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Jan 14, 2016 - Lipid Science and Aging Research Center;. §. Department of Medical. Laboratory Science and Biotechnology, College of Health Sciences;...
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Enhanced Sphingomyelinase Activity Contributes to the Apoptotic Capacity of Electronegative Low-Density Lipoprotein Liang-Yin Ke,¥,†,∥,¶,§ Hua-Chen Chan,¥,†,∥,‡ Chih-Chieh Chen,‡,⊥ Jonathan Lu,∥ Gopal K. Marathe,#,∇ Chih-Sheng Chu,‡,○ Hsiu-Chuan Chan,¶ Chung-Ya Wang,‡ Yi-Ching Tung,△ Thomas M. McIntyre,# Jeng-Hsien Yen,*,†,¶,○ and Chu-Huang Chen*,∥,¶,□,^ †

Graduate Institute of Medicine, College of Medicine; ¶Lipid Science and Aging Research Center; §Department of Medical Laboratory Science and Biotechnology, College of Health Sciences; ‡Center for Lipid Biosciences, KMU Hospital; ○Department of Internal Medicine, KMU Hospital; and △Department of Public Health and Environmental Medicine; and □L5 Research Center, Kaohsiung Medical University (KMU), Kaohsiung, Taiwan 807 ∥ Vascular and Medicinal Research, Texas Heart Institute, Houston, Texas 77030, United States ⊥ Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung, Taiwan 80424 # Departments of Cellular & Molecular Medicine, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio 44195, United States ∇ Department of Studies in Biochemistry, Manasagangothri, University of Mysore, Mysore-570006, India ^ New York Heart Research Foundation, Mineola, New York 11501, United States S Supporting Information *

ABSTRACT: Sphingomyelinase (SMase) catalyzes the degradation of sphingomyelin to ceramide. In patients with metabolic syndrome or diabetes, circulating plasma ceramide levels are significantly higher than in normal individuals. Our data indicate that electronegative low-density lipoprotein (LDL) shows SMase activity, which leads to increased ceramide levels that can produce pro-inflammatory effects and susceptibility to aggregation. According to sequence alignment and protein structure predictions, the putative catalytic site of SMase activity is in the α2 region of apoB-100. To identify specific post-translational modifications of apoB100 near the catalytic region, we performed dataindependent, parallel-fragmentation liquid chromatography/mass spectrometry (LC/MSE), followed by data analysis with ProteinLynx GlobalServer v2.4. Results showed that the serine of apoB100 in electronegative LDL was highly O-glycosylated, including S1732, S1959, S2378, S2408, and S2429. These findings may support the changing of the α-helix/β-pleated sheets ratio in protein structure analysis. Further study is necessary to confirm the activation of SMase activity by electronegative LDL.

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INTRODUCTION

sepsis, or chronic heart failure, serum S-SMase activity is elevated in pathological states.11,12 Usually, 1 mg of low-density lipoprotein (LDL) contains 199 ± 18.8 μg of sphingomyelin.13 Treatment of human LDL with neutral sphingomyelinase from Staphylococcus aureus hydrolyzed more than 95% of the sphingomyelin but had no effect on phosphatidylcholine, cholesteryl esters, free cholesterol, and triglyceride components of the LDL.13 Preincubating LDL with SMase may hydrolyze LDL-carrying sphingomyelin and release phosphocholine and ceramide, which enhances LDL aggregation and foam cell formation.14 In addition, sphingomyelinase may interact with very low-density lipoprotein (VLDL) and intermediate-density lipoprotein (IDL), which increases their

Sphingolipids are important bioactive molecules found in eukaryotic cell membranes and control a wide variety of cellular responses.1−3 Notably, ceramide, a central metabolite of sphingomyelin, plays a key role in regulating cell growth, viability, differentiation, and senescence.4 However, increases in the intracellular level of ceramide lead to aging, inflammation, insulin resistance, and mitochondrial dysfunction.5−7 In addition, circulating plasma ceramide levels are highly correlated to metabolic syndrome, diabetes, and obesity.8,9 The intracellular production of ceramide occurs de novo from the synthetic pathway in the endoplasmic reticulum, the salvage pathway in acidic lysosomes, and the sphingomyelinase (SMase) pathway in the plasma membrane at neutral pH.2,7 In contrast, secreted-SMase (S-SMase) is an alternative form of the lysosomal acid SMase.10 In patients with type II diabetes, © XXXX American Chemical Society

Received: October 1, 2015

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retention to human arterial proteoglycans.15 In vitro studies also show that modifying LDL by treatment with SMase leads to the tendency toward aggregation and subendothelial retention of atherogenic lipoproteins,16 and contributes to inflammatory gene expression from monocytes.17 In animal studies, a reduced aortic area of early foam cell lesions was observed in acid SMase-deficient apoE knockout mice.18 LDL, but not VLDL or HDL, possesses SMase activity, which, importantly, can be abrogated by oxidation.19,20 More specifically, a minor LDL subfractionelectronegative LDL is responsible for the phospholipolytic activities21,22 and is involved in the pro-inflammatory effects of LDL and the susceptibility to aggregation or inflammation.23−26 However, SSMase has not been detected in proteomic studies of electronegative LDL.26−28 Because isolated human LDL catalyzed the formation of ceramide from either the fluorescent sphingomyelin or [14C] sphingomyelin, the putative catalytic site of SMase activity is the His2230-Ser2306-Asp2359 triads in the α2 region of apoB-100.19 ApoB100, a large protein comprising 4536 amino acid residues,29,30 has a pentapartite structure with alternative α-helixes and β-pleated sheets (α1-β1-α2-β2-α3).31 The α1 sheet anchors the protein to the lipid core, and α2 and α3 expand and contract across the phospholipid belt of the LDL particle to stabilize electrostatic interactions, thus maintaining LDL protein structural integrity. The β-sheets are structurally rigid and engaged in electrostatic interactions with the phospholipids.31 With chemical alterations such as oxidation32,33 and nitration,34 apoB100 loses regular secondary elements, increasing the random coil conformation to generate a more flexible structure, which may account for the enzymatic activity. Previously, we have shown that human LDL can be chromatographically resolved into 5 transitional subfractions (L1−L5) with increasing electronegativity and changing protein composition.35,36 LC/MSE analysis of protein composition showed that L1 was mainly composed of apoB100 (99.7%), with trace amounts of apoE and apoAI. In contrast, apoB100 made up only 61.3% of the protein weight of L5, along with several other proteins, including apo(a), apoE, apoAI, apoCIII, platelet-activating factor acetylhydrolase, and paraoxonase 1.27 The concentration of apoE and apoAI gradually increased from L1 to L5, and apo(a) was found only in L5, or electronegative LDL. Using a colorimetric method to analyze lipid components, we have shown that L5 had significantly increased triglyceride content and decreased cholesterol ester content as compared with L1, but there was no difference between the two in the content of phospholipids and cholesterol.36 L5 is highly atherogenic to cardiovascular systems,37 whereas L1, even at very high doses, shows no apoptotic effects on endothelial cells.38 L5 is highly associated with hyperlipidemia, diabetes, metabolic syndrome, and ST-elevation myocardial infarction.35,37−41 Yang et al.35 and Ke et al.27,37 have shown that L5 has reduced cholesterol ester but increased triglyceride content when compared with L1. In this study, we examined the phospholipolytic properties of the L5 and L1 subfractions of LDL and the possible structure of the α2 region in apoB100. In addition, we studied the mechanisms underlying the differences between L5 and L1 by analyzing the posttranslational modifications.

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RESULTS The Neutral Lipid Subfraction from Electronegative LDL (L5) Induces Endothelial Cell Apoptosis. We have previously shown and confirmed here that L1 at concentrations ranging from 50 to 500 μg/mL elicits no cytotoxic effects on endothelial cells (ECs) but that L5 induces EC apoptosis.37,42 To examine the role of lipids on EC apoptosis, we extracted total lipids from L1 and L5. We used 5% fetal bovine serum (FBS) as a carrier of the lipid extracts. After 24 h incubation at 37 °C, ECs were stained with DAPI (4′,6-diamidino-2phenylindole; blue) for cell nuclei, calcein AM (green) for cytoplasm, and propidium iodide (red) for exposed DNA from dead cells. The total lipids from L1, which are representative of normal intact LDL, showed minimal, if any, apoptotic potential (Figure 1B), but L5 lipids showed harmful effects (Figure 1C)

Figure 1. High content of ceramide in L5 induces endothelial apoptosis. BAECs were treated with PBS (A), the total lipid (TL) extract from 50 μg/mL of L1 (B), the total lipid extract from 50 μg/ mL of L5 (C), fatty acids from 50 μg/mL of L5 (D), phospholipids from 50 μg/mL of L5 (E), or neutral lipids from 50 μg/mL of L5 (F) (n = 5). After 24 h incubation at 37 °C, the cells were stained with DAPI (4′,6-diamidino-2-phenylindole; blue) for cell nuclei, calcein AM (green) for cytoplasm, and propidium iodide (red) for exposed DNA from dead cells. Because neutral lipids from L5 showed the most cytotoxic effects on cultured BAECs, we examined the difference in lipid components between L1 and L5 by using LC/MSE (G). Results showed that the concentration of ceramide in L5 was significantly higher than that in L1 (n = 4; **P < 0.01). Incubation with ASAH2 for 2 h significantly decreased the ceramide content in L5 (n = 10; **P < 0.01) but not in L1.

at a concentration of 50 μg/mL (n = 5). Because the total lipids from L5 showed cytotoxic effects on cultured bovine aortic ECs (BAECs), we further divided the total lipids into free fatty acid (FFA), phospholipids (PL), and neutral lipids (NL) by using a NH2 column. The same treatment procedures as described above were applied. Fatty acids from L5 showed no harmful effects (Figure 1D). In contrast, phospholipids damaged some ECs (Figure 1E), and neutral lipids from 50 μg/mL of L5 induced dramatic EC apoptosis (Figure 1F) (n = 5). To further B

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identify the characteristic lipid component in L5, all lipid components in LDL, including diacylglycerol and monoacylglycerol, were analyzed by data-independent, parallel-fragmentation liquid chromatography/mass spectrometry (LC/MSE) and principal component analysis.27 This approach allowed us to analyze the differences in neutral lipids between L1 and L5. We found that the total ceramide content was signficantly higher in L5 than in L1 (n = 4; P < 0.01). Except for ceramide content, there were no other detectable differences between L1 and L5. N-acylsphingosine amidohydrolase (ASAH2) is a nonlysosomal enzyme that specifically degrades ceramide. Incubation with ASAH2 for 2 h significantly decreased ceramide content in L5 (n = 4; P < 0.01) but not in L1 (Figure 1G). Using full mass scan to compare lipid components in L1 versus L5, we showed that ceramide (18:0/16:0), ceramide (18:1/24:1) and ceramide (18:0/24:0) were 64−80% higher in L5 than in L1 (Supplementary Figure 1) (n = 4; P < 0.05). Ceramide (18:1/25:0) was about 98% higher in L5 than in L1 (Supplementary Figure 1) (n = 10; P < 0.05). However, some lipid targets were under the detection limit of full mass scan in certain samples. Electronegative LDL (L5) Shows Sphingomyelinase Activity. SMase activity was detected by using the Amplex Red sphingomyelinase assay kit (Invitrogen). L5 showed strong SMase activity (n = 12), whereas the subfractions, L1 to L4, showed no enzyme activity (Figure 2A). Because desipramine induces the degradation of acid sphingomyelinase and thus abolishes the production of ceramide, we conducted experiments in which we pretreated L5 (50 μg/mL) with 10 uM desipramine and then exposed the mixture to cultured ECs; we found that desipramine attenuates the cytotoxicity of L5 (Figure 2B) (n = 5). L1 Preincubated with Exogenous SMase Induces EC Apoptosis in a Dose-Dependent Manner. Thin layer chromatography (TLC) was used to confirm the presence of sphingomyelin in L1 and L5. Our results showed that L1 contained sphingomyelin and that it can be the reactant for SMase (Figure 3A, n = 3). To mimic the effect of L5 containing SMase activity, we incubated L1 with exogenous SMase. BAECs were treated with (a) PBS; (b) 2U SMase; (c) 25 μg/mL of L5; (d) 50 μg/mL of L5; (e) 50 μg/mL of L1; (f) 50 μg/mL of L1 preincubated with 0.1U SMase; (g) 50 μg/mL of L1 preincubated with 0.5U SMase; or (h) 50 μg/mL of L1 preincubated with 1U SMase. After 24 h of incubation at 37 °C, the cells were stained with DAPI (4′,6-diamidino-2-phenylindole; blue) for cell nuclei, calcein AM (green) for cytoplasm, and propidium iodide (red) for exposed DNA from dead cells (Figure 3B). Our results showed that treatment with PBS (control), L1, or SMase (2U) was not cytotoxic to ECs, whereas preincubation of L1 with exogenous SMase increased apoptosis of ECs in a dose-dependent manner. Again, L5 induced extensive EC apoptosis. Sequence Comparison of apoB100 and SMases. To identify the putative region of LDL responsible for its SMase activity, we compared the amino acid sequence of human apolipoprotein B100 (apoB100) with that of Bacillus cereus SMase (Figure 4). The 3-dimensional structure of SMase from B. cereus was determined in 2006.43 The alignment pair of human apoB100 and B. cereus SMase extracted from the multiple sequence alignment (MSA) was used as the input for model building, and then the following structural comparison was performed with incremental Combinatorial Extension.44 The MSA showed 119 strongly conserved positions out of 360

Figure 2. Sphingomyelinase activity found exclusively in L5. (A) SMase activity was detected by using the Amplex Red sphingomyelinase assay kit (Invitrogen, CA), in which the fluorescent signal represents the amount of product generated by the degradation of sphingomyelin. We used 0.04 U SMase from Bacillus cereus as a positive control and H2O2 as a reagent control. By increasing the fluorescent signal in 20 min, L5 showed strong SMase activity, whereas L1−L4 showed no enzyme activity throughout the 3-h testing period (n = 12) (A). Preincubation of desipramine (10 uM), which inhibits acid sphingomyelinase, with 50 μg/mL of L5 attenuated the cytotoxicity of L5 on cultured endothelial cells (n = 5) (B).

aligned columns (33%). The amino acid residues in the central metal-binding site and in the edge metal-binding site are shown by red boxes and blue boxes, respectively. Almost all of these residues are conserved in bacterial and mammalian SMases and human apoB100. 3-Dimensional Protein Structure Prediction of Human apoB100 (Residues 2021−2377). In protein homology modeling, it is generally necessary to check and edit the alignment by visually inspecting the template structures, especially if the target−template sequence identity is low. Here, we performed minor manual adjustments on the original MUSCLE MSA of the target (apoB100) and template (2ddrA) pair (Figure 4). The putative residues that participate in the SMase activities are shown in boldface. These residues include N2035, E2079, F2081, N2084, E2140, N2251, D2253, D2312, and H2366. The predicted structure of apoB100 (residues 2021−2377) (right panel, Figure 5A) was constructed based on this alignment. As a result, these six amino acid residues are superimposable on the corresponding conserved amino acid residues of SMases (Figure 5A). Electrostatic surface calculations show distinct regions of highly negative patches (red color, Figure 5B), located at the central cleft regions of B. cereus SMase and the apoB100 (residues 2021−2377) model (Figure 5B). These surrounding negatively charged groups probably play a role in interacting with the positively charged C

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Figure 3. Cytotoxicity testing for L1, L5, and L1 with exogenous SMase. (A) Thin layer chromatography comparing the lipid content of sphingomyelin in L1 and L5. Lane 1, 5 μg sphingomyelin (SM); lane 2, 5 μg sphingomyelin incubated with SMase for 2 h at 37 °C; lane 3, lipid extracted from L1; and lane 4, washed lipid extracted from L5. Iodine staining of the lipids showed that L1 contained more sphingomyelin than in L5 (n = 3). Because sphingomyelin is the catabolic target of SMase, we examined the effect of LDL with additional SMase activity (n = 5) (B). BAECs were treated with (a) PBS, (b) 2U SMase, (c) 25 μg/mL of L5, (d) 50 μg/mL of L5, (e) 50 μg/mL of L1, (f) 50 μg/mL of L1 preincubated with 0.1 U SMase, (g) 50 μg/mL of L1 preincubated with 0.5U SMase, or (h) 50 μg/mL of L1 preincubated with 1 U SMase. After 24 h incubation at 37 °C, the cells were stained with DAPI (4′,6-diamidino-2-phenylindole; blue) for cell nuclei, calcein AM (green) for cytoplasm, and propidium iodide (red) for exposed DNA from dead cells.

Figure 5. Structural analysis of the B. cereus SMase and the apoB100 (residues 2021−2377) model. (A, top) Schematic showing the crystal structure of B. cereus SMase and the predicted apoB100 (residues 2021−2377) model. Clusters of metal-binding residues are labeled and depicted as sticks. (B, bottom) Electrostatic potential surfaces of the B. cereus SMase and the apoB100 (residues 2021−2377) model are shown in the same orientation. The electrostatic potential surfaces are shown in blue for positive, white for hydrophobic, and red for negative. High negative potentials are found at the central cleft regions of the two structures. All images were generated by the PyMOL software.

metal ions, such as Co2+, Mn2+, Mg2+, CA2+, or Sr2+. Finally, the main geometric parameters of the best scored structure of the apoB100 (residues 2021−2377) were evaluated by using the PROCHECK software and an analysis of the phi/psi Ramachandran (Figure 6) plot, which showed more than 81.3% for apoB100 (residues 2021−2377) of residues in the most favored regions of the Ramachandran plot. This result is comparable with the crystal structure of SMase; only 5 of the 310 (1.6%) residues of apoB100 (residues 2021−2377) have disallowed geometry. However, because the five residues are

located far from the cleft of the metal-binding site, this does not affect our structure analysis. Based on the model structure, it is reasonable to hypothesize that the apoB100 (residues 2021−

Figure 4. Alignment of apoB100 with sphingomyelinase. The amino acid sequences of the human apoB100 (residues 2021−2377) and Bacillus cereus SMases were aligned by the program MUSCLE. The amino acid residues participating in the central metal-binding site and in the edge metal-binding site are indicated by the red and blue boxes, respectively. D

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Holopainen and colleagues19 have shown that SMase activity is associated with human plasma LDL. Based on a sequence comparison between apoB100 and bacterial SMases, they also suggested that this activity could be an intrinsic property of apoB100 and that the putative functionally important residues for SMase are Glu2079, Asp2237, Asp2193, His2230, Asp2237, Thr2304, Ser2306, and Asp2359 in the sequence of apoB100. In the current study, our goal was to model the apoB100 (residues 2021−2377) in order to understand the structural implications of the catalytic residues that were identified in this study. The atomic coordinates of the model structure for the human apoB100 (residues 2021−2377) were predicted by using B. cereus SMase (PDB entry: 2DDR)43 as the template; therefore, the important residues for the SMase-like activity could be identified. Recent studies show that B. cereus SMase hydrolyzes sphingomyelin to phosphocholine and ceramide48−50 and that the catalytic residues are Asn-16, Asp-195, Asn-197, 253-Asp, and 296-His. In our study, almost all of these residues are conserved in the sequence alignment (Figure 4). Additionally, the divalent metal ion bound at Glu-53 is believed to be essential for hydrolytic activity. Glu-53 was reported previously to be the binding site of the hydrolytically essential Mg2+,51 whereas mutation of Asp-195 or His-296 abolishes the hydrolytic activity.51 It should be noted that the structure of apoB100 (residues 2021−2377) mentioned above is a putative model, and further research is needed to confirm a causal role for SMase-like activity on apoB100. Based on the literature,19,20 model analysis,52,53 and our experimental studies, we conclude that SMase activity is associated with human plasma LDL, especially in the most negatively charged subfraction of LDL (L5). Moreover, this enzymatic activity may be an intrinsic property of apoB100. The amino acid sequence of apoB100 in electronegative LDL was highly O-glycosylated, including S1732, S1959, S2378, S2408, and S2429. These findings may support the changing of the α-helix/ β-pleated sheets ratio in protein structure analysis.

Figure 6. Ramachandran plot of the apoB100 (residues 2021−2377) model. The most favored regions are red, and additional allowed, generously allowed, and disallowed regions are indicated as yellow, light yellow, and white, respectively.

2377) is the most important region, which has the potential for SMase activities. Post-translational Modifications of apoB100. To identify specific post-translational modifications of apoB100 near the catalytic region, we performed LC/MSE. Our results showed that the amino acid sequence of apoB100 in electronegative LDL was highly O-glycosylated, including S1732, S1959, S2378, S2408, and S2429 (Table 1).

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DISCUSSION Our study indicates that electronegative LDL shows SMase activity, which, in turn, can lead to an increase in ceramide levels in the circulation due to the degradation of sphingomyelin. High levels of ceramide in the bloodstream can induce EC apoptosis45,46 and are highly correlated to the development of diabetes, metabolic syndrome, and cardiovascular diseases.8,9,47 Therefore, the SMase activity of L5 is highly atherogenic. LDL carries lipids, including sphingomyelin and ceramide, which serve as the reactant and product, respectively. The SMase activity of L5 may degrade sphingomyelin on the LDL particle; this process generates ceramides, which can then incorporate into cells. The SMase activity of L5 may also hydrolyze sphingomyelin either on the cell membrane or in the cell lysosome, which induces the overproduction of ceramides. Moreover, we have shown that inhibiting the SMase activity of L5 pharmacologically with desipramine attenuates the apoptotic effect, although we cannot rule out a direct antiSMase effect of desipramine on cultured ECs.

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EXPERIMENTAL SECTION

Sample Preparation and L5 Purification. Blood samples were collected from hyperlipidemic (LDL-C > 160 mg/dL) adult subjects with the approval of the internal review board at Baylor College of Medicine, Houston, Texas. The samples were treated with 1% penicillin/streptomycin and citrate phosphate dextrose adenine-1 to protect from bacterial contamination and coagulation. The plasma was obtained and was treated with Complete Protease Inhibitor Cocktail (Roche Diagnostics, Indianapolis, IN, USA; 1 tablet/100 mL plasma) to avoid protein degradation. LDL (d = 1.019−1.063 g/mL) was then isolated by sequential potassium bromide density centrifugation and was treated with 5 mM EDTA and nitrogen to avoid ex vivo oxidation. LDL was further divided into L1 to L5 against graded salt gradient through anion-exchange columns (Uno-Q12) by using the Ä KTA fast protein liquid chromatography system (GE Healthcare Life Sciences, Pittsburgh, PA, USA) as previously described.35,42 The effluent was monitored at 280 nm and protected from ex vivo oxidation with 5 mM EDTA. The LDL subfractions were concentrated by using Centriprep

Table 1. O-Glycosylation of apoB100 in Electronegative LDL Residue

Peptide Sequence

Glycan Sequence

S1732 S1959 S2378 S2408 S2429

VSQEGLK KSISAALEHK LSNVLQQVK KLNELSFK SFDYHQFVDETNDK

Pent3DeoxyHex2Man6HexA1+HexNAc Man1HexNAc1NeuAc1+HexNAc DeoxyHex2Man1HexNAc1+HexNAc Man5HexA1+HexNAc HexNAc3NeuGc3+HexNAc E

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filters (YM-30; EMD Millipore Corp., Billerica, MA, USA) and were sterilized by passing through 0.22-μm filters. We assessed the ability of all L1 and L5 subfractions to induce apoptosis of ECs, as previously described.35 Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 4−20%), we examined the apoplipoprotein content of all L1 and L5 subfractions. The isolated subfractions were N2-sealed and stored at 4 °C during sample characterization.38 Lipid Extraction. We transferred 25 μg of L1 or L5 into a glass tube, added 1 mL of H2O, 2.5 mL of methanol, and 1.25 mL of CHCl3, and vortexed the sample for 15 s. Then, we added an additional 0.9 mL of H2O and 1.25 mL of CHCl3, vortexed the sample for 15 s, and placed it in the centrifuge at 3000 rpm for 10 min. Using a glass syringe, we transferred the bottom layer of organic solvents to a 2.0 mL glass tube as a total lipid sample, or we transferred it to a 6 mL Sep-Pak NH2 column (Waters Corporation, Milford, MA, USA) for further isolation. We used CHCl3/isopropanol to elute neutral lipids, diethyl ether in 2% acetic acid to elute fatty acids, and methanol to elute phospholipids. For cell studies, we flushed each fraction with nitrogen until it became a dry pellet and dissolved it in 500 uL of 5% FBS-containing culture medium. For LC/MSE, the pellets were dissolved in 0.25 mL of sample solution (isopropanol/acetonitrile/ H2O = 2:1:1). Thin Layer Chromatography (TLC). We incubated 5 μg sphingomyelin with or without SMase for 2 h at 37 °C; the lipids extracted from L1 and L5 were applied to a thin layer plate and airdried. The mobile phase comprised solvent that contained heptane: diethyl ether: acetic acid at 70:30:2. After 30 min, the lipids were visualized by using iodine staining. LC/MSE Analysis for Lipid Composition. LC/MSE was used to quantify the lipid content of each subfraction of LDL. The quantitative analysis was performed as previously described.27 Briefly, lipids were chromatographically separated by using an acquity separations module (Waters Corporation) incorporating a CSH 1.7 um, 2.1 mm × 10 cm C-18 column under gradient conditions at a flow rate of 400 uL/min over 18 min at 55 °C. The mobile phase A was composed of 10 mM ammonium formate in ACN/H2O (60/40) and 0.1% formic acid (0.1% v/v); mobile phase B was composed of 10 mM ammonium formate in IPA/ACN (90/10) and 0.1% formic acid (0.1% v/v) for molecule protonation. Mass spectrometry was performed on a Synapt HDMS (Waters Corporation) instrument equipped with a nanoelectrospray ionization interface and operated in the data-independent collection mode (MSE). We programmed the parallel ion fragmentation to switch between low (4 eV) and high (35−55 eV) energy in the collision cell and collected data from 50 to 1600 m/z using leucin as the separate data channel lock mass calibrant. The data were processed with MarkerLynx (Waters Corporation). ASAH2 Transfection of HEK Cells and Protein Purification. ASAH2 was purchased from Origene (OriGene Technologies, Inc., Rockville, MD, USA, RC203706). Genes were amplified by ECOS 101 DH5α Competent Cells (Yeastern Biotech Co., Ltd., Taipei, Taiwan, FYE608) according to the manufacturer’s directions. One day before transfection, we plated 1.25 × 105 HEK293T cells in 500 ul Dulbecco’s Modified Eagle’s medium (DMEM) in 24-well plates. For each well of cells to be transfected, we diluted 1 μg of DNA in 100 ul serum-free medium, added 1.5 μL of Lipofectamine 2000 Transfection Reagent (Invitrogen, Grand Island, NY, USA), and mixed gently. After incubating the mixture for 30 min at room temperature, we added the complexes to each well that already contained the cells and mixed them gently. The cells were incubated at 37 °C in a CO2 incubator for 20 h, and then the transfected cells were lysed by RIPA, which contains a protease inhibitor. To purify the proteins, we equilibrated 80ul ANTI-FLAG M2Magnetic Beads (Sigma-Aldrich, St. Louis, MO, USA) for one-well cell lysate purification. After protein-resin binding at 4 °C overnight, the bound FLAG fusion protein was eluted by competitive elution with 150 μg/mL 3X FLAG peptide for 2 times. We collected the eluate and checked the protein by Western blot. Sphingomyelinase Activities. SMase activity in LDL was monitored indirectly by using coupled enzymes and a sensitive fluorogenic probe, 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red reagent, Life Technologies, Grand Island, NY, USA). Each reaction

contained 50 μM Amplex Red reagent, 1 U/mL HRP, 0.1 U/mL choline oxidase, 4 U/mL of alkaline phosphatase, 0.25 mM sphingomyelin, and 0.04U B. cereus sphingomyelinase as a positive control. SMase hydrolyses sphingomyelin to yield ceramide and phosphorylcholine. After applying alkaline phosphatase, which hydrolyses phosphorylcholine, choline is oxidized by choline oxidase to betaine and the intermediate product, hydrogen peroxide (H2O2). The fluorescent signalresorufinis immediately released by the catalysis of Amplex Red and hydrogen peroxide in the presence of horseradish peroxidase. We used 20 mM H2O2 and 0.04U sphingomyelinase as a reagent control and SMase activity as a positive control. The reactions were incubated at 37 °C for 30 min. We measured the fluorescence at 590 nm.21 Cell Culture and Apoptosis Measurements. Primary BAECs (Cambrex Corp., East Rutherford, NJ, USA) were used after 3 or 4 passages and were maintained in DMEM (Invitrogen) containing 10% FBS. During treatment, FBS was reduced to 5% in DMEM. We seeded 1 × 104 cells in 96-well plates for 24 h, and then exposed the subconfluent cultures of BAEC to PBS (lipoprotein-free, negative control) or graded (25, 50, and 100 μg/mL) LDL subfractions, unfractionated normolipidemic LDL, or Cu2+-oxLDL for 24 h. Apoptosis was assessed visually with the use of a Zeiss Axiovert 200 fluorescence microscope and filters (Carl Zeiss Microscopy GmbH, Jena, Germany) to capture digital images based on Hoechst 33342, propidium iodide (red), and calcein AM (green) staining of nuclear, apoptotic DNA membrane integrity.35 Cytoplasmic histone-associated DNA fragmentation was examined by using the Cell Death Detection ELISA Assay (Roche Diagnostics) according to the manufacturer’s protocol. Sequence Alignment. The amino acid sequences of human apoB100 and B. cereus, Leptospira interrogans, and S. aureus SMase obtained from the Swiss-Prot54 and GenBank55 databases were aligned by MUSCLE.56 Among these sequences, the X-ray crystallographic structure of B. cereus SMase was determined on May 2006 and is available from the Protein Data Bank (PDB code 2DDR). Protein Structure Prediction. The three-dimensional (3D) structure of the human apoB100 (residues 2021−2377) was predicted by using ModellerV9.857 on the target sequence that was derived from its alignment with the template structure. The X-ray crystallographic structure of SMase from B. cereus (PDB code 2DDR)43 was used as a direct template to construct the model of apoB100 (residues 2021− 2377). The target-template alignment was obtained from the results of MUSCLE multiple sequence alignment with minor manual adjustments. The qualities of the model’s structures were checked by using discrete optimized protein energy (DOPE) score58 and PROCHECK Ramachandran plots.59 The model with the highest quality was selected as the final predicted structure for the target. Finally, the protein-metal ion complex was constructed by superimposing the predicted 3D structure of the B. cereus sphingomyelinase-metal ion complex. LC/MSE Analysis for the Post Translational Modifications of L5. Because the proportional increases of these low-pI proteins may contribute to the overall negative charge of L5, we quantified the protein content in L1 to L5 subfractions by using quantitative proteomics techniques utilizing serially coupled LC/MSE.60 This analysis has been shown to be highly quantitative with respect to both relative and/or absolute (when incorporating spiked internal peptide standards in the data collection/analysis procedures) protein abundance in complex protein mixtures.60−62 Quantitative analysis was performed as previously described,62 except we used a Waters Synapt HDMS mass spectrometer (Waters Corporation). In brief, total proteins isolated from each LDL subfraction were first digested with trypsin, and the resulting tryptic peptides were chromatographically separated on a Nano-Acquity separations module (Waters Corporation) incorporating a 50 fmol-on-column tryptic digest of yeast alcohol dehydrogenase as the internally spiked protein quantification standard. Peptide elution was achieved through a 75 μm × 25 cm BEH C-18 column under gradient conditions at a flow rate of 300 nL/min over 30 min at 35 °C. The mobile phase was composed of acetonitrile as the organic modifier and formic acid (0.1% F

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v/v) for molecule protonation. Mass spectrometry was performed on a Synapt HDMS instrument (Waters Corporation) equipped with a nanoelectrospray ionization interface and was operated in the dataindependent collection mode (MSE). Parallel ion fragmentation was programmed to switch between low (4 eV) and high (15−45 eV) energies in the collision cell, and data were collected from 50 to 2000 m/z utilizing glu-fibrinopeptide B as the separate data channel lock mass calibrant. Data were processed with ProteinLynx GlobalServer v2.4 (Waters Corporation).62 Deisotoped results were searched for protein association from the Uniprot (www.uniprot.org) human protein database (v15.12; containing 34,786 entries). Statistical Analysis. The significance of differences was assessed by a paired Student t test, the Mann−Whitney U test, and the χ2 test. P < 0.05 was considered as significant statistically. The Statistical Package for Social Science (version 19.0; IBM SPSS Statistics, New York, NY) was used to perform all statistical analyses.

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ABBREVIATIONS USED BAECs, bovine aortic endothelial cells; ECs, endothelial cells; LDL, low-density lipoprotein; MSA, multiple sequence alignment; ASAH2, N-acylsphingosine amidohydrolase; S-SMase, secreted-SMase; SMase, sphingomyelinase

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ASSOCIATED CONTENT

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b01534. Full mass scan for the comparison of lipid components in L1 versus L5 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Address for Chu-Huang Chen, MD, PhD, Vascular and Medicinal Research, Texas Heart Institute, 6770 Bertner Avenue, Houston, Texas 77030. Phone: 832-355-9026. Fax: 832-355-9333. E-mail: [email protected]. *Address for Jeng-Hsien Yen, MD, PhD: Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. Phone: +886 7 3121101 ext 6088. Fax: +886 7 3118141. E-mail: [email protected]. Author Contributions ¥

REFERENCES

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S Supporting Information *

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Article

L.Y.K. and H.C.C contributed equally.

Author Contributions

All authors have met the requirements for authorship as set forth by the International Committee of Medical Journal Editors. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Rebecca Bartow, Ph.D., of the Texas Heart Institute in Houston, Texas, for editorial assistance. The work described in this review was supported in part by grants from the American Diabetes Association (1-04-RA-13), the National Institutes of Health Heart, Lung, and Blood Institute (HL63364), Kaohsiung Medical University (KMU) grant KMUTP104D02, KMU-Q104019; the Kaohsiung Medical University Alumni Association of America grant KMUH-10402, and Taiwan Ministry of Science and Technology grants MOST 103-2314-B-037−070, 103-2325-B-039−006; the Mao-Kuei Lin Research Fund of Chicony Electronics, the Stroke Biosignature Program Grant of Academia Sinica in Taiwan (BM104010092), Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW104TDU-B-212-113002), and Kaohsiung Medical University Hospital, Taiwan (KMUH102-M209). G

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