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Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide
Biochemical and Biophysical Research Communications xxx (2018) 1e8
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Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide Yohei Ishibashi a, *, Makoto Ito a, Yoshio Hirabayashi b a
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan b Laboratory for Molecular Membrane Neuroscience, RIKEN Brain Science Institute, Wako-shi, Saitama, 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 March 2018 Accepted 4 April 2018 Available online xxx
Phosphoinositides mediate a large number of signaling processes in mammalian cells. Here, we report that phophatidylinositol-4-phosphate (PtdIns(4)P) downregulates the cellular glucosylceramide (GlcCer) level by inhibiting the interaction between GlcCer synthase (UGCG) and UDP-glucose in the Golgi apparatus. In this study, we used two PH domain probes to bind phosphoinositides; one derived from FAPP1 for targeting to the Golgi PtdIns(4)P and the other from PLC d for targeting to the plasma membrane PtdIns(4,5)P2. The levels of GlcCer and lactosylceramide, but not of sphingomyelin (SM), were increased following expression of the FAPP1 PH domain in cells, accompanied by an increase in UGCG activity. However, no elevated GlcCer level was observed after expression of the PLC d PH domain. PtdIns(4)P inhibited UGCG activity, but not SMS activity, in a concentration-dependent manner, and UGCG activity was restored by the addition of UDP-glucose in the reaction mixture. These results indicate that PtdIns(4)P inhibits UGCG activity by competing with UDP-glucose. We conclude that the increase in UGCG activity due to the expression of the FAPP1 PH domain was caused by an attenuation of the inhibitory effect of PtdIns(4)P on UGCG. This study provides new insights into the regulation of GlcCer synthesis by PtdIns(4)P in the Golgi apparatus. © 2018 Elsevier Inc. All rights reserved.
Keywords: Glycosphingolipid Glucosylceramide Phosphoinositide PH domain Sugar nucleotide
1. Introduction Glycosphingolipids (GSLs), amphipathic compounds consisting of oligosaccharides and ceramides, are ubiquitous in the outer leaflet of the plasma membrane and are involved in a large number of cellular processes including signal transduction, membrane trafficking, cytoskeletal organization, and pathogen entry [1,2]. Most mammalian GSLs are generated from glucosylceramide (GlcCer) as a precursor. GlcCer is synthesized by GlcCer synthase (UDP-glucose:ceremide glucosyltransferase; UGCG, EC 2.4.1.80) from ceramide and UDP-glucose (UDP-Glc) at the cytosolic surface of the Golgi apparatus [3]. GlcCer synthesis is an integral step for determining cellular GSL levels; however, how cells regulate GlcCer
Abbreviations: CERT, ceramide transport protein; GlcCer, glucosylceramide; GSL, glycosphingolipid; LacCer, lactosylceramide; MRM, multiple reaction monitoring; NBD, 7-nitro-2,1,3-benzoxadiazole; PH, pleckstrin homology; PtdIns(4)P, phophatidylinositol-4-phosphate; UGCG, UDP-glucose ceramide glucosyltransferase; UDPGlc, UDP-glucose; SM, sphingomyelin. * Corresponding author. E-mail address: [email protected] (Y. Ishibashi).
synthesis through the regulation of UGCG activity is largely unknown. Since abnormal accumulations of GlcCer and GlcCer-based GSLs cause several diseases, such as Garucher disease, Parkinson disease, and polycystic kidney disease [4e7], elucidation of the mechanism regulating UGCG may provide new and more effective treatments for these diseases. Ceramide is transported from the ER to the Golgi apparatus and is converted to GlcCer and sphingomyelin (SM) [8]. Ceramide is transported between these two organelles mainly by membrane vesicles [9]. An alternative transport pathway is mediated by ceramide transport protein (CERT), a protein factor responsible for non-vesicular transport of ceramide [10]. CERT contains a pleckstrin homology (PH) domain that binds phophatidylinositol-4phosphate (PtdIns(4)P), a serine repeat motif, two phenylalanines in an acidic tract (FFAT) motif that bind to the ER resident membranous protein, and a steroidogenic acute regulatory proteinrelated lipid transfer (START) domain that recognizes ceramide. CERT binds to the Golgi-abundant PtdIns(4)P and transfers ceramide from the ER to the trans-Golgi through ER-Golgi membrane contact sites [11]. The interaction between PtdIns(4)P and the PH domain of CERT is indispensable for the transport of ceramide [12].
https://doi.org/10.1016/j.bbrc.2018.04.039 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: Y. Ishibashi, et al., Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.039
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Please cite this article in press as: Y. Ishibashi, et al., Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.039
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Ceramides transported by CERT are mainly utilized for the synthesis of SM [10,13]. Phosphoinositides are important membranous lipids that mediate a large number of membrane-associated signaling processes [14]. PH domains are composed of 100e120 amino acids that recognize phosphoinositides [15]. The fluorescent protein-fused PH domain is utilized to study the dynamics of phophoinositides in living cells [16]. For instance, use of the FAPP1 PH domain, which recognizes PtdIns(4)P, revealed that PtdIns(4)P mainly localizes in the Golgi apparatus [17]. Although these probes are useful, they might capture phosphoinositide and compete with the phosphoinositide binding of endogenous effectors [18]. Since PtdIns(4)P is involved in sphingolipid metabolism [12], we hypothesized that phosphoinositide probes may affect cellular sphingolipid levels. In this study, we found that PtdIns(4)P inhibits UGCG activity and the binding of the FAPP1 PH domain to PtdIns(4)P attenuates the interaction between PtdIns(4)P and UDP-Glc, resulting in an increase in GlcCer synthesis. This paper provides a new insight into the relationship between GlcCer synthesis and phosphoinositides, which may be involved in the regulation of cellular GlcCer and GlcCer-based GSLs levels. 2. Materials and methods 2.1. Materials Pre-coated Silica gel 60 TLC plates were purchased from Merck, Germany. Fluorescent substrate 7-nitro-2,1,3-benzoxadiazole (NBD) C6-Ceramide and NBD C6-Ceramide complexed to BSA was purchased from Life technologies, USA. Phosphatidylinositol (PI) and phophatidylinositol-4-phosphate (PtdIns(4)P) were purchased from Matreya, USA. Antibodies against DYKDDDDK Tag (Rabbit IgG), 6xHis tag (Mouse IgG), and GFP (Mouse IgG) were purchased from Cell Signaling Technology (USA), a-tubulin was purchased from Sigma Aldrich (Germany), anti-b-actin Mouse IgG, Alexa Fluor 568 Anti-Mouse IgG, and Alexa Fluor 488 Anti-Rabbit IgG were purchased from Life Technologies (USA). A plasmid for the expression of the CFP and YFP-tagged FAPP1 PH domain (aa 1e101) was a gift from Dr. Michiyuki Matsuda, Kyoto University [19], and GFP-C1-PLC d-PH was a gift from Tobias Meyer (Addgene plasmid # 21179) [20]. The plasmid pEGFP-N3 was purchased from Takara Bio Inc. (Japan). A plasmid encoding C-terminal FLAG-tagged UGCG was prepared as described in Ref. [21]. 2.2. Construction of expression vectors To make the GFP-tagged FAPP1 probe, the lipid binding domain of GFP-C1-PLC d-PH was replaced by the FAPP1 PH domain (aa 1e101) that was obtained from a plasmid encoding the CFP/YFPtagged FAPP1 PH domain. PCR was carried out using mouse kidney cDNA (GenoStaff, Japan) as a template and the following two primers for amplification of splicing variants of FAPP2: FlagFAPP2v1-S (50 - TGACGATGACAAGCTTGAGGGCGTGCTGTACAAG-30 ), Flag-FAPP2v2-S (50 -TGACGATGACAAGCTTGCAGTCTGTGAGATTCAAGTTC-30 ), and Flag-FAPP2-A (50 -TCGCGGCCGCAAGCTTTCACACCACCTCATCAGATTCC-30 ). The conditions for amplification were 30 cycles (each consisting of denaturation at 98 C for 10 s,
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annealing at 55 C for 30 s, and extension at 72 C for 60 s) using Phusion polymerase (Thermo Scientific). The amplified products were inserted into HindIII-digested p3xFLAG-CMV-10 (Sigma Aldrich) to generate N-terminal 3xFLAG-tagged proteins using an In-Fusion HD Cloning kit (Clontech). 2.3. Cell culture and gene transfection Hek 293 and CHO-K1 cells were cultured in Dulbecco's modified Eagle Medium (DMEM), containing 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 C in 5% CO2. Hek 293 cells were transfected with TurboFect Transfection Reagent (Thermo Scientific), and CHO-K1 cells were transfected with X-tremeGENE 9 DNA Transfection Reagent (Roche Diagnostic) according to the manufacturer's instructions, respectively. 2.4. Immunoblot analysis Transfected cells were lysed in ice-cold lysis buffer (20 mM Tris HCl buffer, pH7.5, containing 150 mM NaCl, 1 mM EDTA, 1% NP-40, phosphatase and protease inhibitor cocktails (Roche diagnostics)) by sonication for 45 s. Cell debris was removed by centrifugation (18,000 g for 10 min at 4 C). The protein amount of the supernatant was determined by a bicinchoninic acid protein assay (Pierce) with bovine serum albumin as a standard. Equal amounts of cellular protein were separated by SDS-PAGE using a MiniPROTEAN TGX Gel (BioRad), and then blotted to a PVDF membrane by Trans Blot Turbo (BioRad). The membrane was blocked in TBS-T containing 5% BSA, then incubated with antibodies against DYKDDDDK tag (1:6000), GFP (1:5000), b-actin (1:10000), and atublin (1:20000) for over 12 h at 4 C. The blot was washed with TBS-T and then incubated with HRP-conjugated anti-mouse or rabbit IgG antibody (1:10000) (Cell Signaling Technology). The blot was washed again with TBS-T. The protein was detected by chemiluminescence using Luminate Forte Western HRP substrate (Millipore) and LAS-3000 Luminescence Image Analyzer (Fujifilm). 2.5. Measurement of the UGCG activity The intracellular UGCG assay was performed as previously described with some modification [21,22]. After transfection, cells were incubated for 24 h in a 24-well plate with DMEM 10% FBS, then were switched to 250 ml of DMEM 10% FBS containing 0.5 mM NBD C6-Ceramide complexed to BSA. After a 1 h incubation at 37 C, cells were rinsed with ice cold PBS, scraped with 50 ml of ice cold water and transferred to a 1.5 ml tube. Lipids were extracted by 190 ml of chloroform/methanol (1/2, v/v). After incubation at room temperature for 10 min, 62.5 ml of water and chloroform were added, then centrifuged at 12,000 g for 5 min. The lower phase was collected and dried with a speed vac concentrator. The dried sample was dissolved in 20 ml of chloroform/methanol (1/2, v/v), then 3 ml of the sample was applied to a TLC plate, which was developed with chloroform/methanol/water (65/25/4, v/v/v). The reaction products from the cell-based assay and in vitro assay were visualized by an LAS-3000 equipped with a blue-light-emitting diode (460 nm EPI) and an Y515-Di filter, and were quantified with Image J 1.43u software (NIH). The extent of the synthesis of
Fig. 1. Effect of FAPP1-PH expression on cellular sphingolipid levels and UGCG activity. (A) Immunoblot analysis showing protein expression of the fluorescent protein-fused FAPP1PH domain in HEK293 cells. (B) Quantification of cellular sphingolipid levels of FAPP1-PH expressing cells by LC-ESI MS/MS analysis. The bar graphs show the amount of cellular GlcCer, LacCer, ceramide, and sphingomyelin. (C) TLC analysis showing intracellular activities of UGCG and SMS in UGCG-FLAG- or FAPP1-PH-expressing cells. TLC was developed with a solution of chloroform/methanol/water (65/25/4, v/v/v) for 25 min. (D) Effect of FAPP1-PH expression on intracellular GlcCer synthase (UGCG, left panel) and sphingomyeline synthase (SMS, right panel) activities. NBD C6-ceramide conjugated to BSA was added to cells and incubated for 60 min, then analyzed by TLC. (D) Sugar nucleotide levels in FAPP1PH-expressing cells. Sugar nucleotides were extracted from FAPP1-PH-expressing cells and then subjected to ion-pair reversed-phase HPLC, as described in the Materials and Methods. Error bars represent means ± S.D. of at least three separate experiments.
Please cite this article in press as: Y. Ishibashi, et al., Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.039
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Fig. 2. Localization of the phosphoinositide probe affecting cellular sphingolipid levels. (A) Localization of GFP, GFP-PLC d-PH, GFP-FAPP1 PH, and UGCG-GFP expressed in CHO-K1 cells. Cells transfected with GFP (control, left upper), GFP-PLC d-PH (right upper), GFP-FAPP1-PH (left lower), or UGCG-GFP (right lower) were observed by fluorescent microscopy. Nuclei were stained with Bisbenzimide H33342 Fluorochrome Trihydrochloride. (B) Immunoblot analysis showing the protein expression of GFP (26.9 kDa), GFP-PLC d-PH (49.6 kDa), and GFP-FAPP1-PH (40.1 kDa) in CHO-K1 cells. (C) Changes of the ratio of GlcCer to sphingomyelin in FAPP1-PH-overexpressing CHO-K1 cells. Expression vectors for GFP, GFP-PLC d-PH, or GFP-FAPP1-PH were transfected into CHO-K1 cells and incubated for 62.5 h, then lipids were extracted. The GlcCer/sphingomyeline ratio was determined by LC-ESI MS/MS analysis. Error bars represent means ± S.D. of six separate experiments. (D) Immunoblot analysis showing the protein expression of FLAG-tagged FAPP2 variant 1 (v1) and variant 2 (v2). (E) Effect of FAPP2_v1 and v2 overexpression on intracellular UGCG activity. Error bars represent means ± S.D. of at least three separate experiments.
Please cite this article in press as: Y. Ishibashi, et al., Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.039
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Fig. 3. Inhibition of UGCG activity by phosphoinositide. (A) Inhibitory effect of PtdIns(4)P on UGCG activity. In vitro activities of UGCG and SMS of cell lysates were measured using fluorescent ceramide as a substrate. Several concentrations of PtdIns(4)P were added to the reaction mixture. (B) Difference in the inhibitory effect on UGCG and SMS activities between PtdIns and PtdIns(4)P. (D) Effect of several phosphoinositides on UGCG and SMS activities. (E) Repairing of UGCG activity inhibited by PtdIns(4)P by adding excessive UDPglucose. Several concentrations of UDP-glucose were added to the reaction mixture of the UGCG assay with or without 20 mM PtdIns(4)P.
NBD-labeled GlcCer was calculated as follows: synthesis (%) ¼ (peak area for NBD C6-GlcCer) x 100/(peak area for NBD C6Cer þ peak area for NBD C6-GlcCer þ peak area for NBD C6-SM). In vitro enzymatic activity was measured by a previously described method [23]. C6 NBD-ceramide and different concentrations of PtdIns(4)P were incubated with HEK293 cell lysate at 37 C for 90 min. 2.6. Observation of cellular localization of phosphoinositide probes and UGCG CHO-K1 cells expressing GFP-tagged phosphoinositide probes and UGCG were observed under a fluorescence microscope DMi8 equipped with a DFC3000G camera (Leica Microsystems). Nuclei was stained using Bisbenzimide H33342 Fluorochrome Trihydrochloride (Nacalai tesque, Japan). CHO-K1 cells were transfected with the CFP/YFP-tagged FAPP1 PH domain and C-terminal FLAGtagged UGCG to check intracellular colocalization. After 24 h of transfection, cells were fixed in 3% paraformaldehyde for 15 min and then washed with PBS. The fixed cells were blocked in Blocking One Histo (Nacalai Tesque), then incubated with antibodies against DYKDDDDK tag (1:500) and GFP (1:500) for 12 h at 4 C. After
incubation, cells were washed by PBS and incubated with Alexa Fluor 568 Anti-Mouse IgG (1:500) and Alexa Fluor 488 Anti-Rabbit IgG (1:500) for 1 h. Cells were mounted in Immu-Mount reagent (Thermo Scientific) and observed under a confocal laser-scanning microscope (FV1000, Olympus). 2.7. Lipid extraction and quantification of sphingolipids by LC-ESI MS/MS Cells were washed once with cold PBS, collected in 100 ml of cold PBS, then homogenized with sonication. Part of the sample (5 ml) was used in the bicinchoninic acid protein assay to determine the amount of protein. Total lipids were extracted by adding 375 ml of chloroform/methanol (1/2, v/v) containing 62.5 pmol of each component of Ceramide/Sphingoid Internal Standard Mixture II (Avanti Polar Lipids, Inc.) from the rest of the sample. The singlephase mixture was incubated at 48 C for 16 h. After cooling, 125 ml of water and chloroform were added. The lower phase was collected and dried with a speed vac concentrator. The dried sample was suspended in chloroform/methanol (2/1, v/v) containing 0.1 N KOH, incubated for 2 h at 37 C, and neutralized with an equal amount of acetic acid, then water was added. The lower
Please cite this article in press as: Y. Ishibashi, et al., Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.039
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Fig. 4. Localization of UGCG and PtdIns(4)P. (A) Localization of UGCG-FLAG and GFP-FAPP1-PH. Cells transfected with UGCG-FLAG and GFP-FAPP1-PH were immunostained with anti-FLAG antibody and anti-GFP antibody, as described in the Materials and Methods. Scale bar represents 10 mm. (B) Predicted mechanism of FAPP1-PH domain expressionemediated activation of UGCG activity. In the normal condition, PtdIns(4)P nearby to UGCG interferes with the utilization of UDP-glucose to synthesize GlcCer by UGCG. GlcCer might by synthesized by UGCG localizing at PtdIns(4)P-less regions in the Golgi apparatus in the normal condition. In the FAPP1-PH domain-expressing condition, PtdIns(4)P is covered by the PH domain that may attenuate the inhibitory effect of PtdIns(4)P on UGCG activity, leading the apparent activation of UGCG activity and increase of the GlcCer level.
phase was withdrawn and dried, then resuspended in 200 ml of methanol, sonicated for 10 s, centrifuged at 14,000 g for 5 min, and the supernatant transferred to vials. The amount of GlcCer, LacCer, Cer, and SM were analyzed by LC-ESI MS/MS using a triple quadruple mass spectrometer 4000Q TRAP (SCIEX) coupled to an Agilent 1100 series HPLC system (Agilent). A binary solvent gradient with a flow rate of 0.2 ml/min was used to separate sphingolipids by normal-phase chromatography using an InertSustain NH2 (2.1 150 mm, 5 mm bead size, GL Science, Japan). The gradient was started at 20% buffer B (methanol/water/formic acid, 89/9/1, v/v/v, with 20 mM ammonium formate) in buffer A (acetonitrile/methanol/formic acid, 97/2/1, v/v/v, with 5 mM ammonium formate). The gradient reached 100% B in 4 min and was maintained at 100% B for 2 min. Finally, the gradient was returned to the starting condition, and the column was equilibrated for 5 min before the next run. Sphingolipids containing C16:0, C18:0, C20:0, C22:0, C24:0, and C24:1 fatty acids were detected using a multiple reaction monitoring (MRM) method, as described in Ref. [24]. 2.8. Quantification of nucleotide sugars The quantification method was performed as previously described with some modification [25]. Cells were washed with cold PBS, collected in 300 ml of cold PBS, and spiked with GDP-Glc as an internal standard, and then sonicated using a Handy Sonic Disruptor for 10 s. A part of the sample (5 ml) was applied to the bicinchoninic acid protein assay. Ice cold ethanol (900 ml) was added to the sample. The extract was centrifuged at 16,000 g for 10 min at 4 C, and supernatant was lyophilized. The freeze-dried sample was applied to an Envi-Carb column (Supelco Inc.), and purified as described in Ref. [25]. After purification, nucleotide
sugars were separated and detected by ion-pair reversed-phase HPLC. 2.9. Statistical analysis All statistical analyses were performed using unpaired twotailed Student's t-tests, and all data are expresses as means and standard deviation for at least three separate experiments. Statistical significance is indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001. 3. Result and discussion Effects of FAPP1-PH domain expression on cellular sphingolipid levels - The fluorescent protein-fused PH domain is utilized to study the dynamics of phophoinositides in living cells [18]. To examine the effect of FAPP1-PH domain expression on sphingolipid metabolism, total lipids were extracted from CFP/YFP-labeled FAPP1-PH expressing cells (Fig. 1A) and subjected to LC-ESI-MS/MS analysis after alkaline treatment. We found that cellular GlcCer and lactosylceramide (LacCer) levels increased in FAPP1-PH-expressing cells (Fig. 1B). Since CERT-mediated ceramide transport is inhibited by FAPP1-PH domain expression [12], we predicted that the SM level may decrease in FAPP1-PH domain-expressing cells. Unexpectedly, however, the SM level was almost the same as that of the mock transfectants (Fig. 1B). On the other hand, the ceramide level increased in FAPP1-PH domain-expressing cells (Fig. 1B), suggesting that ceramide synthesis might be upregulated to compensate for the decrease in CERT-mediated ceramide at Golgi apparatus. Increase of UGCG activity by FAPP1-PH domain expression e First, we examined the possibility that increased GlcCer and LacCer levels are caused by the activation of UGCG. We found that the
Please cite this article in press as: Y. Ishibashi, et al., Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.039
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intracellular UGCG activity significantly increased in the FAPP1-PH domain-expressing cells without affecting sphingomyelin synthase (SMS) activity (Fig. 1C and D). These results suggest that the FAPP1PH domain increased UGCG activity and thereby the GlcCer level increased. Next, we investigated the mechanism of how the FAPP1-PH domain enhances intracellular UGCG activity. Since GlcCer is synthesized by UGCG utilizing ceramide and UDP-Glc as substrates, a change in the level of these substrates could affect UGCG activity [21]. Thus, various sugar nucleotide levels were compared between mock and FAPP1-PH domain-expressing cells. The levels of sugar nucleotides, including UDP-Glc, were not changed by expression of the FAPP1-PH domain (Fig. 1E), indicating that an increase in GlcCer was not caused by changes to sugar nucleotide levels. Effects of the expression of PtdIns(4)P and PtdIns(4,5)P2 recognizing PH probes eWe found that expression of FAPP1-PH domain recognizing PtdIns(4)P increases intracellular UGCG activity and the GlcCer level. In order to investigate whether this effect is specific to the FAPP1-PH domain or common to other phosphoinositide probes, the GFP-tagged phospholipase C (PLC) d-PH domain was expressed in CHO-K1 cells (Fig. 2A and B). The GFP-tagged FAPP1PH domain shows almost the same intracellular distribution of GFP-tagged UGCG; however, the GFP-PLC d-PH domain was detected on the plasma membrane (Fig. 2A), because the FAPP1-PH domain was transported to the PtdIns(4)P-rich Golgi membranes while the PLC d-PH domain was transported to the PtdIns(4,5)P2rich plasma membrane, as previously reported [20]. Importantly, the cellular GlcCer level increased in cells expressing the FAPP1-PH domain, but not the PLC-d-PH domain (Fig. 2C). Next, we examined the effects of FAPP2 PH domains on UGCG activity. FAPP2, known as a GlcCer-transporting protein, also contains a PH domain that recognizes PtdIns(4)P [26]. Mice have two splicing variants of FAPP2 (FAPP2_v1 and v2) (Supplemental Fig. 1). FAPP2_v1 has a full-length PH domain while FAPP2_v2 has a shorter PH domain lacking the PtdIns(4)P-contacting motif (Supplemental Fig. 1). We expressed the FLAG-tagged FAPP2_v1 and v2 independently in Hek293 cells (Fig. 2D). Cell-based UGCG assay revealed that intracellular UGCG activity increased in both FAPP2 PH domain-overexpressing cells (Fig. 2E); however, the increase in UGCG activity by FAPP2_v2 was much weaker than that by FAPP2_v1 (Fig. 2E). These findings indicate that the contacting motif of FAPP2_v1 is important for the increase in UGCG activity, and suggests that the upregulation of UGCG activation requires the interaction between the PH domain and PtdIns(4)P. Inhibition of UGCG activity by PtdIns(4)P e PtdIns(4)P is synthesized from PtdIns at the surface of the Golgi apparatus by phosphtidylinositol 4-kinase (PI4K) [27]. In mammals, GlcCer is also synthesized at the surface of the Golgi apparatus by UGCG, in which a catalytic domain is present at the cytosolic face of the Golgi membrane [3]. Therefore, PtdIns(4)P and UGCG are expected to interact with each other at the Golgi membranes. Interestingly, we found that PtdIns(4)P inhibited in vitro UGCG activity in a concentration-dependent manner (Fig. 3A). However, SMS activity was constant in the presence of PtdIns(4)P. The inhibitory effect of PtdIns(4)P on UGCG was significantly greater than that of PtdIns, indicating that the phosphoryl head group of PtdIns(4)P is important for the suppression of UGCG activity (Fig. 3B). Inhibitory effects of other phosphoinositides, such as PtdIns(3)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 on UGCG activity were similar to PtdIns (4)P (Fig. 3C), indicating that one phosphate group is enough to inhibit UGCG activity even at a different position. The inhibitory effect of PtdIns(4)P on UGCG activity was restored by increasing the UDP-Glc concentration in the reaction mixture (Fig. 3D), suggesting that PtdInd(4)P inhibits UGCG activity by competing with UDP-Glc.
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Localization of UGCG and PtdIns(4)P ePtdIns(4)P inhibited UGCG activity in vitro, and thus, we examined whether UGCG interacts with PtdIns(4)P intracellularly. To clarify the localization of PtdIns(4)P and UGCG, a fluorescent protein-fused FAPP1-PH domain and FLAG-tagged UGCG were co-expressed in CHO-K1 cells. As a result, it was found that a part of UGCG was colocalized with the FAPP1-PH domain, suggesting that PtdIns(4)P was partly colocalized with UGCG (Fig. 4A). These results indicate that a part of UGCG, if not all, can interact with PtdIns(4)P at the Golgi membrane. Why does overexpression of FAPP1-PH increase UGCG activity, and how does PtdIns(4)P disturb the utilization of UDP-Glc by UGCG? We consider that PtdIns(4)P obstructs the use of UDP-Glc by UGCG on the Golgi membrane, because the negative charge of phosphate groups in UDP-Glc and PtdIns(4)P repel each other (Fig. 4B). Overexpression of the PH domain covered the negative charge of PtdIns(4)P, allowing UGCG to use UDP-Glc efficiently (Fig. 4B). Consistent with our results, GlcCer synthesis was upregulated by decreasing the PtdIns(4)P level in the Golgi apparatus through the activation of PKD and PtdIns(4)P phosphatase Sac1 [28]. In this study, we revealed that Golgi-localized PtdIns(4)P inhibits UGCG activity by blocking the interaction of UGCG with UDPGlc, suggesting that PtdIns(4)P is involved in the regulation of cellular GlcCer and GlcCer-based GSLs levels in the Golgi apparatus. Acknowledgements We are grateful to the Support Unit for Bio-material Analysis, RIKEN Brain Science Institute Research Resources Center, for help with the nucleotide sequencing analyses. This work was supported in part by the RIKEN Special Postdoctoral Researchers Program (to Y. I.), and a Grant-in-Aid for Young Scientists (B) (to Y. I.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (24770198). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.04.039. Author contributions YI and YH designed the study; YI performed and analyzed all of the experiments in this study; YI and MI wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript. References [1] R. Jennemann, H.J. Grone, Cell-specific in vivo functions of glycosphingolipids: lessons from genetic deletions of enzymes involved in glycosphingolipid synthesis, Prog. Lipid Res. 52 (2013) 231e248. [2] K. Hanada, Sphingolipids in infectious diseases, Jpn. J. Infect. Dis. 58 (2005) 131e148. [3] Y. Ishibashi, A. Kohyama-Koganeya, Y. Hirabayashi, New insights on glucosylated lipids: metabolism and functions, Biochim. Biophys. Acta 1831 (2013) 1475e1485. [4] G.A. Grabowski, Phenotype, diagnosis, and treatment of Gaucher's disease, Lancet 372 (2008) 1263e1271. [5] E. Sidransky, G. Lopez, The link between the GBA gene and parkinsonism, Lancet Neurol. 11 (2012) 986e998. [6] M. Fuller, Sphingolipids: the nexus between Gaucher disease and insulin resistance, Lipids Health Dis. 9 (2010) 113. [7] T.A. Natoli, L.A. Smith, K.A. Rogers, et al., Inhibition of glucosylceramide accumulation results in effective blockade of polycystic kidney disease in mouse models, Nat. Med. 16 (2010) 788e792. [8] A.H. Futerman, H. Riezman, The ins and outs of sphingolipid synthesis, Trends Cell Biol. 15 (2005) 312e318.
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[9] J.C. Holthuis, T.P. Levine, Lipid traffic: floppy drives and a superhighway, Nat. Rev. Mol. Cell Biol. 6 (2005) 209e220. [10] K. Hanada, K. Kumagai, S. Yasuda, et al., Molecular machinery for nonvesicular trafficking of ceramide, Nature 426 (2003) 803e809. [11] T. Yamaji, K. Hanada, Sphingolipid metabolism and interorganellar transport: localization of sphingolipid enzymes and lipid transfer proteins, Traffic 16 (2015) 101e122. [12] B. Toth, A. Balla, H. Ma, et al., Phosphatidylinositol 4-kinase IIIbeta regulates the transport of ceramide between the endoplasmic reticulum and Golgi, J. Biol. Chem. 281 (2006) 36369e36377. [13] M. Fukasawa, M. Nishijima, K. Hanada, Genetic evidence for ATP-dependent endoplasmic reticulum-to-Golgi apparatus trafficking of ceramide for sphingomyelin synthesis in Chinese hamster ovary cells, J. Cell Biol. 144 (1999) 673e685. [14] T. Balla, Phosphoinositides: tiny lipids with giant impact on cell regulation, Physiol. Rev. 93 (2013) 1019e1137. [15] M.A. Lemmon, Membrane recognition by phospholipid-binding domains, Nat. Rev. Mol. Cell Biol. 9 (2008) 99e111. [16] C.P. Downes, A. Gray, J.M. Lucocq, Probing phosphoinositide functions in signaling and membrane trafficking, Trends Cell Biol. 15 (2005) 259e268. [17] A. Godi, A. Di Campli, A. Konstantakopoulos, et al., FAPPs control Golgi-to-cellsurface membrane traffic by binding to ARF and PtdIns(4)P, Nat. Cell Biol. 6 (2004) 393e404. [18] P. Varnai, T. Balla, Visualization and manipulation of phosphoinositide dynamics in live cells using engineered protein domains, Pflügers Archiv 455 (2007) 69e82. [19] T. Nishioka, K. Aoki, K. Hikake, et al., Rapid turnover rate of phosphoinositides at the front of migrating MDCK cells, Mol. Biol. Cell 19 (2008) 4213e4223.
[20] T.P. Stauffer, S. Ahn, T. Meyer, Receptor-induced transient reduction in plasma membrane PtdIns(4,5)P2 concentration monitored in living cells, Curr. Biol. 8 (1998) 343e346. [21] Y. Ishibashi, Y. Hirabayashi, AMP-activated protein kinase suppresses biosynthesis of glucosylceramide by reducing intracellular sugar nucleotides, J. Biol. Chem. 290 (2015) 18245e18260. [22] V. Gupta, G.A. Patwardhan, Q.J. Zhang, et al., Direct quantitative determination of ceramide glycosylation in vivo: a new approach to evaluate cellular enzyme activity of glucosylceramide synthase, J. Lipid Res. 51 (2010) 866e874. [23] Y. Hayashi, Y. Horibata, K. Sakaguchi, et al., A sensitive and reproducible assay to measure the activity of glucosylceramide synthase and lactosylceramide synthase using HPLC and fluorescent substrates, Anal. Biochem. 345 (2005) 181e186. [24] R.L. Shaner, J.C. Allegood, H. Park, et al., Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers, J. Lipid Res. 50 (2009) 1692e1707. [25] K. Nakajima, S. Kitazume, T. Angata, et al., Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC, Glycobiology 20 (2010) 865e871. [26] G. D'Angelo, T. Uemura, C.C. Chuang, et al., Vesicular and non-vesicular transport feed distinct glycosylation pathways in the Golgi, Nature 501 (2013) 116e120. [27] T.R. Graham, C.G. Burd, Coordination of Golgi functions by phosphatidylinositol 4-kinases, Trends Cell Biol. 21 (2011) 113e121. [28] S. Capasso, L. Sticco, R. Rizzo, et al., Sphingolipid metabolic flow controls phosphoinositide turnover at the trans-Golgi network, EMBO J. 36 (2017) 1736e1754.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide Yohei Ishibashi a, *, Makoto Ito a, Yoshio Hirabayashi b a
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan b Laboratory for Molecular Membrane Neuroscience, RIKEN Brain Science Institute, Wako-shi, Saitama, 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 March 2018 Accepted 4 April 2018 Available online xxx
Phosphoinositides mediate a large number of signaling processes in mammalian cells. Here, we report that phophatidylinositol-4-phosphate (PtdIns(4)P) downregulates the cellular glucosylceramide (GlcCer) level by inhibiting the interaction between GlcCer synthase (UGCG) and UDP-glucose in the Golgi apparatus. In this study, we used two PH domain probes to bind phosphoinositides; one derived from FAPP1 for targeting to the Golgi PtdIns(4)P and the other from PLC d for targeting to the plasma membrane PtdIns(4,5)P2. The levels of GlcCer and lactosylceramide, but not of sphingomyelin (SM), were increased following expression of the FAPP1 PH domain in cells, accompanied by an increase in UGCG activity. However, no elevated GlcCer level was observed after expression of the PLC d PH domain. PtdIns(4)P inhibited UGCG activity, but not SMS activity, in a concentration-dependent manner, and UGCG activity was restored by the addition of UDP-glucose in the reaction mixture. These results indicate that PtdIns(4)P inhibits UGCG activity by competing with UDP-glucose. We conclude that the increase in UGCG activity due to the expression of the FAPP1 PH domain was caused by an attenuation of the inhibitory effect of PtdIns(4)P on UGCG. This study provides new insights into the regulation of GlcCer synthesis by PtdIns(4)P in the Golgi apparatus. © 2018 Elsevier Inc. All rights reserved.
Keywords: Glycosphingolipid Glucosylceramide Phosphoinositide PH domain Sugar nucleotide
1. Introduction Glycosphingolipids (GSLs), amphipathic compounds consisting of oligosaccharides and ceramides, are ubiquitous in the outer leaflet of the plasma membrane and are involved in a large number of cellular processes including signal transduction, membrane trafficking, cytoskeletal organization, and pathogen entry [1,2]. Most mammalian GSLs are generated from glucosylceramide (GlcCer) as a precursor. GlcCer is synthesized by GlcCer synthase (UDP-glucose:ceremide glucosyltransferase; UGCG, EC 2.4.1.80) from ceramide and UDP-glucose (UDP-Glc) at the cytosolic surface of the Golgi apparatus [3]. GlcCer synthesis is an integral step for determining cellular GSL levels; however, how cells regulate GlcCer
Abbreviations: CERT, ceramide transport protein; GlcCer, glucosylceramide; GSL, glycosphingolipid; LacCer, lactosylceramide; MRM, multiple reaction monitoring; NBD, 7-nitro-2,1,3-benzoxadiazole; PH, pleckstrin homology; PtdIns(4)P, phophatidylinositol-4-phosphate; UGCG, UDP-glucose ceramide glucosyltransferase; UDPGlc, UDP-glucose; SM, sphingomyelin. * Corresponding author. E-mail address: [email protected] (Y. Ishibashi).
synthesis through the regulation of UGCG activity is largely unknown. Since abnormal accumulations of GlcCer and GlcCer-based GSLs cause several diseases, such as Garucher disease, Parkinson disease, and polycystic kidney disease [4e7], elucidation of the mechanism regulating UGCG may provide new and more effective treatments for these diseases. Ceramide is transported from the ER to the Golgi apparatus and is converted to GlcCer and sphingomyelin (SM) [8]. Ceramide is transported between these two organelles mainly by membrane vesicles [9]. An alternative transport pathway is mediated by ceramide transport protein (CERT), a protein factor responsible for non-vesicular transport of ceramide [10]. CERT contains a pleckstrin homology (PH) domain that binds phophatidylinositol-4phosphate (PtdIns(4)P), a serine repeat motif, two phenylalanines in an acidic tract (FFAT) motif that bind to the ER resident membranous protein, and a steroidogenic acute regulatory proteinrelated lipid transfer (START) domain that recognizes ceramide. CERT binds to the Golgi-abundant PtdIns(4)P and transfers ceramide from the ER to the trans-Golgi through ER-Golgi membrane contact sites [11]. The interaction between PtdIns(4)P and the PH domain of CERT is indispensable for the transport of ceramide [12].
https://doi.org/10.1016/j.bbrc.2018.04.039 0006-291X/© 2018 Elsevier Inc. All rights reserved.
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Please cite this article in press as: Y. Ishibashi, et al., Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.039
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Ceramides transported by CERT are mainly utilized for the synthesis of SM [10,13]. Phosphoinositides are important membranous lipids that mediate a large number of membrane-associated signaling processes [14]. PH domains are composed of 100e120 amino acids that recognize phosphoinositides [15]. The fluorescent protein-fused PH domain is utilized to study the dynamics of phophoinositides in living cells [16]. For instance, use of the FAPP1 PH domain, which recognizes PtdIns(4)P, revealed that PtdIns(4)P mainly localizes in the Golgi apparatus [17]. Although these probes are useful, they might capture phosphoinositide and compete with the phosphoinositide binding of endogenous effectors [18]. Since PtdIns(4)P is involved in sphingolipid metabolism [12], we hypothesized that phosphoinositide probes may affect cellular sphingolipid levels. In this study, we found that PtdIns(4)P inhibits UGCG activity and the binding of the FAPP1 PH domain to PtdIns(4)P attenuates the interaction between PtdIns(4)P and UDP-Glc, resulting in an increase in GlcCer synthesis. This paper provides a new insight into the relationship between GlcCer synthesis and phosphoinositides, which may be involved in the regulation of cellular GlcCer and GlcCer-based GSLs levels. 2. Materials and methods 2.1. Materials Pre-coated Silica gel 60 TLC plates were purchased from Merck, Germany. Fluorescent substrate 7-nitro-2,1,3-benzoxadiazole (NBD) C6-Ceramide and NBD C6-Ceramide complexed to BSA was purchased from Life technologies, USA. Phosphatidylinositol (PI) and phophatidylinositol-4-phosphate (PtdIns(4)P) were purchased from Matreya, USA. Antibodies against DYKDDDDK Tag (Rabbit IgG), 6xHis tag (Mouse IgG), and GFP (Mouse IgG) were purchased from Cell Signaling Technology (USA), a-tubulin was purchased from Sigma Aldrich (Germany), anti-b-actin Mouse IgG, Alexa Fluor 568 Anti-Mouse IgG, and Alexa Fluor 488 Anti-Rabbit IgG were purchased from Life Technologies (USA). A plasmid for the expression of the CFP and YFP-tagged FAPP1 PH domain (aa 1e101) was a gift from Dr. Michiyuki Matsuda, Kyoto University [19], and GFP-C1-PLC d-PH was a gift from Tobias Meyer (Addgene plasmid # 21179) [20]. The plasmid pEGFP-N3 was purchased from Takara Bio Inc. (Japan). A plasmid encoding C-terminal FLAG-tagged UGCG was prepared as described in Ref. [21]. 2.2. Construction of expression vectors To make the GFP-tagged FAPP1 probe, the lipid binding domain of GFP-C1-PLC d-PH was replaced by the FAPP1 PH domain (aa 1e101) that was obtained from a plasmid encoding the CFP/YFPtagged FAPP1 PH domain. PCR was carried out using mouse kidney cDNA (GenoStaff, Japan) as a template and the following two primers for amplification of splicing variants of FAPP2: FlagFAPP2v1-S (50 - TGACGATGACAAGCTTGAGGGCGTGCTGTACAAG-30 ), Flag-FAPP2v2-S (50 -TGACGATGACAAGCTTGCAGTCTGTGAGATTCAAGTTC-30 ), and Flag-FAPP2-A (50 -TCGCGGCCGCAAGCTTTCACACCACCTCATCAGATTCC-30 ). The conditions for amplification were 30 cycles (each consisting of denaturation at 98 C for 10 s,
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annealing at 55 C for 30 s, and extension at 72 C for 60 s) using Phusion polymerase (Thermo Scientific). The amplified products were inserted into HindIII-digested p3xFLAG-CMV-10 (Sigma Aldrich) to generate N-terminal 3xFLAG-tagged proteins using an In-Fusion HD Cloning kit (Clontech). 2.3. Cell culture and gene transfection Hek 293 and CHO-K1 cells were cultured in Dulbecco's modified Eagle Medium (DMEM), containing 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 C in 5% CO2. Hek 293 cells were transfected with TurboFect Transfection Reagent (Thermo Scientific), and CHO-K1 cells were transfected with X-tremeGENE 9 DNA Transfection Reagent (Roche Diagnostic) according to the manufacturer's instructions, respectively. 2.4. Immunoblot analysis Transfected cells were lysed in ice-cold lysis buffer (20 mM Tris HCl buffer, pH7.5, containing 150 mM NaCl, 1 mM EDTA, 1% NP-40, phosphatase and protease inhibitor cocktails (Roche diagnostics)) by sonication for 45 s. Cell debris was removed by centrifugation (18,000 g for 10 min at 4 C). The protein amount of the supernatant was determined by a bicinchoninic acid protein assay (Pierce) with bovine serum albumin as a standard. Equal amounts of cellular protein were separated by SDS-PAGE using a MiniPROTEAN TGX Gel (BioRad), and then blotted to a PVDF membrane by Trans Blot Turbo (BioRad). The membrane was blocked in TBS-T containing 5% BSA, then incubated with antibodies against DYKDDDDK tag (1:6000), GFP (1:5000), b-actin (1:10000), and atublin (1:20000) for over 12 h at 4 C. The blot was washed with TBS-T and then incubated with HRP-conjugated anti-mouse or rabbit IgG antibody (1:10000) (Cell Signaling Technology). The blot was washed again with TBS-T. The protein was detected by chemiluminescence using Luminate Forte Western HRP substrate (Millipore) and LAS-3000 Luminescence Image Analyzer (Fujifilm). 2.5. Measurement of the UGCG activity The intracellular UGCG assay was performed as previously described with some modification [21,22]. After transfection, cells were incubated for 24 h in a 24-well plate with DMEM 10% FBS, then were switched to 250 ml of DMEM 10% FBS containing 0.5 mM NBD C6-Ceramide complexed to BSA. After a 1 h incubation at 37 C, cells were rinsed with ice cold PBS, scraped with 50 ml of ice cold water and transferred to a 1.5 ml tube. Lipids were extracted by 190 ml of chloroform/methanol (1/2, v/v). After incubation at room temperature for 10 min, 62.5 ml of water and chloroform were added, then centrifuged at 12,000 g for 5 min. The lower phase was collected and dried with a speed vac concentrator. The dried sample was dissolved in 20 ml of chloroform/methanol (1/2, v/v), then 3 ml of the sample was applied to a TLC plate, which was developed with chloroform/methanol/water (65/25/4, v/v/v). The reaction products from the cell-based assay and in vitro assay were visualized by an LAS-3000 equipped with a blue-light-emitting diode (460 nm EPI) and an Y515-Di filter, and were quantified with Image J 1.43u software (NIH). The extent of the synthesis of
Fig. 1. Effect of FAPP1-PH expression on cellular sphingolipid levels and UGCG activity. (A) Immunoblot analysis showing protein expression of the fluorescent protein-fused FAPP1PH domain in HEK293 cells. (B) Quantification of cellular sphingolipid levels of FAPP1-PH expressing cells by LC-ESI MS/MS analysis. The bar graphs show the amount of cellular GlcCer, LacCer, ceramide, and sphingomyelin. (C) TLC analysis showing intracellular activities of UGCG and SMS in UGCG-FLAG- or FAPP1-PH-expressing cells. TLC was developed with a solution of chloroform/methanol/water (65/25/4, v/v/v) for 25 min. (D) Effect of FAPP1-PH expression on intracellular GlcCer synthase (UGCG, left panel) and sphingomyeline synthase (SMS, right panel) activities. NBD C6-ceramide conjugated to BSA was added to cells and incubated for 60 min, then analyzed by TLC. (D) Sugar nucleotide levels in FAPP1PH-expressing cells. Sugar nucleotides were extracted from FAPP1-PH-expressing cells and then subjected to ion-pair reversed-phase HPLC, as described in the Materials and Methods. Error bars represent means ± S.D. of at least three separate experiments.
Please cite this article in press as: Y. Ishibashi, et al., Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.039
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Fig. 2. Localization of the phosphoinositide probe affecting cellular sphingolipid levels. (A) Localization of GFP, GFP-PLC d-PH, GFP-FAPP1 PH, and UGCG-GFP expressed in CHO-K1 cells. Cells transfected with GFP (control, left upper), GFP-PLC d-PH (right upper), GFP-FAPP1-PH (left lower), or UGCG-GFP (right lower) were observed by fluorescent microscopy. Nuclei were stained with Bisbenzimide H33342 Fluorochrome Trihydrochloride. (B) Immunoblot analysis showing the protein expression of GFP (26.9 kDa), GFP-PLC d-PH (49.6 kDa), and GFP-FAPP1-PH (40.1 kDa) in CHO-K1 cells. (C) Changes of the ratio of GlcCer to sphingomyelin in FAPP1-PH-overexpressing CHO-K1 cells. Expression vectors for GFP, GFP-PLC d-PH, or GFP-FAPP1-PH were transfected into CHO-K1 cells and incubated for 62.5 h, then lipids were extracted. The GlcCer/sphingomyeline ratio was determined by LC-ESI MS/MS analysis. Error bars represent means ± S.D. of six separate experiments. (D) Immunoblot analysis showing the protein expression of FLAG-tagged FAPP2 variant 1 (v1) and variant 2 (v2). (E) Effect of FAPP2_v1 and v2 overexpression on intracellular UGCG activity. Error bars represent means ± S.D. of at least three separate experiments.
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Fig. 3. Inhibition of UGCG activity by phosphoinositide. (A) Inhibitory effect of PtdIns(4)P on UGCG activity. In vitro activities of UGCG and SMS of cell lysates were measured using fluorescent ceramide as a substrate. Several concentrations of PtdIns(4)P were added to the reaction mixture. (B) Difference in the inhibitory effect on UGCG and SMS activities between PtdIns and PtdIns(4)P. (D) Effect of several phosphoinositides on UGCG and SMS activities. (E) Repairing of UGCG activity inhibited by PtdIns(4)P by adding excessive UDPglucose. Several concentrations of UDP-glucose were added to the reaction mixture of the UGCG assay with or without 20 mM PtdIns(4)P.
NBD-labeled GlcCer was calculated as follows: synthesis (%) ¼ (peak area for NBD C6-GlcCer) x 100/(peak area for NBD C6Cer þ peak area for NBD C6-GlcCer þ peak area for NBD C6-SM). In vitro enzymatic activity was measured by a previously described method [23]. C6 NBD-ceramide and different concentrations of PtdIns(4)P were incubated with HEK293 cell lysate at 37 C for 90 min. 2.6. Observation of cellular localization of phosphoinositide probes and UGCG CHO-K1 cells expressing GFP-tagged phosphoinositide probes and UGCG were observed under a fluorescence microscope DMi8 equipped with a DFC3000G camera (Leica Microsystems). Nuclei was stained using Bisbenzimide H33342 Fluorochrome Trihydrochloride (Nacalai tesque, Japan). CHO-K1 cells were transfected with the CFP/YFP-tagged FAPP1 PH domain and C-terminal FLAGtagged UGCG to check intracellular colocalization. After 24 h of transfection, cells were fixed in 3% paraformaldehyde for 15 min and then washed with PBS. The fixed cells were blocked in Blocking One Histo (Nacalai Tesque), then incubated with antibodies against DYKDDDDK tag (1:500) and GFP (1:500) for 12 h at 4 C. After
incubation, cells were washed by PBS and incubated with Alexa Fluor 568 Anti-Mouse IgG (1:500) and Alexa Fluor 488 Anti-Rabbit IgG (1:500) for 1 h. Cells were mounted in Immu-Mount reagent (Thermo Scientific) and observed under a confocal laser-scanning microscope (FV1000, Olympus). 2.7. Lipid extraction and quantification of sphingolipids by LC-ESI MS/MS Cells were washed once with cold PBS, collected in 100 ml of cold PBS, then homogenized with sonication. Part of the sample (5 ml) was used in the bicinchoninic acid protein assay to determine the amount of protein. Total lipids were extracted by adding 375 ml of chloroform/methanol (1/2, v/v) containing 62.5 pmol of each component of Ceramide/Sphingoid Internal Standard Mixture II (Avanti Polar Lipids, Inc.) from the rest of the sample. The singlephase mixture was incubated at 48 C for 16 h. After cooling, 125 ml of water and chloroform were added. The lower phase was collected and dried with a speed vac concentrator. The dried sample was suspended in chloroform/methanol (2/1, v/v) containing 0.1 N KOH, incubated for 2 h at 37 C, and neutralized with an equal amount of acetic acid, then water was added. The lower
Please cite this article in press as: Y. Ishibashi, et al., Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.039
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Fig. 4. Localization of UGCG and PtdIns(4)P. (A) Localization of UGCG-FLAG and GFP-FAPP1-PH. Cells transfected with UGCG-FLAG and GFP-FAPP1-PH were immunostained with anti-FLAG antibody and anti-GFP antibody, as described in the Materials and Methods. Scale bar represents 10 mm. (B) Predicted mechanism of FAPP1-PH domain expressionemediated activation of UGCG activity. In the normal condition, PtdIns(4)P nearby to UGCG interferes with the utilization of UDP-glucose to synthesize GlcCer by UGCG. GlcCer might by synthesized by UGCG localizing at PtdIns(4)P-less regions in the Golgi apparatus in the normal condition. In the FAPP1-PH domain-expressing condition, PtdIns(4)P is covered by the PH domain that may attenuate the inhibitory effect of PtdIns(4)P on UGCG activity, leading the apparent activation of UGCG activity and increase of the GlcCer level.
phase was withdrawn and dried, then resuspended in 200 ml of methanol, sonicated for 10 s, centrifuged at 14,000 g for 5 min, and the supernatant transferred to vials. The amount of GlcCer, LacCer, Cer, and SM were analyzed by LC-ESI MS/MS using a triple quadruple mass spectrometer 4000Q TRAP (SCIEX) coupled to an Agilent 1100 series HPLC system (Agilent). A binary solvent gradient with a flow rate of 0.2 ml/min was used to separate sphingolipids by normal-phase chromatography using an InertSustain NH2 (2.1 150 mm, 5 mm bead size, GL Science, Japan). The gradient was started at 20% buffer B (methanol/water/formic acid, 89/9/1, v/v/v, with 20 mM ammonium formate) in buffer A (acetonitrile/methanol/formic acid, 97/2/1, v/v/v, with 5 mM ammonium formate). The gradient reached 100% B in 4 min and was maintained at 100% B for 2 min. Finally, the gradient was returned to the starting condition, and the column was equilibrated for 5 min before the next run. Sphingolipids containing C16:0, C18:0, C20:0, C22:0, C24:0, and C24:1 fatty acids were detected using a multiple reaction monitoring (MRM) method, as described in Ref. [24]. 2.8. Quantification of nucleotide sugars The quantification method was performed as previously described with some modification [25]. Cells were washed with cold PBS, collected in 300 ml of cold PBS, and spiked with GDP-Glc as an internal standard, and then sonicated using a Handy Sonic Disruptor for 10 s. A part of the sample (5 ml) was applied to the bicinchoninic acid protein assay. Ice cold ethanol (900 ml) was added to the sample. The extract was centrifuged at 16,000 g for 10 min at 4 C, and supernatant was lyophilized. The freeze-dried sample was applied to an Envi-Carb column (Supelco Inc.), and purified as described in Ref. [25]. After purification, nucleotide
sugars were separated and detected by ion-pair reversed-phase HPLC. 2.9. Statistical analysis All statistical analyses were performed using unpaired twotailed Student's t-tests, and all data are expresses as means and standard deviation for at least three separate experiments. Statistical significance is indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001. 3. Result and discussion Effects of FAPP1-PH domain expression on cellular sphingolipid levels - The fluorescent protein-fused PH domain is utilized to study the dynamics of phophoinositides in living cells [18]. To examine the effect of FAPP1-PH domain expression on sphingolipid metabolism, total lipids were extracted from CFP/YFP-labeled FAPP1-PH expressing cells (Fig. 1A) and subjected to LC-ESI-MS/MS analysis after alkaline treatment. We found that cellular GlcCer and lactosylceramide (LacCer) levels increased in FAPP1-PH-expressing cells (Fig. 1B). Since CERT-mediated ceramide transport is inhibited by FAPP1-PH domain expression [12], we predicted that the SM level may decrease in FAPP1-PH domain-expressing cells. Unexpectedly, however, the SM level was almost the same as that of the mock transfectants (Fig. 1B). On the other hand, the ceramide level increased in FAPP1-PH domain-expressing cells (Fig. 1B), suggesting that ceramide synthesis might be upregulated to compensate for the decrease in CERT-mediated ceramide at Golgi apparatus. Increase of UGCG activity by FAPP1-PH domain expression e First, we examined the possibility that increased GlcCer and LacCer levels are caused by the activation of UGCG. We found that the
Please cite this article in press as: Y. Ishibashi, et al., Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.039
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intracellular UGCG activity significantly increased in the FAPP1-PH domain-expressing cells without affecting sphingomyelin synthase (SMS) activity (Fig. 1C and D). These results suggest that the FAPP1PH domain increased UGCG activity and thereby the GlcCer level increased. Next, we investigated the mechanism of how the FAPP1-PH domain enhances intracellular UGCG activity. Since GlcCer is synthesized by UGCG utilizing ceramide and UDP-Glc as substrates, a change in the level of these substrates could affect UGCG activity [21]. Thus, various sugar nucleotide levels were compared between mock and FAPP1-PH domain-expressing cells. The levels of sugar nucleotides, including UDP-Glc, were not changed by expression of the FAPP1-PH domain (Fig. 1E), indicating that an increase in GlcCer was not caused by changes to sugar nucleotide levels. Effects of the expression of PtdIns(4)P and PtdIns(4,5)P2 recognizing PH probes eWe found that expression of FAPP1-PH domain recognizing PtdIns(4)P increases intracellular UGCG activity and the GlcCer level. In order to investigate whether this effect is specific to the FAPP1-PH domain or common to other phosphoinositide probes, the GFP-tagged phospholipase C (PLC) d-PH domain was expressed in CHO-K1 cells (Fig. 2A and B). The GFP-tagged FAPP1PH domain shows almost the same intracellular distribution of GFP-tagged UGCG; however, the GFP-PLC d-PH domain was detected on the plasma membrane (Fig. 2A), because the FAPP1-PH domain was transported to the PtdIns(4)P-rich Golgi membranes while the PLC d-PH domain was transported to the PtdIns(4,5)P2rich plasma membrane, as previously reported [20]. Importantly, the cellular GlcCer level increased in cells expressing the FAPP1-PH domain, but not the PLC-d-PH domain (Fig. 2C). Next, we examined the effects of FAPP2 PH domains on UGCG activity. FAPP2, known as a GlcCer-transporting protein, also contains a PH domain that recognizes PtdIns(4)P [26]. Mice have two splicing variants of FAPP2 (FAPP2_v1 and v2) (Supplemental Fig. 1). FAPP2_v1 has a full-length PH domain while FAPP2_v2 has a shorter PH domain lacking the PtdIns(4)P-contacting motif (Supplemental Fig. 1). We expressed the FLAG-tagged FAPP2_v1 and v2 independently in Hek293 cells (Fig. 2D). Cell-based UGCG assay revealed that intracellular UGCG activity increased in both FAPP2 PH domain-overexpressing cells (Fig. 2E); however, the increase in UGCG activity by FAPP2_v2 was much weaker than that by FAPP2_v1 (Fig. 2E). These findings indicate that the contacting motif of FAPP2_v1 is important for the increase in UGCG activity, and suggests that the upregulation of UGCG activation requires the interaction between the PH domain and PtdIns(4)P. Inhibition of UGCG activity by PtdIns(4)P e PtdIns(4)P is synthesized from PtdIns at the surface of the Golgi apparatus by phosphtidylinositol 4-kinase (PI4K) [27]. In mammals, GlcCer is also synthesized at the surface of the Golgi apparatus by UGCG, in which a catalytic domain is present at the cytosolic face of the Golgi membrane [3]. Therefore, PtdIns(4)P and UGCG are expected to interact with each other at the Golgi membranes. Interestingly, we found that PtdIns(4)P inhibited in vitro UGCG activity in a concentration-dependent manner (Fig. 3A). However, SMS activity was constant in the presence of PtdIns(4)P. The inhibitory effect of PtdIns(4)P on UGCG was significantly greater than that of PtdIns, indicating that the phosphoryl head group of PtdIns(4)P is important for the suppression of UGCG activity (Fig. 3B). Inhibitory effects of other phosphoinositides, such as PtdIns(3)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 on UGCG activity were similar to PtdIns (4)P (Fig. 3C), indicating that one phosphate group is enough to inhibit UGCG activity even at a different position. The inhibitory effect of PtdIns(4)P on UGCG activity was restored by increasing the UDP-Glc concentration in the reaction mixture (Fig. 3D), suggesting that PtdInd(4)P inhibits UGCG activity by competing with UDP-Glc.
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Localization of UGCG and PtdIns(4)P ePtdIns(4)P inhibited UGCG activity in vitro, and thus, we examined whether UGCG interacts with PtdIns(4)P intracellularly. To clarify the localization of PtdIns(4)P and UGCG, a fluorescent protein-fused FAPP1-PH domain and FLAG-tagged UGCG were co-expressed in CHO-K1 cells. As a result, it was found that a part of UGCG was colocalized with the FAPP1-PH domain, suggesting that PtdIns(4)P was partly colocalized with UGCG (Fig. 4A). These results indicate that a part of UGCG, if not all, can interact with PtdIns(4)P at the Golgi membrane. Why does overexpression of FAPP1-PH increase UGCG activity, and how does PtdIns(4)P disturb the utilization of UDP-Glc by UGCG? We consider that PtdIns(4)P obstructs the use of UDP-Glc by UGCG on the Golgi membrane, because the negative charge of phosphate groups in UDP-Glc and PtdIns(4)P repel each other (Fig. 4B). Overexpression of the PH domain covered the negative charge of PtdIns(4)P, allowing UGCG to use UDP-Glc efficiently (Fig. 4B). Consistent with our results, GlcCer synthesis was upregulated by decreasing the PtdIns(4)P level in the Golgi apparatus through the activation of PKD and PtdIns(4)P phosphatase Sac1 [28]. In this study, we revealed that Golgi-localized PtdIns(4)P inhibits UGCG activity by blocking the interaction of UGCG with UDPGlc, suggesting that PtdIns(4)P is involved in the regulation of cellular GlcCer and GlcCer-based GSLs levels in the Golgi apparatus. Acknowledgements We are grateful to the Support Unit for Bio-material Analysis, RIKEN Brain Science Institute Research Resources Center, for help with the nucleotide sequencing analyses. This work was supported in part by the RIKEN Special Postdoctoral Researchers Program (to Y. I.), and a Grant-in-Aid for Young Scientists (B) (to Y. I.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (24770198). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.04.039. Author contributions YI and YH designed the study; YI performed and analyzed all of the experiments in this study; YI and MI wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript. References [1] R. Jennemann, H.J. Grone, Cell-specific in vivo functions of glycosphingolipids: lessons from genetic deletions of enzymes involved in glycosphingolipid synthesis, Prog. Lipid Res. 52 (2013) 231e248. [2] K. Hanada, Sphingolipids in infectious diseases, Jpn. J. Infect. Dis. 58 (2005) 131e148. [3] Y. Ishibashi, A. Kohyama-Koganeya, Y. Hirabayashi, New insights on glucosylated lipids: metabolism and functions, Biochim. Biophys. Acta 1831 (2013) 1475e1485. [4] G.A. Grabowski, Phenotype, diagnosis, and treatment of Gaucher's disease, Lancet 372 (2008) 1263e1271. [5] E. Sidransky, G. Lopez, The link between the GBA gene and parkinsonism, Lancet Neurol. 11 (2012) 986e998. [6] M. Fuller, Sphingolipids: the nexus between Gaucher disease and insulin resistance, Lipids Health Dis. 9 (2010) 113. [7] T.A. Natoli, L.A. Smith, K.A. Rogers, et al., Inhibition of glucosylceramide accumulation results in effective blockade of polycystic kidney disease in mouse models, Nat. Med. 16 (2010) 788e792. [8] A.H. Futerman, H. Riezman, The ins and outs of sphingolipid synthesis, Trends Cell Biol. 15 (2005) 312e318.
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Please cite this article in press as: Y. Ishibashi, et al., Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.039