Unidad de Presentación y Regulación Inmunes , Instituto de Salud Carlos III , 28220 Majadahonda (Madrid) , Spain.
Department of Biology , Technion-Israel Institute of Technology , 32000 Haifa , Israel.
J Proteome Res. 2019 Sep 6;18(9):3512-3520. doi: 10.1021/acs.jproteome.9b00416. Epub 2019 Aug 6.
Peptides generated by proteases in the cytosol must be translocated to endoplasmic reticulum lumen by the transporter associated with antigen processing (TAP) prior to their assembly with major histocompatibility complex (MHC) class I molecules. Nonfunctional TAP complexes produce a drastic decrease of the MHC class I/peptide complexes presented on the cell surface. Previously, the cellular MHC class I ligandome from TAP-deficient cell lines was determined, but similar analysis from normal tissues remains incomplete. Using highthroughput mass spectrometry to analyze the MHC-bound peptide pools isolated from ex vivo spleen cells of TAP-deficient mice, we identified 210 TAP-independent ligands naturally presented by murine MHC class I molecules. This ligandome showed increased peptide lengths, presence of multiple nested set peptides, and low theoretical MHC binding affinity. The gene ontology enrichment analysis of parental proteins of this TAP-independent subligandome showed almost exclusively enrichment in tissue-specific biological processes related to the immune system as would be expected. Also, cellular components of the extracellular space (namely proteins outside the cell but still within the organism excluding the extracellular matrix) were specifically associated with TAP-independent antigen processing from these ex vivo mice cells. In addition, functional protein association network analysis revealed low protein-protein interactions between parental proteins from the TAP-independent ligandome. Finally, predominant endoproteolytic peptidase specificity for Leu/Phe residues in the P position of the scissile bond at both ligand termini was found for the ex vivo TAP-independent ligands. These data indicate that the TAP-independent ligandome from ex vivo cells derives from a more diverse collection of both endoprotease activities and parental proteins and where the cell origin and contribution of the extracellular environment are more relevant than in its equivalent cell lines.
细胞溶质中的蛋白酶产生的肽必须通过抗原加工相关转运体（TAP）易位到内质网腔中，然后才能与主要组织相容性复合体（MHC）I 类分子组装。功能失调的 TAP 复合物会导致 MHC I 类/肽复合物在细胞表面的表达急剧减少。以前已经确定了缺乏 TAP 的细胞系的细胞 MHC I 类配体组，但正常组织的类似分析仍然不完整。使用高通量质谱分析从缺乏 TAP 的小鼠体外分离的 MHC 结合肽池，我们鉴定出 210 种由鼠 MHC I 分子自然呈现的 TAP 非依赖性配体。这个配体组显示出肽长度增加、存在多个嵌套集肽和低理论 MHC 结合亲和力。对这个 TAP 非依赖性亚配体组的亲本蛋白进行基因本体论富集分析显示，几乎只富集了与免疫系统相关的组织特异性生物学过程，这是预期的。此外，细胞外空间的细胞成分（即细胞外但仍在生物体内部的蛋白质，不包括细胞外基质）与这些来自体外小鼠细胞的 TAP 非依赖性抗原加工特异性相关。此外，功能蛋白关联网络分析显示，TAP 非依赖性配体组的亲本蛋白之间的蛋白-蛋白相互作用较低。最后，发现体外 TAP 非依赖性配体的裂解键 P 位的 Leu/Phe 残基的内切蛋白酶特异性。这些数据表明，来自体外细胞的 TAP 非依赖性配体组源自更广泛的内切蛋白酶活性和亲本蛋白的集合，并且细胞起源和细胞外环境的贡献比在其等效的细胞系中更为重要。
