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Efficient generation of a hepatitis B virus cytotoxic T lymphocyte epitope requires the structural features of immunoproteasomes.

Sijts AJ, Ruppert T, Rehermann B, Schmidt M, Koszinowski U, Kloetzel PM - J. Exp. Med. (2000)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Charité, Humboldt University Berlin, 10117 Berlin, Germany.

ABSTRACT
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.

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Incorporation of the LMP2 and LMP7 subunits in 20S proteasomes of T2 transfectant lines. 500 ng (A) or 150 ng (B) of purified 20S proteasomes was separated by SDS-PAGE and probed with mouse LMP2–specific (A) or mouse LMP7–specific (B) polyclonal rabbit antisera. Molecular mass markers (kD) are shown at left.
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Figure 5: Incorporation of the LMP2 and LMP7 subunits in 20S proteasomes of T2 transfectant lines. 500 ng (A) or 150 ng (B) of purified 20S proteasomes was separated by SDS-PAGE and probed with mouse LMP2–specific (A) or mouse LMP7–specific (B) polyclonal rabbit antisera. Molecular mass markers (kD) are shown at left.

Mentions: To study the contribution of individual catalytic sites to antigenic peptide generation, we took advantage of a panel of T2 transfectant cells that had been generated in our laboratory. T2 cells lack the endogenous expression of LMP2 and LMP7, whereas MECL-1 is expressed but not incorporated into the 20S proteasome due to the absence of LMP2 37. Proteasomes were isolated from parental T2 cells and from T2 cells transfected with mouse-derived cDNAs encoding LMP2, a mutated, catalytically inactive form of LMP2 (LMP2 T1A), mouse LMP7, LMP2+7, or LMP2 plus catalytically inactive LMP7 T1A. Incorporation of the introduced LMPs was verified by immunoblot analyses using specific rabbit antisera (Fig. 5). As shown in Fig. 5 A, LMP2 was detected as a 21-kD product in proteasome preparations from LMP2, LMP2+7, and LMP2+7 T1A transfectant cells and as a product with slower migration properties in proteasomes from LMP2 T1A transfectant cells. The lower electrophoretic mobility of the T1A mutant forms of LMP2 and LMP7 is due to incomplete processing of the NH2-terminal prosequence, which is an autocatalytic process 33. Likewise, LMP7 (Fig. 5 B) was detected as a 23-kD protein in purified proteasomes from LMP7 and LMP2+7 transfectant cells and as a 26-kD product in proteasomes from LMP2+7 T1A transfectant cells. The exact ratios between constitutive and introduced β subunits in the used T2 proteasome populations have been published recently 34 or have been submitted for publication (Schmidt, M., and P.-M. Kloetzel, manuscript in preparation) and are of minor importance for the interpretation of our results. In summary, all transfected subunits replaced their homologous constitutive subunits with >50% efficiency, with the exception of LMP7 T1A. The incorporation of this subunit could not be quantified due to its unknown migrational behavior in two-dimensional gels but was less efficient than that of LMP7 as judged from immunoblots (Fig. 5 and data not shown). Consistent with our earlier observations 3439, we found that the association of both LMP2 and LMP2 T1A with the proteasome complex induced the simultaneous incorporation of MECL-1 with equal efficiency. Thus, taken together, the isolated set of proteasomes contained the immunosubunits LMP2 or LMP2 T1A, each in combination with MECL-1 and with or without LMP7 or LMP7 T1A.


Efficient generation of a hepatitis B virus cytotoxic T lymphocyte epitope requires the structural features of immunoproteasomes.

Sijts AJ, Ruppert T, Rehermann B, Schmidt M, Koszinowski U, Kloetzel PM - J. Exp. Med. (2000)

Incorporation of the LMP2 and LMP7 subunits in 20S proteasomes of T2 transfectant lines. 500 ng (A) or 150 ng (B) of purified 20S proteasomes was separated by SDS-PAGE and probed with mouse LMP2–specific (A) or mouse LMP7–specific (B) polyclonal rabbit antisera. Molecular mass markers (kD) are shown at left.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195811&req=5

Figure 5: Incorporation of the LMP2 and LMP7 subunits in 20S proteasomes of T2 transfectant lines. 500 ng (A) or 150 ng (B) of purified 20S proteasomes was separated by SDS-PAGE and probed with mouse LMP2–specific (A) or mouse LMP7–specific (B) polyclonal rabbit antisera. Molecular mass markers (kD) are shown at left.
Mentions: To study the contribution of individual catalytic sites to antigenic peptide generation, we took advantage of a panel of T2 transfectant cells that had been generated in our laboratory. T2 cells lack the endogenous expression of LMP2 and LMP7, whereas MECL-1 is expressed but not incorporated into the 20S proteasome due to the absence of LMP2 37. Proteasomes were isolated from parental T2 cells and from T2 cells transfected with mouse-derived cDNAs encoding LMP2, a mutated, catalytically inactive form of LMP2 (LMP2 T1A), mouse LMP7, LMP2+7, or LMP2 plus catalytically inactive LMP7 T1A. Incorporation of the introduced LMPs was verified by immunoblot analyses using specific rabbit antisera (Fig. 5). As shown in Fig. 5 A, LMP2 was detected as a 21-kD product in proteasome preparations from LMP2, LMP2+7, and LMP2+7 T1A transfectant cells and as a product with slower migration properties in proteasomes from LMP2 T1A transfectant cells. The lower electrophoretic mobility of the T1A mutant forms of LMP2 and LMP7 is due to incomplete processing of the NH2-terminal prosequence, which is an autocatalytic process 33. Likewise, LMP7 (Fig. 5 B) was detected as a 23-kD protein in purified proteasomes from LMP7 and LMP2+7 transfectant cells and as a 26-kD product in proteasomes from LMP2+7 T1A transfectant cells. The exact ratios between constitutive and introduced β subunits in the used T2 proteasome populations have been published recently 34 or have been submitted for publication (Schmidt, M., and P.-M. Kloetzel, manuscript in preparation) and are of minor importance for the interpretation of our results. In summary, all transfected subunits replaced their homologous constitutive subunits with >50% efficiency, with the exception of LMP7 T1A. The incorporation of this subunit could not be quantified due to its unknown migrational behavior in two-dimensional gels but was less efficient than that of LMP7 as judged from immunoblots (Fig. 5 and data not shown). Consistent with our earlier observations 3439, we found that the association of both LMP2 and LMP2 T1A with the proteasome complex induced the simultaneous incorporation of MECL-1 with equal efficiency. Thus, taken together, the isolated set of proteasomes contained the immunosubunits LMP2 or LMP2 T1A, each in combination with MECL-1 and with or without LMP7 or LMP7 T1A.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Charité, Humboldt University Berlin, 10117 Berlin, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus