesis results (Miettinen, H. M., Mills, J., Gripentrog, J., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol. 159, 4045-4054) and the photochemical cross-linking data.