Characterization of the Lipidome and Biophysical Properties of Membranes from High Five Insect Cells Expressing Mouse P-Glycoprotein

biomolecules

Article Characterization of the Lipidome and Biophysical Properties of Membranes from High Five Insect Cells Expressing Mouse P-Glycoprotein

Maria João Moreno 1,2,* , Patrícia Alexandra Teles Martins 1, Eva F. Bernardino 1, Biebele Abel 3 and Suresh V. Ambudkar 3

1 Coimbra Chemistry Center, Chemistry Department, FCTUC, University of Coimbra, 3004-535 Coimbra, Portugal; [email protected] (P.A.T.M.); [email protected] (E.F.B.) 2 CNC—Center for Neuroscience and Cell Biology, University of Coimbra, 3004-535 Coimbra, Portugal 3 Laboratory of Cell Biology, CCR, National Cancer Institute, NIH, Bethesda, MD 20892, USA; [email protected] (B.A.); [email protected] (S.V.A.) * Correspondence: [email protected]

Abstract: The lipid composition of biomembranes influences the properties of the lipid bilayer and that of the proteins. In this study, the lipidome and the lipid/protein ratio of membranes from High Five™ insect cells overexpressing mouse P-glycoprotein was characterized. This provides a better understanding of the lipid environment in which P-glycoprotein is embedded, and thus of its functional and structural properties. The relative abundance of the distinct phospholipid classes and their acyl chain composition was characterized. A mass ratio of 0.57 ± 0.11 phospholipids to protein was obtained. Phosphatidylethanolamines are the most abundant phospholipids, followed by phos- Citation: Moreno, M.J.; Teles phatidylcholines. Membranes are also enriched in negatively charged lipids (phosphatidylserines, Martins, P.A.; Bernardino, E.F.; Abel, phosphatidylinositols and phosphatidylglycerols), and contain small amounts of sphingomyelins, B.; Ambudkar, S.V. Characterization ceramides and monoglycosilatedceramides. The most abundant acyl chains are monounsaturated, of the Lipidome and Biophysical with significant amounts of saturated chains. The characterization of the phospholipids by HPLC- Properties of Membranes from High MS allowed identification of the combination of acyl chains, with palmitoyl-oleoyl being the most Five Insect Cells Expressing Mouse representative for all major phospholipid classes except for phosphatidylserines, which are mostly P-Glycoprotein. Biomolecules 2021, 11, saturated. A mixture of POPE:POPC:POPS in the ratio 45:35:20 is proposed for the preparation 426. https://doi.org/10.3390/ biom11030426 of simple representative model membranes. The adequacy of the model membranes was further evaluated by characterizing their surface potential and fluidity.

Academic Editor: Mikhail Bogdanov Keywords: lipidome; High Five insect cells; membrane proteins; biomembranes Received: 5 February 2021 Accepted: 12 March 2021 Published: 14 March 2021 1. Introduction Publisher’s Note: MDPI stays neutral High Five™ insect cells are commonly used to overexpress membrane proteins, as with regard to jurisdictional claims in is the case for the drug efflux transporter P-glycoprotein [1]. When expressed in mam- published maps and institutional affil- malian cell lines, P-glycoprotein is fully glycosylated. On the other hand, P-glycoprotein iations. expressed in High Five cells is not glycosylated. For this reason, ABC transporters including P-glycoprotein expressed in High Five cells are suitable for structural and biochemical/biophysical studies [2,3]. The functional properties of the membrane protein of interest are usually characterized while embedded in the original membranes [1], although some Copyright: © 2021 by the authors. more specific properties and structural characterization requires the isolation of the protein Licensee MDPI, Basel, Switzerland. of interest followed by purification and reconstitution in simpler model systems [4,5]. This article is an open access article The lipid composition of the original membranes is very important to understand distributed under the terms and the results obtained, because membrane properties influence the structure, stability, and conditions of the Creative Commons function of the membrane protein of interest [5–10]. Additionally, the active molecules Attribution (CC BY) license (https:// interacting with membrane proteins are usually lipophilic and the lipid bilayer influences creativecommons.org/licenses/by/ globally observed properties such as binding affinity and activity [8–14]. Since the relative 4.0/).

Biomolecules 2021, 11, 426. https://doi.org/10.3390/biom11030426 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 426 2 of 16

affinity for the lipid bilayer and for the protein are not necessarily directly correlated, ignoring the mediator role of the lipid bilayer may lead to misinterpretations and to incorrect prediction of a protein’s specificity. The association of the active molecules with the lipid bilayer increases the local concentration in areas of the membrane surrounding the protein, orients amphiphilic molecules, and constrains their transversal location in the membrane, thus controlling their interaction with membrane proteins. The lipid composition is the most important property of the native membranes, controlling the global charge, thickness, and membrane fluidity [15–21]. The importance of the lipid fraction of biomembranes has often been overlooked, mostly due to difficulties in characterization. The extraordinary developments achieved in recent decades on gas chromatography with mass spectrometry detection (GC–MS) have made this important task not only possible but also simple and accurate [19,20,22–26]. This has led to the characterization of the lipid composition of a wide variety of biomembranes and the observation of significant variations between membranes obtained from different species and for cells grown under different conditions [15–19,22,23,26–35]. This shows the importance of characterizing the lipid composition of the native membranes in order to improve our understanding of the results obtained for the functional characterization of the proteins of interest when expressed in those systems. In this work we characterized the lipidome and biophysical properties of membranes obtained from High Five insect cells. The major phospholipid classes were characterized by TLC, the fatty acids were analyzed by GC–MS, and the full characterization of the lipidome was performed by HPLC-MS. The membrane vesicles were also characterized by dynamic light scattering (DLS), zeta potential, and fluorescence anisotropy of the membrane probes DPH and NBD-C16. We found the membrane lipid composition to be similar to that obtained previously for other insect cells, although significant variations were revealed. In addition, it was possible to obtain not only the overall acyl chain composition of the major lipid classes but also the characterization of the most abundant lipids. The lipidome characterization completed in this work includes all major lipid classes and minor components such as lysophospholipids, ceramides, and their glycosylates. A biophysical characterization was completed for total membranes and for representative lipid bilayers, allowing a better understanding of the role of the lipids in the overall properties of native membranes.

2. Materials and Methods The lipids 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- L-serine (POPS) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), the fluorescent probe 1,6-Diphenyl-1,3,5-hexatriene (DPH) was acquired from Sigma-Aldrich (Algés, Portugal), and N-hexadecyl-7-nitro-2,1,3-benzoxadiazol-4-amine (NBD-C16) was synthesized and purified as described before [36]. Reagents and solvents used were at the analytical grade or higher purity, and water was distilled and deionized with a final resistance ≥ 18 MΩ. High Five insect cells (Invitrogen) were infected with recombinant baculovirus en- coding (His)6-tagged mouse P-glycoprotein (p-gp). Membrane vesicles were prepared by hypotonic lysis of the insect cells, followed by ultracentrifugation to collect the membrane vesicles, as described in detail in the literature [1]. The membranes obtained were suspended in buffer (pH 7.5) containing 10% glycerol, aliquoted and stored at −80 ◦C until use. The amount of protein in the membranes was quantified by the Sherman and Weis- mann method using Amido Black B dye (acquired to Alfa Aesar, Kandel, Germany) [37]. The lipids were extracted by the method described by Bligh and Dyer [38]. Briefly, CHCl3/MeOH 1:2 (v/v) was added to the sample (3.75 mL per mL of the sample in aqueous media), vortexed, and incubated on ice for 30 min. An additional volume of 1.25 mL CHCl3 and of 1.25 mL H2O were added and samples were vigorously vortexed. The mixture was Biomolecules 2021, 11, 426 3 of 16

then centrifuged at 180× g for 5 min at room temperature to obtain a two-phase system from which lipids were obtained. An aliquot of the total lipid extract was analyzed for total phosphorous quantification [39] to obtain the total amount of phospholipids in the sample. An average molar mass of 750 g was considered to calculate the phospholipid mass. The lipid extract dissolved in CH2Cl2 was analyzed by TLC using silica gel plates to characterize the different phospholipid classes present. Prior to separation, the plates were ◦ washed with CHCl3/MeOH (1:1, v/v) and placed in an oven at 100 C for 15 min to dry. Each plate was developed with CHCl3/EtOH/H2O/triethylamine (30:35:7:35 v/v), allowed to dry and sprayed with primuline (at 0.50 µg/mL in acetone/H2O (80:20)) and lipid spots were visualized under UV radiation (254 and 366 nm; Camag, Berlin, Germany). The spots were scraped off the plates and added to glass tubes for phosphorous quantification [39]. Fatty acid methyl esters (FAMEs) were prepared by reaction with a solution of KOH 2 M in MeOH, according to the methodology described previously [40,41]. Hexane solutions containing the FAMEs (0.75 µg) were analyzed by GC–MS on an Agilent Technologies 6890 N Network gas chromatograph (Santa Clara, CA, USA) equipped with a DB–FFAP column (30 m of length, 0.32 mm internal diameter, and 0.25 µm film thickness, 123-3232, J&W Scientific, Folsom, CA, USA). The GC equipment was connected to an Agilent 5973 Network Mass Selective Detector operating with an electron impact mode at 70 eV and scanning the range m/z 50–550 in a 1 s cycle in a full scan mode acquisition. The oven temperature was programmed from an initial temperature of 80 ◦C for 3 min; a linear increase to 160 ◦C at 25 ◦C min−1; a linear increase at 2 ◦C min−1 to 210 ◦C; and a linear increase at 30 ◦C min−1 to 250 ◦C followed by 10 min at this temperature. The injector and detector temperatures were 220 ◦C and 280 ◦C, respectively. Helium was used as a carrier gas at a flow rate of 1.4 mL min−1. The identification of each fatty acid (FA) was performed considering the retention times and comparison with MS spectra of FA standards (Supelco 37 Component Fame Mix, acquired to Sigma-Aldrich, Algés, Portugal) and those in the Wiley 275 library and AOCS Lipid Library. Internal standard methyl-nonadecanoate from Sigma-Aldrich was used (1 µg/mL). The relative amounts of FAs were calculated by the percent area method with proper normalization, considering the sum of all areas of the identified FAs. Results were expressed as means ± SD and in mass of the internal standard, expressed in micrograms per milligram of phospholipids. Total lipid extracts were analyzed by hydrophilic interaction liquid chromatography on an Ultimate 3000 Dionex (Thermo Fisher Scientific, Bremen, Germany) high- performance liquid chromatography system (HPLC) with an autosampler coupled online to a Q-Exactive hybrid quadrupole mass spectrometer (Thermo Fisher, Scientific, Bremen, Germany). The mobile phase used consisted of a gradient starting at 10:90 to 90:10% v/v of eluents A:B where eluent A was H2O:ACN:MeOH at 1:2:1, and eluent B was ACN:MeOH at 3:2 (both containing 10 mM ammonium acetate). Each injection contained 5 µg of total lipid extract in 91 µL eluent B, and 4 µL of phospholipid standards mix (dMPC—0.02 µg, SM d18:1/17:0—0.02 µg, dMPE—0.02 µg, LPC—0.02 µg, dPPI—0.08 µg, dMPG—0.012 µg, dMPS—0.04 µg, dMPA—0.08 µg and CL—0.08 µg). The stationary phase was an Ascentis Si column HPLC Pore column (10 cm × 1 mm, 3 µm, Sigma-Aldrich), and elution was at a flow rate of 50 µL/min and 35 ◦C. Acquisition in the Orbitrap® mass spectrometer was performed in both positive (electrospray voltage 3.0 kV) and negative (electrospray voltage −2.7 kV) modes, with a high resolution of 70,000 and AGC target of 1e6. Capillary temperature was 250 ◦C and the sheath gas flow was 15 U. For MS/MS determinations, a resolution of 17,500 and AGC target of 105 was used and the cycles consisted of one full scan mass spectrum. Ten data-dependent MS/MS scans were repeated for each of the experiments, with a dynamic exclusion of 60 s and intensity threshold of 104. Normalized collision energy™ (CE) ranged from 20 to 30 eV. Data acquisition was performed using the Xcalibur data system (V3.3, Thermo Fisher Scientific, Waltham, MA, USA). The facility of formation of positive and negatively charged ions depends on the structure of the lipid head group. The phospholipid classes, phosphatidylglycerols (PGs), phosphatidylinositols (PIs), phosphatidylserines (PSs) Biomolecules 2021, 11, 426 4 of 16

and cardiolipines (CLs), easily form negatively charged ions due to the loss of a proton from the polar head group, thus forming the anion [M − H]−. Phosphatidylethanolamines (PEs) may easily gain or lose a proton leading to [M + H]+ or [M − H]− and the mass spectra was thus acquired in both the positive and negative modes. Phosphatidylcholines (PCs) were also analyzed in the positive and negative modes. The cation is produced from protonation of the phosphate and the charge in the choline group [M − H]+, while the anion is the result of an adduct formed between the lipid and acetate from the mobile phase [M + CH3COO]−. The size of the membrane vesicles and their zeta potential was characterized using a Zetasizer Nano ZSP system (Malvern), and the fluorescence intensity and anisotropy was measured in a Cary Eclipse fluorescence spectrophotometer (Varian) equipped with a thermostatted multicell holder accessory and automated polarizers. The fluorescent probes (at a final concentration of 1 µM) were added from a 100 µM stock in DMSO to the membrane vesicles and to preformed vesicles of POPE:POPC:POPS 45:35:20 or pure POPC model membranes. The vesicles plus probe were allowed to equilibrate overnight at 37 ◦C with gentle stirring.

3. Results 3.1. Lipid Composition of the Membranes Isolated from High Five Insect Cells 3.1.1. Relative Abundance of Lipids and Proteins The mass ratio of lipidic material to protein extracted from the membranes from High Five insect cells was 1.1 ± 0.2, of which 52% were phospholipids. This leads to a phospholipid to protein ratio equal to 0.57 ± 0.11 in the total membranes. Neutral lipids were also present in the membranes extract but they were not subjected to a detailed analysis. Small amounts of sterols have been previously found in similar cells [30,42], and were also identiﬁed by TLC in the membranes from High Five insect cells. Ceramides were also identiﬁed and characterized by HPLC-MS (see Section 3.1.5).

3.1.2. Relative Abundance of the Different Phospholipid Classes The different phospholipid classes were separated by TLC, identiﬁed against standards, and quantiﬁed by phosphorous analysis [39]. The results are shown in Table1.

Table 1. Quantiﬁcation of the different phospholipid classes found in membranes isolated from High Five insect cells.

Lipid Class % (w/w) 1 CL 4.8 LysoPL 1.4 PC 24 PE 46 PG 5.1 PI 4.8 PS 8.7 SM 5.0 1 Assuming an average molar mass of 750 g for all phospholipids.

Phosphatidylethanolamines (PEs) were the most abundant, representing 45% of the total phospholipids in the membranes isolated from the High Five insect cells. The second most abundant class of phospholipids was phosphatidylcholines (PCs, 24%) followed by the negatively charged phosphatidylserines (PSs, 9%), phosphatidylinositols (PIs, 5%), and phosphatidylglycerols (PGs, 5%). Sphingomyelins (SMs) and lysophospholipids (Lyso- PLs) were also encountered in the membranes, at 5 and 1%, respectively. The isolated membranes also contain cardiolipins (CLs, 5%), reﬂecting some contamination by mitochondrial membranes. Biomolecules 2021, 11, 426 5 of 16

3.1.3. Quantiﬁcation of the Fatty Acid Composition in the Phospholipid Pool The overall composition of the fatty acids esteriﬁed with phospholipids is shown in Table2. The most abundant is oleic acid (C18:1n9), representing almost half of the fatty acids content. Palmitoleic (C16:1n7), stearic (C18:0), and palmitic (C16:0) acyl chains are also abundant, representing 15–20% each. Small amounts of myristic (C14:0) and C18:1 other than oleic acid are also encountered, and minor quantities of very long fatty acids (arachidic C20:0, and eicosapentanoic C20:5). Monounsaturated fatty acids (MUFA) represented 66% of the total acyl chains, 33% were saturated (SFA) and only 1.4% were polyunsaturated (PUFA).

Table 2. Quantiﬁcation of the most abundant acyl chains from the phospholipids pool of membranes isolated from High Five insect cells.

Acyl Chain % (w/w) 1 C14:0 2.24 ± 0.27 C16:0 14.2 ± 0.16 C16:1n7 18.5 ± 0.14 C18:0 15.7 ± 0.33 C18:1n9 45.5 ± 1.4 C18:1 2.23 ± 0.34 C18:2 0.87 ± 0.25 C20:0 0.88 ± 0.18 C20:5 0.55 ± 0.06 SFA 33 MUFA 66 PUFA 1.4 1 Mean ± standard deviation of 3 independent measurements from the same membrane isolation.

3.1.4. Lipidome Characterization of the Most Abundant Phospholipid Classes Biomolecules 2021, 11, x FOR PEER REVIEW 6 of 17 The relative abundance of distinct acyl chains in the major phospholipid classes was characterized by HPLC-MS. The results obtained are shown in Figures1–3.

40 PE 30 PC 20

10 % (w/w) % 2

0 28:0 28:1 30:0 30:1 30:2 32:1 32:2 32:4 34:0 34:1 34:2 34:3 34:4 34:5 36:0 36:1 36:2 36:3 36:4 36:5 36:6 38:1 38:2 38:5 38:6 38:7 38:8 40:4 40:5 40:6 40:7

42:2/3 FigureFigure 1.1. RelativeRelative abundanceabundance ofof thethe differentdifferent acylacyl chainschains ofof phosphatidylethanolaminephosphatidylethanolamine (PE,(PE, )) andand  + + phosphadidylcholinephosphadidylcholine (PC, ) identified identiﬁed as as [M [M + + H] H] . Results. Results shown shown are are the the mean mean and and standard standard deviationdeviation ofof 33 independentindependent measurementsmeasurements from from the the same same membrane membrane isolation. isolation.

The lipidome of negatively charged phospholipids (PS, PI and PG) is shown in Figure 2. The results obtained for PI and PG were similar to those of PE and PC, with higher amounts of C34:1, and C32:1 and C36:1. However, large amounts are also observed of very long and highly unsaturated acyl chains (38:4–40:6). The acyl chain profile obtained for the PS lipid class is however very distinct from the others, with C38:0 being the most abundant combination of acyl chains.

40 PS 30 PI PG 20

10 % (w/w) % 1

28:0 30:0 30:1 30:2 32:0 32:1 32:2 34:0 34:1 34:2 34:3 36:0 36:1 36:2 36:3 36:4 36:5 36:6 38:0 38:1 38:2 38:3 38:4 38:5 38:6 40:5 40:6 40:7 Figure 2. Relative abundance of the different acyl chains of phosphatidylserine (PS, ), phosphatidylinositol (PI, ), and phosphatidylglycerol (PG, ) identified as [M-H]-. Results shown are the mean and standard deviation of 3 independent measurements from the same membrane isolation.

A comparison of the distinct lipid classes is shown in Figure 3 with respect to the saturation of the acyl chains. The distinct profile of the PS lipids clearly stood out, with totally saturated chains being the most abundant, in contrast with the observation that a single unsaturation was usually found in the other lipid classes. Biomolecules 2021, 11, x FOR PEER REVIEW 6 of 17

40 PE 30 PC 20

10 % (w/w) % 2

0 28:0 28:1 30:0 30:1 30:2 32:1 32:2 32:4 34:0 34:1 34:2 34:3 34:4 34:5 36:0 36:1 36:2 36:3 36:4 36:5 36:6 38:1 38:2 38:5 38:6 38:7 38:8 40:4 40:5 40:6 40:7

42:2/3 Figure 1. Relative abundance of the different acyl chains of phosphatidylethanolamine (PE, ) and phosphadidylcholine (PC, ) identified as [M + H]+ . Results shown are the mean and standard deviation of 3 independent measurements from the same membrane isolation.

The lipidome of negatively charged phospholipids (PS, PI and PG) is shown in Figure 2. The results obtained for PI and PG were similar to those of PE and PC, with higher amounts of C34:1, and C32:1 and C36:1. However, large amounts are also observed of very Biomolecules 2021, 11, 426long and highly unsaturated acyl chains (38:4–40:6). The acyl chain profile obtained for 6 of 16 the PS lipid class is however very distinct from the others, with C38:0 being the most abundant combination of acyl chains.

40 PS 30 PI PG 20

10 % (w/w) % 1

28:0 30:0 30:1 30:2 32:0 32:1 32:2 34:0 34:1 34:2 34:3 36:0 36:1 36:2 36:3 36:4 36:5 36:6 38:0 38:1 38:2 38:3 38:4 38:5 38:6 40:5 40:6 40:7 Figure 2. RelativeFigure abundance 2. Relative of abundance the different of acylthe different chains of acyl phosphatidylserine chains of phosph (PS,atidylserine), phosphatidylinositol (PS, ), phosphati- (PI, ), and Biomolecules 2021, 11, x FOR PEER REVIEW -  - 7 of 17 phosphatidylglyceroldylinositol (PG, (PI, ) identiﬁed), and phosphatidylglycerol as [M-H] . Results (PG, shown ) areidentified the mean as and[M-H] standard. Results deviation shown are of 3the independent measurementsmean from and the samestandard membrane deviation isolation. of 3 independent measurements from the same membrane isolation.

A comparison80 of the distinct lipid classes is shown in Figure 3 with respect to the saturation of the acyl chains. The distinct profile of the PS lipids clearly stood out, with PE totally saturated chains being the most abundant, in contrast with the observation that a 60 PC single unsaturation was usually found in the other lipid classes. PS 40 PI PG

10 % (w/w) %

0 SFA MUFA PUFA HUFA

FigureFigure 3. 3. RelativeRelative abundance abundance of of saturated saturated fatty fatty acids acids (SFA), (SFA), monounsaturated monounsaturated fatty fatty acids acids (MUFA), (MUFA), polyunsaturatedpolyunsaturated fatty fatty acid acidss (PUFA), (PUFA), and and highly highly unsaturated unsaturated fatty fatty acids acids (HUFA) (HUFA) in in the the acyl acyl chains chains of phosphatidylethanolamine (PE, ), phosphatididylcholine (PC, ), phosphatidylserine (PS, ), of phosphatidylethanolamine (PE, ), phosphatididylcholine (PC, ), phosphatidylserine (PS, ), phosphatidylinositol (PI, ), and phosphatidylglycerol (PG, ). phosphatidylinositol (PI, ), and phosphatidylglycerol (PG, ). 3.1.5. TheLipidome most abundant Characterization combination for the of Additional acyl chains Phospholipid for phosphatidylethanolamines Classes and phosphatidylcholinesIn addition to the corresponded detailed characterization to C34:1 (Figure of 1the). Given major the lipid high classes, abundance someof results oleic were(C18:1) also and obtained palmitic for (C16:0) other lipid acids, classes and thefoun prevalenced in the membranes of SFA esterified at relatively with small glycerol quan- at tities.carbon The 1 and results MUFA obtained at carbon are 2shown [43,44 in], theFigures major 4 phospholipidand 5. species are thus identified as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2- oleoyl-glycero-3-phosphocholine80 (POPC). Slightly shorter and longer acyl chains (32 and 36 carbons in both acyl chains) are also abundant in both phospholipid classes, and small SM amounts (below 3%) of even shorter and longer. As expected, very short acyl chains 60 Cer are mostly SFA while very long are highly unsaturated fatty acidsX-Cer (HUFA), in this way contributing40 to a relatively fluid membrane at the temperature of cell growth [29]. The lipidome of negatively charged phospholipids (PS, PI and PG) is shown in Figure202. The results obtained for PI and PG were similar to those of PE and PC, with higher amounts of C34:1, and C32:1 and C36:1. However, large amounts are also observed of very long and highly unsaturated acyl chains (38:4–40:6). The acyl chain profile obtained

% (w/w) % 4

0 30:1 30:2 32:1 32:2 32:3 34:1 34:2 35:1 36:1 36:2 38:1 38:2 32:1 32:1 (Glc) 32:1 (Lac) 36:1 (Lac) 38:1 (Lac) 40:1 (Lac) 42:1 (Lac) 44:0 (Lac) 44:1 (Lac)

38:1 (Glc/Gal) 40:1 (Glc/Gal) 44:1 (Glc/Gal) Figure 4. Relative abundance of the different acyl chains of sphingomyelins (SM, ), ceramides (Cer, ), and monoglycosylceramides (X-Cer, ) identified as [M + H] + . Results shown are the mean and standard deviation of 3 independent measurements from the same membrane isolation.

80 PE 60 PC PS 40 PI PG

10 Biomolecules 2021, 11, 426 (w/w) % 7 of 16 5

for the0 PS lipid class is however very distinct from the others, with C38:0 being the most SFA MUFA PUFA HUFA abundant combination of acyl chains. A comparison of the distinct lipid classes is shown in Figure3 with respect to the saturationFigure 3. Relative of the abundance acyl chains. of saturated The distinct fatty proﬁleacids (SFA), of the monounsaturated PS lipids clearly fatty stood acids out, (MUFA), with polyunsaturated fatty acids (PUFA), and highly unsaturated fatty acids (HUFA) in the acyl chains totally saturated chains being the most abundant, in contrast with the observation that a of phosphatidylethanolamine (PE, ), phosphatididylcholine (PC, ), phosphatidylserine (PS, ), singlephosphatidylinositol unsaturation (PI, was  usually), and phosphatidylglycerol found in the other (PG, lipid ). classes. 3.1.5. Lipidome Characterization for the Additional Phospholipid Classes 3.1.5. Lipidome Characterization for the Additional Phospholipid Classes In addition to the detailed characterization of the major lipid classes, some results were In addition to the detailed characterization of the major lipid classes, some results also obtained for other lipid classes found in the membranes at relatively small quantities. were also obtained for other lipid classes found in the membranes at relatively small quan- The results obtained are shown in Figures4 and5. tities. The results obtained are shown in Figures 4 and 5. Biomolecules 2021, 11, x FOR PEER REVIEW 8 of 17

80 SM given 60that ceramides originate from sphingomyelins through the actionCer of the enzyme sphingomyelinase. Monoglycosylceramides were also identified, withX-Cer glucose, galactose or lactose40 attached to the ceramide moiety. A preference for ceramides with longer acyl chains is clearly observed. Lactosyl derivatives are much more abundant, with lactosyl ceramide20 C36:1 accounting for 50% of all glycosylceramides. Lysolipids that originate from the major phospholipids present have also been identified; the relative abundance of their single acyl chain is represented in Figure 5. % (w/w) % 4 Lysolipids are usually obtained through the action of phospholipase A2, which cleaves the ester2 bond of the acyl chain at the sn-2 position on glycerol [45]. The acyl chain present in the lysolipids is therefore expected to be in the sn-1 position. The results obtained were in good0 agreement with those encountered for the respective phospholipid classes (Figure 1 and 2). The saturated acyl chain C16:0 is the most abundant on lysophosphatidylcholines 30:1 30:2 32:1 32:2 32:3 34:1 34:2 35:1 36:1 36:2 38:1 (LPC), and supports the selection of POPC as the representative38:2 phosphatidylcholine for 32:1 32:1 (Glc) 32:1 (Lac) 36:1 (Lac) 38:1 (Lac) those membranes. The results obtained for lysophosphatidylethanolamine40:1 (Lac) 42:1 (Lac) 44:0 (Lac) 44:1 (Lac) (LPE) show 38:1 (Glc/Gal) that C16:0 was also very abundant at the sn-1 position of glycerol40:1 (Glc/Gal) from phosphatidyleth-44:1 (Glc/Gal) anolamines, but C18:0 and C18:1 were found at high relative abundance. Globally the re- Figure 4. Relative abundance of the different acyl chains of sphingomyelins (SM, ), ceramides Figure 4. Relative abundance of the different acyl chains of sphingomyelins (SM, ), ceramides (Cer, sults(Cer, were ), and similar monoglycosylceramides for LPC and LPE, (X-Cer, with half ) identified of the acyl as [Mchains + H] being+ . Results saturated shown andare the half ), and monoglycosylceramides (X-Cer, ) identiﬁed as [M + H] + . Results shown are the mean and monounsaturated.mean and standard Verydeviation long of acyl 3 independent and saturated measurements acyl chains from were the sameobtained membrane for lysophos- isolation. phatidylserinestandard deviation (LPS), of 3 in independent agreement measurements with the results from obtained the same for membrane this lipid isolation. class (Figure 2). Sphingomyelin accounted for 5% of the total phospholipids, the large majority being 34:1 and100 36:1 (Figure 4). The sphingosine moiety contributes with 18 carbons and a trans LPE double80 bond. Thus, the most abundant fatty acids esterified with sphingosine are palmitoyl (C16:0) and stearoylLPC (C18:0), with small amounts of miristoyl (C14:0) and the longer acyl chain60 C20:0. MonounsaturatedLPS fatty acids (C14:1, C18:1 and C20:1) were also found, although in very small quantities (3% for the sum of all MUFA). Different ceramides could also be40 identified in the lipids extracted and the relative abundance of the different acyl chains20 was very similar to that observed for sphingomyelin. This similarity was expected, % (w/w) 8

0 16:0 16:1 18:0 18:1 20:0 22:0 SFA MUFA FigureFigure 5. 5. RelativeRelative abundance abundance of of the the acyl acyl chain chain of of the lysolipids from phosphatidylethanominesphosphatidylethanomines (LPE, (LPE,), phosphatidylcholines ), phosphatidylcholines (LPC, (LPC,), and ), phosphatidylserinesand phosphatidylserines (LPS, (LPS,). Results ). Results shown shown are the are mean the meanand standardand standard deviation deviation of 3 independent of 3 independent measurements measurements from from the same the same membrane membrane isolation. isolation.

The cardiolipin (CL) class was also characterized, with C64:4 being the most abundant lipid (31% ± 5%), followed by C74:4 and C74:5 (33% ± 3% for the sum of both). Small amounts of the saturated phosphatidic acid with 28 carbons (C28:0) were also identified in the samples analyzed.

3.2. Biophysical Properties of the Membranes Obtained from High Five Insect Cells 3.2.1. Surface charge and size of the vesicles The membranes obtained from High Five insect cells were stored in buffer containing Tris buffer pH = 7.5 and 10 w% glycerol, at a protein concentration of about 5 mg/mL. To decrease the turbidity of the solution and allow the characterization of their biophysical properties, they were diluted to 50 μg/mL in the same solvent. A typical correlogram obtained by DLS analysis is shown in Appendix A, and the size distribution obtained from the best fit is shown in Figure 6. As expected, a very broad distribution was obtained, indicating the presence of vesicles with diameters from 100 nm to above 1 μm (limited by Biomolecules 2021, 11, 426 8 of 16

Sphingomyelin accounted for 5% of the total phospholipids, the large majority being 34:1 and 36:1 (Figure4). The sphingosine moiety contributes with 18 carbons and a trans double bond. Thus, the most abundant fatty acids esterified with sphingosine are palmitoyl (C16:0) and stearoyl (C18:0), with small amounts of miristoyl (C14:0) and the longer acyl chain C20:0. Monounsaturated fatty acids (C14:1, C18:1 and C20:1) were also found, although in very small quantities (3% for the sum of all MUFA). Different ceramides could also be identified in the lipids extracted and the relative abundance of the different acyl chains was very similar to that observed for sphingomyelin. This similarity was expected, given that ceramides originate from sphingomyelins through the action of the enzyme sphingomyelinase. Monoglycosylceramides were also identified, with glucose, galactose or lactose attached to the ceramide moiety. A preference for ceramides with longer acyl chains is clearly observed. Lactosyl derivatives are much more abundant, with lactosyl ceramide C36:1 accounting for 50% of all glycosylceramides. Lysolipids that originate from the major phospholipids present have also been identified; the relative abundance of their single acyl chain is represented in Figure5. Lysolipids are usually obtained through the action of phospholipase A2, which cleaves the ester bond of the acyl chain at the sn-2 position on glycerol [45]. The acyl chain present in the lysolipids is therefore expected to be in the sn-1 position. The results obtained were in good agreement with those encountered for the respective phospholipid classes (Figure1 and 2). The saturated acyl chain C16:0 is the most abundant on lysophosphatidylcholines (LPC), and supports the selection of POPC as the representative phosphatidylcholine for those membranes. The results obtained for lysophosphatidylethanolamine (LPE) show that C16:0 was also very abundant at the sn-1 position of glycerol from phosphatidylethanolamines, but C18:0 and C18:1 were found at high relative abundance. Globally the results were similar for LPC and LPE, with half of the acyl chains being saturated and half monounsaturated. Very long acyl and saturated acyl chains were obtained for lysophosphatidylserine (LPS), in agreement with the results obtained for this lipid class (Figure2). The cardiolipin (CL) class was also characterized, with C64:4 being the most abundant lipid (31% ± 5%), followed by C74:4 and C74:5 (33% ± 3% for the sum of both). Small amounts of the saturated phosphatidic acid with 28 carbons (C28:0) were also identified in the samples analyzed.

3.2. Biophysical Properties of the Membranes Obtained from High Five Insect Cells 3.2.1. Surface Charge and Size of the Vesicles The membranes obtained from High Five insect cells were stored in buffer containing Tris buffer pH = 7.5 and 10 w% glycerol, at a protein concentration of about 5 mg/mL. To decrease the turbidity of the solution and allow the characterization of their biophysical properties, they were diluted to 50 µg/mL in the same solvent. A typical correlogram obtained by DLS analysis is shown in AppendixA, and the size distribution obtained from the best fit is shown in Figure6. As expected, a very broad distribution was obtained, indicating the presence of vesicles with diameters from 100 nm to above 1 µm (limited by the upper limit of the DLS equipment used). Fitting the correlograms with a single characteristic size (cumulant fits, Figure A1B in AppendixA) led to an average diameter of 956 ± 16 nm and a polydispersity index (PDI) close to 0.9 (the high PDI reflecting the broad distribution of vesicle sizes). Those numbers should be taken as indicative only because the best fit of the correlograms was of poor quality due to the polydispersity of the sample (Figure A1 in AppendixA). Biomolecules 2021, 11, x FOR PEER REVIEW 9 of 17

the upper limit of the DLS equipment used). Fitting the correlograms with a single characteristic size (cumulant fits, Figure A1B in Appendix A) led to an average diameter of 956  16 nm and a polydispersity index (PDI) close to 0.9 (the high PDI reflecting the broad Biomolecules 2021, 11, 426 distribution of vesicle sizes). Those numbers should be taken as indicative only because9 of 16 the best fit of the correlograms was of poor quality due to the polydispersity of the sample (Figure A1 in Appendix A).

FigureFigure 6.6. Size distributiondistribution ofof thethe membranemembrane vesicles vesicles obtained obtained from from High High Five Five insect insect cells. cells. The The sample sam- wasple was suspended suspended in buffer in buffer (pH (p =H 7.5) = 7 containing.5) containing 10% 10% glycerol glycerol at aat lipid a lipid concentration concentration of of 50 50µg/mL. Theg/mL. corresponding The corresponding correlogram correlogram and best and ﬁt is best shown fit is in shown Appendix in AppendixA. A.

The zeta potential of of the the vesicles vesicles was was also also characterized, characterized, being being −14−14.4.4  1±.2 1.2mV mV at the at theionic ionic strength strength of the of thesuspension suspension solven solventt (42.5 (42.5 mM) mM);; see seeFigure Figure A2 inA2 A inppendix Appendix A forA forthe thezeta zeta potential potential distribution distribution.. The TheGouy Gouy Chapman Chapman theory theory provides provides a way a way to calculat to calculatee the thesurface surface potential potential and andsurface surface charge charge of the of membranes the membranes [46– [48]46–. 48Considering]. Considering that thatthe zeta the zetapotential potential corresponds corresponds to the to potential the potential at a distance at a distance of 0.2 nm of 0.2from nm the from membrane the membrane surface surface[49], the [calculated49], the calculated surface potential surface was potential −16.5  was 1.4 mV,−16.5 corresponding± 1.4 mV, corresponding to a surface charge to a surfacedensity chargeof −0.011 density  0.001 of −C 0.011m−2. The± 0.001 lipid C portion m−2. The of lipidthese portionmembranes of these contain membraness a high containsfraction of a highnegat fractionively charged of negatively lipids corresponding charged lipids correspondingto an average charge to an average of −0.2 per charge phos- of −pholipid.0.2 per phospholipid. This led to a predicted This led to charge a predicted density charge of − density0.033 C of m−−2 0.033(considering C m−2 (considering an average anarea average per lipid area equal per lipidto 0.64 equal nm2 to) [50] 0.64, which nm2)[ was50], whichthree times was threehigher times than higher what was than meas- what wasured measuredfor the whole for membranes. the whole membranes. The discrepancy The discrepancyindicates that indicates the proteins that present the proteins in the presentmembrane in the shield membrane and/or partially shield and/or compensate partially for compensate the negative for charges the negative of the membrane charges of thelipids. membrane This is supported lipids. This by isthe supported observation by that the the observation whole lipid that extract the whole obtained lipid from extract the obtainedmembranes from shows the membranes a significantly shows more a significantly negative surface more negative potential surface than the potential total mem- than thebranes total, corresponding membranes, correspondingto a surface charge to a density surface twice charge as density high as twice that obtained as high asfor that the obtainedmembranes for. the membranes. 3.2.2. Membrane Polarity and Fluidity 3.2.2. Membrane Polarity and Fluidity Fluorescence anisotropy of the membrane probes DPH and NBD-C16 was used to Fluorescence anisotropy of the membrane probes DPH and NBD-C16 was used to characterize the dynamic properties of the membranes. This parameter depends on the characterize the dynamic properties of the membranes. This parameter depends on the mobility of the probes (higher mobility leading to smaller anisotropy), which in turn mobility of the probes (higher mobility leading to smaller anisotropy), which in turn re- reflects the fluidity of their environment. The fluorophore of DPH is located in the non- flects the fluidity of their environment. The fluorophore of DPH is located in the non-polar polar interior of the lipid bilayer [51–54], thus reporting on the fluidity of this region of interior of the lipid bilayer [51–54], thus reporting on the fluidity of this region of the the membrane. For lipid bilayers in the gel phase, the acyl chains had very little mobility, membrane. For lipid bilayers in the gel phase, the acyl chains had very little mobility, leading to a DPH fluorescence anisotropy around 0.35. A large decrease in anisotropy is leading to a DPH fluorescence anisotropy around 0.35. A large decrease in anisotropy is observed at temperatures above the lipid melting temperature [54], due to the increased observed at temperatures above the lipid melting temperature [54], due to the increased mobility of the lipid acyl chains, which lead to a high fluidity in the region of the bilayer mobility of the lipid acyl chains, which lead to a high fluidity in the region of the bilayer where DPH is located. On the other hand, the fluorophore of NBD-C16 is located at the membranewhere DPH interface, is located [36. On,55, 56the] providingother hand, complementary the fluorophore information. of NBD-C16 The is anisotropy located at was the measuredmembrane at interface, 37 ◦C, which[36,55,56] is the temperatureproviding complementary at which the functional information. characterization The anisotropy of the  membranewas measured proteins at 37 is C performed., which is the The temperature results are shown at which in Table the functional3, for the total charact membranes,erization forof the lipid membrane bilayers withproteins a simple is performed. but representative The results lipid are composition shown in Table (POPE:POPC:POPS 3, for the total 45:35:20),membranes, and for lipid lipid bilayers bilayers with POPC with a only. simple but representative lipid composition (POPE:POPC:POPS 45:35:20), and lipid bilayers with POPC only. Biomolecules 2021, 11, 426 10 of 16

Biomolecules 2021, 11, x FOR PEER REVIEW 10 of 17

Table 3. Fluorescence anisotropy of membranes isolated from High Five insect cells, and lipid bilayers prepared from a representative lipid composition and from POPC.

Fluorescent ProbeProbe Membrane Membrane Anisotropy Anisotropy Hi5 membranes 0.17 ± 0.01 Hi5 membranes 0.17 ± 0.01 DPH PE:PC:PS 45:35:20 0.12 ± 0.01 DPH PE:PC:PS 45:35:20 0.12 ± 0.01 POPCPOPC 0.10 0.10± ± 0.01 Hi5 membranes 0.25 ± 0.05 Hi5 membranes 0.25 ± 0.05 NBD-C16 PE:PC:PS 45:35:20 0.20 ± 0.04 NBD-C16 PE:PC:PS 45:35:20 0.20 ± 0.04 POPCPOPC 0.18 0.18± ± 0.03 The results obtained for the simple POPC membrane compare well with data re- portedThe in results the literature, obtained both for the for simple DPH [57] POPC and membrane for NBD-C16 compare [36]. well The withpresence data reportedof POPE inand the POPS literature, in the both lipid for bilayers DPH [57 led] and to fora small NBD-C16 increase [36]. in The the presence fluorescence of POPE anisotropy and POPS of inboth the probes lipid bilayers (indicating led toa decrease a small increasein membrane in the fluidity). fluorescence This anisotropyresult was expected, of both probes given (indicatingthe relatively a decrease higher temperature in membrane of fluidity).the main Thisphase result transition was expected, for those givenlipids theas compared relatively higherto POPC temperature (Tm(POPE) of = the9 °C, main Tm(POPS) phase transition= 14 °C, and for Tm(POPC) those lipids = as−2 compared°C) [58]. The to anisot- POPC ◦ ◦ ◦ (Tm(POPE)ropy is still =relatively 9 C, Tm(POPS) small, as = expected 14 C, and for Tm(POPC) membranes = − in2 theC) [fluid58]. Thestate. anisotropy A most signifi- is still relativelycant increase small, is observed as expected when for the membranes probes were in the inserted fluid state. in Hi5 A membranes, most significant although increase the isfluorescence observed when anisotropy the probes indicates were that inserted the me inmbranes Hi5 membranes, were also although in a fluid the state fluorescence [54]. anisotropy indicates that the membranes were also in a fluid state [54]. The fluorescencefluorescence spectra of the probes inserted in the different membranes are pre- sentedsented inin FigureFigure7 7.. DPHDPH shows shows the the same same spectra spectra in in all all three three membranes, membranes, indicating indicating that that thethe probeprobe waswas inin aa mediamedia withwith similarsimilar polarity.polarity. ThisThis waswas expectedexpected fromfrom thethe locationlocation ofof thethe fluorophorefluorophore inin thethe non-polarnon-polar portionportion ofof thethe membrane.membrane. AA smallsmall blueblue shiftshift waswas howeverhowever observed for NBD-C16NBD-C16 inserted in thethe Hi5Hi5 membranesmembranes asas comparedcompared withwith thethe purepure lipidlipid bilayers. This suggests that the membrane interface was less polar in the Hi5 membranes, thethe NBDNBD fluorescentfluorescent groupgroup beingbeing inin aa lessless hydratedhydrated environmentenvironment [59[59].]. TheThe membranemembrane BiomoleculesBiomoleculesBiomolecules 2021 2021, 202111,, 11x, FOR,11 x ,FOR x PEERFOR PEER PEER REVIEW REVIEW REVIEW proteins present in the membranes might explain the different properties11 of11 1118of of18 18of the mem- proteins present in the membranes might explain the different properties of the membrane– waterbrane–water interface. interface.

1.0 1.0 1.01.01.0 1.01.01.0 A AA A B BB B

0.5 0.5

0.50.50.5 0.50.50.5 Fluorescence Intensity (a.u.) Fluorescence Intensity(a.u.) Fluorescence Intensity(a.u.) Fluorescence Intensity(a.u.) Fluorescence Intensity (a.u.) Intensity Fluorescence Fluorescence Intensity (a.u.) Fluorescence Intensity (a.u.) 0.0 Fluorescence Intensity (a.u.) 0.0 0.00.00.0 0.00.00.0 400 440 480 510 530 550 570 400400400 440 440 440 480 480 480 510510510 530 530 530 550 550 550 570 570 570 Wavelength (nm) Wavelength (nm) WavelengthWavelengthWavelength (nm) (nm) (nm) WavelengthWavelengthWavelength (nm) (nm) (nm) 365365 365 Figure 7. Fluorescence spectra of DPH (Plot A) and NBD-C16 (Plot B) inserted in lipid bilayers 366366 366 FigureFigureFigure 7. Fluorescence7. 7.Fluorescence FluorescenceFigure spectra 7. spectraFluorescence spectra of DPHof ofDPH DPH (Plot spectra (Plot (PlotA) A)and of A) and DPH NBDand NBD ( NBDA‐C16)‐ andC16 ‐(PlotC16 NBD-C16(Plot (PlotB) B)inserted B)inserted ( insertedB) inserted in lipidin inlipid lipid inbilayers lipidbilayers bilayers bilayers pre pre‐ pre‐ prepared‐ from prepared from POPC ( ), POPE:POPC:POPS in the ratio 45:35:20 ( ), and in the membranes from 367367 367 paredparedpared from from from POPC POPC POPCPOPC ( (), (POPE:POPC:POPS), POPE:POPC:POPS), POPE:POPC:POPS in inthe in inthe the ratiothe ratio ratio ratio45:35:20 45:35:20 45:35:20 45:35:20 ( (), (and), and), inand ininthe the inthe membranesthe membranes membranes membranes from from from from High Five insect High Five insect cells ( ). The fluorescence intensity was normalized at the wavelength of maxi- 368368 368 HighHighHigh‐Five‐Five‐ Fiveinsect insect insect cells cells cells ( (). (The ). The). ﬂuorescencefluorescenceThe fluorescence fluorescence intensityintensity intensity intensity waswas was normalized normalizedwas normalized normalized at at the atthe at wavelengththe wavelength the wavelength wavelength of of maximum ofmaxi ofmaxi maxi‐ ‐ emission.‐ mum emission. 369369 369 mummummum emission. emission. emission. 4. Discussion 4. Discussion 370370 370 The structural and functional properties of membrane proteins were strongly affected by theirThe solvating structural lipids. and Thisfunctional is particularly properties relevant of membrane for the efﬂux proteins protein were P-gp, strongly for which af- 371371 371 somefected molecules by their solvating may act lipids. as substrates This is orparticularly inhibitors relevant depending for onthe the efflux environment protein P-gp, where for the protein is embedded [5]. Therefore, to interpret the protein functional properties in 372372 372 4. Discussion4. 4.Discussion Discussion which some molecules may act as substrates or inhibitors depending on the environment where the protein is embedded [5]. Therefore, to interpret the protein functional proper- 373373 373 TheThe overallThe overall overall lipid lipid lipid composition composition composition of ofHigh ofHigh High Five Five Fiveinsect insect insect cells cells cells obtained obtained obtained in inthis inthis workthis work work is similaris issimilar similar ties in native membranes it is necessary to know their composition and properties. The 374374 374 to tothat tothat thatreported reported reported previously previously previously for for Spodoptera for Spodoptera Spodoptera frugiperda frugiperda frugiperda (Sf9) (Sf9) (Sf9) insect insect insect cells cells cells and and andTrichoplusia Trichoplusia Trichoplusia 375375 375 ni ni(Tn),[27,28,40] ni(Tn),[27,28,40] (Tn),[27,28,40] although although although some some some significant significant significant differences differences differences were were were observed. observed. observed. 376376 376 TheThe ratioThe ratio ratio of ofphospholipids ofphospholipids phospholipids to toproteins toproteins proteins obtained obtained obtained (0.57 (0.57 (0.57  0.11) 0.11) 0.11) corresponds corresponds corresponds to to64 to 64% 64 %pro %pro‐ pro‐ ‐ 377377 377 teinsteinsteins and and and36 36% 36 %phospholipids. %phospholipids. phospholipids. A Asomewhat Asomewhat somewhat higher higher higher weight weight weight fraction fraction fraction of ofphospholipids ofphospholipids phospholipids was was was 378378 378 obtainedobtainedobtained than than than previously previously previously reported reported reported for for Sf9 for Sf9 insect Sf9 insect insect cells, cells, cells, which which which may may may reflect reflect reflect differences differences differences in inthe inthe the 379379 379 cellcell typecell type type and/or and/or and/or in inthe inthe protein the protein protein being being being overexpressed overexpressed overexpressed [40]. [40]. [40]. The The Thephospholipid phospholipid phospholipid ratio ratio ratio obtained obtained obtained 380380 380 is inis isinthe inthe lower the lower lower range range range of ofvalues ofvalues values encountered encountered encountered for for plasma for plasma plasma membranes membranes membranes from from from mammalian mammalian mammalian cells, cells, cells, 381381 381 andand andis ishigher ishigher higher than than than the the ratiothe ratio ratio encountered encountered encountered for for mitochondrialfor mitochondrial mitochondrial and and andbacterial bacterial bacterial membranes membranes membranes 382382 382 [58,59].[58,59].[58,59]. 383383 383 SomeSomeSome differences differences differences are are observedare observed observed in inthe inthe relativethe relative relative abundance abundance abundance of ofthe ofthe differentthe different different phospho phospho phospho‐ ‐ ‐ 384384 384 lipidlipidlipid classes classes classes when when when compared compared compared with with with those those those reported reported reported in inthe inthe literaturethe literature literature for for Sf9for Sf9 andSf9 and andTn Tn cellsTn cells cells 385385 385 [27,28,40].[27,28,40].[27,28,40]. The The Theamount amount amount of ofPE ofPE is PE somewhatis issomewhat somewhat higher higher higher in inthe inthe cells the cells cells analyzed analyzed analyzed in inthis inthis work,this work, work, and and andis is is 386386 386 compensatedcompensatedcompensated by by the by the smallerthe smaller smaller amount amount amount of ofPC. ofPC. ChangesPC. Changes Changes in inthe inthe relativethe relative relative proportion proportion proportion of ofthe ofthe difthe dif‐ dif‐ ‐ 387387 387 ferentferentferent lipid lipid lipid classes classes classes have have have been been been observed observed observed when when when Sf9 Sf9 [28,40]Sf9 [28,40] [28,40] or orTn or Tn [28] Tn [28] [28]cells cells cells are are infectedare infected infected by by by 388388 388 recombinantrecombinantrecombinant baculovirus. baculovirus. baculovirus. The The Thedifferences differences differences observed observed observed in inthis inthis thiswork work work may may may thus thus thus be be due be due dueto toin to‐in ‐in‐ 389389 389 trinsictrinsictrinsic differences differences differences in inthe inthe cell the cell types,cell types, types, the the procedure the procedure procedure of ofinfection ofinfection infection and/or and/or and/or proteins proteins proteins being being being over over over‐ ‐ ‐ 390390 390 expressed,expressed,expressed, or orsmall or small small differences differences differences in inthe inthe temperature the temperature temperature maintained maintained maintained during during during cell cell culture.cell culture. culture. A Ahigh Ahigh high 391391 391 fractionfractionfraction of of ofnegatively negativelynegatively charged chargedcharged phospholipids phospholipidsphospholipids was was was obtained obtainedobtained in in inthis this this work workwork 392392 392 (PI+PS+PG+PA=19(PI+PS+PG+PA=19(PI+PS+PG+PA=19 %), %), as%), asobtained as obtained obtained before before before for for Sf9 for Sf9 cellsSf9 cells cells (15 (15 to(15 to21 to 21%,[27] 21 %,[27] %,[27] 23%,[28] 23%,[28] 23%,[28] and and and34 34% 34 % % 393393 393 [40])[40])[40]) and and andfor for Tn for Tn cells Tn cells cells (14 (14 to(14 to17 to 17%)[28]. 17 %)[28]. %)[28]. Significant Significant Significant differences differences differences are are howeverare however however observed observed observed con con‐ con‐ ‐ 394394 394 cerningcerningcerning the the relativethe relative relative abundance abundance abundance of ofthe ofthe distinctthe distinct distinct lipid lipid lipid classes. classes. classes. Only Only Only PI PIwas PI was wasidentified identified identified in inthe inthe the 395395 395 membranesmembranesmembranes from from from Sf9 Sf9 and Sf9 and andTn Tn cells Tn cells cells by by Marheineke by Marheineke Marheineke et alet,[28] etal ,[28]al,[28] while while while Gerbal Gerbal Gerbal et alet [27]etal al[27] identified[27] identified identified 396396 396 PI PIandPI and andPS, PS, andPS, and andParathath Parathath Parathath et etal etal[40] al[40] [40]identified identified identified PI, PI, PS PI, PS and PS and andPA PA inPA inSf9 inSf9 membranes.Sf9 membranes. membranes. All All fourAll four four 397397 397 negativelynegativelynegatively charged charged charged lipids lipids lipids were were were found found found in inthe inthe membranes the membranes membranes from from from High High High Five Five Fivecells cells cells analyzed analyzed analyzed in in in 398398 398 thisthis work,this work, work, although although although PA PA was PA was wasfound found found only only only in invery invery very small small small quantities. quantities. quantities. The The differencesThe differences differences observed observed observed 399399 399 in inthe inthe negativelythe negatively negatively charged charged charged lipids lipids lipids detected detected detected may may may be be related be related related to todifficulties todifficulties difficulties in intheir intheir their separa separa separa‐ ‐ ‐ 400400 400 tion.[32]tion.[32]tion.[32] When When When the the methodthe method method allows allows allows the the separationthe separation separation of ofPS, ofPS, thisPS, this thislipid lipid lipid is usuallyis isusually usually identified identified identified in in in 401401 401 highhighhigh quantities.[27,29,31,40] quantities.[27,29,31,40] quantities.[27,29,31,40] Phosphatidylglycerol Phosphatidylglycerol Phosphatidylglycerol was was wasalso also alsoencountered encountered encountered in insignificant insignificant significant Biomolecules 2021, 11, 426 11 of 16

native membranes it is necessary to know their composition and properties. The interplay between the protein and lipid composition of biomembranes is still poorly understood, although some general relationships have been identified. This work represents a contribution to this goal. As expected, the overall lipid composition of High Five insect cells overexpressing P-gp is similar to that reported previously for Spodoptera frugiperda (Sf9) insect cells and Trichoplusia ni (Tn) [29,30,42]. Significant differences are however observed, namely in the relative abundance of phospholipids and proteins, and in the relative proportion of the distinct phospholipids. A higher ratio of phospholipid to protein is observed (0.57 ± 0.11 as compared to 0.4 [42]), which is in the lower range of values encountered for plasma membranes, and higher than for mitochondrial and bacterial membranes [60,61]. The membranes characterized in this work are enriched in PEs, compensated by a smaller fraction of PCs. The fraction of negatively charged lipids is similar to that found previously in insect cells, but with different relative proportions of the distinct classes (PS > PG ≈ PI > PA). This is most likely due to difficulties in their separation [28,34]. In fact, the TLC separation performed in this work did not allow us to distinguish between PG and PA, and their quantification was only possible from the results obtained by HPLC-MS. Accordingly, more recent reports identify a large variety of negatively charged lipids, and PS stands out as the most abundant one [42]. The relative abundance of each acyl chain combination in the distinct lipid classes has not been previously reported for High Five or for any other insect cell line, being an important outcome from this work. We observe that the most abundant PEs and PCs were 34:1, 34:2 and 36:1, which is in close agreement with the higher abundance of C34 and C36 [42], and with the high percentage of C18:1, C16:1 and C18:0 [30]. From the overall acyl chain composition identified in previous reports, it was not however possible to predict class specific differences. The full lipidome characterization performed in this work shows that PIs were enriched in long and polyunsaturated acyl chains, while PSs show a predominance of unsaturated chains. The acyl chain composition of the different lipid classes may be a general property of the lipids, reflect cell type specificities, or be influenced by the membrane proteins. It is interesting to note that deviations from the average acyl chain compositions were mostly observed for negatively charged lipids, which are enriched in the annular fraction around P-gp [6,7,62]. In this work, some lipids present in the High Five membranes in low quantities were also characterized, including ceramides and their monoglycosylates (X-Cer). Interestingly, the acyl chain composition of X-Cer does not reflect that of the precursor, with a higher abundance of very long acyl chains. These long acyl chain ceramides cause interdigitation of the membrane leaflets with effects on the phase behavior [63] and with important consequences in cell signaling [64–66]. Elevated levels of X-Cer have also been associated with the development of multidrug resistance in cancer cells [67–69]. It would be relevant to study if overexpression of P-gp has some impact on the abundance and properties of X-Cer. From the lipidome obtained with the membranes of Hi5 insect cells overexpressing P-gp, one may define the lipid composition for representative model lipid bilayers. This knowledge is very important to understand the properties of the membranes where the protein of interest was embedded, and to allow the preparation of proteoliposomes enriched in the protein of interest but with the lipid composition of the native membrane. It is also important to characterize the interaction of membrane protein substrates/inhibitors or modulators with the lipid portion of the membrane, allowing the discrimination between direct and membrane-mediated effects. As a first approximation, a representative model membrane may be prepared from POPE, POPC and POPS at the relative proportions of 45:35:20, where POPE and POPC are the most abundant lipids in each lipid class, and POPS represent all the negatively charged lipids in the membrane. The same acyl chain combination in all three lipid classes facilitates lipid mixing. More complex lipid compositions will preferably introduce variability in the acyl chain composition of the Biomolecules 2021, 11, 426 12 of 16

major lipid classes, for example using extracts from natural sources such as E. coli (enriched in PE and PG), Egg PC, and brain extract (enriched in PE, PC, PI, and PS). In this work we also characterized the size, zeta potential, and fluidity of the Hi5 membrane vesicles, and this was compared with model membranes with the representative lipid composition. As expected from their lipid composition, the membranes were characterized by a negative surface potential. However, this was significantly less negative than predicted from the lipid composition of the membranes. This difference was partially explained by the dilution of the charged lipids in the Hi5 membranes due to the presence of neutral lipids and proteins. However, the difference observed might be the result of limitations in the method used to convert the zeta potential (at the slipping plane) into the surface potential. The 0.2 nm usually assumed between the surface and the slipping plane [49], may be a reasonable approach for pure lipid membranes, but not for protein containing membranes due to protein protrusion. This parameter should be re-evaluated for the case of protein containing membranes, to allow a more accurate characterization of their surface potential from the measured zeta potential. The results obtained from the fluorescence anisotropy of the probes DPH and NBD-C16 show that the Hi5 membranes are in the fluid phase at 37 ◦C, as expected. Their fluidity was however lower than that of the lipid model membranes prepared from POPE:POPC:POPS (45.35:20). This reflected the presence of some additional lipids with longer and/or saturated acyl chains in the Hi5 membranes. The difference was however small, supporting the adequacy of this simple lipid mixture as a model for the lipid portion of the membranes obtained from High Five insect cells.

Author Contributions: Conceptualization, M.J.M. and P.A.T.M.; methodology, M.J.M. and P.A.T.M.; formal analysis, M.J.M.; investigation, M.J.M., E.F.B., and B.A.; resources, M.J.M. and S.V.A.; writing— original draft preparation, M.J.M.; writing—review and editing, P.A.T.M. and S.V.A.; supervision, M.J.M. and P.A.T.M.; project administration, M.J.M.; funding acquisition, M.J.M. and S.V.A. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Portuguese “Fundação para a Ciência e a Tecnologia” (FCT), grant number UIDB/00313/2020, co-funded by COMPETE2020-UE, grant PT2020_PTDC_DTP- FTO_2784_2014, and by Programa Operacional Regional do Centro” CENTRO2020, grant CENTRO- 01-0145-FEDER-000014. SVA was funded by the Intramural Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Original data will be provided when requested. Acknowledgments: The authors acknowledge Maria Rosário G.R. Marques Domingues from the Mass Spectrometry Centre, Department of Chemistry and QOPNA, University of Aveiro, for the support in obtaining the lipidome data and analysis. We also thank George Leiman (Laboratory of Cell Biology, CCR, NCI, NIH) for editorial assistance. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Appendix A Raw data of the characterization of the size and zeta potential of Hi5 membrane vesicles. Biomolecules 2021, 11, 426 13 of 16 Biomolecules 2021, 11, x FOR PEER REVIEW 13 of 17

FigureFigure A1. DDynamicynamic light light scattering scattering (DLS) correlogram (DLS) correlogram of vesicles prepared of vesicles from High prepared Five insect from High Five insect cells following the procedure described in the materials and methods section. The sample was cellssuspended following in buffer the (pH procedure = 7.5) containing described 10% glycerol in the at a materials lipid concentration and methods of 50 g/m section.L. × The The sample was suspendedsymbols are inthebuffer experimental (pH =results 7.5) containingand the lines are 10% the glycerol best fit: when at a considering lipid concentration that the sam- of 50 µg/mL. × The ple is polydisperse (Plot A), please see Figure 6 for the size distribution; or assuming that the sam- symbolsple has a arechar theacteristic experimental size (Plot B), results corresponding and the to an lines average are thediameter best of ﬁt: 967 when nm and considering a poly- that the sample isdispersity polydisperse index of ( A0.921), please. see Figure6 for the size distribution; or assuming that the sample has a characteristic size (B), corresponding to an average diameter of 967 nm and a polydispersity index Biomolecules 2021, 11, x FOR PEER REVIEW 14 of 17 of 0.921.

FigureFigure A2. ZetaZeta potential potential distribution distribution of vesicles of prepared vesicles from prepared High Five from insect High cells. FiveThe sample insect cells. The sample was suspended in buffer (pH = 7.5) containing 10% glycerol at a lipid concentration of 50 g/mL. wasThe suspendedcolored lines incorrespond buffer (pH to three = 7.5) consecutive containing measurements 10% glycerol on the at same a lipid sample. concentration green, blue, of 50 µg/mL. The coloredyellow lines correspond to three consecutive measurements on the same sample. green, blue, yellow.

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