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Abstract

The proteasome generates exact major histocompatibility complex (MHC) class I ligands as well as NH2-terminal-extended precursor peptides. The proteases responsible for the final NH2-terminal trimming of the precursor peptides had, until now, not been determined. By using specific selective criteria we purified two cytosolic proteolytic activities, puromycin-sensitive aminopeptidase and bleomycin hydrolase. These proteases could remove NH 2-terminal amino acids from the vesicular stomatitis virus nucleoprotein cytotoxic T cell epitope 52–59 (RGYVYQGL) resulting, in combination with proteasomes, in the generation of the correct epitope. Our data provide evidence for the existence of redundant systems acting downstream of the proteasome in the antigen-processing pathway for MHC class I molecules.

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Acknowledgements

We thank L. Yakes for reading the manuscript. Supported by the Deutsche Forschungsgemeinschaft (Leibnizprogram Ra369/4-1, to H. G. R.); Sonderforschungsbereich 510 (to H. S.); the European Union (Biomed 95-0263), Merck KGaA (Darmstadt, Germany), CTL Immunotherapies Corporation (Chatsworth, CA, USA) and a grant from the NIH/NIDA, DA 02243 (to L. B. H.).

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  1. Institute for Cell Biology, Department of Immunology, University of Tübingen, Auf der Morgenstelle 15, D-72076 Tübingen, Germany.

    • Lars Stoltze
    • , Markus Schirle
    • , Stefan Stevanovic
    • , Hans-Georg Rammensee
    •  & Hansjörg Schild
  2. Institute of Physiological Chemistry, Hoppe-Seyler Str. 4, D-72076 Tübingen, Germany.

    • Gerold Schwarz
    • , Christian Schröter
    •  & Hubert Kalbacher
  3. Department of Biochemistry, 800 Rose Lexington, University of Kentucky, Lexington KY 40536, USA

    • Michael W. Thompson
    •  & Louis B. Hersh

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Image files

  1. 1.

    Web Figure 1

    Effect of AAF-CMK on SIINFEKL processing and effect of E64 on RGYVYQGL processing. (a) Recognition of EG-7 in a 51 Cr-release assay by LS-O, specific for OVA(257-264), after treatment with acid wash and lactacystin and AAF-CMK. Cells were incubated (2 h, 37 °C) with 10 μM lactacystin and 50 μM AAF-CMK (σ), followed by an acid wash and a further 2 h of incubation with lactacystin and AAF-CMK. Targets were EG-7 cells treated with 50 μM AAF-CMK and the addition of 100 nM SIINFEKL during 51Cr labeling (open triangle), acid wash only (solid circle), untreated EG-7 (open circle) or EL-4 (open square). CTLs were used at the given E:T. (b) As for a but using MC-5v transfectants instead and 10 μM lactacystin and 100 μM E64. A standard 4-h 51 Cr-release assay with LS-Vn, specific for VSV(NP52-59) (RGYVYQGL), as effector cells followed

  2. 2.

    Web Figure 2

    Inhibitor profile of BH. Purified BH was preincubated for 30 min at 37 °C with 50 μM of various inhibitors (butabindide 5 μM). L-AMC (50 μM) was added and the percentage inhibition shown.

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https://doi.org/10.1038/80852

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