26 S proteasome-mediated production of an authentic major histocompatibility class I-restricted epitope from an intact protein substrate.
Peptides displayed on the cell surface by major histocompatibility class I molecules (MHC class I) are generated by proteolytic processing of protein-antigens in the cytoplasm. Initially, antigens are degraded by the 26 S proteasome, most probably following ubiquitination. However, it is unclear whether this proteolysis results in the generation of MHC class I ligands or if further processing is required. To investigate the role of the 26 S proteasome in antigen presentation, we analyzed the processing of an intact antigen by purified 26 S proteasome. A recombinant ornithine decarboxylase was produced harboring the H-2K(b)-restricted peptide epitope, derived from ovalbumin SIINFEKL (termed ODC-ova). Utilizing recombinant antizyme to target the antigen to the 26 S proteasome, we found that proteolysis of ODC-ova by the 26 S proteasome resulted in the generation of the K(b)-ligand. Mass spectrometry analysis indicated that in addition to SIINFEKL, the N-terminally extended ligand, HSIINFEKL, was also generated. Production of SIINFEKL was linear with time and directly proportional to the rate of ODC-ova degradation. The overall yield of SIINFEKL was approximately 5% of the amount of ODC-ova degraded. The addition of PA28, the 20 S, or the 20 S-PA28 complex to the 26 S proteasome did not significantly affect the yield of the antigenic peptide. These findings demonstrate that the 26 S proteasome can efficiently digest an intact physiological substrate and generate an authentic MHC class I-restricted epitope.
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
S
S
Ben-Shahar
A
A
Komlosh
E
E
Nadav
I
I
Shaked
T
T
Ziv
A
A
Admon
G N
GN
DeMartino
Y
Y
Reiss
D000595:Amino Acid Sequence; D000818:Animals; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D000939:Epitopes / Q000378:metabolism*; D006183:H-2 Antigens / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D016254:Mutagenesis, Insertional; D009955:Ornithine Decarboxylase / Q000235:genetics / Q000378:metabolism; D010047:Ovalbumin / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping
D000939:Epitopes; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D015395:Histocompatibility Antigens Class I; D008024:Ligands; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010047:Ovalbumin; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D009955:Ornithine Decarboxylase