|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Hsp70 binds to PrPC in the process of PrPC release via exosomes from THP-1 monocytes
Gui‑hua Wang*†1, Xiang‑mei Zhou*1, Yu Bai*, Xiao‑min Yin*, Li‑feng Yang* and Deming Zhao*2
*State Key Laboratories for Agrobiotechnology, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, Peoples Republic of China, and †College of Animal Science and Veterinary Medicine, Shandong Agricultural Univerisity, Taian 271018, Peoples Republic of China
PrPC (cellular prion protein) is a GPI (glycophosphatidylinositol)-anchored protein present on the surface of a number of peripheral blood cells. PrPC must be present for the generation and propagation of pathogenic conformer [PrPSc (scrapie prion protein)], which is a conformational conversion form of PrPC and has a central role in transmissible spongiform encephalopathies. It is important to determine the transportation mechanism of normal PrPC between cells. Exosomes are membrane vesicles released into the extracellular space upon fusion of multivesicular endosomes with the plasma membrane. We have identified that THP-1 monocytes can secrete exosomes to culture medium, and the secreted exosomes can bear PrPC. We also found that Hsp70 interacts with PrPC not only in intracellular environment, but in the secreted exosomes. However, the specific markers of exosomes, Tsg101 and flotillin-1, were found with no interaction with PrPC. Our results demonstrated that PrPC can be released from THP-1 monocytes via secreted exosomes, and in this process, Hsp70 binds to PrPC, which suggests that Hsp70 may play a potential functional role in the release of PrPC.
Key words: exosome, flotillin-1, interaction, Hsp70, PrPC, Tsg101
Abbreviations: CNS, central nervous system, Co-IP, co-immunoprecipitation, EM, electron microscopy, FBS, fetal bovine serum, GPI, glycophosphatidylinositol, IEM, immunoelectron microscopy, PrPC, cellular prion protein, PrPSc, scrapie prion protein, RT, room temperature, siRNA, small interfering RNA, TBST, TBS/ 0.05% Tween 20
1Gui-hua Wang and Xiang-mei Zhou contributed equally to this work.
2To whom correspondence should be addressed (email firstname.lastname@example.org).
PrPC (cellular prion protein) is a GPI (glycophosphatidylinositol)-anchored protein found in a number of tissues throughout the body (Robertson et al., 2006). PrPC is the key protein for prion diseases, which are a family of neurodegenerative disorders that affect both humans and animals (Johnson, 2005; Prusiner, 1998). According to the widely accepted ‘prion-only’ hypothesis, the pathogenic conformer [PrPSc (scrapie prion protein)], which arises by a conformational conversion of PrPC has a central role in prion diseases (Prusiner, 1998). Replication of PrPSc depends critically on the presence of PrPC. The deposition of PrPSc in the CNS (central nervous system) of affected individuals is an important pathological feature. However, CNS is not the first site at which PrPSc is apparent. PrPSc must first replicate and be transported to the CNS after peripheral infection (Weissmann et al., 2002), but the mechanism of transferring from peripheral tissue to CNS is not elucidated. Some previous studies have demonstrated that the scrapie and vCJD can be transmitted by blood transfusions (Fischer et al., 2000; Houston et al., 2000). There is, therefore, a significant concern that blood transfusions may represent a portal for the transmission of prion diseases (Robertson et al., 2006). Thus, it is important to determine the transportation mechanism of normal PrPC in blood cells.
Exosomes are small membrane vesicles originating from late endosomes and are released in the extracellular space by a broad array of cells through the endocytic pathway (Andre et al., 2004; Kim et al., 2007). A large number of proteins and lipids are associated with exosomes, which include members of the tetraspan protein family, the immunoglobulin supergene family, GPI-anchored proteins and cytosolic proteins (Denzer et al., 2000). Exosomes may function as a refined intercellular exchange device allowing transfer of proteins between cells (Couzin, 2005).
Several investigators working with different cell types have reported the association of PrPC but also PrPSc with exosomes (Fevrier et al., 2004; Robertson et al., 2006). Indeed how PrP is targeted to exosomes is unclear. We showed that THP-1 monocytes, derived from human peripheral blood, could secrete exosomes, and PrPC was released via the secreted exosomes. Importantly, we found that Hsp70 has the interaction with PrPC not only in cell lysates but in the secreted exosomes. However, the interactions between Tsg101 and flotillin-1, the markers of exosomes, with PrPC were not detected.
2. Materials and methods
2.1. Cell culture and lysis preparation
FBS (fetal bovine serum) (Invitrogen) used for cell culture was ultracentrifuged at 100000 g for 1.5 h to remove exosomes present in FBS, according to the methods previously described (Wubbolts et al., 2003). THP-1 monocytes derived from human peripheral blood (ATCC, TIB-202TM) were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS and standard antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin), in 37°C, 5% CO2 in air incubator. The medium was replaced with fresh medium containing 10% FBS and standard antibiotics every 48–72 h.
After 48–72 h culture, the cells were pelleted and washed in PBS. The PBS-washed cell pellets were lysated with pre-chilled RIPA buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS) (Applygen) and proteinase inhibitor cocktail (Novasygen) on ice for 30 min. The cell lysates were collected and centrifuged at 10000 g at 4°C for 15 min to remove cell debris. The supernatants were immediately transferred to clean tubes and stored at −20°C.
2.2. Isolation and purification of exosomes
Exosomes secreted by THP-1 monocytes were isolated from medium by differential centrifugation and purified by sucrose gradient centrifugation as described previously (Rajendran et al., 2006; Bhatnagar and Schorey, 2007). Culture medium was harvested and centrifuged at 300 g for 10 min, 1000 g for 20 min and 10000 g for 30 min to eliminate cells and cell debris. Then, exosomes were pelleted at 100000 g for 1.5 h. The pelleted exosomes were resuspended in 2.5 M sucrose in 20 mM Hepes (pH 7.4) and floated onto a linear gradient [2.25, 2.0, 1.75, 1.5, 1.25, 1.0, 0.75, 0.5 and 0.25 M sucrose in 20 mM Hepes (pH 7.4)]. The sucrose gradient was ultracentrifuged at 100000 g for 15 h. Gradient fractions were collected from top to bottom of the tube, diluted with PBS and centrifuged at 100000 g for 1.5 h. The pellets were resuspended in PBS containing proteinase inhibitor cocktail, and suspensions were stored at −20°C for further analysis.
2.3. Western blot analysis
Protein quantifications of whole cell lysates and gradient fractions, purified from the same cell culture supernatants, were performed with the Bradford assay kit (Bio-Rad). Equivalents of cell lysates and exosomes were mixed with 2× SDS loading buffer (100 mM Tris/HCl, pH 6.8, 200 mM DTT (dithiothreitol), 4% SDS, 0.2% Bromophenol Blue, 20% glycerol) and boiled at 95°C for 5 min. Protein samples were separated by 12% SDS/PAGE gels and electrotransferred onto PVDF membranes (Millipore). The membranes were blocked with 5% (w/v) non-fat dry milk in TBST (TBS-0.05% Tween 20) at RT (room temperature) for 1 h. Primary antibodies as mouse anti-Tsg101 (1:500), rabbit anti-flotillin-1 (1:500), mouse anti-Hsp70 (1:500), rabbit anti-calnexin (1:500) (Santa Cruz Biotechnology) or anti-PrP antibody AH6 (1:2000) (Compton) were added to the membranes, respectively, and incubated at 37°C for 2 h. After washing three times briefly with TBST, binding of the primary antibodies was probed with horseradish peroxidise-conjugated goat anti-mouse or goat anti-rabbit IgG (both at 1:5000) (Santa Cruz Biotechnology) for 1 h incubation. After six washes, the membrane-bound proteins were visualized with DAB (3,3′-diaminobenzidine) (Roche).
2.4. Electron microscopy
The purified exosomes were applied to formvar–carbon-coated EM (electron microscopy) grids, negatively stained with 1% uranyl acetate and directly observed by EM for characteristic morphology.
Samples were deposited to EM grids for 10 min. The grids were fixed with 2% paraformaldehyde in PBS at RT for 15 min and blocked with 1% BSA in PBS for 20 min. After blocking, the grids were incubated with primary antibodies: mouse anti-Tsg101 (1:100), rabbit anti-flotillin-1 (1:100) and mouse anti-PrP (1:500) at RT for 1.5 h. The unbound antibodies were removed by three washes with 0.1% BSA in PBS and then labelled with 15 nm goat anti-mouse and goat anti-rabbit IgG-gold particles (1:50) (ZSGB). After extensive washes with PBS, the grids were stained with 1% uranyl acetate at RT for 3 min and observed under transmission electron microscope.
One microgram of AH6 was mixed with 500 μl of cell lysates and exosomes separately and incubated with agitation at 4°C for 2 h. For non-specific IP (immunoprecipitation), non-immune mouse IgG was used as negative control. Subsequently, 15 μl of proteins A/G plus-agarose beads (Santa Cruz Biotechnology) were added to the mixture and incubated at 4°C for 2 h. Beads were pelleted by centrifugation at 1000 g at 4°C for 5 min, and supernatant was carefully aspirated and discarded. The pellets were washed three times with 1.0 ml of ice-cold RIPA buffer. After the final wash, pelleted beads were resuspended in 20 μl of 2× SDS loading buffer and boiled at 95°C for 5 min to dissociate the immunocomplex from the beads. Protein samples were centrifuged at 10000 g for 2 min to pellet the agarose beads and subjected to Western blot as described above, using antibodies of Tsg101, flotillin-1 and Hsp70.
Reverse co-IP was performed by using the antibodies against Tsg101, flotillin-1 and Hsp70 for IP and AH6 to develop the Western blot.
3.1. THP-1 monocytes secrete exosomes
To investigate whether THP-1 monocytes secrete exosomes, differential centrifugation and sucrose gradient centrifugation (Rajendran et al., 2006; Zhang et al., 2006; Bhatnagar and Schorey, 2007) were carried out to purify exosomes form THP-1 monocytes culture medium. The density gradient fractions were then submitted to Western blot. We analysed the distribution of Tsg101 and flotillin-1, typical markers of exosomes to identify exosome-containing gradient fractions. The bands of Tsg101 of 43 kDa and flotillin-1 of 45 kDa were found in fourth to fifth fractions (Figure 1A), with corresponding densities of 1.13 and 1.16 g/ml. The fractions containing Tsg101 and flotillin-1 were adsorbed on formvar–carbon-coated grids and contrasted in uranyl acetate for direct EM observation. The vesicles were present in culture medium with average diameter of 30–100 nm and cup-shaped morphology (Figure 1B). The density, shape and size of these vesicles are similar to the previous reports for exosomes released from other cells (Raposo et al., 1996; Caby et al., 2005). These results demonstrate that the culture medium from THP-1 monocytes contain exosomes.
We further characterized the exosome-containing gradient fractions by immunoelectron microscopy and Western blot. Tsg101 and flotillin-1 on exosomes were labelled by 15 nm gold particles (Figures 2A, 2B), which confirmed the presence of exosome-specific markers again. To rule out the possibility of organelle release as a consequence of cell lysis, the samples were tested for the presence of calnexin, a luminal marker of the endoplasmic reticulum. Calnexin was detected in the cell lysates but absent in exosomes. Compared with cell lysates, both Tsg101 and flotillin-1 were found to be enriched in exosomes. Molecular chaperones, Hsp70, were also contained in the component of exosomes, but were not as enriched relative to cell lysates as Tsg101 and flotillin-1 (Figure 2C). The data further confirm that THP-1 monocytes release exosomes, and the purified exosomes are not contaminated by organelles.
3.2. THP-1 monocytes release PrPC in association with exosomes
Cell lysates and the exosome-containing gradient fractions by Western blot using anti-PrP antibody AH6 for the presence of PrPC were initially analysed. The bands between 20 and 37 kDa detected in cell lysates suggested that THP-1 monocytes express PrPC (Figure 3A, lane 1), which is consistent with Fontes et al. (2005). A similar result was obtained with the 20- to 37-kDa bands appearance in exosomes, which showed positive labelling of PrPC for exosomes (Figure 3A, lane 2). To confirm exosomes bearing PrPC, immunogold labelled, the exosome-containing gradient fractions for PrPC was detected. Immunogold was obviously observed on exosomes (Figure 3B). These results indicate that the presence of PrPC in culture medium is associated with exosomes.
3.3. Hsp70 interact with PrPC
Co-IP was used to study the association of PrPC with Tsg101, flotillin-1 and Hsp70. There were no differences in the experimental groups from the controls (Figure 4A). There were no bands of Tsg101 at 43 kDa found in lysates and exosomes, which indicated that Tsg101 was not present in the PrP–AH6 complex of cell lysates and exosomes. Only bands of heavy chain at 24 kDa and light chain 55 kDa of IgG were visible in all lanes. A similar result is seen with the immunoblot of flotillin-1. Except for heavy chain and light chain of IgG, no bands of flotillin-1 were visible at 45 kDa (Figure 4B) in PrP–AH6 complex of cell lysates and exosomes. These negative results demonstrate that there are no interactions between Tsg101 and flotillin-1 with PrPC in cell lysates and exosomes. However, Hsp70 interact with PrPC both in intracellular environment and exosomes since the appearance of bands of Hsp70 at 70 kDa in PrP–AH6 complex of cell lysates and exosomes (Figure 4C).
To confirm these results, we performed reverse co-IP. Bands of PrPC between 20 and 37 kDa were neither found in the immunoprecipitate of Tsg101 nor of flotillin-1 (Figures 4D–4E). However, PrPC was found in the immunoprecipitate of Hsp70 (Figure 4F). These data further confirmed that Hsp70 interact with PrPC (Figures 4A–4C).
Exosomes are membrane vesicles released into the extracellular space upon fusion of multivesicular endosomes with the plasma membrane. In order to separate exosomes from aggregates and nucleosomal fragments released by fragmented cells, sucrose gradient centrifugation was used to purify exosomes. The pellets purified from THP-1 monocytes cell culture medium were characterized as exosomes based on both physical and biochemical criteria. First, the density of fractions containing exosomes was between 1.13 and 1.16 g/ml. Secondly, as observed by EM, the pellets were 30–100 nm in size and with cup-shaped morphology. Third, IEM (immunoelectron microscopy) observation and Western blot analysis showed that Tsg101 and flotillin-1, hallmarks of exosomes, were positive, while calnexin, an integral membrane marker, only existed in cell lysates, not in exosomes. The possibility of contamination by organelles released by fragmented cells was excluded.
We obtained evidence that PrPC is released by THP-1 monocytes in association with exosomes by Western blot analysis and IEM. Exosomes have been implicated in cell-to-cell communication mechanisms by transfer of proteins directly from the exosomes to target cells. Monocytes can enter into organs in body through blood circulation; therefore, monocyte-derived exosomes could potentially act as an important transportation vehicle for PrPC transferring between cells. PrPC can be converted into PrPSc, so a hypothesis on whether exosomes can transport the PrPSc released from monocytes to other cells or not was proposed, which needed further studies to confirm this.
Vella et al. (2008) presumed that PrP incorporation into exosome may be a result of recycling of molecules, but there were no reported studies about why PrP is present in exosomes. Fisher rat thyroid cells releasing PrPC into cell culture medium is in a soluble form, not by secreted exosomes (Campana et al., 2007). So, PrP incorporating into exosomes appears to be dependent on host cell type.
Exosomes are secreted upon fusion of multivesicular endosomes with the plasma membrane. PrPC is a membrane-bound, GPI-anchored protein associated with lipid rafts at the plasma menmbrane. Therefore, the presence of PrPC on exosomes is consistent with the subcellular localization. Flotillin-1 was shown to be present in lipid rafts in late endosomes (Fivaz et al., 2002). PrPC and flotillin-1 were simultaneously associated with lipid rafts and exosomes. We assumed that PrPC has an association with flotillin-1. However, our data showed no interaction between PrPC and flotillin-1. Tsg101, an important component of exosomes, was considered to be associated with the forming of endosomes (Lu et al., 2003) and the pathogenesis of spongiform encephalopathy (Jiao et al., 2009). To our disappointment, the interaction between PrPC and Tsg101 was not found either. So flotillin-1 and Tsg101 in our experiment are markers for exosomes. There is no connection between flotillin-1 and Tsg101 with PrPC.
Hsp70, contained in most exosomes (Keller et al., 2006), is a major player in protein transport across membranes, although the mechanism of transport remained elusive (Gastpar et al., 2005). Furthermore, Hsp70 has been detected in lipid rafts in normal cells, a plasma membrane microdomain critical for PrP biology. In this study, we showed that Hsp70 interact with both intracellular PrPC and exosome-bearing PrPC. Our results suggest that Hsp70 play a potential role in the process of PrPC released via exosomes from THP-1 monocytes. The lipid rafts may provide a physical site for the interaction of PrPC with Hsp70. Stress conditions increase the amount of Hsp70, but further studies are required to determine whether the amount change of Hsp70 have an effect on the Hsp70/PrPC interaction, which may regulate the release of exosome-bearing PrPC. Stable transfection of a siRNA (small interfering RNA) to Hsp70 completely abrogated the endogenous levels of Hsp70 (Guo et al., 2005). siRNA of Hsp70 could be applied to address the functional role of Hsp70 in the release of PrPC. Regardless of the effect of Hsp70/PrPC interaction on release of PrPC via exosomes secreted from THP-1 monocytes, this is the first evidence that Hsp70 interact with PrPC.
Gui-hua Wang and Xiang-mei Zhou conceived and designed the experiments. Gui-hua Wang and Yu Bai performed the experiments. Xiaomin Yin isolated and purified exosomes. Li-feng Yang did the Western blotting. Deming Zhao supervised the study.
This work was supported by
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Received 4 November 2009/18 August 2010; accepted 22 October 2010
Published as Cell Biology International Immediate Publication 22 October 2010, doi:10.1042/CBI20090391
© The Author(s) Journal compilation © 2011 Portland Press Limited
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 2 Exosome-containing gradient fractions were further characterized by immunoelectron microscopy and Western blot