The Journal of Experimental Medicine
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© The Rockefeller University Press, 0022-1007/1998/5/1641/ $5.00
The Journal of Experimental Medicine, Volume 187, Number 10, May 18, 1998 1641-1646

Articles

Inactivation of a Defined Active Site in the Mouse 20S Proteasome Complex Enhances Major Histocompatibility Complex Class I Antigen Presentation of a Murine Cytomegalovirus Protein

Gunter Schmidtke*, Maren Eggers{ddagger}, Thomas Ruppert{ddagger}, Marcus Groettrup*, Ulrich H. Koszinowski{ddagger}, and Peter-M. Kloetzel*

From the * Zentrum für Experimentelle Medizin (ZEM), Institut für Biochemie, Charité, Humboldt Universität zu Berlin, 10117 Berlin, Germany; and the {ddagger} Max von Pettenkofer Institut, 80336 München, Germany

Proteasomes generate peptides bound by major histocompatibility complex (MHC) class I molecules. Avoiding proteasome inhibitors, which in most cases do not distinguish between individual active sites within the cell, we used a molecular genetic approach that allowed for the first time the in vivo analysis of defined proteasomal active sites with regard to their significance for antigen processing. Functional elimination of the {delta}/low molecular weight protein (LMP) 2 sites by substitution with a mutated inactive LMP2 T1A subunit results in reduced cell surface expression of the MHC class I H-2Ld and H-2Dd molecules. Surface levels of H-2Ld and H-2Dd molecules were restored by external loading with peptides. However, as a result of the active site mutation, MHC class I presentation of a 9-mer peptide derived from a protein of murine cytomegalovirus was enhanced about three- to fivefold. Our experiments provide evidence that the {delta}/LMP2 active site elimination limits the processing and presentation of several peptides, but may be, nonetheless, beneficial for the generation and presentation of others.

Key Words: proteosome • antigen processing • mutation • active site • low molecular weight protein 2 T1A

Address correspondence to P.-M. Kloetzel, Zentrum für Experimentelle Medizin, Institut für Biochemie– Charité, Humboldt Universität zu Berlin, Monbijoustrabe 2, 10117 Berlin, Germany. Phone: 030-2802-6382; Fax: 030-2802-6608; E-mail: kloetzel{at}rz.charite.hu-berlin.de

The present address of Marcus Groettrup is Kantonsspital St. Gallen, Laborforschungsabteilung, 9007 St. Gallen, Switzerland.

Abbreviations used: LMP, low molecular weight protein; MCA, amido-4-methylcoumarin; MCMV, murine CMV.


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