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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 Proteases¹

Michelle L. Altrich-VanLith^2,*, Marina Ostankovitch^*, Joy M. Polefrone, Claudio A. Mosse^3,*, Jeffrey Shabanowitz, Donald F. Hunt^, and Victor H. Engelhard^4,*

^* Carter Immunology Center and Department of Microbiology, Department of Chemistry, and Department of Pathology, University of Virginia, Charlottesville, VA 22908

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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by U.S. Public Health Service Grants AI20963 (to V.H.E.) and AI33993 (to D.F.H.). M.L.A.-V. was supported by American Cancer Society Grant PF-03-128-01-LIB.

² Current address: IBT Reference Laboratory, 11274 Renner Boulevard, Lenexa, KS 66219.

³ Current address: Department of Pathology, Vanderbilt University, Nashville, TN 37212.

⁴ Address correspondence and reprint requests to Dr. Victor H. Engelhard, Carter Immunology Center, University of Virginia, Box 801386, Charlottesville, VA 22908-1386. E-mail address: vhe{at}virginia.edu

⁵ Abbreviations used in this paper: Tyr₃₆₉, tyrosinase 369–377; LCMV, lymphocytic choriomeningitis virus; ER, endoplasmic reticulum; PNGase, peptide-N-glycanase; TPPII, tripeptidyl peptidase II; ERAP, ER aminopeptidase; LAP, leucine aminopeptidase; PSA, puromycin-sensitive aminopeptidase; BH, bleomycin hydrolase; TOP, thimet oligopeptidase; EGFP, enhanced GFP; ICS, intracellular cytokine staining; BFA, brefeldin A; siRNA, small interfering RNA; FTMS, Fourier transform mass spectrometer.

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