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And Cytosolic Proteases Endoplasmic Reticulum Aminopeptidase 1 Processing of a Class I-Restricted Epitope from Tyrosinase Requires Peptide N -Glycanase and the Cooperative Action of Endoplasmic Reticulum Aminopeptidase 1 This information is current as and Cytosolic Proteases of September 28, 2021. Michelle L. Altrich-VanLith, Marina Ostankovitch, Joy M. Polefrone, Claudio A. Mosse, Jeffrey Shabanowitz, Donald F. Hunt and Victor H. Engelhard J Immunol 2006; 177:5440-5450; ; Downloaded from doi: 10.4049/jimmunol.177.8.5440 http://www.jimmunol.org/content/177/8/5440 http://www.jimmunol.org/ References This article cites 45 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/177/8/5440.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 28, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Processing of a Class I-Restricted Epitope from Tyrosinase Requires Peptide N-Glycanase and the Cooperative Action of Endoplasmic Reticulum Aminopeptidase 1 and Cytosolic Proteases1 Michelle L. Altrich-VanLith,2* Marina Ostankovitch,* Joy M. Polefrone,† Claudio A. Mosse,3* Jeffrey Shabanowitz,† Donald F. Hunt,†‡ and Victor H. Engelhard4* 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 Downloaded from 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. The Journal of Immunology, 2006, 177: http://www.jimmunol.org/ 5440–5450. ultiple posttranslational modifications have been de- cells developed against cells expressing the endogenous sequences scribed that alter the repertoire of MHC class I pep- preferentially recognize synthetic peptides containing Asp in place M tides displayed by cells. One of these modifications of Asn. In addition, both Asn- and Asp-containing forms of an involves the deamidation of Asn to Asp (1). The first epitope de- epitope derived from lymphocytic choriomeningitis virus (LCMV) scribed to undergo this process encompassed residues 369–377 GP1 are presented on cells infected with LCMV (6). In all four 5 from tyrosinase (Tyr369), a melanocyte differentiation protein that epitopes, the Asn residues in the original sequences are part of is also a target for melanoma-reactive T cells. In melanoma cells Asn-linked glycosylation sites, and it has been suggested that de- by guest on September 28, 2021 expressing full-length tyrosinase, the deamidated (Asp-containing) amidated epitopes would arise following glycosylation in the en- form of this epitope is presented at high copy number by HLA- doplasmic reticulum (ER) and subsequent deglycosylation. A cy- A*0201, whereas the Asn-containing form encoded directly by the tosolic enzyme, peptide-N-glycanase (PNGase), was recently gene is not detectable by either T cells or mass spectrometry (2, 3). shown to remove N-linked sugars from proteins or peptides during Later, two additional epitopes from HIV-1 env (4) and hepatitis C protein degradation (7–9). An obligatory step in this enzymatic E1 (5) proteins have been suggested to be deamidated, because T reaction is hydrolysis, which converts Asn to Asp (10). Despite suggestive evidence (3, 5), there has not yet been a direct demon- *Carter Immunology Center and Department of Microbiology, †Department of Chem- stration that a deamidated epitope is produced as a result of protein istry, and ‡Department of Pathology, University of Virginia, Charlottesville, VA glycosylation and deglycosylation, nor has the involvement of PN- 22908 Gase in such a process been evaluated. Received for publication April 24, 2006. Accepted for publication July 31, 2006. Because glycosylation occurs during translation of tyrosinase in The costs of publication of this article were defrayed in part by the payment of page the ER, the necessity of this process for deamidation of Tyr369 was charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. examined by altering the site of protein expression. Consistent 1 This work was supported by U.S. Public Health Service Grants AI20963 (to V.H.E.) with the involvement of glycosylation in deamidation, we previ- and AI33993 (to D.F.H.). M.L.A.-V. was supported by American Cancer Society ously established that the minimal 9-mer peptide representing Grant PF-03-128-01-LIB. Tyr369 did not undergo deamidation when translated in the cytosol 2 Current address: IBT Reference Laboratory, 11274 Renner Boulevard, Lenexa, KS (3). However, cytosolic translation of a fragment encoding resi- 66219. dues 144–378 of tyrosinase resulted in presentation of both the 3 Current address: Department of Pathology, Vanderbilt University, Nashville, TN 37212. Asp (Tyr369(D)) and Asn (Tyr369(N)) forms of the epitope. We 4 Address correspondence and reprint requests to Dr. Victor H. Engelhard, Carter hypothesized that this large fragment was processed in the cytosol Immunology Center, University of Virginia, Box 801386, Charlottesville, VA 22908- to give both minimal epitope and extended precursor peptides, 1386. E-mail address: [email protected] both of which were transported by TAP. We further hypothesized 5 Abbreviations used in this paper: Tyr369, tyrosinase 369–377; LCMV, lymphocytic that the additional length in the extended precursors prevented im- choriomeningitis virus; ER, endoplasmic reticulum; PNGase, peptide-N-glycanase; TPPII, tripeptidyl peptidase II; ERAP, ER aminopeptidase; LAP, leucine aminopep- mediate binding to HLA-A*0201 after TAP transport, enabling the tidase; PSA, puromycin-sensitive aminopeptidase; BH, bleomycin hydrolase; TOP, peptide to become glycosylated and to exit the ER into the cytosol thimet oligopeptidase; EGFP, enhanced GFP; ICS, intracellular cytokine staining; BFA, brefeldin A; siRNA, small interfering RNA; FTMS, Fourier transform mass for both deglycosylation and processing to the mature epitope. In spectrometer. the present paper, we have investigated these hypotheses directly. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 5441 In considering the hypothesis outlined above, it became of in- T cell activity terest to understand which proteases were involved in the process- Tyr369(D)- and Tyr369(N)-specific T cells were generated as described pre- ing of Tyr369 epitope precursors. The proteasome has been shown viously (3) and restimulated weekly with peptide-pulsed splenocytes in to be responsible for proteolytic cleavage to generate the final C RPMI 1640 supplemented with 10% FBS, 1ϫ essential and nonessential terminus of many epitopes (11–13), whereas tripeptidyl peptidase amino acids, 10 U/ml IL-2, 15 mM HEPES, and 50 ␮M 2-ME (T cell medium). Presentation of Tyr (N) and Tyr (D) was evaluated either by II (TPPII) has been shown to play a more limited role (14). How- 369 369 intracellular cytokine staining (ICS) or ELISA. For ICS, T cells and stim- ever, the peptides that are generated by these proteases frequently ulator cells were incubated at various ratios for4hinTcell medium contain one or more N-terminal residues beyond that of the mature containing 50 U/ml IL-2 and 10 ␮g/ml brefeldin A (BFA) (Sigma-Aldrich). epitope (11–13, 15, 16). Several aminopeptidases in the cytosol Cells were stained with anti-CD8-PE (eBioscience), fixed and permeabil- and the ER have been implicated in the removal of these N-ter- ized with Cytofix/Cytoperm (BD Pharmingen), and stained with anti-IFN- ␥-allophycocyanin (eBioscience). Background IFN-␥ production was sub- minal extensions. One aminopeptidase localized in the ER, ER tracted out using unstimulated parallel cultures. In some ICS assays, aminopeptidase 1 (ERAP1), trims precursors to their final anti- deamidation was quantified by as the ratio of the percentage of IFN-␥ϩ ␥ϩ genic form in vitro, and exerts a strong influence on epitope gen- Tyr369(D) T cells over the sum of the percentage
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