Chiritoiu Gabriela N, Jandus Camilla, Munteanu Cristian V A, Ghenea Simona, Gannon Philippe O, Romero Pedro, Petrescu Stefana M
Institute of Biochemistry, Romanian Academy, Bucharest, Romania.
Ludwig Cancer Research Center, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland.
Electrophoresis. 2016 Jun;37(11):1448-60. doi: 10.1002/elps.201500449. Epub 2016 Feb 3.
The degradation process of the antigens specific to MHC-I presentation depends mainly on the proteasomal proteases in the cytosol. However, since many antigens are glycoproteins, including tumor antigens or viruses envelope proteins, their glycosylation status could also affect their processing and presentation. Here, we investigate the processing of tyrosinase, a multiple glycosylated tumor antigen overexpressed in human malignant melanoma. By LC-MS/MS analysis of human tyrosinase expressed in a melanoma cell, we show that all seven sites of tyrosinase are at least partially N-glycosylated. Using human CD8+ T-cell clones specific for the tyrosinase epitope YMDGTMSQV (369-377), including an N-glycosylation site, we found that transfectants of single and triple N-glycosylation mutants are recognized by specific T cells. Importantly, single, triple, and the aglycosylated tyrosinase mutants lacking the epitope located N-glycosylation site (N371D) were able to trigger higher CD8+ T-cell activation. The LC/MS analysis showed significant increase of the amount of YMDGTMSQV peptide resulted from accelerated oligomerization and degradation of aglycosylated mutants. The generation of the antigenic peptide by the antigen processing machinery is therefore largely independent of tyrosinase N-glycosylation. However, while distal N-glycans had no effect on the epitope generation, the mutants lacking the N371 glycan generated the antigenic peptide more efficiently. We conclude that epitope located N-glycans limit the ability of human tyrosinase to provide HLA-A2-restricted antigen for recognition by specific CD8+ T cells.
MHC-I呈递所特有的抗原的降解过程主要取决于胞质溶胶中的蛋白酶体蛋白酶。然而，由于许多抗原是糖蛋白，包括肿瘤抗原或病毒包膜蛋白，它们的糖基化状态也可能影响其加工和呈递。在此，我们研究了酪氨酸酶的加工过程，酪氨酸酶是一种在人类恶性黑色素瘤中过度表达的多重糖基化肿瘤抗原。通过对黑色素瘤细胞中表达的人类酪氨酸酶进行液相色谱-串联质谱分析，我们发现酪氨酸酶的所有七个位点至少部分被N-糖基化。使用对酪氨酸酶表位YMDGTMSQV（369-377）具有特异性的人类CD8+T细胞克隆，包括一个N-糖基化位点，我们发现单N-糖基化突变体和三N-糖基化突变体的转染子能被特异性T细胞识别。重要的是，缺乏位于N-糖基化位点（N371D）的表位的单、三及无糖基化酪氨酸酶突变体能够引发更高的CD8+T细胞活化。液相色谱/质谱分析显示，无糖基化突变体加速寡聚化和降解导致YMDGTMSQV肽的量显著增加。因此，抗原加工机制对抗原肽的产生在很大程度上独立于酪氨酸酶的N-糖基化。然而，虽然远端N-聚糖对表位产生没有影响，但缺乏N371聚糖的突变体更有效地产生了抗原肽。我们得出结论，位于表位的N-聚糖限制了人类酪氨酸酶为特异性CD8+T细胞识别提供HLA-A2限制性抗原的能力。
