Tsumagari Kazuya, Chang Chih-Hsiang, Ishihama Yasushi
Department of Molecular and Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
Eisai-Keio Innovation Laboratory for Dementia, Center for Integrated Medical Research, Keio University School of Medicine, Tokyo 160-8582, Japan.
iScience. 2021 Mar 2;24(4):102259. doi: 10.1016/j.isci.2021.102259. eCollection 2021 Apr 23.
Ectodomain shedding is a proteolytic process that regulates the levels and functions of membrane proteins. Dysregulated shedding is linked to severe diseases, including cancer and Alzheimer's disease. However, the exact cleavage sites of shedding substrates remain largely unknown. Here, we explore the landscape of ectodomain shedding by generating large-scale, cell-type-specific maps of shedding cleavage sites. By means of N- and C-terminal peptide enrichment and quantitative mass spectrometry, we quantified protein termini in the culture media of 10 human cell lines and identified 489 cleavage sites on 163 membrane proteins whose proteolytic terminal fragments are downregulated in the presence of a broad-spectrum metalloprotease inhibitor. A major fraction of the presented cleavage sites was identified in a cell-type-specific manner and mapped onto receptors, cell adhesion molecules, and protein kinases and phosphatases. We confidently identified 86 cleavage sites as metalloprotease substrates by means of knowledge-based scoring.
胞外域脱落是一种蛋白水解过程，可调节膜蛋白的水平和功能。脱落失调与包括癌症和阿尔茨海默病在内的严重疾病有关。然而，脱落底物的确切切割位点在很大程度上仍然未知。在此，我们通过生成大规模、细胞类型特异性的脱落切割位点图谱来探索胞外域脱落的全貌。借助N端和C端肽富集以及定量质谱分析，我们对10种人类细胞系培养基中的蛋白质末端进行了定量，并在163种膜蛋白上鉴定出489个切割位点，其蛋白水解末端片段在广谱金属蛋白酶抑制剂存在下表达下调。所呈现的切割位点大部分是以细胞类型特异性方式鉴定出来的，并定位到受体、细胞粘附分子以及蛋白激酶和磷酸酶上。我们通过基于知识的评分方法，可靠地鉴定出86个切割位点为金属蛋白酶底物。
