Processing of a Class I-Restricted Epitope from Tyrosinase Requires Peptide <i>N</i>-Glycanase and the Cooperative Action of Endoplasmic Reticulum Aminopeptidase 1 and Cytosolic Proteases

Altrich-VanLith, Michelle L.; Ostankovitch, Marina; Polefrone, Joy M.; Mosse, Claudio A.; Shabanowitz, Jeffrey; Hunt, Donald F.; Engelhard, Víctor H.

doi:10.4049/jimmunol.177.8.5440

Cited by 39 publications

(43 citation statements)

References 45 publications

(57 reference statements)

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“…This posttranslational deamidation was already described for the Asn residue at position 371 during the processing of the tyrosinase 369-377 epitope (22). It was shown that deamidation of this Asn was partially caused by its glycosylation in the ER and subsequent deglycosylation by PNGase in the cytosol (28). Here, by inhibiting PNGase activity with z-VAD-fmk in melanoma cells naturally expressing tyrosinase, we confirmed the involvement of this enzyme in the processing of the tyrosinase 369-377 epitope and showed that PNGase also contributes to the processing of the spliced antigenic peptide IYMDGTADFSF.…”

Section: Discussionmentioning

confidence: 86%

“…We then evaluated the role of PNGase in the deamidation process leading to spliced peptide IYMDGTADFSF. We treated melanoma cells LB39-MEL with the caspase inhibitor z-VAD-fmk, which is also a potent inhibitor of PNGase (28,40), and tested them for recognition by the three CTL clones used previously. Treatment with z-VADfmk strongly inhibited presentation of peptide IYMDGTADFSF and to a lesser extent, peptide YMDGTMSQV (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Last, peptides were found to be modified at the posttranslational level undergoing, for example, deamidation of asparagine residues (20)(21)(22), phosphorylation of serine and threonine residues (23,24), or peptide splicing (25)(26)(27). Deamidation was found to occur on asparagine residues corresponding to an N-glycosylation site and is thought to involve retrotranslocation from the ER into the cytosol and deglycosylation by peptide-N-glycanase (PNGase) (28). Peptide splicing allows the production of antigenic peptides made of two noncontiguous fragments of a given protein that are linked together after the excision of an intervening segment.…”

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confidence: 99%

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An antigenic peptide produced by reverse splicing and double asparagine deamidation

Dalet

Robbins

Stroobant

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

120

122

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A variety of unconventional translational and posttranslational mechanisms contribute to the production of antigenic peptides, thereby increasing the diversity of the peptide repertoire presented by MHC class I molecules. Here, we describe a class I-restricted peptide that combines several posttranslational modifications. It is derived from tyrosinase and recognized by tumor-infiltrating lymphocytes isolated from a melanoma patient. This unusual antigenic peptide is made of two noncontiguous tyrosinase fragments that are spliced together in the reverse order. In addition, it contains two aspartate residues that replace the asparagines encoded in the tyrosinase sequence. We confirmed that this peptide is naturally presented at the surface of melanoma cells, and we showed that its processing sequentially requires translation of tyrosinase into the endoplasmic reticulum and its retrotranslocation into the cytosol, where deglycosylation of the two asparagines by peptide-N-glycanase turns them into aspartates by deamidation. This process is followed by cleavage and splicing of the appropriate fragments by the standard proteasome and additional transport of the resulting peptide into the endoplasmic reticulum through the transporter associated with antigen processing (TAP).antigen processing | peptide splicing | tumor antigen C D8 + cytotoxic T lymphocytes (CTLs) are the principal effectors recognizing, through their specific T cell receptor, peptide fragments bound to MHC class I molecules present on the surface of malignant cells. Most of the genes encoding class I-restricted tumor antigenic peptides were discovered by isolating T lymphocytes from melanoma patients. After in vitro stimulation by autologous melanoma cells, antitumor T lymphocytes were obtained and used to clone the genes encoding the tumor antigens. These genes included cancer germ-line genes, such as those of the melanoma antigen (MAGE) family, which encode tumor-specific antigens expressed in various tumors (1-4), but also differentiation genes encoding antigens expressed in both melanomas and normal melanocytes, such as Melan-A (MART-1), gp100, and tyrosinase (5-8). Precise identification of the antigenic peptides present at the surface of tumor cells is crucial to develop efficient immunotherapy strategies. These peptides can be used, for example, to generate melanoma-specific T cells for adoptive immunotherapy or in strategies using epitope-based vaccination. Understanding the processing and presentation pathways of such antigenic peptides may also help in designing appropriate protocols of immunotherapy. Class I-restricted antigenic peptides are usually made up of fragments of 8-11 aa directly derived from the degradation of the parental protein by the proteasome (9). Proteasomal substrates mostly comprise nuclear and cytosolic proteins but also include endoplasmic reticulum (ER) proteins returned to the cytosol during the process of ER-associated degradation (ERAD) (10, 11). In most cases, the sequence of the antigenic peptides can be easily p...

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Section: Discussionmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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An antigenic peptide produced by reverse splicing and double asparagine deamidation

Dalet

Robbins

Stroobant

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

120

122

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“…Subsequently, 3 additional deamidated peptide antigens from viral proteins has been reported [121][122][123]. In all four cases, the modified residues are Asn-linked glycosylation sites, and it has subsequently been shown that deamidation is a consequence of the removal of the carbohydrate by peptide-N-glycanase [124], which normally accompanies protein degradation [125][126][127].…”

Section: Identification Of Post-translationally Modified Peptides Prementioning

confidence: 99%

“…Among these, O-linked glycosylation of Lys [129], N-linked glycosylation of Asn [124,130], N-terminal acetylation [131], methylation of Arg [130], and phosphorylation of Ser and Thr [61,129] have all been identified through the use of mass spectrometry. Hunt and colleagues have also identified several peptides containing cysteine residues that have been altered by the attachment of another cysteine residue via a disulfide bond [53,132,133].…”

Section: Identification Of Post-translationally Modified Peptides Prementioning

confidence: 99%

The contributions of mass spectrometry to understanding of immune recognition by T lymphocytes

Engelhard

2007

International Journal of Mass Spectrometry

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Over the last 15 years, the ability of mass spectrometry to analyze complex peptide mixtures and identify individual species has provided unprecedented insights into the repertoire of peptide antigens displayed by MHC molecules and recognized by T lymphocytes. These include: understanding the peptide binding specificity of MHC molecules; understanding of roles of different intracellular components of the antigen processing pathways in determining the peptide display; and identification of a large number of individual peptide antigens associated with infectious diseases, cancer, and transplant rejection that have provided the basis for new immunologically based therapies. This review will summarize the impact that the application of mass spectrometry has had on these advances, with particular attention to the contributions of Professor Donald Hunt and members of his laboratory, and point out the opportunities for future work. Keywordsantigen processing; post-translational modification; peptides; MHC molecules THE NATURE OF ANTIGEN RECOGNITION BY T LYMPHOCYTEST lymphocytes are a branch of the immune system concerned with recognition of antigens that are displayed on the surface of host cells. These antigens are produced through intracellular proteolytic mechanisms that create small peptides, which are then rescued and presented at the cell surface by adaptor proteins called MHC molecules [reviewed in [1-6]. The binding site of each MHC molecule enables it to bind to a wide range of different peptides, and results in the display of a repertoire of such antigens, each recognized by a distinct T lymphocyte. This "antigen processing and presentation" mechanism results in the display of peptides derived from the proteins of pathogens that have infected the cell or been taken up by endocytosis. Their recognition by T lymphocytes results in the development of an immune response, and results in clearance of the pathogen and infected cells from the body. However, antigen processing and presentation pathways operate constitutively on normal cellular proteins as well, resulting in the display of peptide antigens that can be distinguished on tumors and transplanted tissues. Some of these "self-peptides" are also involved in the development of T lymphocytes in the thymus, and their recognition also controls the balance between selftolerance and autoimmune disease. Correspondence: phone: 434-924-2423; fax: 434-924-1221; email: vhe@virginia.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In most individuals, molecules of each MHC class are expressed from 3 genetic loci that ...

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Peptide Presentation to T Cells: Solving the Immunogenic Puzzle

Croft

2020

BioEssays

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The vertebrate immune system uses an impressive arsenal of mechanisms to combat harmful cellular states such as infection. One way is via cells delivering real‐time snapshots of their protein content to the cell surface in the form of short peptides. Specialized immune cells (T cells) sample these peptides and assess whether they are foreign, warranting an action such as destruction of the infected cell. The delivery of peptides to the cell surface is termed antigen processing and presentation, and decades of research have provided unprecedented understanding of this process. However, predicting the capacity for a given peptide to be immunogenic—to elicit a T cell response—has remained both enigmatic and a long sought‐after goal. In the era of big data, a point is being approached where the steps of antigen processing and presentation can be quantified and assessed against peptide immunogenicity in order to build predictive models. This review presents new findings in this area and contemplates challenges ahead.

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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

Cited by 39 publications

References 45 publications

An antigenic peptide produced by reverse splicing and double asparagine deamidation

An antigenic peptide produced by reverse splicing and double asparagine deamidation

The contributions of mass spectrometry to understanding of immune recognition by T lymphocytes

Peptide Presentation to T Cells: Solving the Immunogenic Puzzle

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