International Journal of Molecular Sciences Article Presence of Ceramidase Activity in Electronegative LDL Núria Puig 1,2 , Jose Rives 1,2 , Montserrat Estruch 3 , Ana Aguilera-Simon 4 , Noemi Rotllan 5,6 , Mercedes Camacho 7,8 , Núria Colomé 9 , Francesc Canals 9 , José Luis Sánchez-Quesada 1,6, * and Sonia Benitez 1,6, * 1 Cardiovascular Biochemistry Group, Research Institute of the Hospital de la Santa Creu i Sant Pau (IIB Sant Pau), 08041 Barcelona, Spain 2 Biochemistry and Molecular Biology Department, Universitat Autònoma de Barcelona, 08193 Cerdanyola, Spain 3 Biotech Research and Innovation Centre, Faculty of Health and Medical Sciences, University of Copenhagen BRIC, DK-1350 Copenhagen, Denmark 4 Vascular Brain Diseases, IIB Sant Pau, 08041 Barcelona, Spain 5 Molecular Basis of Cardiovascular Risk, IIB Sant Pau, 08041 Barcelona, Spain 6 CIBER of Diabetes and Related Metabolic Diseases (CIBERDEM), Instituto de Salud Carlos III, 28029 Madrid, Spain 7 Genomics of Complex Diseases Unit, IIB Sant Pau, 08041 Barcelona, Spain 8 CIBER of Cardiovascular Disease (CIBERCV), Instituto de Salud Carlos III, 28029 Madrid, Spain 9 Proteomics Laboratory, Vall d’Hebron Institute of Oncology (VHIO), 08035 Barcelona, Spain * Correspondence: jsanchezq@santpau.cat (J.L.S.-Q.); sbenitez@santpau.cat (S.B.) Abstract: Electronegative low-density lipoprotein (LDL(−)) is a minor modified fraction of human plasma LDL with several atherogenic properties. Among them is increased bioactive lipid mediator content, such as lysophosphatidylcholine (LPC), non-esterified fatty acids (NEFA), ceramide (Cer), and sphingosine (Sph), which are related to the presence of some phospholipolytic activities, including platelet-activating factor acetylhydrolase (PAF-AH), phospholipase C (PLC), and sphingomyelinase (SMase), in LDL(−). However, these enzymes’ activities do not explain the increased Sph content, which typically derives from Cer degradation. In the present study, we analyzed the putative Citation: Puig, N.; Rives, J.; Estruch, presence of ceramidase (CDase) activity, which could explain the increased Sph content. Thin layer M.; Aguilera-Simon, A.; Rotllan, N.; chromatography (TLC) and lipidomic analysis showed that Cer, Sph, and NEFA spontaneously Camacho, M.; Colomé, N.; Canals, F.; increased in LDL(−) incubated alone at 37 ◦ C, in contrast with native LDL(+). An inhibitor of neutral Sánchez-Quesada, J.L.; Benitez, S. CDase prevented the formation of Sph and, in turn, increased Cer content in LDL(−). In addition, Presence of Ceramidase Activity in LDL(−) efficiently degraded fluorescently labeled Cer (NBD-Cer) to form Sph and NEFA. These Electronegative LDL. Int. J. Mol. Sci. observations defend the existence of the CDase-like activity’s association with LDL(−). However, 2023, 24, 165. https://doi.org/ neither the proteomic analysis nor the Western blot detected the presence of an enzyme with known 10.3390/ijms24010165 CDase activity. Further studies are thus warranted to define the origin of the CDase-like activity Academic Editors: Philip W. Wertz detected in LDL(−). and Lih Kuo Keywords: electronegative LDL; ceramide; sphingosine; ceramidase; sphingomyelinase Received: 5 September 2022 Revised: 25 November 2022 Accepted: 17 December 2022 Published: 22 December 2022 1. Introduction Atherosclerosis involves chronic inflammation in the intima layer of the arterial wall, as triggered by the slow and progressive accumulation of lipids [1]. The main sources Copyright: © 2022 by the authors. of lipids in this process are low-density lipoproteins (LDL), which undergo chemical Licensee MDPI, Basel, Switzerland. modification in the subendothelial microenvironment, primarily from lipolytic enzymes This article is an open access article and oxidative stress. Modified LDL triggers a strong inflammatory response in monocyte- distributed under the terms and derived macrophages that infiltrate the arterial wall. conditions of the Creative Commons Electronegative LDL (LDL(−)) is a minor modified form of LDL found in the circula- Attribution (CC BY) license (https:// tion that accounts for 2–3% of total LDL in healthy subjects. This proportion increases in creativecommons.org/licenses/by/ 4.0/). pathologies associated with cardiovascular disease, such as dyslipemia, diabetes, obesity, Int. J. Mol. Sci. 2023, 24, 165. https://doi.org/10.3390/ijms24010165 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 165 2 of 13 metabolic syndrome, or non-alcoholic fatty acid disease [2–4], suggesting an active role of LDL(−) in atherogenesis. The atherogenic role of LDL(−) is supported by in vitro studies showing its strong binding with proteoglycans, high susceptibility to aggregation, and induction of a portfolio of inflammatory and apoptotic effects in different cell types from the arterial wall, such as monocytes/macrophages [5–8], and also lipid accumulation in macrophages [6]. These atherogenic effects seem to be due to an abnormal lipid composition compared to native LDL (LDL(+)). In this context, lipid alterations include increased bioac- tive lipid mediator content, such as lysophosphatidylcholine (LPC) [9,10], non-esterified fatty acids (NEFA) [9,11], ceramide (Cer), and sphingosine (Sph) [12,13]. Previous studies of ours have described that chemical modifications of total LDL that increase its LPC, NEFA, Cer, and Sph content endow it with inflammatory properties similar to those displayed by LDL(−) [9,11–13]. In addition, Cer and Sph have especially been connected to atheroscle- rosis and CV risk [14–16], as well as described to act as crucial signaling molecules that regulate processes such as inflammation, apoptosis, and cell differentiation [17–19]. The lipid alterations in LDL(−) are, in part, a consequence of at least two phospho- lipolytic activities present in this particle type. LDL(−) shows increased content and activity of the enzyme platelet-activating factor acetylhydrolase (PAF-AH, also known as lipoprotein-associated phospholipase A2 , or Lp-PLA2 ) [20], which could be responsible for the particle’s higher LPC content compared to LDL(+)). PAF-AH could be involved in LDL(−)’s increased NEFA content as well, although a fraction of these lipids could also come from its binding to LDL during circulation in situations in which the capacity of albu- min for NEFA buffering is exceeded. LDL(−) also shows phospholipase C-like (PLC-like) activity that efficiently hydrolyzes the polar head of choline-containing phospholipids, primarily sphingomyelin (SM) and LPC [21–23]. The effect of the PLC-like activity on SM renders phosphorylcholine, a water-soluble molecule that presumably leaves the LDL particle, and Cer, which due to its hydrophobicity is retained in the LDL particles and causes the particle to aggregate [12,24,25]. However, none of these activities explain the increased Sph content in LDL(−). Besides sphingomyelinase (SMase), a key enzyme controlling sphingolipid metabolism is ceramidase (CDase), which hydrolyzes Cer to yield Sph and NEFA. Therefore, CDases (acid, neutral, and alkaline) are pivotal enzymes that regulate the intracellular homeostasis of Cer/Sph and are critical regulators of signaling pathways within cells [26]. The involve- ment and potential therapeutic roles of these enzymes in illnesses such as diabetes, obesity, cancer, and neurodegenerative diseases have been extensively reviewed [26,27]. Although there are no data reporting the presence of CDase activity associated with lipoproteins, previous observations have suggested the putative involvement of CDase-like activity in yielding Sph in LDL(−) [13]. Therefore, the aim of the current study was to assess whether Sph content in LDL(−) is a product of intrinsic CDase-like activity. 2. Results 2.1. Sph and Cer Content in LDL(−) Increase at 37 ◦ C In a previous study, we reported that the amount of Cer and NEFA in LDL(−), but not LDL(+), was exacerbated when samples were incubated at 37 ◦ C [12]. In the current study, we similarly evaluated the effect of incubation at 37 ◦ C on Sph content. Figure 1 shows that Sph content in LDL(−) strongly increased after 24 h of incubation compared with LDL(+), as measured by thin layer chromatography (TLC) and liquid chromatography- mass spectrometry (LC-MS). In parallel with the Sph increase, LDL(−)’s Cer content also increased during incubation at 37 ◦ C compared with LDL(+), as previously described [12]. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 3 of Int. J. Mol. Sci. 2023, 24, 165 3 of 13 Figure 1. Ceramide (Cer) and sphingosine (Sph) contents in LDL(+) and LDL(−), basal and after 24 h Figure of incubation at 37 ◦(Cer) 1. Ceramide C. (a,b)and Datasphingosine obtained by TLC(Sph) contents (n = in LDL(+) 9). (c,d) Data and obtained by LDL(−), lipidomicbasal and after 2 analysis h of(nincubation at 37 = 4). * p < 0.05 °C. LDL( versus (a,b)−Data ) at 0obtained byversus h; # p < 0.05 TLC (n = 9). (c,d) LDL(+) Data obtained by lipidomic analys at 24 h. (n = 4). * p < 0.05 versus LDL(−) at◦ 0 h; # p < 0.05 versus LDL(+) at 24 h. 2.2. NEFA Species Increase at 37 C in LDL(−) If the increased Sph in LDL(−) is due to CDase activity, the relative NEFA quantity 2.2. NEFA Species Increase at 37 °C in LDL(−) should also increase and have the same order of magnitude as Sph. To test this possibility, weIfquantified the increased Sph in total NEFAs viaLDL(−) is due to CDase an enzymocolorimetric activity, assay the relative and determined NEFA quantit the different should NEFAalso increase species with a and haveanalysis. lipidomic the same order These data ofcorroborated magnitudeaas Sph. To previous test this possibilit observation of increased NEFA content in LDL( − ) when the particle was incubated at 37 ◦ C [12]. Figure 2a we quantified total NEFAs via an enzymocolorimetric assay and determined the differen shows the total NEFA in LDL(−) after incubation for 24 h at 37 ◦ C. As expected, the NEFA species with a lipidomic analysis. These data corroborated a previous observatio increases in both Sph (Figure 1b,d) and total NEFA (Figure 2a) were similar, approximately of increased two-fold afterNEFA content 24 h. This in LDL(−) observation when supports thethe roleparticle of CDasewas incubated activity as beingat 37 °C [12]. Figur responsible 2a for shows the totalofNEFA the generation in LDL(−) after incubation for 24 h at 37 °C. As expected, th both lipids. increases in both Regarding theSph NEFA (Figure species,1b,d) and total their increase NEFA in LDL( (Figure −) was found2a)to bewere similar, similar regard- approx less oftwo-fold mately their classification after 24ash.saturated (SFA), monounsaturated This observation supports the (MUFA), role oforCDase polyunsaturated activity as bein fatty acids (PUFA). responsible Figure 2b shows for the generation that the of both relative quantities of NEFA in LDL(−) versus lipids. LDL(+) were similar among the different species (two–three-fold increases), suggesting that Regarding the NEFA species, their increase in LDL(−) was found to be similar regard the putative CDase-like activity in LDL(−) is able to hydrolyze different ceramide species. less of their classification as saturated (SFA), monounsaturated (MUFA), or polyunsatu rated fatty acids (PUFA). Figure 2b shows that the relative quantities of NEFA in LDL( versus LDL(+) were similar among the different species (two–three-fold increases), sug gesting that the putative CDase-like activity in LDL(−) is able to hydrolyze differen ceramide species. . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 o Int. J. Mol. Sci. 2023, 24, 165 4 of 13 NEFA (mol/mol apoB) h h h h 0 24 0 24 -) +) -) L( +) L( L( LD L( LD LD LD Figure 2. Non-esterified fatty acid (NEFA) content in LDL(+) and LDL(−), basal and after 24 h of incubation at 37 ◦ C. (a) Total NEFA content measured via an enzymocolorimetric assay (n = 6). Figure 2. Non-esterified fatty acid (NEFA) content in LDL(+) and LDL(−), basal and after 24 (b) LDL(−)/LDL(+) ratio of the different NEFAs generated after lipoprotein incubation at 37 ◦ C, as incubation at 37 °C. (a) Total NEFA content measured via an enzymocolorimetric assay (n = 6) determined by a lipidomic analysis (n = 4). * p < 0.05 versus LDL(−) at 0 h; # p < 0.05 versus LDL(+) LDL(−)/LDL(+) ratio of the different NEFAs generated after lipoprotein incubation at 37 °C, as at 24 h. termined by a lipidomic analysis (n = 4). * p < 0.05 versus LDL(−) at 0 h; # p < 0.05 versus LDL(+ 24 h. 2.3. Effect of CDase Inhibitor on Sph, Cer, and NEFA Content in LDL(−) Next, we analyzed the effect of the neutral/alkaline CDase inhibitor MAPP (D-erythro- 2-(N-Myristoylamino)-1-phenyl-1-propanol). 2.3. Effect of CDase Inhibitor on Sph, Cer, andSMase-like and PAF-AH NEFA Content activities in LDL(−) in LDL(−) were not altered in the presence of MAPP, meaning this inhibitor did not have an effect on Next, these we analyzed enzymatic activitiesthe effect (Table 1). of the neutral/alkaline CDase inhibitor MAPP erythro-2-(N-Myristoylamino)-1-phenyl-1-propanol). SMase-like and PAF-AH activi Table 1. Effect of D-D-erytro-2-(N-myristoylamino)-1-phenyl-1-propanolerytro-MAPP (MAPP) in LDL(−) were not altered in the presence of MAPP, meaning this inhibitor did not h on platelet-activating factor acetylhydrolase (PAF-AH) and sphingomyelinase (SMase) activities an effect on in LDL(−). these enzymatic activities (Table 1). PAF-AH Activity SMase Activity Table 1. Effect of D-D-erytro-2-(N-myristoylamino)-1-phenyl-1-propanolerytro-MAPP (MAPP (mmol/min/mL) (mU/mg apoB) platelet-activating factor acetylhydrolase (PAF-AH) and sphingomyelinase (SMase) activitie LDL(−). LDL(−) 24 h 1.25 ± 0.35 205.85 ± 57.77 LDL(−) 24 h plus MAPP 1.29 ± 0.30 215.92 ± 34.48 PAF-AH Data represent the means ± SDs of seven independent Activity experiments. SMase Activity (mmol/min/mL) (mU/mg apoB) In contrast, Sph and NEFA content in LDL(−) was altered after 24 h of incubation, LDL(−)by24TLC as measured h (Figure 3a), enzymocolorimetry 1.25 ± 0.35(Figure 3b), or LC-MS205.85 (Figure ± 57.77 3c,d). LDL(−) 24 h plus MAPP partially MAPPthe formation of1.29 inhibited Sph; ± in0.30 215.92 turn, LDL(−)’s Cer content ± 34.48 was higher Data in the presence represent of MAPP. the means Accordingly, ± SDs of seventhere was a strong independent decrease in the Sph/Cer ratio in experiments. LDL(−) in the presence of MAPP, from 0.78 ± 0.19 to 0.42 ± 0.19 (p < 0.05, as measured by TLC/densitometry, n = 9). This supports the inhibitory effect of MAPP on CDase- In contrast, like activity in Sph LDL(and NEFA content −). Lipidomic analysisinconfirmed LDL(−) was altered the TLC after results, 24 h ofa non- revealing incubation measured by increase significant TLC (Figure 3a),aenzymocolorimetry in Cer and decrease in Sph (Figure (Figure 3b), or LC-MS 3c). The decreased (Figure 3c– Sph content MAPP partially in LDL( −) wasinhibited thea formation paralleled by of Sph;NEFA similarly decreased in turn, LDL(−)’s content, Cer content as measured by LC-MSwas hig (Figure 3d) and enzymocolorimetry (Figure 3b). MAPP did not alter in the presence of MAPP. Accordingly, there was a strong decrease in the the Cer, Sph, orSph/Cer NEFA ratio contents in LDL(+) in any case. LDL(−) in the presence of MAPP, from 0.78 ± 0.19 to 0.42 ± 0.19 (p < 0.05, as measured TLC/densitometry, n = 9). This supports the inhibitory effect of MAPP on CDase-like tivity in LDL(−). Lipidomic analysis confirmed the TLC results, revealing a non-signific increase in Cer and a decrease in Sph (Figure 3c). The decreased Sph content in LDL was paralleled by a similarly decreased NEFA content, as measured by LC-MS (Fig 3d) and enzymocolorimetry (Figure 3b). MAPP did not alter the Cer, Sph, or NEFA c tents in LDL(+) in any case. Int. Int. J. Mol. Sci. J. Mol. 2023,24, Sci.2023, 24, 165 x FOR PEER REVIEW 5 of 14 5 of 13 Figure 3. Effect of the ceramidase inhibitor MAPP on the formation of Cer, Sph, and NEFA in LDL(−) Figure 3. Effect of the ceramidase inhibitor MAPP on the formation of Cer, Sph, and NEFA in LDL(−) ◦ C. (a) Cer after after 2424 h incubation h of of incubation at 37 at °C.37 (a) Cer and Sphand Sphmeasured content content measured by TLC (n =by 9). TLC (n = content (b) NEFA 9). (b) NEFA content measured measured viavia enzymocolorimetry enzymocolorimetry (n = 6).(n (c)=Cer 6). and (c) Cer and Sph Sph content contentby measured measured byanalysis a lipidomic a lipidomic analysis (n(n= = 4).4). (d)(d) NEFA NEFA content measured content by a lipidomic measured analysis analysis by a lipidomic (n = 4). * p(n < 0.05 = 4).versus LDL(−) * p < 0.05 in theLDL(−) in the versus absence of MAPP. absence of MAPP. 2.4. 2.4.LDL(−) LDL(Is−Able to Degrade ) Is Able Cer from to Degrade anfrom Cer External Source Source an External We also investigated whether the putative CDase-like activity associated with We also investigated whether the putative CDase-like activity associated with LDL(−) LDL(−) has the ability to degrade Cer molecules external to LDL(−). For this purpose, we has the ability to used fluorescently degrade labeled Cer molecules Cer (NBD-Cer) external as a substrate. toNBD The LDL( −). For this fluorescent probepurpose, was we used fluorescently labeled Cer (NBD-Cer) as a substrate. The NBD fluorescent bound to Cer’s fatty acid moiety. As a result of the incubation with LDL(−), the NBD-Cerprobe was bound to Cer’s fatty acid moiety. As a result of the incubation with LDL( − was hydrolyzed and yielded NBD-fatty acid (NBD-FA), whose release was partly inhib- ), the NBD-Cer was hydrolyzed ited Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW and yielded in the presence NBD-fatty of MAPP. Figure 4bacid shows(NBD-FA), whose release that the NBD-Cer was(measured hydrolysis partly inhibited 6 of 14 in the aspresence the formation of NBD-FA) of MAPP. Figurepromoted 4b showsby LDL(−) that was partially the NBD-Cer inhibited(measured hydrolysis in the presence as the formation ofofMAPP. NBD-FA) promoted by LDL(−) was partially inhibited in the presence of MAPP. Figure Figure 4.4. Degradation Degradation of NBD-labeled of NBD-labeled CerCer by LDL(−). −). NBD-Cer by LDL(NBD-Cer was incubated was incubated with LDL(+), with LDL(+), LDL(−), LDL(−), LDL(−)+MAPP andLDL(−)+MAPP ◦ and for for 24 h24ath37 at°C. 37 Degradation C. Degradation of NBD-Cer of NBD-Cer was measured was measured as theas the formation formation of of NBD-FA NBD-FA(n(n= 7). * p*