title: Purification of the subcellular compartment in which exogenous antigens undergo endoplasmic reticulum-associated degradation from dendritic cells.
abstract: Dendritic cells (DCs) are capable of processing and presenting exogenous antigens using MHC class I molecules. This pathway is called antigen cross-presentation and plays an important role in the stimulation of naïve CD8(+) T cells for infectious and tumor immunity. Our previous studies in DC2.4 cells and bone marrow-derived DCs revealed that exogenously added ovalbumin (OVA) is processed through endoplasmic reticulum (ER)-associated degradation (ERAD) for cross-presentation. In this study, we aimed to further confirm these results by purification of the subcellular compartment in which exogenous antigens undergo ERAD from homogenates of DC2.4 cells pretreated with biotinylated OVA (bOVA). bOVA-containing vesicles were purified using streptavidin (SA)-magnetic beads from cell homogenates and were found to contain ER chaperones and ERAD components together with proteins for antigen presentation. In purified microsomes, bOVA was retained in membranous fractions and degraded by the ubiquitin proteasome system in presence reticulocyte lysates and ATP. These results strongly suggested that DCs processed and degraded exogenous antigens through ERAD for cross-presentation in this purified subcellular compartment.
authors.affiliation: Laboratory of Physiological Chemistry, Faculty of Pharmacy, Tkasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Imai
authors.affiliation: Laboratory of Physiological Chemistry, Faculty of Pharmacy, Tkasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033, Japan.
authors.forename: Mayu
authors.initials: M
authors.lastname: Otani
authors.affiliation: Laboratory of Physiological Chemistry, Faculty of Pharmacy, Tkasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Sakai
authors.affiliation: Laboratory of Physiological Chemistry, Faculty of Pharmacy, Tkasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Hatta
keywords: Cell biology; Immunology