Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Podsumowanie

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

Streszczenie

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of naïve CD8⁺ T cells and memory CD8⁺ T cells for infectious and tumor immunity but also in the inactivation of self-acting naïve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

Wprowadzenie

The MHC I molecules are expressed on the surface of all nucleated cells, with short antigenic peptides derived from endogenous antigens, which are processed by the ubiquitin-proteasome system in the cytosol¹. After processing, antigenic peptides are transported into the endoplasmic reticulum (ER) lumen by the peptide transporter TAP. In the ER lumen, a series of specific chaperones assist the peptide loading and the correct folding of the MHC I complex. This series of molecules is called the peptide-loading complex (PLC), indicating that the ER is a central compartment for peptide loading upon MHC I². After peptide loading, the MHC I molecules are transported to the cell surface and play a key role in the adaptive immune system as self-markers, and enables the CD8⁺ cytotoxic T lymphocytes (CTLs) to detect cancer cells or infectious agents by antigenic peptides from non-self proteins³.

In antigen presenting cells (APC), antigenic peptides from exogenous antigens are also presented upon MHC I^4,5,6,7,8 via CP, which is mainly carried out by DCs^9,10,11. CP is essential both for the activation of naïve CD8⁺ T cells and memory CD8⁺ T cells into anti-infectious and anti-tumoral CTLs^12,13, and in the maintenance of immune tolerance by the inactivating of self-acting naïve T cells^14,15. The CP plays many important roles in the adaptive immune system, however the molecular mechanisms of CP have yet to be described in detail. Previous studies of CP revealed that exogenous antigens were localized both in the ER and the endosome and were processed by ERAD, suggesting that exogenous antigens are transported from the endosome to the ER for ERAD-like processing and peptide loading¹⁶. However, accumulating evidence indicates that the peptide loading of CP is carried out not in the ER but rather in non-classical endocytic compartments, which also have distinctive features of the ER (Figure 1)^{17,18,19,20,21}. To avoid degradation of the antigenic peptide precursors by the high activity of aminopeptidase²² in the cytosol, processing and peptide loading in CP occurs in the proximal area of these non-classical endocytic compartments (Figure 1). Though the characterizations of these endocytic compartments are controversial, there are no existing specific molecules other than exogenous antigens in this compartment.

ERAD is a cellular pathway, which specifically removes misfolded proteins from the ER. In the ERAD pathway, misfolded proteins are retrogradely transported through the ER membrane to the cytoplasm and processed by the ubiquitin-proteasome system^23,24,25. When large molecules, such as proteins, are transported through the lipid bilayer, these molecules pass through a molecular apparatus called a translocon, such as the Sec61 complex and Derlin complex in the ER²⁶, and the Tom complex and Tim complex in the mitochondria²⁷. When exogenously-added antigens are transported through the ER membrane, they must penetrate the lipid bilayer in complex with translocons, such as the Sec61 complex. The method described here purified the targeted vesicle by utilizing these membrane-penetrating molecules as markers for the endocytic compartments.

The method described here is a new vesicle purification protocol using the DC-like cell line DC2.4²⁸ and biotinylated ovalbumin (bOVA) as an exogenous antigen. The endocytic compartments were purified by streptavidin (SA)-magnetic beads using the membrane-penetrating bOVA as a maker. In this purified microsome, some exogenously added bOVA was still preserved in membrane fractions but were transported to the outside of microsome, and then ubiquitinated and processed in vitro²⁹. This purified microsome contained not only endocytic compartment-specific proteins but also ER-resident proteins for ERAD and the peptide loading complex; suggesting that the cellular compartment is the prospective endocytic compartment for CP²⁹. This protocol is not dependent on the kind of exogenous antigens, and is also applicable for other DC subsets and other cell types, such as macrophages, B cells, and endothelial cells, to clarify the precise molecular mechanism of DCs for proficient CP.

Protokół

1. Growing Cells and Addition of Exogenous Antigens

1. Prepare bOVA using a biotin-protein labeling kit following the manufacturer's protocol.
   NOTE: Ordinarily, bOVA contains 2 M biotin per 1 M OVA on average.
2. Grow DC2.4 cells in RPMI-1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/mL penicillin-streptomycin, 55 mM 2-mercaptoethanol, 10 mM HEPES (pH 7.5), and 10% fetal calf serum (hereafter RPMI) at 37 °C in 5% CO₂ in a humidified incubator (hereafter without mention, cells are incubated at this condition). It is also possible to use DMEM supplemented with 10% fetal calf serum, 3.7 g/L NaHCO₃ (hereafter DMEM) in place of RPMI.
3. A day before purification, split the cells into RPMI to 1 x 10⁵ cells/mL in a tissue culture plate. Avoid keeping the cells at a confluent state. DC2.4 cells can highly incorporate exogenous antigens until a semi-confluent state, but rapidly lose the ability after reaching an over-confluent state.
4. Before the DC2.4 cells are semi-confluent, add 250 µg/mL of bOVA for 1 x 10⁶ cells/mL and incubate for 2 - 4 h.
   NOTE: During downstream experiments, all buffers and reagents are kept at 4 °C unless otherwise indicated.

2. Preparation of Microsomes

1. Harvest the DC2.4 cells by gentle pipetting from the tissue plate into a new 50 mL conical tube.
2. Centrifuge at 1,000 x g for 5 min, 4 °C and carefully remove the medium with bOVA by aspiration.
3. Wash the DC2.4 cells twice with 40 mL of PBS buffer (1.37 mM NaCl, 8.1 mM Na₂HPO₄, 2.68 mM KCl, 1.47 mM KH₂PO₄) by centrifugation at 1,000 x g for 5 min, 4 °C and discard the supernatant each time by aspiration.
4. Resuspend the pellet in step 2.3 in 1 - 2 mL (1/20-1/10 volume of culture medium) of homogenization medium (0.25 M sucrose, 1 mM EDTA, 10 mM HEPES-NaOH [pH 7.4]) with 1/1,000 volume of protease inhibitor cocktails and transfer the resuspended DC2.4 cells into an ice-cold Dounce homogenizer.
5. Disrupt the resuspended DC2.4 cells gently by 10 - 20 strokes with the ice-cold Dounce homogenizer.
   NOTE: During the disruption step, the Dounce homogenizer is cooled on ice.
6. Transfer the cell-disrupted suspension to a new 15 mL conical tube.
7. Add 8 - 9 mL homogenization medium to 10 mL total and centrifuge the conical tube for 10 min at 2,000 x g and 4 °C.
8. Transfer the supernatant to a new 15-mL conical tube to remove unbroken cells and nuclei, and centrifuge the conical tube again for 10 min at 2,000 x g and 4 °C.
9. Transfer the supernatant into a new ultracentrifugation tube and centrifuge for 45 min at 100,000 x g and 4 °C.
10. Aspirate the supernatant and resuspend the pellet carefully in 1 - 2 mL of homogenization medium with 1/1,000 volume of protease inhibitor cocktails by pipetting the solution up and down several times to make a microsome fraction for downstream experiments.
11. Transfer the microsome fraction into a new 5-mL round bottom tube.
    NOTE: After this step, it is possible to enrich objective microsomes by iodixanol density gradient centrifugation according to the manufacturer's protocol, to remove non-specific microsomes. The peak fractions for exogenous antigens are collected and then subjected to the next step of purification (step 3).

3. Purification of Microsomes with bOVA Undergoing ERAD

1. Add 1/100 volume of fresh SA-magnetic beads to the microsome fraction of step 2.11 in the 5 mL round bottom tube.
   NOTE: Before this step, it is possible to pre-clear the microsome fraction by control-magnetic beads to reduce contaminations of non-specific microsomes.
2. Gently mix well and rotate the round bottom tube slowly for 30 min at 4 °C.
3. Add 2-3 mL of homogenization medium to 4 mL total into the round bottom tube and gently mix well.
4. Place the round bottom tube on a magnetic stand and incubate for 10 min at 4 °C. Since the beads bound to the vesicles are attracted to the magnet, brown micro-beads will gradually accumulate to the tube wall closest to the magnet.
5. With the tube remaining in the magnetic stand, carefully discard the supernatants by aspiration to remove the unbound vesicles.
6. Wash the magnetic beads bound to the vesicles twice with 5 mL of homogenization medium by the magnetic stand for 10 min at 4 °C and discard the supernatant each time by carefully aspiration.
   NOTE: Without the magnetic stand, purification by centrifugations at 2,000 x g for 10 min, 4 °C is also available.
7. Resuspend the magnetic beads bound to the vesicles carefully in 100 µL homogenization medium by pipetting the solution up and down several times.
8. Transfer the resuspended vesicles into a new microtube as the purified microsome for downstream experiments.
   NOTE: Typically, 50 µL microsome fraction containing 5 - 20 µg proteins from 1 x 10⁷ cells can be isolated.

4. Analysis of the Purified Microsomes

1. Resuspend the magnetic beads bound to the vesicles from step 3.8 by 100 - 200 µL of TNE buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5 M EDTA, 1% Nonidet P-40) with 1/1,000 volume of protease inhibitor cocktails instead of the homogenization medium.
2. Transfer the lysate from step 4.1 to a new 1.5-mL microtube.
3. Determine the protein concentration of step 4.2 by using a BCA assay kit per the manufacturer's protocol.
   NOTE: Typically, 5 - 10 µL lysate from step 4.2 is enough to determine the protein concentration.
4. Transfer the lysate from step 4.2 (2 µg of protein for silver staining and 10 µg of protein for Western blotting) into new microtubes.
5. Put the microtubes on a heat block at 95 °C and boil proteins in 1x SDS gel-loading buffer (100 mM Tris-HCl pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) for 5 min.
6. Centrifuge the microtubes for 10 min at 21,500 x g and 4 °C. Collect the supernatants into new microtubes to remove the insoluble fractions.
7. Analyze the resolved proteins in step 4.6 (2 µg) by standard SD-PAGE.
8. Visualize the protein bands by the silver staining using silver staining kit following the manufacturer's protocol.
9. Analyze the resolved proteins in step 4.6 (10 µg) by standard SD-PAGE and Western-blotting. Use the reagent (SA-HRP) per the manufacturer's protocol and visualize by fluorography.

5. In Vitro Reconstitution of ERAD Ubiquitination of bOVA Using Purified Microsomes

1. Transfer the purified microsomes (5 - 10 µg of protein) from step 3.8 with a 50% volume of reticulocyte lysate (RL), in 1x reaction buffer (50 mM Tris pH 7.4, 3 mM ATP, 0.5 mM MgCl₂), and 0.2 pM Flag-tagged ubiquitin in a new microtube. The final volume of this experiment is 20 - 40 µL.
2. Incubate the microtube for 2 h at 37 °C.
   NOTE: If the amount of ubiquitinated bOVA in step 5.12 is too small to detect by Western blotting, add 10 µM of MG132 or 2 µM of lactacystin to inhibit the processing of the ubiquitinated bOVA by proteasomes.
3. Stop the reaction by placing the microtube on ice.
4. Resolve the microsomes by adding 500 µL TNE buffer with 1/1,000 volume of protease inhibitor cocktails.
5. Centrifuge the microtube for 10 min at 21,500 x g and 4 °C. Collect the supernatants into a new microtube to remove the insoluble fractions, which contain ubiquitinated bOVA in DC2.4 before the in vitro ubiquitination assay.
6. Transfer the supernatant to a new microtube and add 1/100 volume of new SA-magnetic beads (usually 5 µL for 500 µL of supernatants).
7. Gently mix well and rotate the microtube slowly for 30 min at 4 °C.
8. Centrifuge the microtube for 10 min at 21,500 x g and 4 °C. Discard the supernatant by aspiration to recover the bOVA and ubiquitinated bOVA bound with the SA-magnetic beads.
9. Wash twice the collected SA-magnetic beads with 1 mL of TNE buffer by centrifugation for 10 min at 21,500 x g and 4 °C. Discard the supernatant each time by aspiration.
10. Boil the SA-magnetic beads in 1x SDS gel loading buffer for 5 min at 95 °C on a heat-block to resolve the purified proteins by the SA-magnetic beads.
11. Centrifuge the microtube for 10 min at 21,500 x g and 4 °C. Collect the supernatants into a new microtube to remove the insoluble fractions.
12. Analyze the SA-magnetic beads bound to the proteins by standard SD-PAGE and Western-blotting. The use of antibodies (anti-Flag, anti-multi-Ub, and anti-mouse IgG-HRP) and the reagent (SA-HRP) is per the manufacturer's protocol and visualized by fluorography.

Wyniki

To elucidate the molecular mechanism of CP, it is necessary to identify the cellular compartments, where exogenous antigens undergo ERAD-like transport and processing. While observations by immunofluorescent microscopy or by electron microscopy identified the cellular compartment where exogenous antigens accumulated^16,17,18,19,...

Dyskusje

In previous studies of CP, the incorporated exogenous antigens accumulated in the restricted area of the late endosome or ER by immunofluorescent microscopy^16,30,31,32. It is estimated that ERAD-like transport and processing of exogenous antigens are carried out in these specialized areas of the ER or late endosome, as the cellular compartment was identified by sucrose or iodixanol density grad...

Materiały

Name	Company	Catalog Number	Comments
RPMI 1640	gibco by life technologies	11875-093
Fetal bovine serum	Equitech bio	SFB30
Sodium pyruvate	gibco by life technologies	11360-070
MEM non-essential amino acids	gibco by life technologies	11140-050
HEPES	gibco by life technologies	15630-080
2-mercaptoethanol	gibco by life technologies	21985-023
L-glutamine	gibco by life technologies	25030-164
Penisicillin-Sreptomycin	gibco by life technologies	15140-122
DMEM	gibco by life technologies	12100-46
OVA	SIGMA	A5503
Biotin-protein labelling kit	Thermo Fisher Scientific	F6347
MG-132	Santa Cruz Biotechnology	201270
lactacystin	SIGMA	L6785
Dounce homogenizer	IUCHI	131703
protease inhibitor cocktails	SIGMA	P8340
iodixanol	Cosmo bio	1114542
SA-magnetic beads	New England Biolabs	201270
control magnetic beads	Chemagen	M-PVA012
magnetic stand	BD Biosciences	552311
BCA protein assay kit	Thermo Fisher Scientific	23225
silver staining kits	Cosmo bio	423413
Reticulocyte Lysate	Promega	1730714
Flag-tagged ubiquitin	SIGMA	U5382
anti-ovalbumin (OVA,mouse)	Antibody Shop	HYB 094-06
ant-multi-ubiquitin (mouse)	MBL	D058−3
anti-Flag (mouse)	SIGMA	F3165
trypsin	SIGMA	85450C