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Original Article

Maria Lucchiari-Hartz^a, Peter M. van Endert^b, Grégoire Lauvau^b, Reinhard Maier^c, Andreas Meyerhans^c, Derek Mann^d, Klaus Eichmann^a, and Gabriele Niedermann^a

^a Max-Planck Institute of Immunobiology, D-79108 Freiburg, Germany
^b Institut National de la Santé et de la Recherche Médicale (INSERM) U25, Hôpital Necker, 75743 Paris Cedex 15, France
^c Institute for Microbiology and Hygiene, Department of Virology, The Saarland University Hospital, D-66421 Homburg, Germany
^d Department of Clinical Biochemistry, University of Southampton School of Medicine, Southampton SO16 7PX, United Kingdom
Max-Planck Institute of Immunobiology, Stübeweg 51, D-79108 Freiburg, Germany.49-761-5108-54549-761-5108-435

niedermann{at}immunbio.mpg.de

Although a pivotal role of proteasomes in the proteolytic generation of epitopes for major histocompatibility complex (MHC) class I presentation is undisputed, their precise function is currently the subject of an active debate: do proteasomes generate many epitopes in definitive form, or do they merely generate the COOH termini, whereas the definitive NH₂ termini are cleaved by aminopeptidases? We determined five naturally processed MHC class I ligands derived from HIV-1 Nef. Unexpectedly, the five ligands correspond to only three cytotoxic T lymphocyte (CTL) epitopes, two of which occur in two COOH-terminal length variants. Parallel analyses of proteasomal digests of a Nef fragment encompassing the epitopes revealed that all five ligands are direct products of proteasomes. Moreover, in four of the five ligands, the NH₂ termini correspond to major proteasome cleavage sites, and putative NH₂-terminally extended precursor fragments were detected for only one of the five ligands. All ligands are transported by the transporter associated with antigen processing (TAP). The combined results from these five ligands provide strong evidence that many definitive MHC class I ligands are precisely cleaved at both ends by proteasomes. Additional evidence supporting this conclusion is discussed, along with contrasting results of others who propose a strong role for NH₂-terminal trimming with direct proteasomal epitope generation being a rare event.

Key Words: proteasome • HIV Nef • cytotoxic T lymphocyte epitopes • antigen processing • naturally processed peptides

Abbreviations used in this paper: aa, amino acid(s); ER, endoplasmatic reticulum; FR, fluorescence ratio; IC₅₀, 50% inhibition of specific binding; LAP, leucine aminopeptidase; Nt, NH₂-terminally; PA28, proteasome activator 28; rp-HPLC, reversed phase HPLC; TAP, transporter associated with antigen processing; TPP II, tripeptidyl peptidase II.

© 2000 The Rockefeller University Press

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