Interferon-gamma can stimulate post-proteasomal trimming of the N terminus of an antigenic peptide by inducing leucine aminopeptidase.
Most antigenic peptides presented on major histocompatibility complex class I molecules are generated during protein breakdown by proteasomes, whose specificity is altered by interferon-gamma (IFN-gamma). When extended versions of the ovalbumin-derived epitope SIINFEKL are expressed in vivo, the correct C terminus is generated by proteasomal cleavage, but distinct cytosolic protease(s) generate its N terminus. To identify the other protease(s) involved in antigen processing, we incubated soluble extracts of HeLa cells with the 11-mer QLESIINFEKL, which in vivo is processed to the antigenic 8-mer (SIINFEKL) by a proteasome-independent pathway. This 11-mer was converted to the 9-mer by sequential removal of the N-terminal residues, but surprisingly the extract showed little or no endopeptidase or carboxypeptidase activity against this precursor. After treatment of cells with IFN-gamma, this N-terminal trimming was severalfold faster and proceeded to the antigenic 8-mer. The IFN-treated cells also showed greater aminopeptidase activity against many model fluorogenic substrates. Upon extract fractionation, three bestatin-sensitive aminopeptidase peaks were detected. One was induced by IFN-gamma and was identified immunologically as leucine aminopeptidase (LAP). Purified LAP, like the extracts of IFN-gamma-treated cells, processed the 11-mer peptide to SIINFEKL. Thus, IFN-gamma not only promotes proteasomal cleavages that determine the C termini of antigenic peptides, but also can stimulate formation of their N termini by inducing LAP. This enzyme appears to catalyze the trimming of the N terminus of this and presumably other proteasome-derived precursors. Thus, susceptibility to LAP may be an important influence on the generation on immunodominant epitopes.
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
J
J
Beninga
K L
KL
Rock
A L
AL
Goldberg
D000941:Antigens / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004790:Enzyme Induction; D004792:Enzyme Precursors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016056:Immunodominant Epitopes / Q000096:biosynthesis*; D007371:Interferon-gamma / Q000494:pharmacology*; D007931:Leucyl Aminopeptidase / Q000096:biosynthesis*; D009097:Multienzyme Complexes / Q000378:metabolism*; D010047:Ovalbumin / Q000031:analogs & derivatives; D046988:Proteasome Endopeptidase Complex; D014407:Tumor Cells, Cultured
D000941:Antigens; D000970:Antineoplastic Agents; D004792:Enzyme Precursors; D016056:Immunodominant Epitopes; D009097:Multienzyme Complexes; D007371:Interferon-gamma; D010047:Ovalbumin; D007931:Leucyl Aminopeptidase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex