Three immunoproteasome-associated subunits cooperatively generate a cytotoxic T-lymphocyte epitope of Epstein-Barr virus LMP2A by overcoming specific structures resistant to epitope liberation.
The precise roles of gamma interferon-inducible immunoproteasome-associated molecules in generation of cytotoxic T-lymphocyte (CTL) epitopes have yet to be fully elucidated. We describe here a unique epitope derived from the Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) presented by HLA-A*2402 molecules. Generation of the epitope, designated LMP2A(222-230), from the full-length protein requires the immunoproteasome subunit low-molecular-weight protein 7 (ip-LMP7) and the proteasome activator 28-alpha subunit and is accelerated by ip-LMP2, as revealed by gene expression experiments using an LMP2A(222-230)-specific CTL clone as a responder in enzyme-linked immunospot assays. The unequivocal involvement of all three components was confirmed by RNA interference gene silencing. Interestingly, the LMP2A(222-230) epitope could be efficiently generated from incomplete EBV-LMP2A fragments that were produced by puromycin treatment or gene-engineered shortened EBV-LMP2A lacking some of its hydrophobic domains. In addition, epitope generation was increased by a single amino acid substitution from leucine to alanine immediately flanking the C terminus, this being predicted by a web-accessible program to increase the cleavage strength. Taken together, the data indicate that the generation of LMP2A(222-230) is influenced not only by extrinsic factors such as immunoproteasomes but also by intrinsic factors such as the length of the EBV-LMP2A protein and proteasomal cleavage strength at specific positions in the source antigen.
Ito
Yoshinori
Y
Division of Immunology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan.
Kondo
Eisei
E
Demachi-Okamura
Ayako
A
Akatsuka
Yoshiki
Y
Tsujimura
Kunio
K
Tanimoto
Mitsune
M
Morishima
Yasuo
Y
Takahashi
Toshitada
T
Kuzushima
Kiyotaka
K
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D017951:Antigen Presentation; D003546:Cysteine Endopeptidases / Q000276:immunology / Q000378:metabolism; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D020031:Epstein-Barr Virus Infections / Q000276:immunology*; D015234:HLA-A Antigens; D004854:Herpesvirus 4, Human / Q000276:immunology*; D007930:Leucine / Q000235:genetics; D009097:Multienzyme Complexes / Q000276:immunology / Q000378:metabolism; D009124:Muscle Proteins / Q000276:immunology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000276:immunology* / Q000378:metabolism; D037182:T-Cell Antigen Receptor Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000276:immunology*
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D018984:Epitopes, T-Lymphocyte; D015234:HLA-A Antigens; D009097:Multienzyme Complexes; D009124:Muscle Proteins; C085047:PSME1 protein, human; D014763:Viral Matrix Proteins; C071313:LMP-2 protein; D003546:Cysteine Endopeptidases; C077436:LMP7 protein; D046988:Proteasome Endopeptidase Complex; D007930:Leucine; D000409:Alanine