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J. Biol. Chem., Vol. 283, Issue 26, 17891-17897, June 27, 2008

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Antitopes Define Preferential Proteasomal Cleavage Site Usage^*

Britta Strehl, Kathrin Textoris-Taube, Sandra Jäkel, Antje Voigt, Peter Henklein, Ulrich Steinhoff^¶, Peter-Michael Kloetzel¹, and Ulrike Kuckelkorn

From the Institut für Biochemie and Klinik für Kardiologie und Pulmologie, Charité-Universitätsmedizin, and ^¶Max-Planck-Institut für Infektionsbiologie, D-10117 Berlin, Germany

Protein degradation by proteasomes is a major source of peptides presented by major histocompatibility v complex class I proteins. Importantly, interferon -induced immunoproteasomes in many cases strongly enhance the generation of antigenic peptides both in vitro and in vivo. Whether this is due to enhanced substrate turnover or to a change in proteasomal cleavage specificity is, however, largely unresolved. To overcome the problems of peptide quantification inherent to mass spectrometry, we introduced the "antitope" as substrate-specific internal standard. The antitope is a non-functional peptide that is generated by proteasomal cleavage within the epitope, resulting in partial overlaps with the functional epitope. Using antitopes as internal standards we demonstrate that the observed enhanced immunoproteasome-dependent presentation of the bacterial listeriolysin O T-cell epitope LLO(296–304) is indeed due to altered cleavage preferences. This method is also applicable to other major histocompatibility class I epitopes as is shown for two potential epitopes derived from Coxsackievirus.

Received for publication, December 10, 2007 , and in revised form, March 6, 2008.

^* This work was supported in part by Sonderforschungsbereich (SFB) Grants 421 (to P.-M. K.) and SFB/TR 19 (to U. K.), and Deutsche Forschungsgemeinschaft Grant Ku1261 (to U. K. and U. S.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Monbijoustr. 2, D-10117 Berlin, Germany. Tel.: 49-30-450-528071; Fax: 49-30-450-528921; E-mail: p-m.kloetzel{at}charite.de.

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