N-linked carbohydrates in tyrosinase are required for its recognition by human MHC class II-restricted CD4(+) T cells.
Glycosylation of mammalian proteins is known to influence their intracellular trafficking, half life, and susceptibility to enzymatic degradation. Rare instances of natural T cell epitopes dependent upon glycosylation for recognition have been described. We report here on human CD4(+) T lymphocyte cultures and clones from two melanoma patients that recognize the melanoma-associated Ag tyrosinase in the context of HLA-DR4 and -DR8. These T cells recognize tyrosinase, normally a heavily glycosylated molecule, when expressed constitutively in melanoma cells or in COS-7 transfectants pulsed as lysates onto autologous APC. However, these T cells fail to recognize tyrosinase expressed in bacteria, nor do they react with overlapping peptides covering full-length tyrosinase, suggesting a critical role for glycosylation in the processing and / or composition of the stimulatory epitopes. The requirement for glycosylation was demonstrated by the failure of tyrosinase-specific CD4(+) T cells to recognize tyrosinase synthesized in the presence of glycosylation inhibitors, or deglycosylated enzymatically. Site-directed mutagenesis of each of seven potential N-glycosylation sites showed that four sites were required to generate forms of tyrosinase that could be recognized by individual T cell clones. These data indicate that certain carbohydrate moieties are required for processing the tyrosinase peptides recognized by CD4(+) T cells. Post-translational modifications of human tumor-associated proteins such as tyrosinase could be a critical factor for the development of antitumor immune responses.
Housseau
F
F
The Surgery Branch, Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Moorthy
A
A
Langer
D A
DA
Robbins
P F
PF
Gonzales
M I
MI
Topalian
S L
SL
D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D019556:COS Cells; D002241:Carbohydrates / Q000502:physiology*; D002460:Cell Line; D002999:Clone Cells; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000276:immunology; D006023:Glycoproteins / Q000096:biosynthesis; D006031:Glycosylation; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D006801:Humans; D008545:Melanoma / Q000276:immunology*; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D002241:Carbohydrates; D018984:Epitopes, T-Lymphocyte; D006023:Glycoproteins; D000949:Histocompatibility Antigens Class II; D014442:Monophenol Monooxygenase