Proteolytic processing of ovalbumin and beta-galactosidase by the proteasome to a yield antigenic peptides.
The identification of genes in the class II region of the MHC that are homologous to genes encoding subunits of the proteasome has led to intense interest in the possible role of this enzyme in the proteolytic processing of polypeptide Ags. We have tested the ability of the 20S proteasome to produce peptides that can be presented by class I molecules as targets for killing by OVA-specific and beta-galactosidase-specific CTL clones. Samples of intact OVA and beta-galactosidase were subjected to digestion in vitro by 20S proteasome purified from bovine red cells and the resulting peptide mixtures were fractionated by reverse-phase HPLC. The fractions were tested for their ability to sensitize appropriate mouse target cells for lysis by specific CTL clones. In both cases, components that under all chromatographic conditions eluted with retention times indistinguishable from synthetic peptides representing known epitopes of the naturally processed proteins were found to be able to sensitize the target cells. Moreover, in the case of OVA, the presence of the expected target peptides was demonstrated directly by amino acid sequence and mass spectrometric analysis. The results demonstrate that the pure 20S proteasome is capable of generating antigenic peptides from two proteins for presentation by class I molecules without the participation of additional components of the protein degradation system. This finding is consistent with the hypothesis of proteasome involvement in Ag processing in vivo.
Dick
L R
LR
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9050.
Aldrich
C
C
Jameson
S C
SC
Moomaw
C R
CR
Pramanik
B C
BC
Doyle
C K
CK
DeMartino
G N
GN
Bevan
M J
MJ
Forman
J M
JM
Slaughter
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D010047:Ovalbumin / Q000276:immunology / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology*; D046988:Proteasome Endopeptidase Complex; D001616:beta-Galactosidase / Q000276:immunology / Q000378:metabolism*
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D010047:Ovalbumin; D001616:beta-Galactosidase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex