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. 2016 Sep 8;2(9):e00151.
doi: 10.1016/j.heliyon.2016.e00151. eCollection 2016 Sep.

Purification of the subcellular compartment in which exogenous antigens undergo endoplasmic reticulum-associated degradation from dendritic cells

Jun Imai  1 , Mayu Otani  1 , Takahiro Sakai  1 , Shinichi Hatta  1
Affiliations

Purification of the subcellular compartment in which exogenous antigens undergo endoplasmic reticulum-associated degradation from dendritic cells

Jun Imai et al. Heliyon. .

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.

Keywords: Cell biology; Immunology.

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Figures

Fig. 1
Fig. 1
In vitro reconstitution of retrotransport and degradation using OVA in microsomes (A), Microsomes with (+) or without (−) prior addition of bOVA were treated with (+) or without (−) trypsin and Triton X–100 (TX100). Proteins (2 μg) were resolved on 7.5–15% SDS-PAGE and subjected to western blotting with the indicated antibodies. (B), Microsomes with (+) or without (−) prior addition of bOVA were treated with (+) or without (−) RL and ATP for 1 h. Proteins (2 μg) were subjected to western blotting with SA-HRP. Asterisks in the right indicate non-specific bands with SA-HRP. Equivalent results were attained at least three independent assays. (C), Quantification of the results shown in (B). Bars, mean ± S.D. (error bars) of three independents experiments. Student's test was used to compare (bOVA + RL − ATP −) sample with (bOVA + RL + ATP −) sample, (bOVA + RL − ATP +) sample, and (bOVA + RL + ATP +). **, ρ < 0.01, *,ρ < 0.1, NS, not significant, n = 3. (D), Microsomes with prior addition of bOVA were treated with RL, ATP, and the indicated inhibitors: MG132 (MG: 10 μM), lactacystine (LC: 2 μM), and chloroquine (CQ: 10 μM). Microsomes without prior addition of bOVA were incubated with 2.5 μg of bOVA after fractionation (after) and were treated with RL and ATP. Proteins (2 μg) were subjected to western blotting with SA-HRP. (E), Quantification of the results shown in (D). Bars, mean ± S.D. (error bars) of three independents experiments. Student's test was used to compare (Before, RL + reagent −) sample with (Before RL + reagent +) samples and (After, RL − reagent −) sample with (After, RL + reagent −) sample. *, ρ < 0.01, NS, not significant, n = 3. (F), Microsomes with (+) or without (−) prior addition of bOVA were treated with (+) or without (−) RL in presence of ATP and MG132 (10 μM) for 1 h. Samples were then separated into supernatants and pellets by centrifugation at 100,000 × g for 45 min. Proteins (2 μg) were subjected to western blotting with SA-HRP. Asterisks in the right indicate non-specific bands with SA-HRP. To show the band of bOVA in lane 4, exposure time of this figure is longer than Fig. 1B, results in enhancements of non-specific bands with SA-HRP. Equivalent results were attained at least three independent assays. (G), Microsomes with (bOVA before +) or without (bOVA before −) prior addition of bOVA were treated with RL and ATP in presence or absence of and MG132 (10 μM) for 1 h. Samples without prior addition of bOVA (bOVA before −) were added with 1.0 mg/mL bOVA (bOVA after +) or the same volume of PBS (bOVA after −). Supernatants were subjected to immunoprecipitation (IP) with anti-Hsp70. After SDS-PAGE, blotting was performed with SA-HRP and anti-HSP70. Equivalent results were attained at least three independent assays. Full versions of all sliced images are provided in the Supplementary Figures.
Fig. 2
Fig. 2
In vitro reconstitution of ubiquitination using OVA in microsomes (A), Microsomes with prior addition of bOVA were treated with (+) or without (−) RL and Flag-tagged ubiquitin (Flag-Ub) for 1 h and were then solubilized with TNE (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.5 M EDTA, 1% Nonidet P-40). bOVA was purified with SA-magnetic beads and subjected to western blotting with the indicated antibodies. Asterisks in the right indicate non-specific bands with SA-HRP. Equivalent results were attained at least three independent assays. (B), Microsomes with prior addition of bOVA were treated with (+) or without (−) RL, Flag-Ub, and MG132 for 1 h and solubilized using TNE. bOVA was purified with SA-magnetic beads and subjected to western blotting with SA-Flag. Equivalent results were attained at least three independent assays. Full versions of all sliced images are provided in the Supplementary Figures.
Fig. 3
Fig. 3
Purification of microsomes with bOVA undergoing ERAD (A), Schematic model of purification of microsomes with bOVA undergoing ERAD. bOVA is associated with the membrane through the Sec61 translocon and targeted with SA-magnetic beads. (B), Microsomes with (+) or without (−) prior addition of bOVA were purified with (+) or without (−) SA-magnetic beads. Proteins (2 μg) or corresponding volumes of purified proteins were resolved on 7.5–15% SDS-PAGE, and silver staining was used to visualize protein bands. Triangles on the right side indicate nonspecific proteins binding to the SA-magnetic beads. Triangles with asterisks indicate unique proteins found only in the presence of exogenously added bOVA and SA-magnetic beads. Arrow indicates bOVA. P.N., post nuclear fraction. Asterisks in the right indicate non-specific bands with SA-HRP. Equivalent results were attained at least three independent assays. (C), Western blotting results of the samples shown in (B) using SA-HRP. Equivalent results were attained at least three independent assays. (D), Microsomes with prior addition of bOVA were purified with (+) or without (−) SA-magnetic beads after addition of 2.5 mg/mL bOVA (+) or the same volume of PBS (−). Proteins (2 μg) or corresponding volumes of purified proteins were resolved on 7.5–15% SDS-PAGE, and silver staining was used to visualize protein bands. Equivalent results were attained at least three independent assays. (E), Western blotting results of the samples shown in (D) using SA-HRP. Equivalent results were attained at least three independent assays. (F), Microsomes with prior addition of bOVA were purified with SA-magnetic beads. Microsomes were treated with (+) or without (−) trypsin and TX-100 before purification (left two lanes) or after purification (right two lanes). Proteins (2 μg) or corresponding volumes of purified proteins were resolved on 7.5–15% SDS-PAGE, and silver staining was used to visualize protein bands. Triangles on the right side indicate nonspecific proteins binding to the SA-magnetic beads. Triangles with asterisks indicate unique proteins found only in the presence of exogenously added bOVA and SA-magnetic beads. Equivalent results were attained at least three independent assays. (G), Western blotting results of the samples shown in (F) using SA-HRP. Equivalent results were attained at least three independent assays. Full versions of all sliced images are provided in the Supplementary Figures.
Fig. 4
Fig. 4
In vitro reconstitution of degradation and ubiquitination using OVA in purified microsomes (A), Purified microsomes were treated with RL, ATP, and the indicated inhibitors. Proteins (2 μg) were subjected to western blotting with SA-HRP. Equivalent results were attained at least three independent assays. (B), Quantification of the results shown in (A). Student's test was used to compare (Before, RL + reagent −) sample with (RL + reagent +) samples. *, ρ < 0.01, NS, not significant, n = 3. (C), Purified microsomes with (+) or without (−) prior addition of bOVA were treated with (+) or without (−) RL and Flag-Ub for 1 h and were solubilized using TNE. bOVA was purified with SA-magnetic beads and subjected to western blotting with the indicated antibodies. Asterisks in the right indicate non-specific bands with SA-HRP. Equivalent results were attained at least three independent assays. (D), Purified microsomes were treated with (+) or without (−) RL, Flag-Ub, and MG132 for 1 h and were then solubilized using TNE. bOVA was purified with SA-magnetic beads and subjected to western blotting with SA-Flag. Asterisks in the right indicate non-specific bands with SA-HRP. Equivalent results were attained at least three independent assays. Full versions of all sliced images are provided in the Supplementary Figures.
Fig. 5
Fig. 5
Proteins in purified microsomes. (A–E), Proteins (2 μg) from purified microsomes with (+) or without (−) prior addition of bOVA were subjected to western blotting with the indicated antibodies. Equivalent results were attained at least three independent assays. (F), Proteins (10 μg) from purified microsomes without (−) prior addition of bOVA were resolved by two-dimensional DIGE. Silver staining was used to visualize protein spots. Triangles on the right side indicate nonspecific proteins binding to the SA-magnetic beads. Equivalent results were attained at least three independent assays. (G), Proteins (10 μg) from purified microsomes with (+) prior addition of bOVA were resolved by two-dimensional DIGE. Silver staining was used to visualize protein spots. Triangles on the right side indicate nonspecific proteins binding to the SA-magnetic beads. Triangles with asterisks indicate unique proteins found only in the presence of exogenously added bOVA and SA-magnetic beads. Equivalent results were attained at least three independent assays. (H), The silver stained image of purified microsomes with (+) prior addition of bOVA was pseudo-colored green (Fig. 5F), and that of control microsomes without (−) prior addition of bOVA was pseudo-colored red (Fig. 5G). Triangles on the right side indicate nonspecific proteins binding to the SA-magnetic beads. Triangles with asterisks indicate unique proteins found only in the presence of exogenously added bOVA and SA-magnetic beads. The two images were merged. A black arrow indicates BiP, and a dotted arrow indicates actin. Full versions of all sliced images are provided in the Supplementary Figures.
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