Antitopes Define Preferential Proteasomal Cleavage Site Usage

• Published in: The Journal of biological chemistry Vol. 283; no. 26; pp. 17891 - 17897
• Main Authors: Strehl, Britta, Textoris-Taube, Kathrin, Jäkel, Sandra, Voigt, Antje, Henklein, Peter, Steinhoff, Ulrich, Kloetzel, Peter-Michael, Kuckelkorn, Ulrike
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 27.06.2008; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Chaperonin 60 - chemistry; Coxsackievirus; Enterovirus - metabolism; Epitopes - chemistry; Interferon gamma Receptor; Mass Spectrometry - methods; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Multienzyme Complexes - chemistry; Peptides - chemistry; Proteasome Endopeptidase Complex - chemistry; Protein Binding; Receptors, Interferon - genetics; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M710042200

Protein degradation by proteasomes is a major source of peptides presented by major histocompatibility v complex class I proteins. Importantly, interferon γ-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.