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N-Glycosylation Enhances Presentation of a MHC Class I-Restricted Epitope from Tyrosinase ¹

Carter Immunology Center and Department of Microbiology, University of Virginia, Charlottesville, VA 22908

We recently demonstrated that the mechanism of processing of an HLA-A*0201-restricted peptide epitope, Tyr₃₆₉(D), derived from the membrane protein tyrosinase, involves retrotranslocation of glycosylated molecules from the endoplasmic reticulum to the cytosol, removal of an N-linked carbohydrate from Asn₃₇₁ by peptide N-glycanase, proteolysis by the proteasome and other proteases, and retransport of the resulting peptides into the endoplasmic reticulum for association with HLA-A*0201. Carbohydrate removal results in deamidation of Asn₃₇₁ to aspartic acid. The asparagine-containing homolog of this peptide, Tyr₃₆₉(N), is not presented by tyrosinase-expressing cells, and this has been presumed to be due to quantitative glycosylation of Asn₃₇₁. Although examining cytosolic intermediates that accumulated in human melanoma cells treated with proteasome inhibitors, we were surprised to find both molecules that had been deglycosylated by peptide N-glycanase and a large number of molecules that had not been previously glycosylated. The failure of Tyr₃₆₉(N) to be processed and presented from these latter molecules may be partially due to a process of deamidation independent of glycosylation. However, we also established that proteasomes degrade tyrosinase molecules that are still glycosylated, giving rise to a set of discrete intermediates that are not observed when unglycosylated molecules are degraded. We propose that Tyr₃₆₉(N) fails to be presented because unglycosylated tyrosinase is degraded rapidly and relatively nonselectively. In contrast, glycosylation alters the selectivity of tyrosinase processing by the proteasome, enhancing the production or survival of Tyr₃₆₉(D).

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¹ This work was supported by U.S. Public Health Service Grants AI20963 and AI33134 (to V.H.E.).

² Current address: Miriam and Sheldon G. Adelson Medical Research Foundation, 300 1st Avenue, Needham, MA 02494.

³ Current address: IBT Reference Laboratory, 11274 Renner Boulevard, Lenexa, KS 66219.

⁴ Address correspondence and reprint requests to Dr. Victor Engelhard, Beirne Carter Center for Immunology Research, Box 801386, University of Virginia School of Medicine, Charlottesville, VA 22908. E-mail address: vhe{at}virginia.edu.

⁵ Abbreviations used in this paper: Tyr, tyrosinase; ER, endoplasmic reticulum; WT, wild type; PNGase, peptide N-glycanase; siRNA, small interfering RNA.