N-glycosylation enhances presentation of a MHC class I-restricted epitope from tyrosinase.

We recently demonstrated that the mechanism of processing of an HLA-A*0201-restricted peptide epitope, Tyr(369)(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 Asn(371) 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 Asn(371) to aspartic acid. The asparagine-containing homolog of this peptide, Tyr(369)(N), is not presented by tyrosinase-expressing cells, and this has been presumed to be due to quantitative glycosylation of Asn(371). 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 Tyr(369)(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 Tyr(369)(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 Tyr(369)(D).

MHC class II presentation of gp100 epitopes in melanoma cells requires the function of conventional endosomes and is influenced by melanosomes.

Many human solid tumors express MHC class II (MHC-II) molecules, and proteins normally localized to melanosomes give rise to MHC-II-restricted epitopes in melanoma. However, the pathways by which this response occurs have not been defined. We analyzed the processing of one such epitope, gp100(44-59), derived from gp100/Pmel17. In melanomas that have down-regulated components of the melanosomal pathway, but constitutively express HLA-DR*0401, the majority of gp100 is sorted to LAMP-1(high)/MHC-II(+) late endosomes. Using mutant gp100 molecules with altered intracellular trafficking, we demonstrate that endosomal localization is necessary for gp100(44-59) presentation. By depletion of the AP-2 adaptor protein using small interfering RNA, we demonstrate that gp100 protein internalized from the plasma membrane to such endosomes is a major source for gp100(44-59) epitope production. The gp100 trapped in early endosomes gives rise to epitopes that are indistinguishable from those produced in late endosomes but their production is less sensitive to inhibition of lysosomal proteases. In melanomas containing melanosomes, gp100 is underrepresented in late endosomes, and accumulates in stage II melanosomes devoid of MHC-II molecules. The gp100(44-59) presentation is dramatically reduced, and processing occurs entirely in early endosomes or stage I melanosomes. This occurrence suggests that melanosomes are inefficient Ag-processing compartments. Thus, melanoma de-differentiation may be accompanied by increased presentation of MHC-II restricted epitopes from gp100 and other melanosome-localized proteins, leading to enhanced immune recognition.

Processing of a class I-restricted epitope from tyrosinase requires peptide N-glycanase and the cooperative action of endoplasmic reticulum aminopeptidase 1 and cytosolic proteases.

Although multiple components of the class I MHC processing pathway have been elucidated, the participation of nonproteasomal cytosolic enzymes has been largely unexplored. In this study, we provide evidence for multiple cytosolic mechanisms in the generation of an HLA-A*0201-associated epitope from tyrosinase. This epitope is presented in two isoforms containing either Asn or Asp, depending on the structure of the tyrosinase precursor. We show that deamidation of Asn to Asp is dependent on glycosylation in the endoplasmic reticulum (ER), and subsequent deglycosylation by peptide-N-glycanase in the cytosol. Epitope precursors with N-terminal extensions undergo a similar process. This is linked to an inability of ER aminopeptidase 1 to efficiently remove N-terminal residues, necessitating processing by nonproteasomal peptidases in the cytosol. Our work demonstrates that processing of this tyrosinase epitope involves recycling between the ER and cytosol, and an obligatory interplay between enzymes involved in proteolysis and glycosylation/deglycosylation located in both compartments.

Post-translational modifications of naturally processed MHC-binding epitopes.

A variety of different post-translational modifications of peptides displayed by class I and II MHC molecules have now been described. Some modifications promote the binding of peptides to MHC molecules, and might also influence the ability of the peptide to be produced by antigen processing pathways. In some instances, the antigen processing components themselves are actually responsible for generating post-translational modifications. Finally, evidence is accumulating that modifications can be altered as a consequence of inflammation, transformation, apoptosis and aging. This leads to altered repertories of MHC-associated peptides, which may be important in immune responses associated with autoimmune diseases, infection and cancer.

Regulated folding of tyrosinase in the endoplasmic reticulum demonstrates that misfolded full-length proteins are efficient substrates for class I processing and presentation.

Short-lived protein translation products have been proposed to be the principal substrates that enter the class I MHC processing and presentation pathway. However, the biochemical nature of these substrates is poorly defined. Whether the major processing substrates are misfolded full-length proteins, or alternatively, aberrantly initiated or truncated polypeptides still remains to be addressed. To examine this, we used melanoma in which one-third of wild-type tyrosinase molecules were correctly folded and localized beyond the Golgi, while the remainder were present in the endoplasmic reticulum in an unfolded/misfolded state. Increasing the efficiency of tyrosinase folding using chemical chaperones led to a reduction in the level of substrate available to the proteasome and decreased the expression of a tyrosinase-derived epitope. Conversely, in transfectants expressing tyrosinase mutants that are completely misfolded, both proteasome substrate and epitope presentation were significantly enhanced. Proteasome substrate availability was a consequence of misfolding and not simply due to retention in the endoplasmic reticulum. Thus, the extent of folding/misfolding of a full-length protein is an important determinant of the level of epitope presentation.