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Electrophoresis. 2016 Jun;37(11):1448-60. doi: 10.1002/elps.201500449. Epub 2016 Feb 3.

Epitope located N-glycans impair the MHC-I epitope generation and presentation.

Author information

1
Institute of Biochemistry, Romanian Academy, Bucharest, Romania.
2
Ludwig Cancer Research Center, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland.
3
Department of Oncology, Lausanne University Hospital (CHUV), University of Lausanne, Lausanne, Switzerland.

Abstract

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.

KEYWORDS:

Antigen processing; Epitope generation; LC/MS; N-glycosylation; Tyrosinase

PMID:
26701645
DOI:
10.1002/elps.201500449
[Indexed for MEDLINE]

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