Diversity of Natural Self-Derived Ligands Presented by Different HLA Class I Molecules in Transporter Antigen Processing-Deficient Cells

The transporter associated with antigen processing (TAP) translocates the cytosol-derived proteolytic peptides to the endoplasmic reticulum lumen where they complex with nascent human leukocyte antigen (HLA) class I molecules. Non-functional TAP complexes and viral or tumoral blocking of these transporters leads to reduced HLA class I surface expression and a drastic change in the available peptide repertoire. Using mass spectrometry to analyze complex human leukocyte antigen HLA-bound peptide pools isolated from large numbers of TAP-deficient cells, we identified 334 TAP-independent ligands naturally presented by four different HLA-A, -B, and -C class I molecules with very different TAP dependency from the same cell line. The repertoire of TAP-independent peptides examined favored increased peptide lengths and a lack of strict binding motifs for all four HLA class I molecules studied. The TAP-independent peptidome arose from 182 parental proteins, the majority of which yielded one HLA ligand. In contrast, TAP-independent antigen processing of very few cellular proteins generated multiple HLA ligands. Comparison between TAP-independent peptidome and proteome of several subcellular locations suggests that the secretory vesicle-like organelles could be a relevant source of parental proteins for TAP-independent HLA ligands. Finally, a predominant endoproteolytic peptidase specificity for Arg/Lys or Leu/Phe residues in the P1 position of the scissile bond was found for the TAP-independent ligands. These data draw a new and intricate picture of TAP-independent pathways.抗原加工相关转运体（transporter associated with antigen processing, TAP）可将胞浆来源的蛋白水解肽段转运至内质网腔（endoplasmic reticulum lumen），在此处这些肽段与新生人类白细胞抗原（human leukocyte antigen, HLA）I类分子结合形成复合物。功能缺陷的TAP复合物以及病毒或肿瘤对该转运体的阻断作用，会导致HLA I类分子的表面表达水平降低，且可获取的肽库发生显著改变。本研究通过质谱法（mass spectrometry）分析从大量TAP缺陷细胞中分离得到的HLA结合肽复合物池，从同一细胞系的四种TAP依赖度差异极大的HLA-A、-B、-C I类分子中，共鉴定出334种天然呈递的TAP非依赖性配体。本次分析的TAP非依赖性肽库倾向于拥有更长的肽段长度，且对于所研究的四种HLA I类分子均缺乏严格的结合基序。该TAP非依赖性肽组（peptidome）源自182种亲本蛋白，其中大多数仅产生一种HLA配体；与之相反，仅有极少数细胞蛋白可通过TAP非依赖性抗原加工途径生成多种HLA配体。通过对比TAP非依赖性肽组与多个亚细胞区域的蛋白质组（proteome），结果提示分泌囊泡样细胞器可能是TAP非依赖性HLA配体的亲本蛋白的重要来源。最后，本研究发现，针对TAP非依赖性配体的剪切位点P1位的精氨酸/赖氨酸（Arg/Lys）或亮氨酸/苯丙氨酸（Leu/Phe）残基，存在显著的内切蛋白酶（endoproteolytic peptidase）特异性偏好。上述数据为TAP非依赖性抗原加工通路描绘了一幅全新且复杂的图景。