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Mouse Apolipoprotein L9 is a phosphatidylethanolamine-binding protein
Biochemical and Biophysical Research Communications xxx (2016) 1e7
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Mouse Apolipoprotein L9 is a phosphatidylethanolamine-binding protein Thekkinghat Anantharaman Arvind, Pundi N. Rangarajan* Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 September 2016 Accepted 29 September 2016 Available online xxx
Mouse Apolipoprotein L9 (ApoL9) is an understudied cytoplasmic, interferon-inducible protein. The details of its intracellular localization and normal cellular functions are unclear. We report here that ApoL9 localizes to small puncta diffusely distributed in the cytoplasm, as well as to larger granules of varying size and number that are similar to aggresome-like induced structures (ALIS) and contain the autophagy receptor Sqstm1/p62, the autophagosome marker Lc3, and ubiquitin. Transfection of B16F10 mouse melanoma cells stably expressing ApoL9 (B16F10L9) with certain liposome-based transfection reagents causes dramatic disturbances in its subcellular distribution. We reasoned that these disturbances may be due to the interaction of ApoL9 with dioleoylphosphatidylethanolamine (DOPE), the helper lipid component of several transfection reagents. Recombinant ApoL9 produced in E. coli, as well as ApoL9 expressed in HEK293T cells, specifically bind phosphatidylethanolamine (PE) in vitro. ApoL9 is expressed at high levels in liver and brain, organs enriched in PE. Since PE is known to facilitate replication of positive strand RNA viruses, we examined the role of ApoL9 during replication of Japanese encephalitis virus (JEV), a positive strand virus of the family Flaviviridae. JEV titres in B16F10L9 cells are higher than those in B16F10 cells. We propose that ApoL9 is a PE-binding protein that may have important roles in several cellular processes that involve this phospholipid. © 2016 Elsevier Inc. All rights reserved.
Keywords: Apolipoprotein L9 Phosphatidylethanolamine B16F10 melanoma Japanese encephalitis virus Aggresome-like induced structures
1. Introduction The Apolipoprotein L (ApoL) family comprises six genes in humans and twelve genes in mice [1,2]. The genes of this family are well conserved in several mammals. However, apart from the secreted human trypanosome lytic factor ApoL1 which is also genetically linked to kidney disease [3], the functions of other ApoL proteins, which are projected to have primarily intracellular functions [4], remain poorly understood. ApoL proteins have previously been shown to regulate apoptotic and autophagic cell death [5,6]. Independent genes encode mouse ApoL9a and ApoL9b, which contain 310 amino acid residues and share 97% identity. Their human homologues are not yet documented. Kreit et al. (2015) reported that ApoL9 has a cytoplasmic localization and inhibits the replication of Theiler's murine encephalomyelitis virus (TMEV) through its interaction with cellular prohibitins (2). It was proposed that inefficient interferon-inducible expression of Apol9 in primary
* Corresponding author. Department of Biochemistry, FE15, Biology Building, Indian Institute of Science, Bangalore 560012, India E-mail address: [email protected] (P.N. Rangarajan).
mouse neurons may contribute to the susceptibility of these cells to TMEV infection [7]. Sun et al. (2015) reported that small quantities of ApoL9a/b secreted by macrophages during type I interferon signalling stimulate epithelial cell proliferation [8]. However, the mechanism of secretion isn't clear and the bulk of the protein is retained within the cell, suggesting it could have intracellular functions. Apart from those involving ApoL1, most studies on ApoL proteins have so far relied on transcriptomic analysis or quantification of mRNA levels. Quantifying the protein levels of this family in their biological milieu would be both a necessary and important step in the context of future studies that investigate these proteins. In this study, we generated a B16F10 mouse melanoma cell line stably expressing ApoL9 (B16F10L9) and examined its intracellular distribution. Using an anti-ApoL9 antibody, we show that ApoL9 specifically binds the phospholipid phosphatidylethanolamine (PE). Finally, we demonstrate that JEV titres in B16F10L9 cells are higher than those in control B16F10 cells. We believe that this study unearths the single major function of ApoL9 in cells, i.e. PE-binding, and could open doors for discovering potentially important roles for ApoL9 in various cellular pathways that involve this phospholipid.
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2. Materials and methods
anti-Sqstm1 (ab56416), Abcam. Anti- (mouse IFIT3) and antiMBP-Apol9 were polyclonal antibodies made in rabbit.
2.1. Plasmids, reagents, and antibodies 2.2. Cell lines The Apol9a gene was amplified from total RNA isolated from B16F10 cells treated with mouse interferon b (Calbiochem, Cat. No.407298). Apol9-V5, V5-Apol9, and Apol9-3XFLAG constructs were made using the plasmid pBApo-EF1a pur (Takara Bio Inc.). The 3 FLAG sequence was amplified from p3XFLAG-CMV-10 expression vector (Sigma-Aldrich). Apol9-GFP and GFP-Apol9 were made using pEGFP-N1 and pEGFP-C1 (Clontech Laboratories Inc.), respectively. MBP-Apol9 was made by cloning Apol9a into pMAL-c2X. MBP and MBP-ApoL9 were purified according to the manufacturer's instructions (NEB). Mouse cDNAs encoding Map1lc3b and Dcp1a were cloned into pEGFP-C1. The cDNA encoding eEF1a was cloned into p3XFLAG-CMV-10. MG132 was purchased from Merck Millipore. Lipofectamine 2000 was from Thermo Fisher Scientific. Purified phospholipids and Poly (I:C) were from Sigma-Aldrich. Calf thymus DNA was from Amersham Life Science. Antibodies used were: anti-V5 (R960-25), anti-JEV NS2B (PA532237), anti-JEV E (MA1-71256), all Thermo Fisher Scientific; antiV5 (V8137); anti-FLAG M2 (F1804), anti-ubiquitin (U0508), antiFLAG (F7425), anti-b-Actin-Peroxidase (A3854), all Sigma-Aldrich;
B16F10 mouse melanoma was obtained from National Institute of Immunology, New Delhi, India. BHK-21 was purchased from ATCC. HEK293T cells were a gift from Prof. Ganesh Nagaraju, Indian Institute of Science (IISc), Bangalore, India. PS Porcine kidney cell line was a gift from Prof. R. Manjunath, IISc. B16F10 and HEK293T were cultured in DMEM (Gibco) with 10% FBS; BHK-21 and PS cells were cultured in MEM Alpha (Gibco) with 10% FBS, at 37 C in a 5% CO2 environment. Transfections with Lipofectamine 2000 were performed according to the manufacturer's instructions. B16F10 cells were electroporated at 240 V using the Gene Pulser Xcell system (BioRad). Calcium phosphate-mediated transfection was performed as described in Ref. [27]. 2.3. Creation of B16F10L9 cell line stably expressing ApoL9 The Apol9-V5 construct was linearized and transfected into B16F10 cells using Lipofectamine 2000. 48 h later, the cells were subcultured into six-well plates and selected with 2 mg mL1
Fig. 1. Subcellular localization of ApoL9. A. Localization of ApoL9 tagged with different epitopes as indicated. Plasmids expressing ApoL9 were electroporated into B16F10 cells. V5and FLAG-tagged ApoL9 were visualized using anti-V5 and anti-FLAG antibodies. B. Localization of ApoL9-V5 in B16F10L9 cells. C. Quantification of relative Apol9 mRNA levels in B16F10 and B16F10L9 by qPCR. Error bars indicate mean ± S.D (n ¼ 3). D. Detection of ApoL9-V5 protein in B16F10L9 cells by western blotting using anti-V5 antibody. a. ApoL9-V5, b. b-actin. E, F, G. Colocalization of ApoL9-V5 in granules with Sqstm1, Lc3b and ubiquitin. Nuclei are stained using DAPI. H. Perinuclear localization of ApoL9 in B16F10L9 cells following treatment with MG132 for 4 h. Two different cells are shown. I. Western blotting of ApoL9-V5 in MG132 treatment using anti-V5 antibody J. Quantification of data in I. Error bars indicate mean ± S.D (n ¼ 2). K. Localization of ApoL9-V5 and 3XFLAG-eEF1a in untreated and MG132-treated B16F10L9 cells.
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puromycin. Ten days later, single colonies were picked and expanded, screened for ApoL9 expression by indirect immunofluorescence and clones with an optimum level of expression were further expanded and frozen. 2.4. Viruses and plaque assay JEV P20778 strain was amplified in brains of suckling mice and propagated in BHK-21 cells. Infections for experiments were carried out at an m.o.i of 10. Plaque assay was performed on porcine kidney (PS) cells. Briefly, ten-fold serial dilutions of virus stock or supernatants from infected cells were prepared in medium without serum and inoculated on to confluent monolayers of PS cells. After 2 h of incubation with intermittent shaking, the inoculum was removed and replaced with DMEM with 2% FBS and 1% low-melting agarose. 72 h later, the monolayers were fixed with 3.7% formaldehyde, the agarose plugs were removed, and the cells were stained with 0.1% crystal violet solution in 20% ethanol. Wells with countable plaques were selected and viral titres were calculated considering the appropriate dilution factors.
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2.5. Animal experiments Animal experiments performed in this study were approved by the Institutional Animal Ethics Committee (IAEC) of the Indian Institute of Science, Bangalore (CAF/Ethics/192/2010). For infection with JEV, BALB/c mice were injected intracranially with 103 p.f.u of virus. Control mice were injected with cell culture medium. Animals were euthanised 4e5 days following infection, when the typical symptoms of advanced encephalitis were evident. Brains and other organs were aseptically harvested and processed for RNA or protein extraction. 2.6. qPCR and western blotting Two-step qPCR was performed by preparing cDNA from RNA samples. RNA was isolated using Tri Reagent (Sigma-Aldrich) and cDNA was synthesised using RevertAid reverse transcriptase (Thermo Fisher Scientific). cDNA samples were appropriately diluted and used for qPCR on the StepOnePlus real time PCR system (Thermo Fisher Scientific). iQ SYBR Green Supermix (Bio-Rad
Fig. 2. Identification of ApoL9 as a PE-binding protein. A. B16F10L9 cells were transfected with Lipofectamine 2000 alone or together with 1 mg of the indicated nucleic acids. The plasmid DNA pBApo-EF1a pur was transfected for 30 min, 1 h or 4 h. ApoL9-V5 was stained with anti-V5 antibody. B. Transfection of pEGFP-C1, expressing EGFP, into B16F10L9 cells using Lipofectamine 2000. C. Schematic representation of MBP-ApoL9 construct. D. Visualization of MBP-ApoL9 in a Coomassie blue-stained SDS-PAGE gel before and after partial cleavage by Factor Xa. E. Demonstration of immunoreactivity of anti-MBP-ApoL9 antibody by western blotting against MBP-ApoL9 (1), MBP (2), ApoL9-V5 present in lysate of B16F10L9 cells (3). Lysate of B16F10 cells (4) served as negative control. FeH. Protein-lipid overlay (PLO) assays. F. Lipofectamine 2000 was serially diluted and spotted on to nitrocellulose and its ability to interact with MBP and MBP-ApoL9 was examined. Quantities in mg are indicated above the blot. G. Different phospholipids were spotted on to nitrocellulose and their ability to interact with MBP and MBP-ApoL9 was examined. Order of lipids: 1. Phosphatidylserine, 2. Phosphatidic acid, 3. Phosphatidylethanolamine, 4. Phosphatidylcholine, 5. Phosphatidylglycerol, 6. Lysophosphatidylethanolamine. The quantities of lipid (mg) are indicated on the left. MBP control bound phosphatidic acid. H. PLO assay of lysates of HEK293T cells transfected with vector alone, vector encoding ApoL9-V5 or ApoL9-GFP by calcium phosphate-mediated transfection. Order of lipids: 1. Dipalmitoylphosphatidylethanolamine, 2. Distearoylphosphatidylethanolamine, 3. Phosphatidylcholine. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Analysis of ApoL9 expression in mouse organs and its effect on JEV titres. A. Quantification of ApoL9 levels in various organs from mice by western blotting using anti-MBPApoL9 antibody (upper panel). Blots stained with Ponceau S (lower panel) served as loading controls. B. Quantification of data in A (upper panel). Data is the average of two independent experiments (n ¼ 2). C. Analysis of Apol9 mRNA levels in different mouse organs by semi-quantitative RT-PCR. D. Quantification of Apol9 mRNA levels in mouse organs by qPCR, normalized to 18s rRNA. Error bars indicate mean ± S.D (n ¼ 2). E. Demonstration of interferon-induced expression of ApoL9-V5 by western blotting in B16F10 cells with
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Laboratories) was used, and relative quantification was done using the DDCt method. Western blotting procedures were performed essentially as in Ref. [26] with minor modifications. Blots were visualized using the ImageQuant LAS 4000 system (GE Healthcare), and quantified using the software ImageJ. 2.7. Immunofluorescence and confocal microscopy Cells grown on coverslips were fixed with 3.7% formaldehyde, washed twice with PBS, permeabilized with 0.1% Triton X-100. After blocking with 5% BSA in PBS, they were incubated with primary antibodies in blocking solution for 2 h. After 3 washes with PBS containing 0.05% Triton X-100, they were incubated with secondary fluorophore-conjugated antibodies (Alexa Fluor 555/488; Thermo Fisher Scientific) in blocking solution for 2 h. After 3 more washes, they were stained with 1 mg/mL DAPI, washed and mounted on antifade. Imaging was done on a Zeiss LSM 510 Meta confocal microscope. Intensity and colour contrast of images were balanced using LSM Image Browser (Carl Zeiss Microimaging) and Adobe Photoshop. 2.8. Protein-lipid overlay assay Purified phospholipids were dissolved in appropriate organic solvents according to the manufacturers' instructions. They were serially diluted in chloroform:methanol:water (1:2:0.8) as described in Takahashi et al. (2006). 1 ml of each dilution was spotted on a nitrocellulose membrane and allowed to dry for 1 h. The membrane was blocked in TBST containing 3% fatty acid-free BSA overnight, followed by incubation with purified protein (2 mg/mL) or cell lysate (50 mg total protein/mL) for 2 h. After 3 washes in TBST, the membranes were incubated with primary and secondary antibodies as in the procedure for western blotting and viewed in a chemiluminescence imager. For assays with cell lysates, HEK293T cells were transfected with vector alone or vectors expressing ApoL9-V5 or ApoL9-GFP by calcium phosphate-mediated transfection. The cells were lysed with a buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, and 0.1% Triton X-100 and the supernatants were clarified by centrifugation. ApoL9 was detected using anti-V5 or anti-GFP antibodies. 3. Results 3.1. ApoL9 localizes to structures similar to aggresome-like induced structures (ALIS) To study the intracellular distribution of ApoL9, plasmids encoding ApoL9 with C- or N-terminal V5 tags, C-terminal 3X-FLAG tag, and C- or N-terminal GFP were electroporated into B16F10 melanoma cells and localization of ApoL9 was examined by indirect immunofluorescence or by GFP fluorescence. ApoL9 containing Nor C-terminal V5 or FLAG tags showed similar subcellular distribution (Fig. 1A). It localized to small puncta scattered diffusely in the cytoplasm, and to sparse larger granules whose number and size varied in different cells. ApoL9 GFP fusion proteins exhibited a slightly different pattern, appearing as larger, more numerous,
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hollow structures scattered throughout the cytoplasm. In view of the relatively large size of GFP, and because the smaller epitope tags displayed identical subcellular distribution patterns, all subsequent studies were carried out with the C-terminal V5-tagged form of ApoL9 (ApoL9-V5). A B16F10 cell line stably expressing ApoL9-V5 was generated (B16F10L9). Indirect immunofluorescence studies indicated that the distribution pattern of ApoL9-V5 in B16F10L9 was similar to that observed by transient transfection (Fig. 1B). The levels of Apol9 mRNA in B16F10L9 cells were quantified by qPCR (Fig. 1C). Protein expression was verified by western blotting (Fig. 1D). Colocalization studies using organelle markers indicated that the small ApoL9 puncta or the large ApoL9-containing granule-like structures do not localize to lysosomes, mitochondria, Golgi apparatus, P-bodies, lipid droplets, or peroxisomes (data not shown). Interestingly, ApoL9 in the large granule-like structures colocalizes with sequestosome-1 (Sqstm1/p62) and Lc3b (the autophagosome marker) (Fig. 1E and F) suggesting that these may be aggresomelike induced structures (ALIS) or similar structures, since the latter proteins are known to localize to ALIS [9,10]. Since ALIS are known to be storage compartments for polyubiquitinated defective ribosomal products, we examined whether these granules stain for ubiquitin. The results indicate that they contain ubiquitin (Fig. 1G). On arresting proteasomal degradation using the inhibitor MG132, there was an increase in granule size as well as congregation of the granules in the perinuclear region (Fig. 1H). Further, MG132 treatment resulted in an increase in ApoL9 protein (Fig. 1I and J), suggesting that the protein might be degraded in normal conditions by the ubiquitin-proteasome system. Eukaryotic translation elongation factor 1A (eEF1a), known to associate with ubiquitinated defective ribosomal products [11], accumulated in these granules (Fig. 1K). Though ALIS and related granules are usually induced by stress conditions, they have been reported to be present at a relatively high background level in some cell lines under non-stress conditions [9]. The case with B16F10 seems to be similar.
3.2. Transfection of nucleic acids using Lipofectamine 2000 dramatically alters ApoL9 distribution in B16F10L9 cells During subcellular localization studies of ApoL9, we observed that transfection of any plasmid into B16F10L9 cells using Lipofectamine 2000 immediately induced rapid, progressive, and dramatic aggregation of ApoL9 (Fig. 2A). ApoL9 localization was altered by lipofection of plasmids, polyinosinic:polycytidilic acid (poly I:C), or sonicated calf thymus DNA but not by transfection reagent alone (Fig. 2A). Since these effects manifested themselves as early as 30 min after transfection, we thought it unlikely that it was a consequence of any transfected protein being expressed. Introduction of plasmids by electroporation or calcium phosphatemediated transfection did not alter ApoL9 localization. Lipofection of a GFP expression vector into B16F10L9 cells led to aggregation of ApoL9 but not GFP (Fig. 2B). Since lipofection of such varied nucleic acid substrates and even empty plasmids resulted in similar changes in ApoL9 distribution, we hypothesised that the protein was likely reacting with some component of the transfection reagent itself, rather than with any of the molecules being transfected. We also observed the same effect when using a few other
anti-MBP-ApoL9 antibody. F. Demonstration of JEV-induced expression of Apol9 mRNA in mouse brain by qPCR. Data is the average of two independent experiments. G. Western blotting forApoL9 in uninfected and JEV-infected brains using anti-MBP-ApoL9 antibody. Virus replication in JEV-infected brains was confirmed by the presence of JEV-NS2b. IFIT3 is a positive control JEV-induced protein [28]. H. Quantification of data in G. I. Demonstration of increased JEV titres in the medium of B16F10L9 cells by plaque assay. One representative plate is shown. J. Quantification of plaques. Data from two individual experiments (1, 2) are shown. Error bars indicate mean ± S.D. *, p 0.05; **, p 0.01; ***, p 0.001. Statistical significance was calculated using an unpaired t-test. K. Immunostaining of ApoL9-V5 in JEV-infected B16F10L9 cells. Virus replication in cells is confirmed by staining for JEV envelope protein.
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liposome-based transfection reagents whose compositions are proprietary. We hypothesised that ApoL9 was interacting with the lipoplexes that are formed bycomplexing nucleic acid with liposomes prior to transfection, whereas transfection reagent alone diffused rapidly through the cytoplasm. Since the neutral lipid DOPE (dioleoylphosphatidylethanolamine) is the most common helper lipid component in several different cationic lipid-based transfection reagents [12], including Lipofectamine (the predecessor of Lipofectamine 2000) [13], we hypothesised that interactions between ApoL9 and DOPE, or more specifically phosphatidylethanolamine (PE), might be responsible for the observed changes in ApoL9 distribution in cells. 3.3. ApoL9 binds phosphatidylethanolamine ApoL9 was expressed as a maltose binding protein fusion (MBPApoL9) in E. coli (Fig. 2C and D) and the purified protein was injected into rabbits to obtain anti-MBP-ApoL9 polyclonal antibodies. Western blotting showed that recombinant MBP-ApoL9 expressed in E. coli, and ApoL9-V5 expressed in B16F10L9 cells were immunoreactive to this antibody (Fig. 2E). To test if ApoL9 binds a component of Lipofectamine 2000, the reagent was serially diluted and spotted on nitrocellulose and a protein-lipid overlay assay was performed. MBP-ApoL9 (but not MBP) bound to Lipofectamine 2000 (Fig. 2F). To examine the ability of ApoL9 to bind to PE, different phospholipids were spotted onto nitrocellulose and the membranes were incubated with either MBP or MBP-ApoL9. After washing the membrane, the protein-lipid complex was detected using the anti-MBP-ApoL9 antibody and HRP-conjugated anti-rabbit antibody. MBP-ApoL9, but not MBP, interacted specifically with PE but not with other phospholipids (Fig. 2G). ApoL9-V5 or ApoL9-GFP overexpressed in HEK293T cells also interacted specifically with both dipalmitoyl-PE and distearoyl-PE (Fig. 2H). 3.4. Some organs show poor correlation between ApoL9 RNA and protein levels Using the anti-MBP-L9 antibody, we examined protein levels of ApoL9 in various mouse organs. Liver and brain expressed high levels of ApoL9 compared with other organs. (Fig. 3A and B). Surprisingly, there was poor correlation between mRNA and protein levels in organs such as brain and kidney. Apol9 mRNA levels in brain are extremely low compared with that of liver (Fig. 3C and D), yet the brain expresses moderately high amounts of ApoL9 protein. Similarly, kidney expresses almost ten times the amount of Apol9 mRNA that is found in the brain, yet the protein levels are very modest (compare Fig. 3B and D). 3.5. ApoL9-expressing cells show increased JEV titres to control cells Apol9 is an interferon- and virus-inducible gene [2]. We demonstrate that ApoL9 protein levels increase several fold in B16F10 cells following treatment with IFN-b (Fig. 3E). In view of ApoL9 binding PE, which was recently shown to be indispensable for the replication of certain positive strand RNA viruses [14], we examined the role of ApoL9 during infection by other such viruses. We chose JEV, a positive strand RNA virus of Flaviviridae. It is relevant to note here that JEV primarily infects the brain, which contains significant amounts of ApoL9 protein (Fig. 3A). JEVinfected mice show strong upregulation of Apol9 mRNA in the brain (Fig. 3F), though protein levels increase only moderately (Fig. 3G and H). To study the effect of ApoL9 on JEV infection, B16F10 cells and B16F10L9 cells were infected by JEV and virus titres in the postinfection culture supernatants were measured by performing
plaque assays on porcine kidney cells. JEV titres were consistently two-fold higher in supernatants from B16F10L9 cells than from B16F10 cells (Fig. 3I and J), suggesting that ApoL9 might function as a weak proviral host factor during JEV infection. Immunostaining indicates that the intracellular distribution pattern of ApoL9 is not significantly altered during JEV infection (Fig. 3K). 4. Discussion Here we report for the first time a key intracellular function for ApoL9, i.e., binding to PE. PE is not only a major component of cell membranes but plays key roles in diverse cellular processes such as autophagy [15], and sorting and trafficking of proteins inhabiting lipid rafts [16]. Some previously described PE-binding proteins bind the hydrophilic head group of PE by means of a cavity formed by the tertiary structure of the protein [17]. Elaborate studies on ApoL9 structure will be required to identify how it binds PE. A role for ApoL9 in autophagy is highly likely, not only because of the mandatory role of PE in autophagy [18] but also because ApoL9 colocalizes with Sqstm1 and Lc3, key proteins involved in autophagy [19]. ApoL9 might also possibly function as a lipid transfer protein involved in trafficking PE inside the cell. It is important to exercise caution while using transfection reagents that may contain DOPE in experiments involving ApoL9, as they inevitably lead to the formation of biological artefacts. Alternate transfection methods should be employed. ApoL9 contributed to increasing JEV titres in infected cells despite its subcellular distribution not being significantly altered during JEV infection, suggesting that it might be a player in some high-turnover, dynamic cellular process that is co-opted by the virus for completing its life cycle. It will be interesting to examine whether the PE-binding property of ApoL9 is essential for its proviral function, because it was reported [14] that certainpositive strand RNA viruses subvert PE to support their replication. Kreit et al. [2] report a narrow antiviral range for ApoL9, based on its effect on various viruses. Since it is possible that the magnitude of the pro- or antiviral effect of ApoL9 varies according to the virus species, it will be interesting to study if ApoL9 has a role during infection by other flaviviruses such as West Nile Virus, Dengue virus and Zika virus. The high levels of ApoL9 protein in liver and brain suggest that ApoL9 might have some housekeeping role in these organs. It is noteworthy that both these organs have a high content of PE and that lipid metabolism plays an important role in their functioning [20,21]. The PEBP (phosphatidylethanolamine-binding protein) family represents the most well-known class of PE-binding proteins. Proteins of this family have been implicated in inhibiting serine proteases [22], regulating the MAP kinase pathway [23], muopioid receptor signalling [24] and abnormal expression in some types of cancer [25]. The PEBP family is well conserved across flowering plants, parasites, nematodes, insects, and mammals. The ApoL proteins, on the other hand, possess a BH3 domain [26] and are only present in higher eukaryotes. It is likely that the members of this family could have specialized, as-yet-undiscovered, functions in these organisms. Since PE is involved in numerous processes in the cell, it is only logical that ApoL9 could have roles in influencing at least some of these processes. This discovery offers incentives to further study this protein and explore its potential functions, and provides new impetus to characterize other members of the ApoL family and investigate if a human homologue of ApoL9 exists. Funding This study was supported by the J. C. Bose fellowship grant # SB/
Please cite this article in press as: T.A. Arvind, P.N. Rangarajan, Mouse Apolipoprotein L9 is a phosphatidylethanolamine-binding protein, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.09.161
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S2/JCB-025/2015 by the Science and Engineering Research Board, New Delhi, India awarded to P.N.R. Institutional support from the Departments of Science and Technology and Biotechnology, New Delhi are acknowledged. Acknowledgements We acknowledge the Confocal Microscopy facility, Biological Sciences, IISc, Bangalore. We thank Mr.Iyappan and Prof. Ram Rajasekharan for help with the protein-lipid overlay assay. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.09.161. References [1] N.M. Page, D.J. Butlin, K. Lomthaisong, et al., The human Apolipoprotein L gene cluster: identification, classification, and sites of distribution, Genomics 74 (2001) 71e78. [2] M. Kreit, D. Vertommen, L. Gillet, et al., The interferon-inducible mouse Apolipoprotein L9 and prohibitins cooperate to restrict Theiler's virus replication, PLoS One 7 (2015) e0133190. [3] E. Kruzel-Davila, W.G. Wasser, S. Aviram, et al., ApoL1nephropathy: from gene to mechanisms of kidney injury, Nephrol. Dial. Transplant. 31 (2015) 349e358. [4] B. Vanhollebeke, E. Pays, The function of apolipoproteins L, Cell. Mol. Life Sci. 63 (2006) 1937e1944. [5] G. Wan, S. Zhaorigetu, Z. Liu et al. Apolipoprotein L1, a novel Bcl-2 Homology domain 3-only lipid-binding protein, induces Autophagic cell death, J. Biol. Chem. (283) 21540e21549. [6] Z. Liu, H. Lu, Z. Jiang, et al., Apolipoprotein L6, a Novel Proapoptotic Bcl-2 homology 3-Only protein, induces mitochondria-mediated apoptosis in cancer cells, Mol. Cancer Res. 3 (2005) 21e31. [7] M. Kreit, S. Paul, L. Knoops, et al., Inefficient type I interferon-mediated antiviral protection of primary mouse neurons is associated with the lack of Apolipoprotein L9 expression, J. Virol. 88 (2014) 3874e3884. [8] L. Sun, H. Miyoshi, S. Origanti, et al., Type I interferons link viral infection to enhanced epithelial turnover and repair, Cell Host Microbe 17 (2015) 85e97. [9] J. Szeto, N. A. Kaniuk, V. Canadien, et al., ALIS are stress-induced protein storage compartments for substrates of the proteasome and Autophagy, Autophagy 2 (2006) 189e199. [10] G. Bjørkøy, T. Lamark, A. Brech, et al., p62/Sqstm1 forms protein aggregates degraded by autophagy and has a protective effect onhuntingtin-induced cell death, J. Cell Biol. (2005) 603e614. [11] A.B. Meriin, N. Zaarur, M.Y. Sherman, et al., Association of translation factor eEF1A with defective ribosomal products generates a signal for aggresome formation, J. Cell Sci. 125 (2012) 2665e2674.
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[12] Z. Du, M.M. Munye, A.D. Tagalakis, et al., The role of the helper lipid on the DNA transfection efficiency of lipopolyplex formulations, Sci. Rep. 4 (2014) 7107. [13] Lipofectamine® Reagent for the Transfection of DNA into Eukaryotic Cells, 2016 (accessed 21.9.16), https://www.thermofisher.com/in/en/home/ references/protocols/cell-culture/transfection-protocol/lipofectaminereagent-suitable-for-the-transfection-of-dna-into-eukaryotic-cells.html. [14] K. Xu, P.D. Nagy, RNA virus replication depends on enrichment of phosphatidylethanolamine at replication sites in subcellular membranes, P. Natl. Acad. Sci. U. S. A. 112 (2015) E1782eE1791. [15] C. Wilson-Zbinden, A.X. dos Santos, I. Stoffel-Studer, et al., Autophagy competes for a common phosphatidylethanolamine pool with major cellular PEconsuming pathways in Saccharomyces cerevisiae, Genetics 199 (2014) 475e485. , K. Malínsk , et al., Differential effect of phos[16] M. Opekarova a, L. Nov akova phatidylethanolamine depletion on raft proteins, Biochim. Biophys. Acta 1711 (2005) 87e95. e, N. Bureaud, et al., Crystal structure of the [17] L. Serre, B. Valle phosphatidylethanolamine-binding protein from bovine brain: a novel structural class of phospholipid-binding proteins, Structure 6 (1998) 1255e1265. [18] Y. Kabeya, N. Mizushima, A. Yamamoto, et al., LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation, J. Cell Sci. 117 (2004) 2805e2812. [19] S. Pankiv, T.H. Clausen, T. Lamark, et al., P62/SQSTM1 binds directly to Atg8/ LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy, J. Biol. Chem. 282 (2007) 24131e24145. [20] Z. Li, L.B. Agellon, T.M. Allen, et al., The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis, Cell Metab. 3 (2006) 321e331. € derberg, C. Edlund, K. Kristensson, et al., Fatty acid composition of brain [21] M. So phospholipids in aging and in Alzheimer's disease, Lipids 26 (1991) 421e425. [22] U. Hengst, H. Albrecht, D. Hess, et al., The Phosphatidylethanolamine-binding protein is the prototype of a novel family of serine protease inhibitors, J. Biol. Chem. 276 (2000) 535e540. [23] X. Wang, N. Li, B. Liu, et al., A novel human phosphatidylethanolaminebinding protein resists tumor necrosis factor -induced apoptosis by inhibiting mitogen-activated protein kinase pathway activation and phosphatidylethanolamine externalization, J. Biol. Chem. 279 (2004) 45855e45864. [24] K. Edwards, J. Bogusz, J. LaVigne, et al., Phosphatidylethanolamine-binding protein regulates Mu opioid receptor induced barrestin2 recruitment and downstream signaling, J. Pain 17 (2016) S47. [25] R. Huang, D. Shi, W. Huang, et al., Increased expression of phosphatidylethanolamine-binding protein 4 (PEBP4) strongly associates with human gliomas grade, J. Neuro Oncol. 127 (2016) 235e242. [26] J. Galindo-Moreno, R. Iurlaro, N. El Mjiyad, et al., Apolipoprotein L2 contains a BH3-like domain but it does not behave as a BH3-only protein, Cell Death Dis. 5 (2014) e1275. [27] J. Sambrook, D. Russell, Calcium-phosphate-mediated transfection of eukaryotic cells with plasmid DNAs, Cold Spring Harb. Protoc. 1 (2006) p. pdb.prot3871. [28] S. Saha, P.N. Rangarajan, Common host genes are activated in mouse brain by Japanese encephalitis and rabies viruses, J. Gen. Virol. 84 (2003) 1729e1735.
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Mouse Apolipoprotein L9 is a phosphatidylethanolamine-binding protein Thekkinghat Anantharaman Arvind, Pundi N. Rangarajan* Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 September 2016 Accepted 29 September 2016 Available online xxx
Mouse Apolipoprotein L9 (ApoL9) is an understudied cytoplasmic, interferon-inducible protein. The details of its intracellular localization and normal cellular functions are unclear. We report here that ApoL9 localizes to small puncta diffusely distributed in the cytoplasm, as well as to larger granules of varying size and number that are similar to aggresome-like induced structures (ALIS) and contain the autophagy receptor Sqstm1/p62, the autophagosome marker Lc3, and ubiquitin. Transfection of B16F10 mouse melanoma cells stably expressing ApoL9 (B16F10L9) with certain liposome-based transfection reagents causes dramatic disturbances in its subcellular distribution. We reasoned that these disturbances may be due to the interaction of ApoL9 with dioleoylphosphatidylethanolamine (DOPE), the helper lipid component of several transfection reagents. Recombinant ApoL9 produced in E. coli, as well as ApoL9 expressed in HEK293T cells, specifically bind phosphatidylethanolamine (PE) in vitro. ApoL9 is expressed at high levels in liver and brain, organs enriched in PE. Since PE is known to facilitate replication of positive strand RNA viruses, we examined the role of ApoL9 during replication of Japanese encephalitis virus (JEV), a positive strand virus of the family Flaviviridae. JEV titres in B16F10L9 cells are higher than those in B16F10 cells. We propose that ApoL9 is a PE-binding protein that may have important roles in several cellular processes that involve this phospholipid. © 2016 Elsevier Inc. All rights reserved.
Keywords: Apolipoprotein L9 Phosphatidylethanolamine B16F10 melanoma Japanese encephalitis virus Aggresome-like induced structures
1. Introduction The Apolipoprotein L (ApoL) family comprises six genes in humans and twelve genes in mice [1,2]. The genes of this family are well conserved in several mammals. However, apart from the secreted human trypanosome lytic factor ApoL1 which is also genetically linked to kidney disease [3], the functions of other ApoL proteins, which are projected to have primarily intracellular functions [4], remain poorly understood. ApoL proteins have previously been shown to regulate apoptotic and autophagic cell death [5,6]. Independent genes encode mouse ApoL9a and ApoL9b, which contain 310 amino acid residues and share 97% identity. Their human homologues are not yet documented. Kreit et al. (2015) reported that ApoL9 has a cytoplasmic localization and inhibits the replication of Theiler's murine encephalomyelitis virus (TMEV) through its interaction with cellular prohibitins (2). It was proposed that inefficient interferon-inducible expression of Apol9 in primary
* Corresponding author. Department of Biochemistry, FE15, Biology Building, Indian Institute of Science, Bangalore 560012, India E-mail address: [email protected] (P.N. Rangarajan).
mouse neurons may contribute to the susceptibility of these cells to TMEV infection [7]. Sun et al. (2015) reported that small quantities of ApoL9a/b secreted by macrophages during type I interferon signalling stimulate epithelial cell proliferation [8]. However, the mechanism of secretion isn't clear and the bulk of the protein is retained within the cell, suggesting it could have intracellular functions. Apart from those involving ApoL1, most studies on ApoL proteins have so far relied on transcriptomic analysis or quantification of mRNA levels. Quantifying the protein levels of this family in their biological milieu would be both a necessary and important step in the context of future studies that investigate these proteins. In this study, we generated a B16F10 mouse melanoma cell line stably expressing ApoL9 (B16F10L9) and examined its intracellular distribution. Using an anti-ApoL9 antibody, we show that ApoL9 specifically binds the phospholipid phosphatidylethanolamine (PE). Finally, we demonstrate that JEV titres in B16F10L9 cells are higher than those in control B16F10 cells. We believe that this study unearths the single major function of ApoL9 in cells, i.e. PE-binding, and could open doors for discovering potentially important roles for ApoL9 in various cellular pathways that involve this phospholipid.
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2. Materials and methods
anti-Sqstm1 (ab56416), Abcam. Anti- (mouse IFIT3) and antiMBP-Apol9 were polyclonal antibodies made in rabbit.
2.1. Plasmids, reagents, and antibodies 2.2. Cell lines The Apol9a gene was amplified from total RNA isolated from B16F10 cells treated with mouse interferon b (Calbiochem, Cat. No.407298). Apol9-V5, V5-Apol9, and Apol9-3XFLAG constructs were made using the plasmid pBApo-EF1a pur (Takara Bio Inc.). The 3 FLAG sequence was amplified from p3XFLAG-CMV-10 expression vector (Sigma-Aldrich). Apol9-GFP and GFP-Apol9 were made using pEGFP-N1 and pEGFP-C1 (Clontech Laboratories Inc.), respectively. MBP-Apol9 was made by cloning Apol9a into pMAL-c2X. MBP and MBP-ApoL9 were purified according to the manufacturer's instructions (NEB). Mouse cDNAs encoding Map1lc3b and Dcp1a were cloned into pEGFP-C1. The cDNA encoding eEF1a was cloned into p3XFLAG-CMV-10. MG132 was purchased from Merck Millipore. Lipofectamine 2000 was from Thermo Fisher Scientific. Purified phospholipids and Poly (I:C) were from Sigma-Aldrich. Calf thymus DNA was from Amersham Life Science. Antibodies used were: anti-V5 (R960-25), anti-JEV NS2B (PA532237), anti-JEV E (MA1-71256), all Thermo Fisher Scientific; antiV5 (V8137); anti-FLAG M2 (F1804), anti-ubiquitin (U0508), antiFLAG (F7425), anti-b-Actin-Peroxidase (A3854), all Sigma-Aldrich;
B16F10 mouse melanoma was obtained from National Institute of Immunology, New Delhi, India. BHK-21 was purchased from ATCC. HEK293T cells were a gift from Prof. Ganesh Nagaraju, Indian Institute of Science (IISc), Bangalore, India. PS Porcine kidney cell line was a gift from Prof. R. Manjunath, IISc. B16F10 and HEK293T were cultured in DMEM (Gibco) with 10% FBS; BHK-21 and PS cells were cultured in MEM Alpha (Gibco) with 10% FBS, at 37 C in a 5% CO2 environment. Transfections with Lipofectamine 2000 were performed according to the manufacturer's instructions. B16F10 cells were electroporated at 240 V using the Gene Pulser Xcell system (BioRad). Calcium phosphate-mediated transfection was performed as described in Ref. [27]. 2.3. Creation of B16F10L9 cell line stably expressing ApoL9 The Apol9-V5 construct was linearized and transfected into B16F10 cells using Lipofectamine 2000. 48 h later, the cells were subcultured into six-well plates and selected with 2 mg mL1
Fig. 1. Subcellular localization of ApoL9. A. Localization of ApoL9 tagged with different epitopes as indicated. Plasmids expressing ApoL9 were electroporated into B16F10 cells. V5and FLAG-tagged ApoL9 were visualized using anti-V5 and anti-FLAG antibodies. B. Localization of ApoL9-V5 in B16F10L9 cells. C. Quantification of relative Apol9 mRNA levels in B16F10 and B16F10L9 by qPCR. Error bars indicate mean ± S.D (n ¼ 3). D. Detection of ApoL9-V5 protein in B16F10L9 cells by western blotting using anti-V5 antibody. a. ApoL9-V5, b. b-actin. E, F, G. Colocalization of ApoL9-V5 in granules with Sqstm1, Lc3b and ubiquitin. Nuclei are stained using DAPI. H. Perinuclear localization of ApoL9 in B16F10L9 cells following treatment with MG132 for 4 h. Two different cells are shown. I. Western blotting of ApoL9-V5 in MG132 treatment using anti-V5 antibody J. Quantification of data in I. Error bars indicate mean ± S.D (n ¼ 2). K. Localization of ApoL9-V5 and 3XFLAG-eEF1a in untreated and MG132-treated B16F10L9 cells.
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puromycin. Ten days later, single colonies were picked and expanded, screened for ApoL9 expression by indirect immunofluorescence and clones with an optimum level of expression were further expanded and frozen. 2.4. Viruses and plaque assay JEV P20778 strain was amplified in brains of suckling mice and propagated in BHK-21 cells. Infections for experiments were carried out at an m.o.i of 10. Plaque assay was performed on porcine kidney (PS) cells. Briefly, ten-fold serial dilutions of virus stock or supernatants from infected cells were prepared in medium without serum and inoculated on to confluent monolayers of PS cells. After 2 h of incubation with intermittent shaking, the inoculum was removed and replaced with DMEM with 2% FBS and 1% low-melting agarose. 72 h later, the monolayers were fixed with 3.7% formaldehyde, the agarose plugs were removed, and the cells were stained with 0.1% crystal violet solution in 20% ethanol. Wells with countable plaques were selected and viral titres were calculated considering the appropriate dilution factors.
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2.5. Animal experiments Animal experiments performed in this study were approved by the Institutional Animal Ethics Committee (IAEC) of the Indian Institute of Science, Bangalore (CAF/Ethics/192/2010). For infection with JEV, BALB/c mice were injected intracranially with 103 p.f.u of virus. Control mice were injected with cell culture medium. Animals were euthanised 4e5 days following infection, when the typical symptoms of advanced encephalitis were evident. Brains and other organs were aseptically harvested and processed for RNA or protein extraction. 2.6. qPCR and western blotting Two-step qPCR was performed by preparing cDNA from RNA samples. RNA was isolated using Tri Reagent (Sigma-Aldrich) and cDNA was synthesised using RevertAid reverse transcriptase (Thermo Fisher Scientific). cDNA samples were appropriately diluted and used for qPCR on the StepOnePlus real time PCR system (Thermo Fisher Scientific). iQ SYBR Green Supermix (Bio-Rad
Fig. 2. Identification of ApoL9 as a PE-binding protein. A. B16F10L9 cells were transfected with Lipofectamine 2000 alone or together with 1 mg of the indicated nucleic acids. The plasmid DNA pBApo-EF1a pur was transfected for 30 min, 1 h or 4 h. ApoL9-V5 was stained with anti-V5 antibody. B. Transfection of pEGFP-C1, expressing EGFP, into B16F10L9 cells using Lipofectamine 2000. C. Schematic representation of MBP-ApoL9 construct. D. Visualization of MBP-ApoL9 in a Coomassie blue-stained SDS-PAGE gel before and after partial cleavage by Factor Xa. E. Demonstration of immunoreactivity of anti-MBP-ApoL9 antibody by western blotting against MBP-ApoL9 (1), MBP (2), ApoL9-V5 present in lysate of B16F10L9 cells (3). Lysate of B16F10 cells (4) served as negative control. FeH. Protein-lipid overlay (PLO) assays. F. Lipofectamine 2000 was serially diluted and spotted on to nitrocellulose and its ability to interact with MBP and MBP-ApoL9 was examined. Quantities in mg are indicated above the blot. G. Different phospholipids were spotted on to nitrocellulose and their ability to interact with MBP and MBP-ApoL9 was examined. Order of lipids: 1. Phosphatidylserine, 2. Phosphatidic acid, 3. Phosphatidylethanolamine, 4. Phosphatidylcholine, 5. Phosphatidylglycerol, 6. Lysophosphatidylethanolamine. The quantities of lipid (mg) are indicated on the left. MBP control bound phosphatidic acid. H. PLO assay of lysates of HEK293T cells transfected with vector alone, vector encoding ApoL9-V5 or ApoL9-GFP by calcium phosphate-mediated transfection. Order of lipids: 1. Dipalmitoylphosphatidylethanolamine, 2. Distearoylphosphatidylethanolamine, 3. Phosphatidylcholine. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Analysis of ApoL9 expression in mouse organs and its effect on JEV titres. A. Quantification of ApoL9 levels in various organs from mice by western blotting using anti-MBPApoL9 antibody (upper panel). Blots stained with Ponceau S (lower panel) served as loading controls. B. Quantification of data in A (upper panel). Data is the average of two independent experiments (n ¼ 2). C. Analysis of Apol9 mRNA levels in different mouse organs by semi-quantitative RT-PCR. D. Quantification of Apol9 mRNA levels in mouse organs by qPCR, normalized to 18s rRNA. Error bars indicate mean ± S.D (n ¼ 2). E. Demonstration of interferon-induced expression of ApoL9-V5 by western blotting in B16F10 cells with
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Laboratories) was used, and relative quantification was done using the DDCt method. Western blotting procedures were performed essentially as in Ref. [26] with minor modifications. Blots were visualized using the ImageQuant LAS 4000 system (GE Healthcare), and quantified using the software ImageJ. 2.7. Immunofluorescence and confocal microscopy Cells grown on coverslips were fixed with 3.7% formaldehyde, washed twice with PBS, permeabilized with 0.1% Triton X-100. After blocking with 5% BSA in PBS, they were incubated with primary antibodies in blocking solution for 2 h. After 3 washes with PBS containing 0.05% Triton X-100, they were incubated with secondary fluorophore-conjugated antibodies (Alexa Fluor 555/488; Thermo Fisher Scientific) in blocking solution for 2 h. After 3 more washes, they were stained with 1 mg/mL DAPI, washed and mounted on antifade. Imaging was done on a Zeiss LSM 510 Meta confocal microscope. Intensity and colour contrast of images were balanced using LSM Image Browser (Carl Zeiss Microimaging) and Adobe Photoshop. 2.8. Protein-lipid overlay assay Purified phospholipids were dissolved in appropriate organic solvents according to the manufacturers' instructions. They were serially diluted in chloroform:methanol:water (1:2:0.8) as described in Takahashi et al. (2006). 1 ml of each dilution was spotted on a nitrocellulose membrane and allowed to dry for 1 h. The membrane was blocked in TBST containing 3% fatty acid-free BSA overnight, followed by incubation with purified protein (2 mg/mL) or cell lysate (50 mg total protein/mL) for 2 h. After 3 washes in TBST, the membranes were incubated with primary and secondary antibodies as in the procedure for western blotting and viewed in a chemiluminescence imager. For assays with cell lysates, HEK293T cells were transfected with vector alone or vectors expressing ApoL9-V5 or ApoL9-GFP by calcium phosphate-mediated transfection. The cells were lysed with a buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, and 0.1% Triton X-100 and the supernatants were clarified by centrifugation. ApoL9 was detected using anti-V5 or anti-GFP antibodies. 3. Results 3.1. ApoL9 localizes to structures similar to aggresome-like induced structures (ALIS) To study the intracellular distribution of ApoL9, plasmids encoding ApoL9 with C- or N-terminal V5 tags, C-terminal 3X-FLAG tag, and C- or N-terminal GFP were electroporated into B16F10 melanoma cells and localization of ApoL9 was examined by indirect immunofluorescence or by GFP fluorescence. ApoL9 containing Nor C-terminal V5 or FLAG tags showed similar subcellular distribution (Fig. 1A). It localized to small puncta scattered diffusely in the cytoplasm, and to sparse larger granules whose number and size varied in different cells. ApoL9 GFP fusion proteins exhibited a slightly different pattern, appearing as larger, more numerous,
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hollow structures scattered throughout the cytoplasm. In view of the relatively large size of GFP, and because the smaller epitope tags displayed identical subcellular distribution patterns, all subsequent studies were carried out with the C-terminal V5-tagged form of ApoL9 (ApoL9-V5). A B16F10 cell line stably expressing ApoL9-V5 was generated (B16F10L9). Indirect immunofluorescence studies indicated that the distribution pattern of ApoL9-V5 in B16F10L9 was similar to that observed by transient transfection (Fig. 1B). The levels of Apol9 mRNA in B16F10L9 cells were quantified by qPCR (Fig. 1C). Protein expression was verified by western blotting (Fig. 1D). Colocalization studies using organelle markers indicated that the small ApoL9 puncta or the large ApoL9-containing granule-like structures do not localize to lysosomes, mitochondria, Golgi apparatus, P-bodies, lipid droplets, or peroxisomes (data not shown). Interestingly, ApoL9 in the large granule-like structures colocalizes with sequestosome-1 (Sqstm1/p62) and Lc3b (the autophagosome marker) (Fig. 1E and F) suggesting that these may be aggresomelike induced structures (ALIS) or similar structures, since the latter proteins are known to localize to ALIS [9,10]. Since ALIS are known to be storage compartments for polyubiquitinated defective ribosomal products, we examined whether these granules stain for ubiquitin. The results indicate that they contain ubiquitin (Fig. 1G). On arresting proteasomal degradation using the inhibitor MG132, there was an increase in granule size as well as congregation of the granules in the perinuclear region (Fig. 1H). Further, MG132 treatment resulted in an increase in ApoL9 protein (Fig. 1I and J), suggesting that the protein might be degraded in normal conditions by the ubiquitin-proteasome system. Eukaryotic translation elongation factor 1A (eEF1a), known to associate with ubiquitinated defective ribosomal products [11], accumulated in these granules (Fig. 1K). Though ALIS and related granules are usually induced by stress conditions, they have been reported to be present at a relatively high background level in some cell lines under non-stress conditions [9]. The case with B16F10 seems to be similar.
3.2. Transfection of nucleic acids using Lipofectamine 2000 dramatically alters ApoL9 distribution in B16F10L9 cells During subcellular localization studies of ApoL9, we observed that transfection of any plasmid into B16F10L9 cells using Lipofectamine 2000 immediately induced rapid, progressive, and dramatic aggregation of ApoL9 (Fig. 2A). ApoL9 localization was altered by lipofection of plasmids, polyinosinic:polycytidilic acid (poly I:C), or sonicated calf thymus DNA but not by transfection reagent alone (Fig. 2A). Since these effects manifested themselves as early as 30 min after transfection, we thought it unlikely that it was a consequence of any transfected protein being expressed. Introduction of plasmids by electroporation or calcium phosphatemediated transfection did not alter ApoL9 localization. Lipofection of a GFP expression vector into B16F10L9 cells led to aggregation of ApoL9 but not GFP (Fig. 2B). Since lipofection of such varied nucleic acid substrates and even empty plasmids resulted in similar changes in ApoL9 distribution, we hypothesised that the protein was likely reacting with some component of the transfection reagent itself, rather than with any of the molecules being transfected. We also observed the same effect when using a few other
anti-MBP-ApoL9 antibody. F. Demonstration of JEV-induced expression of Apol9 mRNA in mouse brain by qPCR. Data is the average of two independent experiments. G. Western blotting forApoL9 in uninfected and JEV-infected brains using anti-MBP-ApoL9 antibody. Virus replication in JEV-infected brains was confirmed by the presence of JEV-NS2b. IFIT3 is a positive control JEV-induced protein [28]. H. Quantification of data in G. I. Demonstration of increased JEV titres in the medium of B16F10L9 cells by plaque assay. One representative plate is shown. J. Quantification of plaques. Data from two individual experiments (1, 2) are shown. Error bars indicate mean ± S.D. *, p 0.05; **, p 0.01; ***, p 0.001. Statistical significance was calculated using an unpaired t-test. K. Immunostaining of ApoL9-V5 in JEV-infected B16F10L9 cells. Virus replication in cells is confirmed by staining for JEV envelope protein.
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liposome-based transfection reagents whose compositions are proprietary. We hypothesised that ApoL9 was interacting with the lipoplexes that are formed bycomplexing nucleic acid with liposomes prior to transfection, whereas transfection reagent alone diffused rapidly through the cytoplasm. Since the neutral lipid DOPE (dioleoylphosphatidylethanolamine) is the most common helper lipid component in several different cationic lipid-based transfection reagents [12], including Lipofectamine (the predecessor of Lipofectamine 2000) [13], we hypothesised that interactions between ApoL9 and DOPE, or more specifically phosphatidylethanolamine (PE), might be responsible for the observed changes in ApoL9 distribution in cells. 3.3. ApoL9 binds phosphatidylethanolamine ApoL9 was expressed as a maltose binding protein fusion (MBPApoL9) in E. coli (Fig. 2C and D) and the purified protein was injected into rabbits to obtain anti-MBP-ApoL9 polyclonal antibodies. Western blotting showed that recombinant MBP-ApoL9 expressed in E. coli, and ApoL9-V5 expressed in B16F10L9 cells were immunoreactive to this antibody (Fig. 2E). To test if ApoL9 binds a component of Lipofectamine 2000, the reagent was serially diluted and spotted on nitrocellulose and a protein-lipid overlay assay was performed. MBP-ApoL9 (but not MBP) bound to Lipofectamine 2000 (Fig. 2F). To examine the ability of ApoL9 to bind to PE, different phospholipids were spotted onto nitrocellulose and the membranes were incubated with either MBP or MBP-ApoL9. After washing the membrane, the protein-lipid complex was detected using the anti-MBP-ApoL9 antibody and HRP-conjugated anti-rabbit antibody. MBP-ApoL9, but not MBP, interacted specifically with PE but not with other phospholipids (Fig. 2G). ApoL9-V5 or ApoL9-GFP overexpressed in HEK293T cells also interacted specifically with both dipalmitoyl-PE and distearoyl-PE (Fig. 2H). 3.4. Some organs show poor correlation between ApoL9 RNA and protein levels Using the anti-MBP-L9 antibody, we examined protein levels of ApoL9 in various mouse organs. Liver and brain expressed high levels of ApoL9 compared with other organs. (Fig. 3A and B). Surprisingly, there was poor correlation between mRNA and protein levels in organs such as brain and kidney. Apol9 mRNA levels in brain are extremely low compared with that of liver (Fig. 3C and D), yet the brain expresses moderately high amounts of ApoL9 protein. Similarly, kidney expresses almost ten times the amount of Apol9 mRNA that is found in the brain, yet the protein levels are very modest (compare Fig. 3B and D). 3.5. ApoL9-expressing cells show increased JEV titres to control cells Apol9 is an interferon- and virus-inducible gene [2]. We demonstrate that ApoL9 protein levels increase several fold in B16F10 cells following treatment with IFN-b (Fig. 3E). In view of ApoL9 binding PE, which was recently shown to be indispensable for the replication of certain positive strand RNA viruses [14], we examined the role of ApoL9 during infection by other such viruses. We chose JEV, a positive strand RNA virus of Flaviviridae. It is relevant to note here that JEV primarily infects the brain, which contains significant amounts of ApoL9 protein (Fig. 3A). JEVinfected mice show strong upregulation of Apol9 mRNA in the brain (Fig. 3F), though protein levels increase only moderately (Fig. 3G and H). To study the effect of ApoL9 on JEV infection, B16F10 cells and B16F10L9 cells were infected by JEV and virus titres in the postinfection culture supernatants were measured by performing
plaque assays on porcine kidney cells. JEV titres were consistently two-fold higher in supernatants from B16F10L9 cells than from B16F10 cells (Fig. 3I and J), suggesting that ApoL9 might function as a weak proviral host factor during JEV infection. Immunostaining indicates that the intracellular distribution pattern of ApoL9 is not significantly altered during JEV infection (Fig. 3K). 4. Discussion Here we report for the first time a key intracellular function for ApoL9, i.e., binding to PE. PE is not only a major component of cell membranes but plays key roles in diverse cellular processes such as autophagy [15], and sorting and trafficking of proteins inhabiting lipid rafts [16]. Some previously described PE-binding proteins bind the hydrophilic head group of PE by means of a cavity formed by the tertiary structure of the protein [17]. Elaborate studies on ApoL9 structure will be required to identify how it binds PE. A role for ApoL9 in autophagy is highly likely, not only because of the mandatory role of PE in autophagy [18] but also because ApoL9 colocalizes with Sqstm1 and Lc3, key proteins involved in autophagy [19]. ApoL9 might also possibly function as a lipid transfer protein involved in trafficking PE inside the cell. It is important to exercise caution while using transfection reagents that may contain DOPE in experiments involving ApoL9, as they inevitably lead to the formation of biological artefacts. Alternate transfection methods should be employed. ApoL9 contributed to increasing JEV titres in infected cells despite its subcellular distribution not being significantly altered during JEV infection, suggesting that it might be a player in some high-turnover, dynamic cellular process that is co-opted by the virus for completing its life cycle. It will be interesting to examine whether the PE-binding property of ApoL9 is essential for its proviral function, because it was reported [14] that certainpositive strand RNA viruses subvert PE to support their replication. Kreit et al. [2] report a narrow antiviral range for ApoL9, based on its effect on various viruses. Since it is possible that the magnitude of the pro- or antiviral effect of ApoL9 varies according to the virus species, it will be interesting to study if ApoL9 has a role during infection by other flaviviruses such as West Nile Virus, Dengue virus and Zika virus. The high levels of ApoL9 protein in liver and brain suggest that ApoL9 might have some housekeeping role in these organs. It is noteworthy that both these organs have a high content of PE and that lipid metabolism plays an important role in their functioning [20,21]. The PEBP (phosphatidylethanolamine-binding protein) family represents the most well-known class of PE-binding proteins. Proteins of this family have been implicated in inhibiting serine proteases [22], regulating the MAP kinase pathway [23], muopioid receptor signalling [24] and abnormal expression in some types of cancer [25]. The PEBP family is well conserved across flowering plants, parasites, nematodes, insects, and mammals. The ApoL proteins, on the other hand, possess a BH3 domain [26] and are only present in higher eukaryotes. It is likely that the members of this family could have specialized, as-yet-undiscovered, functions in these organisms. Since PE is involved in numerous processes in the cell, it is only logical that ApoL9 could have roles in influencing at least some of these processes. This discovery offers incentives to further study this protein and explore its potential functions, and provides new impetus to characterize other members of the ApoL family and investigate if a human homologue of ApoL9 exists. Funding This study was supported by the J. C. Bose fellowship grant # SB/
Please cite this article in press as: T.A. Arvind, P.N. Rangarajan, Mouse Apolipoprotein L9 is a phosphatidylethanolamine-binding protein, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.09.161
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S2/JCB-025/2015 by the Science and Engineering Research Board, New Delhi, India awarded to P.N.R. Institutional support from the Departments of Science and Technology and Biotechnology, New Delhi are acknowledged. Acknowledgements We acknowledge the Confocal Microscopy facility, Biological Sciences, IISc, Bangalore. We thank Mr.Iyappan and Prof. Ram Rajasekharan for help with the protein-lipid overlay assay. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.09.161. References [1] N.M. Page, D.J. Butlin, K. Lomthaisong, et al., The human Apolipoprotein L gene cluster: identification, classification, and sites of distribution, Genomics 74 (2001) 71e78. [2] M. Kreit, D. Vertommen, L. Gillet, et al., The interferon-inducible mouse Apolipoprotein L9 and prohibitins cooperate to restrict Theiler's virus replication, PLoS One 7 (2015) e0133190. [3] E. Kruzel-Davila, W.G. Wasser, S. Aviram, et al., ApoL1nephropathy: from gene to mechanisms of kidney injury, Nephrol. Dial. Transplant. 31 (2015) 349e358. [4] B. Vanhollebeke, E. Pays, The function of apolipoproteins L, Cell. Mol. Life Sci. 63 (2006) 1937e1944. [5] G. Wan, S. Zhaorigetu, Z. Liu et al. Apolipoprotein L1, a novel Bcl-2 Homology domain 3-only lipid-binding protein, induces Autophagic cell death, J. Biol. Chem. (283) 21540e21549. [6] Z. Liu, H. Lu, Z. Jiang, et al., Apolipoprotein L6, a Novel Proapoptotic Bcl-2 homology 3-Only protein, induces mitochondria-mediated apoptosis in cancer cells, Mol. Cancer Res. 3 (2005) 21e31. [7] M. Kreit, S. Paul, L. Knoops, et al., Inefficient type I interferon-mediated antiviral protection of primary mouse neurons is associated with the lack of Apolipoprotein L9 expression, J. Virol. 88 (2014) 3874e3884. [8] L. Sun, H. Miyoshi, S. Origanti, et al., Type I interferons link viral infection to enhanced epithelial turnover and repair, Cell Host Microbe 17 (2015) 85e97. [9] J. Szeto, N. A. Kaniuk, V. Canadien, et al., ALIS are stress-induced protein storage compartments for substrates of the proteasome and Autophagy, Autophagy 2 (2006) 189e199. [10] G. Bjørkøy, T. Lamark, A. Brech, et al., p62/Sqstm1 forms protein aggregates degraded by autophagy and has a protective effect onhuntingtin-induced cell death, J. Cell Biol. (2005) 603e614. [11] A.B. Meriin, N. Zaarur, M.Y. Sherman, et al., Association of translation factor eEF1A with defective ribosomal products generates a signal for aggresome formation, J. Cell Sci. 125 (2012) 2665e2674.
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Please cite this article in press as: T.A. Arvind, P.N. Rangarajan, Mouse Apolipoprotein L9 is a phosphatidylethanolamine-binding protein, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.09.161