Characterization of Proteasome-Generated Spliced Peptides Detected by Mass Spectrometry
Characterization of Proteasome-Generated Spliced Peptides Detected by Mass Spectrometry
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DOI：
10.4049/jimmunol.2100717
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发表时间：
2022-06-15
影响因子：
4.4
通讯作者：
Torigoe, Toshihiko
中科院分区：
文献类型：
--
作者：
Kato, Koji;Nakatsugawa, Munehide;Torigoe, Toshihiko
CD8（+）T细胞识别HLA I类分子展示的肽并监测细胞内肽库。已知蛋白酶体拼接两个短肽片段。最近的研究使用质谱（MS）和生物信息学分析表明，蛋白酶体产生的剪接肽（PSP）可能占HLA I类配体的相当大的比例。然而，使用生物信息学方法鉴定的PSP的真实性仍然模糊不清。在这项研究中，我们采用基于MS的从头测序直接捕获隐藏的HLA配体，没有在基因组中的模板。我们在一个具有微卫星不稳定性的人结直肠癌细胞系中发现了两个来源于相同蛋白质的PSP。健康供者来源的CD8（+）T细胞对两种PSP很容易应答，显示其天然的HLA呈递和抗原性。使用minigene构建体的实验表明，分别通过标准和反向顺式剪接产生的两个PSP的蛋白酶体依赖性加工。我们的研究结果表明，蛋白酶体剪接产生的HLA I类抗原库的多样性更广泛，支持基于MS的方法的优势，全面识别PSP。
CD8(+) T cells recognize peptides displayed by HLA class I molecules and monitor intracellular peptide pools. It is known that the proteasome splices two short peptide fragments. Recent studies using mass spectrometry (MS) and bioinformatics analysis have suggested that proteasome-generated spliced peptides (PSPs) may account for a substantial proportion of HLA class I ligands. However, the authenticity of the PSPs identified using bioinformatics approaches remain ambiguous. In this study, we employed MS-based de novo sequencing to directly capture cryptic HLA ligands that were not templated in the genome. We identified two PSPs originating from the same protein in a human colorectal cancer line with microsatellite instability. Healthy donor-derived CD8(+) T cells readily responded to the two PSPs, showing their natural HLA presentation and antigenicity. Experiments using minigene constructs demonstrated proteasome-dependent processing of two PSPs generated by standard and reverse cis splicing, respectively. Our results suggest a broader diversity of HLA class I Ag repertoires generated by proteasomal splicing, supporting the advantage of MS-based approaches for the comprehensive identification of PSPs.