Presentation of endogenous and exogenous antigens is not affected by inactivation of E1 ubiquitin-activating enzyme in temperature-sensitive cell lines.
Little is known regarding the mechanism by which MHC class I-associated peptides are generated. Proteins can be targeted for degradation by the covalent attachment of ubiquitin. The first step in ubiquitin conjugation to proteins is its binding to E1 ubiquitin-activating enzyme. To study the role of ubiquitin-targeted protein degradation in Ag processing, we used two mutant cell lines with temperature-sensitive E1 proteins, and a recombinant vaccinia virus expressing wild-type human E1. One of the cell lines examined (hamster ts20 cells) was previously reported to have a minimal capacity after a 1-h incubation at 41 degrees C to present osmotically loaded OVA to a T cell hybridoma, as assessed by IL-2 release. Even after incubating the same cells for 1 h at 43 degrees C, we failed to detect an E1-related decrease in the presentation of biosynthesized or osmotically loaded OVA to splenic T cells, as measured by target cell lysis. We introduce the use of mouse tsA1S9 cells to Ag-processing studies and provide the initial biochemical characterization of their defect in protein ubiquitination. Relative to parental L929 cells, after thermal inactivation of E1, these cells actually demonstrate enhanced presentation of endogenous or exogenous viral Ags to T cells. Our findings do not support a role for protein ubiquitination in Ag processing, and indicate that either the temperature-sensitive cell lines examined do not exhibit a sufficient reduction in ubiquitin-conjugating activity to affect the generation of antigenic peptides, or that ubiquitin-targeted proteolysis is not essential for processing the two exogenous and six endogenous Ags examined.
Cox
J H
JH
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.
Galardy
P
P
Bennink
J R
JR
Yewdell
J W
JW
D000818:Animals; D017951:Antigen Presentation / Q000502:physiology*; D000956:Antigens, Viral / Q000276:immunology; D015153:Blotting, Western; D002460:Cell Line; D003601:Cytotoxicity Tests, Immunologic; D006358:Hot Temperature; D008025:Ligases / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D010047:Ovalbumin / Q000276:immunology; D014162:Transfection / Q000235:genetics; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases
D000956:Antigens, Viral; D010047:Ovalbumin; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes