S. Ben-shahar et al., 26 S proteasome-mediated production of an authentic major histocompatibility class I-restricted epitope from an intact protein substrate, J BIOL CHEM, 274(31), 1999, pp. 21963-21972
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 pro
teasome, most probably following ubiquitination, However, it is unclear whe
ther this proteolysis results in the generation of MHC class I ligands or i
f further processing is required. To investigate the role of the 26 S prote
asome in antigen presentation, we analyzed the processing of an intact anti
gen by purified 26 S proteasome. A recombinant ornithine decarboxylase was
produced harboring the H-2K(b)-restricted peptide epitope, derived from ova
lbumin 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 spe
ctrometry 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 degradat
ion. The overall yield of SIINFEKL was approximately 5% of the amount of OD
C-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 antigeni
c peptide. These findings demonstrate that the 26 S proteasome can efficien
tly digest an intact physiological substrate and generate an authentic MHC
class I-restricted epitope.