Co-determination of Sphingomyeline and Cholesterol in Cellular

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Co-determination of Sphingomyeline and Cholesterol in Cellular Plasma Membrane in Sphingomyeline-depletion Induced Cholesterol Efflux Shuohan Huang, Kang Liu, Dechen Jiang, and Danjun Fang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04593 • Publication Date (Web): 14 Dec 2018 Downloaded from http://pubs.acs.org on December 18, 2018

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Analytical Chemistry

Co-determination of Sphingomyeline and Cholesterol in Cellular Plasma Membrane in Sphingomyeline-depletion Induced Cholesterol Efflux

Shuohan Huang1, Kang Liu1, Dechen Jiang2, Danjun Fang1*

1

School of Pharmacy and Key Laboratory of Targeted Intervention of Cardiovascular Disease,

Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University , Nanjing , Jiangsu China , 211126. 2

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and

Chemical Engineering, Nanjing University , Nanjing, Jiangsu, 210093, China

Email: [email protected]

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract: The quantification of multiple lipids with different contents in plasma membrane at single cells is significant, but challenging, for the investigation of lipid interactions and the role of the dominant protein transporters. In this paper, co-monitoring the alteration of low-content sphingomyelin (SM) and high-content cholesterol in plasma membrane at one living cell is realized using luminol electrochemiluminescence (ECL) at the first time. The concentration of SM as low as 0.5 µM is detected that permits the measurement of low-content membrane SM at single cells.

More

membrane cholesterol is observed at individual cells after the depletion of membrane SM providing direct evidence about SM-depletion induced cholesterol efflux. The up-regulation of ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1) at SM-depleted cells induces the further increase of membrane cholesterol. These results imply that higher expression of ABCA1/G1 promotes cholesterol trafficking, which offers additional information to solve the debate about ABC transporters in cholesterol efflux. Moreover, the established approach offers a special strategy to investigate the correlation of membrane lipids and the role of transporters in cholesterol trafficking.

Key words: Membrane cholesterol, sphingomyelin depletion, ABC transporter, single cells, luminol electrochemiluminescence


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Introduction. Recent progress in biophysics, chemistry and genetics has attracted renewed attention to the biological roles of lipids at cellular plasma membrane.1 More specially, pathological processes have been unexpectedly connected to specific membrane lipids and their biophysical properties. Therefore, the integrated study of cellular lipids at living cells is critical.2,3 An important example is to understand the relation of membrane cholesterol and sphingomyelin (SM) in the process of cholesterol trafficking. Cholesterol is a major structural component in plasma membrane that plays an important role in the development of atherosclerosis.4 Promoting cholesterol efflux to extracellular acceptors is of great importance in the maintenance for cellular cholesterol homeostasis.5,6 SM, as a lowcontent structural component of cellular membrane, constitutes 2–15% of total organ phospholipid that is suggested to be a predictive value for atherosclerosis.7-9 Both clinical and animal studies have revealed that SM levels in plasma are positively and directly linked to coronary artery disease.7,10 Recent evidences show that abundant cholesterol and low-content SM form lipid micro-environments on cell surface,11,12 and the deficiency of sphingomyelin synthase 2 (SMS2) in macrophage causes a notable induction of cholesterol efflux both in vitro and in vivo.13 Despite the important functions of membrane cholesterol and SM, the detail relation between them in cellular plasma membrane is not clear. Most importantly, ABC transporters, including ABCA1 and ABCG1, are known to be involved in the interactions between cholesterol and SM,13,14 but, a long debate about their roles in SM-depletion induced cholesterol efflux exists.13,15 This vague understanding is partially caused by the missing approach to co-determine membrane cholesterol and SM at one living cell.

Accordingly, the current methodology to separately

examine these two lipids at the cell population could not offer the accurate information. Many advances in optical microscopy and mass spectrometry have catalysed precipitous developments in high-sensitivity and high-throughput lipid analysis at single cells.16 However, the obvious difference in the contents of cholesterol (50% of phospholipid) and SM (2–15% of



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phospholipid) in plasma membrane results in the challenging to co-assay these two molecules at one living cell. In the past years, our group has developed a luminol electrochemiluminescence (ECL) strategy to analyze membrane lipids, including cholesterol and phosphatidylserine (PS), at single cells.17-19 ECL is a highly sensitive electrochemical method whereby the luminescence probe is electrochemically excited to the intermediates and transits to the ground state emitting the luminescence.20-24 In presence of kit components for a special lipid, enzyme cocktail in the buffer react with this lipid at cellular membrane to generate hydrogen peroxide that enhances ECL from luminol. The increased intensity is proportional to the amount of membrane lipid from one cell so that the quantification at single cell level is achieved.25 By means of high detection sensitivity using ECL and high detection specificity using bio-specific enzymes, the measurement of lowcontent lipid at single living cells, even in presence of other high-content lipids, is feasible. In this paper, low-content SM and high-content cholesterol in plasma membrane are attempted to be co-quantified at one living cell. As shown in Figure 1, membrane cholesterol reacts firstly with cholesterol oxidase to generate hydrogen peroxide that enhances luminol ECL. The removal of membrane cholesterol by cholesterol oxidase does not break the membrane structure,17 and thus, an enzyme cocktail, including sphingomyelinase (SMase), alkaline phosphatase (ALP) and choline oxidase, is introduced at the cells to react with membrane SM. The produced hydrogen peroxide leads to the further ECL increase. After validating the codetermination of membrane cholesterol and SM at single cells, the fluctuation of membrane cholesterol and SM at one cell with up-regulated ABCA1 and ABCG1 is investigated. The obtained result should provide direct evidence about the interaction between membrane cholesterol and SM, and the role of ABC transporters in cholesterol trafficking.

EXPERIMENTAL SECTION Chemical.

8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione

(L012)

was

obtained from Wako Chemical (VA, USA). Myriocin was obtained from Cayman Chemical (MI,


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USA). T0901317 was purchased from MedChem Express (NJ, USA). All other chemicals were from Sigma-Aldrich, unless indicated otherwise. Buffer solutions were sterilized. Cell culture. Raw264.7 cells were grown in DMEM/high glucose medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin). Cultures were maintained at 37 °C under a 5% CO2 humidified atmosphere. SM content and ABC transporter levels at plasma membrane. To decrease SM content at plasma membrane, the cells were treated with 10 µM myriocin for 16 h or 100 µM tricyclodecan9-yl-xanthogenate (D609) for 4 h at 37 °C.14,26 To induce ABCA1 expression, the cells were pretreated in presence of 0.3 mM 8-Br-cAMP for 16 h. In addition, the cells were exposed to 10 µM T0901317 for 20 h to upregulate ABCG1 expression mainly (note: some upregulation of ABCA1 expression occurs as well).27-29 Assay of SM and cholesterol at cells. To assay cholesterol at plasma membrane, the cells were cultured in 0.5 mM phosphate-buffered saline (PBS, pH 7.4) with 310 mM sucrose at 37 °C for 1 h to activate membrane cholesterol. Then, the cells were washed and re-cultured in 10 mM PBS. 2 μL cholesterol oxidase (100 U/mL) was added in 200 μL buffer with final concentration of 1 U/mL for the reaction of membrane cholesterol.

Afterwards the luminescence detection of

cholesterol, the cells were washed and pretreated with choline oxidase at 37℃ for 10 min to avoid possible interference of membrane choline during the following detection of membrane SM. After washing the cells with PBS for three times, an enzyme cocktail including 4 μL bacterial sphingomyelinase (SMase, 25 U/mL), 4 μL alkaline phosphatase (ALP, 500 U/mL), 2 μL choline oxidase (100 U/mL) and 10 μL 0.2% Triton X-100 was added into 200 μL buffer to initial the reaction with membrane SM. The final concentrations of SMase, ALP, choline oxidase and Triton X-100 were 0.5 U/mL, 10 U/mL, 1 U/mL and 0.01%, respectively. ECL detection. Indium tin oxide (ITO) slides cultured with the cells were used as the working electrode. An Ag/AgCl and Pt wires were connected as a reference and counter electrode, respectively. To measure membrane cholesterol and SM from cell population, ~ 80,000 cells were



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cultured on ITO slides, and treated with enzyme cocktails in serial at 37 °C. The voltage of photomultiplier tube (PMT) was set at 600 V. For single cell analysis, the cell density was adjusted to ~ 2000 cells/cm2 at ITO slide and the distances between the adjacent cells were ranged from 80 to 1000 μm.17,18 A 100 µm diameter pinhole was placed between ITO slide and PMT so that only one cell was exposed to PMT. The voltage of PMT was set as 900 V.

RESULTS AND DISCUSSIONS ECL detection of SM in cell population and single cells The enzymatic detection of SM in cellular membrane through a serial reaction with SMase, ALP and choline has been validated using fluorescence assay, as demonstrated in Fig 1.30-32 In this process, aqueous SMase hydrolyzes SM into ceramide and phosphorylcholine.

Then,

phosphorylcholine is degraded by ALP to choline and phosphate, followed by the oxidation of choline by choline oxidase to generate hydrogen peroxide for enhanced luminol ECL. To increase ECL intensity, L012 as a luminol analog with a higher ECL efficiency is used.19 Experimentally, SM in PBS solution was firstly reacted with the enzyme cocktail, and remarkable ECL increase is observed (Figure 2A). This increase is linearly correlated with the concentration of SM from 0.5 to 80 μM with a correlation coefficient of 0.993 (Figure 2B), which supports the quantitative measurement of aqueous SM. Using this protocol, the cells cultured at ITO slides were exposed to the enzyme cocktail. The addition of 0.01% Triton X-100 is reported to inhibit SMase activity toward membrane sphingosyl-phosphocholine (SPC), and 1-palmitoyl-2-hydroxy-sn-glycerol-3-phosphorylcholine (LPC).30,33 Moreover, to avoid a possible interference of membrane choline in the detection of membrane SM, the cells were pretreated with choline oxidase. Similar to the ECL increase in the determination of aqueous SM, a higher ECL intensity is observed after the introduction of enzyme cocktail (Figure S1, supporting information). The removal of SMase from the enzyme cocktail did not induce the ECL increase in this process. All these results confirm the measurement of SM


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in cellular plasma membrane. After validating the assay of membrane SM, ECL from one target cell is collected through the pinhole. The ECL intensity increased after the introduction of enzyme cocktail, as demonstrated in Figure 2C, exhibiting single cell SM analysis. Figure 2D lists the ECL intensity ratios from 12 individual cells after and before the enzymatic reaction. The average ECL ratio from single cells is calculated to be 0.62 ± 0.22, and the relative standard deviation from single cells is 35.36%. The deviation is larger than that from cell population (10.40%) reflecting cell heterogeneity in membrane SM. To the best of our knowledge, this is the first report on the assay of membrane SM at single cell level. Co-detection of membrane cholesterol and SM in individual cells To realize the reaction of cholesterol oxidase and membrane cholesterol, the cells are pretreated with low ionic strength buffer (0.5 mM PBS with 310 mM sucrose) to activate cholesterol in plasma membrane. This pretreatment is believed to rearrange the location of cholesterol at cellular membrane creating cholesterol-rich phase, in which cholesterol is more accessible to the oxidase.34 Since cholesterol oxidase removes active cholesterol from plasma membrane only and retains membrane structure, it is feasible to apply cholesterol oxidase and the enzyme cocktail for SM in serial at the same cell. The serial reactions of cholesterol with cholesterol oxidase and SM with enzyme cocktail produce hydrogen peroxide successively to generate the ECL, respectively, for the co-determination of membrane SM and cholesterol. The typical ECL trace recorded from one cell is shown in Figure 3A. The ECL intensities after and before the introduction of enzymes are recorded as Ib and Ia (for cholesterol), and Id and Ic (for SM). Higher ECL intensities observed after the addition of enzymes exhibited the detection of membrane cholesterol and SM at one cell. To characterize the amount of cholesterol and SM in the plasma membrane, these two luminescence ratios, (Ib-Ia)/Ia and (Id-Ic)/Ic, are calculated. Based on individual analysis from 10 single cells (Figure 3B), the average ECL ratios for cholesterol and SM are 3.94 ± 0.96 and 0.62 ± 0.20. This ECL ratio for SM is almost same as that from the separation measurement of SM as



mentioned above, exhibiting the accuracy of our serial detection.

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Moreover, the ratio of

membrane cholesterol/SM is calculated to be 13.31± 3.44, which matches the ratio of their contents in the membrane.1 This consistence with the biological information validates our assay for the co-determination of membrane cholesterol and SM. Myriocin, a potential inhibitor of sphingolipids biosynthesis in cell, is used to decrease the content of SM in cellular membrane. After the treatment of cells with 10 μM myriocin,14 the ECL intensity associated with SM and cholesterol from individual cells are shown in Figure 3C. The ECL ratio for SM decreases to 0.50 ± 0.12; while, the ECL ratio for cholesterol increases to 15.99 ± 3.7, as listed in Figure 3D. Although large deviations on membrane cholesterol and SM are present, these results at single cell level reveal more membrane cholesterol after the depletion of membrane SM. Role of ABC transporter in the SM depletion induced cholesterol efflux pathway As two dominate and unidirectional transporters, ABCA1 and ABCG1 are suggested to be involved in the macrophage-specific cholesterol efflux.6,35 The upregulation of ABCA1 in SMS2 inhibited murine macrophage leads to a remarkably increased cholesterol efflux supporting ABCA1- mediated pathway.14 However, the role of ABCG1 in this process remains controversial. The considerable increase in ABCG1 expression in SMS2 deficiency mice macrophage indicates that ABCG1-mediated cholesterol efflux pathway may contribute to SM related cholesterol efflux.13 While, Remaley et al. reported that a decrease in plasma membrane SM did not change net cholesterol efflux in ABCG1-GFP cells, suggesting that ABCG1-mediated cholesterol efflux was SM-independent.15 In our experiment, ABCA1 transporter in RAW264.7 cell line is transiently upregulated by cyclic adenosine monophosphate (cAMP).27,36 Figure 4A displays the ECL ratio for membrane cholesterol and SM at individual ABCA1 (+) cells. As compared with the ECL ratios from the untreated cell (cholesterol: 3.94 ± 0.96; SM: 0.62 ± 0.20), the ECL ratios for membrane cholesterol and SM increase to 9.41 ± 2.38 and 2.15 ± 0.79. The increased amounts of both


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membrane cholesterol and SM at individual cells are ascribed to more transportation of cholesterol and SM from the cytoplasmic to the membrane through the up-regulated ABCA1 transporters that enhances cholesterol efflux.37 When the cells were co-incubated with cAMP and D609, the up-regulation of ABCA1 and the depletion of SM in plasma membrane happened at the same time. As compared to that from ABCA1 (+) cells, the depletion of SM results in the reduction of membrane SM to 0.60 ± 0.29, (Figure 4B). Meanwhile, the ECL ratio for membrane cholesterol increases to 20.62 ± 10.58, which is higher than that (15.99 ± 3.7) in absence of ABCA1 expression. The elevation of membrane cholesterol suggests that ABCA1 transporter could enhance intracellular cholesterol trafficking to plasma membrane in presence of SM-depletion. Synthetic liver X receptor (LXR) activation is reported to induce the expression of the ABCG1 mainly.38 Therefore, the cells were exposed to LXR agonist, T0901317, to realize the upregulation of ABCG1 mainly in the cells. The ECL ratios for cholesterol and SM from individual ABCG1 (+) cells, as shown in Figure 4C, are 19.97 ± 8.8 and 3.0 ± 2.12. As compared with enhanced intracellular cholesterol and SM trafficking in presence of ABCA1, the expression of ABCG1 induces more cholesterol trafficking to membrane revealing the dominant role of ABCG1. After the co-treatment of the cells with T0901317 and D609, the up-regulation of ABCG1 and the depletion of SM occur at the cells. Figure 4D lists the ECL ratios of cholesterol and SM (71.54 ± 46.91 for cholesterol and 1.17 ± 0.72 for SM) from individual cells. As compared with the ECL ratio from ABCA1 (+) cells (20.62 ± 10.58), ~ 3.5 fold enhancement of membrane cholesterol is observed with the transportation of ABCG1.

All these results support the

conclusion that ABCG1 could induce more cholesterol trafficking with depleted SM in plasma membrane. Overall, our work suggests the important role of ABC transporters in SM-depletion induced cholesterol efflux, even when the contents of cholesterol and SM are 1-2 orders of magnitude different.



CONCLUSION. In this paper, cholesterol and SM with remarkably different contents are co-determined at one living cells using luminol ECL. The high detection sensitivity permits the measurement of lowcontent membrane SM after the depletion, and thus, the alterations of membrane cholesterol and SM in presence of ABC transporters could be investigated. The results at single cell level provide strong evidence about the important role of ABCA1/G1 in the process of SM-depletion induced cholesterol efflux. More work needs to be performed at primary macrophage cells for deeply understanding the role of these transporters in cholesterol trafficking, which will benefit the drug development for cholesterol related diseases. Also, novel materials at the electrode should be selected to enhance the ECL intensity, and thus, co-mapping SM and cholesterol at cellular membrane is feasible.

ACKNOWLEDGEMENTS. This work was supported by National Natural Science Foundation of China (Nos. 2157050130).

Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. More luminescence data about the analysis of SM at cell population.

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Figures and Captions. Figure 1. The schematic co-detection of cholesterol and SM in plasma membrane at single cells, and the simplified trafficking pathways of intracellular cholesterol and SM to plasma membrane. Figure 2. (A) The typical ECL traces for the detection of aqueous SM in 10 mM PBS (pH 7.4) with 200 µM L012. (B) the calibration curve of ECL ratio with different concentrations of aqueous SM. (C) the typical ECL traces for the detection of membrane SM at one living cell in 10 mM PBS (pH 7.4) with 200 µM L012. (D) the ECL ratio for membrane SM from 12 individual cells. The curves labeled with “background” and “enzymes” present the luminescence from the solution or individual cell before and after the addition of enzyme cocktail, respectively. The potential is scanned from -1 to 1 V with a scan rate of 0.1 V/s. Figure 3. (A) The typical ECL traces for the serial determination of membrane cholesterol and SM at one living cells. The background curves (trace b and d) present the background ECL, and the red curves present the ECL after the addition of cholesterol oxidase (trace a) and the enzyme cocktail for SM (trace c); (B) the ECL ratio for membrane cholesterol and SM from 10 individual cells. (C) the ECL ratio for membrane cholesterol and SM from 8 individual cells after the depletion of membrane SM. (D) the comparison of membrane cholesterol and SM in the absence and presence of SM depletion. Figure 4. (A) The ECL ratio for membrane cholesterol and SM from 9 individual ABCA1 (+) cells. (B) the ECL ratio for membrane cholesterol and SM from 6 individual ABCA1 (+) cells after the depletion of membrane SM. (C) the ECL ratio for membrane cholesterol and SM from 8 individual ABCG1(+) cells. (D) the ECL ratio for membrane cholesterol and SM from 10 individual ABCG1 (+) cells after the depletion of membrane SM.



Figure 1.


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Figure 2.

B

150

6 4 2

background

Intensity ratio

8

+ enzymes

A

100

50

0 -1.0

300

-0.5

0.0 Voltage /V

0.5

+enzymes

C

200 background

100 0 -1.0

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0.0 0.5 Voltage /V

1.0

1.0 Intensity ratio

ECL intensity /1000 a.u.

10

ECL intensity /a.u.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

20

40 SM/ M

60

D

0.5

0.0

1

3


5

7 Cell

9

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Figure 3.

8

A

d c

200 a

0 0

1 -1 Voltage /V

2 0

1

0

4

20

10

2

4 6 SM depleted Cell

8

Intensity ratio for Cholesterol

cholesterol SM

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D

C

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20 15

Cell

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8

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* Normal ** SM-depletion

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5 0.0

0 Cholesterol


SM

Intensity ratio for SM

400

Intensity ratio

ECL intensity /a.u.

6

b

-1

cholesterol SM

B

SM

Cholesterol

600

Intensity ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 4. cholesterol SM

A

15

cholesterol SM

B

10

Intensity ratio

Intensity ratio

30

5

0

0

2

4

6

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20 10 1 0

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+ Cell ABCA1Cell

cholesterol SM

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4 6 + ABCG1 Cell

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120 40 20 0

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Co-determination of Sphingomyeline and Cholesterol in Cellular

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