Efficient generation of a hepatitis B virus cytotoxic T lymphocyte epitope requires the structural features of immunoproteasomes.
Interferon (IFN)-gamma-induced cells express the proteasome subunits low molecular weight protein (LMP)2, LMP7, and MECL-1 (multicatalytic endopeptidase complex-like 1), leading to the formation of immunoproteasomes. Although these subunits are thought to optimize MHC class I antigen processing, the extent of their role and the mechanistic aspects involved remain unclear. Herein, we study the proteolytic generation of an human histocompatibility leukocyte antigen (HLA)-Aw68-restricted hepatitis B virus core antigen (HBcAg) cytotoxic T lymphocyte (CTL) epitope that is recognized by peripheral blood lymphocytes from patients with acute self-limited but not chronic hepatitis B virus (HBV). Immunological data suggest that IFN-gamma-induced rather than uninduced HeLa cells process and present the HBV CTL epitope upon infection with HBcAg-expressing vaccinia viruses. Analyses of 20S proteasome digests of synthetic polypeptides covering the antigenic HBcAg peptide demonstrate that only immunoproteasomes efficiently perform the cleavages needed for the liberation of this HBV CTL epitope. Although the concerted presence of the three immunosubunits appears essential, we find that both catalytically active LMP7 and inactive LMP7 T1A support CTL epitope generation. We conclude that LMP7 influences the structural features of 20S proteasomes, thereby enhancing the activity of the LMP2 and MECL-1 catalytic sites, which provide cleavage specificity. Thus, LMP7 incorporation is of greater functional importance for the generation of an HBV CTL epitope than cleavage specificity.
Sijts
A J
AJ
Institute of Biochemistry, Charité, Humboldt University Berlin, 10117 Berlin, Germany.
Ruppert
T
T
Rehermann
B
B
Schmidt
M
M
Koszinowski
U
U
Kloetzel
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000737:chemistry; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000276:immunology*; D006367:HeLa Cells; D006509:Hepatitis B / Q000276:immunology; D006512:Hepatitis B Core Antigens / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007700:Kinetics; D008285:Major Histocompatibility Complex; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex; D011506:Proteins; D013329:Structure-Activity Relationship; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014162:Transfection; D014763:Viral Matrix Proteins
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D018984:Epitopes, T-Lymphocyte; D006512:Hepatitis B Core Antigens; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011506:Proteins; D014763:Viral Matrix Proteins; D007371:Interferon-gamma; D003546:Cysteine Endopeptidases; C077436:LMP7 protein; D046988:Proteasome Endopeptidase Complex