Effect of Phosphatidylcholine Unsaturation on the Lateral Segregation

Subscriber access provided by LAURENTIAN UNIV

Article

Effect of Phosphatidylcholine Unsaturation on the Lateral Segregation of Palmitoyl Ceramide and Palmitoyl Dihydroceramide in Bilayer Membranes Md. Abdullah Al Sazzad, and J.Peter Slotte Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00859 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on May 30, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23

Langmuir

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

Submission to ACS Langmuir

Effect of Phosphatidylcholine Unsaturation on the Lateral Segregation of Palmitoyl Ceramide and Palmitoyl Dihydroceramide in Bilayer Membranes

Md. Abdullah Al Sazzad and J.Peter Slotte* Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland

ABSTRACT: To better understand the interactions of saturated ceramides with unsaturated glycerophospholipids in bilayer membranes, we measured how palmitoyl ceramide (PCer) and dihydroceramide (dihydro-PCer – lacking the trans 4 double bond of the sphingoid base of ceramide) can interact with phosphatidylcholines (PCs) with palmitic acid in the sn-1 position and increasingly unsaturated acyl chains in the sn-2 position. The PCs were 16:0/18:1 (POPC), 16:0/18:2 (PLPC), 16:0/20:4 (PAPC) and 16:0(22:6 (PDPC). We also included di-18:1-PC (DOPC) to compare it with POPC. Since the ceramides were expected to segregate laterally to an ordered ceramide-rich phase, we determined the formation of the ordered phase using lifetime analysis of trans-parinaric acid (tPA) fluorescence. The presence of ordered domains, as indicated by tPA lifetime analysis, was verified by analysis of tPA anisotropy as a function of temperature. The interaction between PCer and POPC was clearly more favored than interactions with DOPC, as seen from a more thermostable gel phase in POPC than in DOPC at equal ceramide content. The concentration needed for PCer gel phase formation was also lower in POPC than in the DOPC bilayers, suggesting that POPC had a better miscibility in the ordered phase. The increased unsaturation of the sn-2 acyl chains of the PCs had more clear effects of dihydro-PCer segregation than on PCer segregation and the dihydro-PCer gel phase became more thermostable as the unsaturation in the PC increased. We conclude that the interactions between ceramides and PCs were complex and affected both by the trans 4 double bond of PCer by the palmitoyl acyl in sn-1 position and by the overall degree of unsaturation of the sn-2 acyl chain of the PCs.

ACS Paragon Plus Environment

Langmuir

Page 2 of 23

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

INTRODUCTION The importance of sphingolipids for cell function and membrane specialization is well-known (for reviews, see (1-4)). Of the sphingolipids, ceramide plays a central role as a precursor of most of the complex sphingolipids found in human cells and tissues (5). Ceramide is also a very important constituent in human skin, where it may contribute to making skin impermeable to both water (dehydration) and exogenous compounds (6, 7). Ceramide is synthesized in the endoplasmic reticulum (ER) by different ceramide synthases, each of which have some specificity regarding the N-linked acyl chain length (8-10). Consequently, ceramides with a highly heterogeneous distribution of N-linked acyl chains are found in the lipidome (11). Ceramide is directly formed from dihydroceramide which itself is formed following the N-acylation of a long-chain sphingoid base, which often is sphinganine (2amino-4-octadecane-1,3-diol), but the long chain base can also be longer or shorter than the 18 carbon sphinganine, and double bonds, hydroxylations, and methyl branching also occur (12, 13). The conversion of dihydro-ceramide to ceramide is catalyzed by a dihydroceramide desaturase which introduces a trans 4 double bond to the long-chain base (14). However, not all dihydro-ceramides are converted to ceramides, so the lipidome of any cell or tissue is likely to contain both dihydro-ceramide and ceramide molecular species (15). The structure of ceramide is very hydrophobic since the only polar groups are the hydroxyls found on the long-chain base (position C1, C3, and sometimes C4 in phytoceramide (16)). The long-chain base also contains the amide group on carbon 2, which together with hydroxyls provide ceramides (and sphingolipids in general), with efficient hydrogen bonding capabilities. A discussion on the importance of hydrogen bonding for ceramide, glycosphingolipid, and sphingomyelin properties can be found elsewhere (5, 17, 18). The fact that ceramide acyl chains are mostly saturated and long (at least 16-18 carbons) and lack a proper large head group gives them some unique membrane properties: their monomer solubility in the bilayer is very low (only a few mol%) and hence they tend to form laterally segregated domains or even a ceramide-enriched gel phase (19). Since a gel phase is not a preferred structure in fluid biological membranes, care is taken by the cells to limit the ceramide concentration in membranes (to approximately 0.1 mol% (20, 21)). However, in skin tissue the ceramide content can be rather high, but then again it is not in a form of hydrated bilayers (22). The low concentration of ceramide in the membranes of the ER is believed to be accomplished by regulated synthesis and by efficient conversion of ceramides to more complex sphingolipids, such as glycosphingolipids, sphingomyelin, and gangliosides. However, ceramides are known to accumulate locally, since they can be acutely formed by ACS Paragon Plus Environment

Page 3 of 23

Langmuir

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

rapid degradation of sphingomyelin by sphingomyelinases (23), an event that may be part of stress-induced physiological changes in cells, possibly including apoptosis (21, 24, 25). The lateral segregation of ceramides in fluid bilayers has been studied previously (26-31). Their segregation properties in neat POPC bilayers is known to depend on temperature, longchain base properties, acyl chain properties (length and unsaturation), and hydrogen bonding. Many studies have examined how saturated ceramides interact with saturated sphingomyelin in fluid phosphatidylcholine (PC) bilayers and how this interaction affects, for example, cholesterol distribution within the bilayer (32-34). Biological membranes contain many types of glycerophospholipids which themselves contain acyl chains with unsaturations. While there is some information about how glycerophospholipid, cholesterol, and sphingomyelin miscibility is affected in increasingly unsaturated glycerophospholipid bilayers (35-39), similar information is not available for how ceramide properties are influenced by increasingly unsaturated glycerophospholipids. To fill this information gap, we have in this study examined how the lateral segregation of palmitoyl ceramide (PCer) and the fully saturated dihydro-PCer is influenced by increasingly unsaturated PCs. Ceramides may well encounter unsaturated PCs in the ER before being transported away and PC-facilitated ceramide segregation may be one part of regulated ceramide transport from the ER by proteins or vesicles. In our study, we have used PCs which had an sn-1 palmitoyl residue and the sn-2 position contained one of the following acyl chains: 18:1, 18:2, 20:4 or 22:6 (POPC, PLPC, PAPC, and PDPC, respectively). We also included DOPC as a di-monounsaturated PC in the study. The lateral segregation of the ceramides was determined by lifetime analysis of tPA fluorescence as a function of ceramide bilayer concentration. tPA is a suitable probe for ceramide-enriched domains since it has a high partitioning preference for ceramide-domains (19, 26, 40). We observed that the saturated ceramides formed gel phases at lower bilayer concentration when the unsaturated PC had palmitic acid in the sn-1 position (POPC, as opposite to di-unsaturated DOPC). The thermostability of the ceramide-rich phase was generally higher when more unsaturated PCs were used in the mixed bilayer. We conclude that acyl chain unsaturation in PC markedly affected the lateral segregation of saturated ceramides and the thermostability of their gel phases.

MATERIALS AND METHODS


Langmuir

Page 4 of 23

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

Materials. All unsaturated phosphatidylcholines and palmitoylated ceramides used in the study were obtained from Avanti Polar Lipids (Alabaster, AL). Trans-parinaric acid was synthesized from the methyl ester of α-linolenic acid, as described previously (41). All stock solutions with unsaturated lipids contained 0.5 mol% butylated hydroxytoluene as an antioxidant and the lipids were dissolved in argon-purged methanol. Preparation of multilamellar vesicles. The required lipids (200 nmol lipid in total) were combined in glass tubes, mixed, and the solvent evaporated with argon gas until completely dry. Lipids were hydrated with 2 ml argon-purged pure water (18.2 MΩ,cm, MilliQ) for 60 min at 65 °C. After this step, the tubes were vortexed for several minutes, followed by a 5 min bath sonication treatment at 80 °C (FinnSonic M3 Bath Sonicator, FinnSonic Oy, Finland). Immediately after sonication, tPA was added from a concentrated methanol stock solution to a final probe concentration of 1 mol%. Addition was done at constant stirring and no more than 0.2 vol% of methanol was added to any sample. Differential scanning calorimetry. The multilamellar lipid vesicle sample contained 7.2 mol POPC and 0.8 mol PCer and was hydrated in 1 ml of argon-purged pure water for 60 min at 60 °C. The multilamellar vesicles were prepared as described above. The sample was degassed before being loaded into a VP-DSC instrument (MicroCal, Northampton, MA). 5 up and down scans were obtained with a temperature gradient of 1 °C/min. Data analysis was performed with Origin software (OriginLab, Northampton, MA). Steady-state anisotropy of tPA. Steady-state fluorescence measurements were performed in quartz cuvettes on a PTI QuantaMaster-spectrofluorometer (Photon Technology International, Lawrenceville, NJ) operating in the T-format, equipped with polarizers in all light paths. The samples were constantly stirred and the temperature in the samples was controlled by a Peltier element, using a temperature probe immersed in the sample. The sample was purged with argon for 2 min before starting the measurements. The fluorescent emissions were recorded continuously at 405 nm (tPA) after excitation at 305 nm. The samples were heated at a rate of 5°C/min between 10 and 60 °C. The steady-state anisotropy r was determined as described previously (42), using the Felix32 software (Photon Technology International). Fluorescence lifetime analysis of tPA. The fluorescence-lifetime analysis was performed at 23oC (or at indicated temperatures) with a FluoTime 200-spectrofluorimeter, using a PicoHarp 300E time-correlated single photon counting module (PicoQuant GmbH, Berlin, Germany). The tPA was excited with a 298 nm LED laser source (PLS300, PicoQuant) and the emission was measured at 405 nm. samples kept under constant stirring during the ACSThe Paragon Pluswere Environment

Page 5 of 23

Langmuir

5

measurements and were purged with argon for 2 min before starting measurements. Data were analyzed with FluoFit Pro software (PicoQuant). The decay was described by the sum of the exponentials, where αi was the normalized pre-exponential, and τi was the lifetime of the decay component i. The intensity-weighted lifetime was given by: < τ > = Σi αi τi2 / Σi αi τi (42). The decay of tPA emissions in binary or more complex bilayers is often characterized by multiple (2-3) lifetime components (43). The longest lifetime component is associated with tPA localized in the most ordered environment, where non-radiative processes are lowest (44).

RESULTS Gel phase detection in bilayers with PCer and POPC. In this study, we measured the lateral segregation of PCer and dihydro-PCer based on their formation of an ordered (gel) phase in increasingly unsaturated PC bilayers. We determined both the thermal stability of the ceramide-enriched gel phase in binary systems (PCer or dihydro-PCer in PXPC bilayers at 1:9 molar ratio; PXPC being POPC, PLPC, PAPC or PDPC), as well as the concentration-dependent formation of the ceramide-enriched gel phase in PXPC bilayers. The thermal stability of the ordered ceramide-enriched phase was determined from tPA steady-state anisotropy since tPA is known to partition favorably into ceramide gel phases (19, 29, 32, 40). Although pure ceramide aggregates may show metastability (45), we have not observed metastablity at low concentration of saturated ceramides in fully hydrated fluid PC bilayers with the sample preparation methods we use (data not shown).

60 o

Tm 25.0 C

C

10

Average Longest lifetime

50 0.30

8 6 Tm 30.7 oC

0.26

4

30

0.24

20

2 0.22

10

0 0.20

5th upscan 5th downscan

end-point temp of melting

-2 0

5

10 15 20 25 30 35 40 5 Temperature oC

10 15 20

25 30 35 40

20

o

Temperature, C


25

30

35

40

Temperature (oC)

45

0 50

tPA lifetime (ns)

40

0.28

o

Steady-state anisotropy of tPA

B

A

0.32

kJ/mol, C

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

Langmuir

Page 6 of 23

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

Figure 1. Phase transition in POPC/PCer bilayers. Panel A shows the anisotropy function of tPA (1 mol%) which reveals the end-point temperature of the gel phase melting (indicated by arrow) in the 1:9:by mol PCer/POPC bilayer (scan rate 2 °C/min). Each value is the mean + SEM for n=3. In Panel B, a DSC thermogram is shown for the same bilayer system (but w/o tPA). The enthalpy of gel melting during the up scan was +27+4 kJ/mol and 26+5 kJ/mol for the crystallization (down scan).The red line shows the 5th up scan and the blue line is the 5th down scan (scan rate 1 °C/min). Panel C shows the average lifetime and longest lifetime component of tPA as a function of temperature for the 1:9 by mol PCer/POPC system. Each value is mean + SEM for n=3.

To verify that tPA anisotropy reveals gel phase melting similar to DSC, we compared the two methods in the PCer/POPC 1:9 system (Fig.1). tPA anisotropy showed that, at low temperatures (i.e., 10 °C), the degree of order in the ceramide-rich phase was high and typical of a gel phase [Fig.1A, (19)]. As the sample was heated (2 °C/min), the tPA anisotropy gradually decreased until reaching its lowest value at about 34+1 °C. At this temperature, we assumed that the gel phase melting was complete. The tPA anisotropy data agree with previously published data for the PCer/POPC system (19). The gel phase melting profile reported by tPA anisotropy was verified by DSC which showed a complex gel phase endotherm during the heating phase (1 °C/min). According to DSC, the gel phase was fully melted at 34+1 °C (Fig.1B), in complete agreement with the tPA anisotropy measurement, and with a previous DSC study of a similar bilayer composition (46). The formation of the ceramide-rich gel phase during a cooling cycle occurred more cooperatively and at a lower temperature as compared to the melting behavior of the gel phase (Fig.1B). Finally, the temperature-dependent lifetime of tPA was examined in PCer/POPC (1:9 by mol) bilayers (Fig.1C). Both the average lifetime and the longest lifetime component showed a discontinuity in the slope as the temperature approached the end-point temperature of gel melting (at 3335 °C). Concentration-dependent lateral segregation of PCer and dihydro-PCer in POPC and DOPC bilayers. It is well-established that when PCer is added to a POPC bilayer, it forms ordered domains already at a very low concentration (around 3-5 mol%) and these domains obtain gel phase-like characteristics as the PCer content increases (19, 26, 27). Analysis of tPA lifetime is a sensitive method to detect the formation of an ordered ceramide phase in a fluid bilayer, since tPA partitions favorably into the ceramide-rich phase and its fluorescence lifetime increases as the ceramide-rich phase becomes more ordered (19, 26, 40). When PCer was added to POPC and DOPC bilayers under identical conditions, we observed that the intensity weighted average lifetime of tPA started to increase above 5 mol% PCer in POPC and above 10 mol% PCer in DOPC bilayers (Fig.2A). The observation that more PCer was needed in DOPC bilayers before PCer segregated into ordered domains suggests that unfavorable ACS Paragon Plus Environment

Page 7 of 23

Langmuir

7

60

60 A

Average tPA lifetime, ns

POPC/PCer DOPC/PCer

B

POPC/dihydro-PCer DOPC/dihydro-PCer

50

50

40

40

30

30

20

20

10

10

0 0

5

10

15

20

25

30

Concentration of PCer, mol%

0

10

20


0 30

Concentration of dihydro-PCer, mol%

Figure 2. Average tPA fluorescence lifetime in POPC or DOPC bilayers with increasing concentrations of PCer (Panel A) or dihydro-PCer (Panel B). The experiment was carried out at 23 °C with 1 mol% tPA. Each value is the mean + SEM for n=3.

acyl chain interactions led to this difference since the sn-1 palmitoyl residue in POPC would be a more favorable interaction partner with a saturated ceramide as compared to sn-1 oleoyl residue in DOPC. As the PCer concentration increased above the threshold concentration of lateral segregation, the PCer-rich phase almost immediately acquired gel-like characteristics as evidenced by the longest lifetime component of tPA (Fig.3A). It is also evident that the gel phase of PCer in DOPC gave slightly longer lifetimes than were observed at identical PCer concentrations in the POPC bilayer (Fig.3A), suggesting higher packing density of the PCer phase in the DOPC bilayer. This result would suggest that the DOPC content in the PCer-rich phase was lower than the comparable POPC content in the PCer-rich phase of the POPC bilayers.

80

80 B

A

Longest tPA lifetime comp, ns

POPC DOPC

60

60

40

40

20

20

0 0

5

10

15

20

25


30

0

10

20


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

0 30


Figure 3. Longest lifetime component of tPA fluorescence in POPC or DOPC bilayers with increasing concentrations of PCer (Panel A) or dihydro-PCer (Panel B). The experiment was carried out at 23 °C with 1 mol% tPA. Each value is the mean + SEM for n=3.


Langmuir

Page 8 of 23

8

When the fully saturated dihydro-PCer was added to either POPC or DOPC bilayers, it also segregated laterally into an ordered phase (Fig. 2B). This segregation occurred at a slightly lower dihydro-PCer concentration than was seen with PCer in the two bilayer systems. When we compared segregation of dihydro-PCer in DOPC with POPC bilayers, the concentration needed for gel phase formation was larger in DOPC than in POPC bilayers, similar to that observed for PCer (Fig.2A and B). The longest lifetime component of tPA fluorescence was again much longer in dihydro-PCer/DOPC bilayers as compared to dihydro-PCer/POPC bilayers (Fig.3B). The thermostability of the PCer and dihydro-PCer gel phases in POPC and DOPC bilayers was determined using tPA anisotropy measurements, to obtain the end-point temperature of the gel melting (see Fig.1A). For 10 mol% PCer in POPC, the gel phase was completely melted above a temperature of 34 + 1 °C (Fig.1A and Fig.4). In DOPC bilayers at 10 mol% PCer, the ceramide-rich phase was fully melted above 24 + 1 °C (Fig.4), showing that the PCer gel phase was destabilized more by DOPC than by POPC. This destabilization is also indicated by the much lower lifetime of the longest component of tPA fluorescence (Table S1) in DOPC versus POPC bilayers. With dihydro-PCer in POPC, the ceramide phase had a similar thermostability as the PCer phase in POPC (Table S1). However, the dihydro-PCer gel phase was much more stable in the DOPC bilayers as compared to the POPC bilayers (the end-point temperature of the gel melting was 42+2 and 35 + 1 °C in the DOPC and POPC bilayers, respectively). The higher thermostability of the dihydro-PCer phase in the DOPC bilayers was also indicated by the longer longest lifetime component of tPA which was 65.8 + 3.5 ns in DOPC and 52.9 + 0.5 ns in POPC bilayers (Table S1).

50 PCer dihydro-PCer o

End of gel phase melting temp, C

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

40

30

20

10

0 POPC PLPC PAPC PDPC

DOPC

Phospholipid type

Figure 4. The end-point temperature of the gel melting of PCer or dihydro-PCer in unsaturated PC bilayers. The end-point temperature of gel melting is defined in Fig.1A. The compositions were 1:9 mole ratio of ceramide to PC. Each value is the mean + SEM for n=3.


Page 9 of 23

Langmuir

9

Concentration-dependent lateral segregation of PCer and dihydro-PCer in increasingly unsaturated PXPC bilayers. Next, we measured the concentration-dependent lateral segregation of PCer and dihydro-PCer in bilayers of phosphatidylcholines which had a palmitoyl residue at sn-1 and either an oleoyl, linoleoyl, arachidonyl, or docosahexanoyl residue in the sn-2 position. The formation of the ordered phase was determined by lifetime analysis of tPA fluorescence. The lateral segregation of PCer occurred at a higher PCer concentration when the bilayer was prepared from PLPC, instead of POPC (Fig.5A). The concentration shift, based on average tPA lifetime, was about 5 mol%, similar to what was observed for PCer segregation in DOPC (Fig.2A). However, when the PCs contained 20:4 (PAPC) or 22:6 (PDPC) in the sn-2 position, the concentration of PCer needed for lateral segregation decreased and became close to that seen for PCer in POPC. The longest lifetime component of tPA fluorescence was higher in the PDPC bilayers as compared to the other PXPC bilayers (Fig.6A), suggesting a slightly higher lateral packing in the PCer phase of PDPC bilayers.

70 A

POPC PLPC PAPC PDPC


60

B 60 50 40

40

30 20

20


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

10 0

0 0

5

10

15

20


25

0

5

10

15

20

25


Figure 5. Average tPA fluorescence lifetime in PXPC unsaturated bilayers containing increasing concentrations of PCer (Panel A) or dihydro-PCer (Panel B). The experiment was carried out at 23 °C with 1 mol% tPA. Each value is the mean + SEM for n=3.

With dihydro-PCer, the concentration needed for the formation of an ordered phase was slightly higher in the PLPC bilayers as compared to the other three PXPC bilayers (Fig.5B), following the same trend as observed for PCer. Interestingly, the order in the dihydro-PCer phase increased substantially with increasing unsaturation and chain length (Fig.5B and Fig.6B), as evidenced by the increased lifetime of tPA in more unsaturated bilayers. Based on the longest lifetime component of tPA fluorescence, the order in the dihydro-PCer phase was significantly higher than seen in the PCer gel phase, with one exception: in POPC the longest lifetime component was similar for both PCer and dihydro-PCer (Fig.6A and B). It should be noted that while tPA average lifetime results (Fig.5) include contributions from all ceramideACS Paragon Plus Environment

Langmuir

Page 10 of 23

10

rich and ceramide-poor domains in the bilayer, the longest component (Fig.6) only reports from the most ordered phase present.

A

POPC PLPC PAPC PDPC


80

B 80

60

60

40

40

20

20

0


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

0 0

5

10

15

20


25

0

5

10

15

20

25


Figure 5. Longest lifetime component of tPA fluorescence in PXPC unsaturated bilayers with increasing concentrations of PCer (Panel A) or dihydro-PCer (Panel B). The experiment was carried out at 23 °C with 1 mol% tPA. Each value is the mean + SEM for n=3.

Thermostability of PCer and dihydro-PCer gel phases in PXPC bilayers. Finally, we compared gel phase melting for PCer and dihydro-PCer in PXPC bilayers at a 1:9 molar ratio, using the tPA anisotropy approach. With PCer in PXPC, we could observe that the thermostability of gel domains in PLPC was slightly lower than in POPC. However, as the number of cis double bonds and chain length increased (in PAPC and PDPC), the thermostability of the PCer gel phase started to increase, relative to the stability seen in PLPC bilayers (Fig.4). However, the thermostability of the PCer gel phase in the PLPC, PAPC or PDPC bilayers did not reach to the same level as seen with POPC bilayers. With dihydro-PCer (1:9 with PXPC), the gel phases became more thermostable as the unsaturation and chain length of the PCs increased (Fig.4), going from 35.1 + 1 °C in the POPC bilayers up to 44 + 1 °C in the PDPC bilayers. We also measured tPA fluorescence lifetime (both intensity based average lifetime and the longest lifetime component) in the 1:9 ceramide/PXPC bilayers at 23 °C. As seen from the results presented in Table S1, the average lifetime data followed the same trends as the thermostability data reported by tPA anisotropy measurements. Low thermostability was accompanied by low average tPA lifetime and higher thermostability was accompanied by increased tPA average lifetime values. This was true for both the PCer and dihydro-PCer gel phases. The longest lifetime component was diagnostic of gel phase properties and was long or very long in all systems, except PCer/PLPC, suggesting that the lateral packing was the least ordered in this composition at 23 °C, in good agreement with the lowest thermostability measured for this composition. ACS Paragon Plus Environment

Page 11 of 23

Langmuir

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

DISCUSSION The study of the behavior of ceramides in unsaturated glycerophospholipid bilayers is of interest not only for a better understanding of biological interface properties, but also for a general understanding of how a very hydrophobic molecule with no large head group, save for interfacial hydrogen bonding groups (1-OH, 2-NH and 3-OH), behaves in different co-lipid environments. We chose to study PCer since it is one of the most prevalent ceramide molecular species in many cell types. Dihydro-PCer was included because it is a structural analog of PCer, lacking the trans 4 double bond of the long-chain base. Removal of the trans double bond affects both the dynamic mobility of the 3-OH and intermolecular hydrogen bonding involving the 3-OH (47, 48). Fully hydrated ceramides in bulk are known to form a well-ordered lamellar phase (at 26 °C) with a bilayer periodicity d=46.9 Å (45). However, even though the fully hydrated ceramide aggregates have lamellar internal structures, they are not similar to bilayer membranes formed by phospholipids (49). Since ceramides lack a large head group, they need to interact with large head group (phospho)lipids in bilayers in order to achieve a thermodynamically stable bilayer existence. This is also true for cholesterol (50, 51) which lacks a large head group. The DSC trace shown in Fig.1B, with the complex gel melting transition, actually indicated that the PCer gel phase (at 10 mol% PCer in POPC) is a mixture of POPC and PCer in which POPC provides the large head group for PCer – otherwise the melting cooperativity would be larger, and the melting at higher temperature (45). A similar DSC trace for porcine brain ceramide in POPC at 1:9 molar ratio has been published (46). The transition is very uncooperative and multicomponent, suggesting that the PCer/POPC gel phase goes through different compositions (i.e., different Cer/PC molar ratios) during the melting. Since a large head group phospholipid is part of the formed ceramide gel phase, it is not surprising that phospholipid acyl chain properties have large effects on ceramide gel phase properties. Comparing POPC and DOPC with regard to the lateral segregation of PCer and dihydroPCer, it was evident that the sn-1 palmitoyl residue of POPC stabilized PCer gel phase formation when compared to the sn-1 oleoyl residue in DOPC. The stabilization was evident both from the lower concentration of PCer needed to form a gel phase in the POPC vs DOPC bilayers (Fig.2A and 3A) and from the increased thermostability of the formed gel phase in the POPC bilayers (Fig. 4). POPC is a hybrid lipid since its saturated chain can favorably interact with another saturated acyl chain in a neighboring lipid (or with cholesterol), whereas the unsaturated chain of POPC can be assumed to disfavor interactions with saturated acyl chains ACS Paragon Plus Environment

Langmuir

Page 12 of 23

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

(52, 53). This hybrid nature of POPC most likely explains why PCer prefers this co-lipid over the di-unsaturated DOPC as a large head group interaction partner. Dihydro-PCer behaved slightly differently in the POPC and DOPC bilayers as compared to PCer: the onset-set of the gel phase formation of dihydro-PCer was at slightly lower dihydro-PCer concentration in the POPC bilayers when compared to PCer (as judged by the appearance of the longest tPA lifetime component, Fig.3B). Also, the thermostability of the gel phase formed by dihydroPCer was higher in both the POPC and DOPC bilayers as compared to the situation with PCer (Fig.4). This suggests that the miscibility of either PC was less ideal with dihydro-PCer as compared to PCer, and could result from stronger intermolecular hydrogen bonding among dihydro-PCer molecules, analogous to the situation reported for palmitoyl SM and its dihydro analogs (47). However, it has also been suggested that ceramides would have stronger hydrogen bonding than dihydro ceramides (48) so this remains an unresolved matter. The poor miscibility of PC in the dihydro-PCer gel phase was indicated by the very long longest lifetime component of tPA fluorescence (about 72 ns for dihydro-PCer at 20 mol% as compared to about 58 ns for PCer at similar conditions – Fig.3B), indicating a higher degree of order or lateral packing in the dihydro-PCer gel phase than in the corresponding PCer phase. Surprisingly, in monolayers ceramides have been reported to form more condensed lateral packing (at least at lower surface pressures), than acyl chain-matched dihydroceramides (54, 55). It is possible that interactions between ceramide or dihydroceramides and PCs obscure packing properties seen in neat ceramide/dihydro ceramide monolayers. Although the longest lifetime component of tPA fluorescence was assumed to only originate from the ceramide-rich gel phase, it is not possible to use that information to compare the nature of the ceramide/PC gel phase with a pure hydrated ceramide gel phase, since ceramide crystals do not incorporate tPA (19). Even though the gel phase of dihydro-PCer in the DOPC showed a very long longest lifetime component of tPA fluorescence (about 72 ns – Fig.3B), suggesting a very gel-like nature, DOPC must have been included in the phase as a large head group provider since the ceramide solubility limit in PC bilayers is about 67 mol%, similar to cholesterol (56). PCer is known to order the acyl chains of POPC in the mixed gel phase (27) and a similar ordering of DOPC is likely to take place. When ceramide interactions with increasingly unsaturated PCs were examined, PCer and dihydro-PCer did not behave entirely similarly. Whereas dihydro-PCer formed increasingly ordered gel phases as the degree of PC unsaturation increased (as is clearly shown by the thermostability of the formed gel phase – Fig.4), the gel phase formed by PCer/POPC showed higher thermostability than was the case with PCer in the other three unsaturated PCs (PLPC,PAPC and PDPC). It is likely that the changed dynamics of the 3-OH and possibly the ACS Paragon Plus Environment

Page 13 of 23

Langmuir

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

altered hydrogen bonding properties in dihydro-PCer favored lateral segregation as the acyl chains of the unsaturated PCs became more and more unfavorable for interactions with the fully saturated ceramide analog. Hydrogen bonding has recently been shown to markedly influence lateral segregation of ceramides in POPC bilayers (26). Since PAPC and PDPC have longer acyl chains than POPC and PLPC, some of the effects we have seen with the PXPC bilayers are likely to arise from the chain length, in addition to being caused by the number of double bonds. The gel-to-liquid crystalline phase transition temperature (Tm) of all the unsaturated PCs used in this study were below 0 °C (Tm for fully hydrated POPC is -5 °C (57), for DOPC is -20°C (58), for PAPC is -22°C (53), and for PDPC is -3°C (53)). The low Tm of these unsaturated PCs ensured that the sn-2 acyl chain was highly disordered and its interactions with the saturated ceramides were unfavorable, especially as compared to the saturated sn-1 acyl chain (where present). Our present study has not included cholesterol in the bilayers, even though cholesterol is known to affect the formation of ceramide gel phase domains in at least POPC and DOPC bilayers (32, 34, 46, 56). It is not easy to foresee the effects of cholesterol on ceramide domain formation in polyunsaturated phospholipid bilayers, since the polyunsaturations themselves have such dramatic effects on the lateral distribution of cholesterol (36, 59, 60), which may differ significantly from effects on ceramide distribution.

CONCLUSIONS We have shown that PCer and dihydro-PCer behavior in unsaturated PC bilayers depends markedly on (i) the presence of a saturated acyl chain in sn-1 position, and (ii) on the number of double bonds and chain length of the acyl chain in the sn-2 position. Furthermore, (iii) PCer and dihydro-PCer behaved differently in the polyunsaturated PC bilayers. PCer and dihydroPCer have differing hydrogen bonding properties, since the trans double bond (or its absence) influences hydrogen bonding to and from the 3-OH in the long-chain base of sphingolipids (47). Therefore, we suggest that both hydrogen bonding (mainly between ceramides, but also between ceramides and PCs) and acyl chain interactions (influenced by length and number of cis double bonds) influenced markedly how the ceramides behaved in and interacted with the unsaturated PCs. The results of this study may be of relevance for ceramide or dihydroceramide behavior in membranes of ER – the site of their biosynthesis – since those membranes are believed to be rich in unsaturated glycerophospholipids (including phosphatidylcholines). The lateral segregation of saturated ceramides in ER membranes is probably hindered by efficient regulation of synthesis coupled with efficient transport of ACS Paragon Plus Environment

Langmuir

Page 14 of 23

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

ceramides from the ER to the Golgi membranes by CERT (61). After the ceramides have been transported to the Golgi apparatus, other sphingolipids formed from the ceramides start to influence unmodified ceramide behavior more dominantly.

■ SUPPLEMENTAL INFORMATION Table S1 presents data for tPA anisotropy and tPA fluorescence lifetimes in PCer and dihydro-PCer mixed bilayers with unsaturated PCs. It is available on line free of charge.

■ AUTHOR INFORMATION Corresponding author: [email protected] Notes The authors declare no competing financial interests.

■ ACKNOWLEDGMENTS We thank Dr. Thomas Nyholm for helpful discussions. Funding was generously provided by the Sigrid Juselius Foundation, the Academy of Finland, and the Magnus Ehrnrooth Foundation.

■ REFERENCES Reference List

1. Merrill, A. H., Jr., and Sandhoff, K. Sphingolipids: Metabolism and cell signaling. In: Vance and Vance: Biochemistry of Lipids, Lipoproteins and Membranes (4th ed) 2002, 373-407.

2. Zheng, W.; Kollmeyer, J.; Symolon, H.; Momin, A.; Munter, E.; Wang, E.; Kelly, S.; Allegood, J. C.; Liu, Y.; Peng, Q.; Ramaraju, H.; Sullards, M. C.; Cabot, M. Merrill, A. H., Jr. Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy. Biochim Biophys Acta 2006, 1758, 18641884.

3. Merrill, A. H., Jr. De novo sphingolipid biosynthesis: a necessary, but dangerous, pathway. J Biol. Chem 2002, 277, 25843-25846.

ACS Plus Environment 4. Slotte, J. P. Biological functions of Paragon sphingomyelins. Prog. Lipid Res. 2013, 52, 424-437.

Page 15 of 23

Langmuir

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

5. Castro, B. M.; Prieto, M. Silva, L. C. Ceramide: a simple sphingolipid with unique biophysical properties. Prog. Lipid Res. 2014, 54, 53-67.

6. Bouwstra, J. A.; Dubbelaar, F. E.; Gooris, G. S.; Weerheim, A. M. Ponec, M. The role of ceramide composition in the lipid organisation of the skin barrier. Biochim Biophys Acta 1999, 1419, 127-136.

7. Choi, M. J., and Maibach, H. I. Role of ceramides in barrier function of healthy and diseased skin. Am J Clin. Dermatol. 2005, 6, 215-223.

8. Ben-David, O., and Futerman, A. H. The role of the ceramide acyl chain length in neurodegeneration: involvement of ceramide synthases. Neuromolecular. Med. 2010, 12, 341-350.

9. Lahiri, S.; Park, H.; Laviad, E. L.; Lu, X.; Bittman, R. Futerman, A. H. Ceramide synthesis is modulated by the sphingosine analog FTY720 via a mixture of uncompetitive and noncompetitive inhibition in an Acyl-CoA chain length-dependent manner. J. Biol. Chem. 2009, 284, 16090-16098.

10. Pewzner-Jung, Y.; Ben-Dor, S. Futerman, A. H. When do Lasses (longevity assurance genes) become CerS (ceramide synthases)?: Insights into the regulation of ceramide synthesis. J. Biol. Chem 2006, 281, 25001-25005.

11. Hannun, Y. A., and Obeid, L. M. Many ceramides. J Biol Chem 2011, 286, 27855-27862.

12. Yasugi, E.; Kasama, T.; Shibahara, M. Seyama, Y. Composition of long-chain bases in sphingomyelin of the guinea pig Harderian gland. Biochem Cell Biol 1990, 68, 154-60.

13. Yasugi, E.; Kasama, T. Seyama, Y. Composition of long chain bases in ceramide of the guinea pig Harderian gland. J. Biochem. 1991, 110, 202-206.

14. Fabrias, G.; Munoz-Olaya, J.; Cingolani, F.; Signorelli, P.; Casas, J.; Gagliostro, V. Ghidoni, R. Dihydroceramide desaturase and dihydrosphingolipids: debutant players in the sphingolipid arena. Prog. Lipid Res. 2012, 51, 82-94.


Langmuir

Page 16 of 23

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

15. Kok, J. W.; Nikolova-Karakashian, M.; Klappe, K.; Alexander, C. Merrill, A. H., Jr. Dihydroceramide biology. Structure-specific metabolism and intracellular localization. J Biol Chem 1997, 272, 2112836.

16. Omae, F.; Miyazaki, M.; Enomoto, A.; Suzuki, M.; Suzuki, Y. Suzuki, A. DES2 protein is responsible for phytoceramide biosynthesis in the mouse small intestine. Biochem. J 2004, 379, 687-695.

17. Boggs, J. M. Lipid intermolecular hydrogen bonding: influence on structural organization and membrane function. Biochim Biophys Acta 1987, 906, 353-404.

18. Slotte, J. P. The importance of hydrogen bonding in sphingomyelin's membrane interactions with co-lipids. Biochim Biophys Acta 2016, 1858, 304-310.

19. Silva, L.; de Almeida, R. F.; Fedorov, A.; Matos, A. P. Prieto, M. Ceramide-platform formation and induced biophysical changes in a fluid phospholipid membrane. Mol. Membr. Biol. 2006, 23, 137148.

20. Kolesnick, R., and Hannun, Y. A. Ceramide and apoptosis. Trends Biochem. Sci. 1999, 24, 224-225.

21. Obeid, L. M.; Linardic, C. M.; Karolak, L. A. Hannun, Y. A. Programmed cell death induced by ceramide. Science 1993, 259, 1769-1771.

22. Wertz, P. W. Epidermal lipids. Semin. Dermatol. 1992, 11, 106-113.

23. Milhas, D.; Clarke, C. J. Hannun, Y. A. Sphingomyelin metabolism at the plasma membrane: implications for bioactive sphingolipids. FEBS Lett. 2010, 584, 1887-1894.

24. Grassme, H.; Riethmuller, J. Gulbins, E. Biological aspects of ceramide-enriched membrane domains. Prog. Lipid Res. 2007, 46, 161-170.

25. Mullen, T. D., and Obeid, L. M. Ceramide and apoptosis: exploring the enigmatic connections between sphingolipid metabolism and programmed cell death. Anticancer Agents Med. Chem. 2012, 12, 340-363. ACS Paragon Plus Environment

Page 17 of 23

Langmuir

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

26. Ekman, P.; Maula, T.; Yamaguchi, S.; Yamamoto, T.; Nyholm, T. K.; Katsumura, S. Slotte, J. P. Formation of an ordered phase by ceramides and diacylglycerols in a fluid phosphatidylcholine bilayer--Correlation with structure and hydrogen bonding capacity. Biochim Biophys Acta 2015, 1848, 2111-2117.

27. Hsueh, Y. W.; Giles, R.; Kitson, N. Thewalt, J. The effect of ceramide on phosphatidylcholine membranes: a deuterium NMR study. Biophys. J. 2002, 82, 3089-3095.

28. Pinto, S. N.; Silva, L. C.; Futerman, A. H. Prieto, M. Effect of ceramide structure on membrane biophysical properties: the role of acyl chain length and unsaturation. Biochim Biophys Acta 2011, 1808, 2753-2760.

29. Silva, L. C.; de Almeida, R. F.; Castro, B. M.; Fedorov, A. Prieto, M. J. Ceramide-domain formation and collapse in lipid rafts: membrane reorganization by an apoptotic lipid. Biophys J. 2006, 92, 502516.

30. Sot, J.; Aranda, F. J.; Collado, M. I.; Goni, F. M. Alonso, A. Different effects of long- and short-chain ceramides on the gel-fluid and lamellar-hexagonal transitions of phospholipids. A calorimetric, NMR and X-ray diffraction study. Biophys J 2005.

31. Veiga, M. P.; Arrondo, J. L.; Goni, F. M. Alonso, A. Ceramides in phospholipid membranes: effects on bilayer stability and transition to nonlamellar phases. Biophys. J. 1999, 76, 342-350.

32. Castro, B. M.; Silva, L. C.; Fedorov, A.; de Almeida, R. F. Prieto, M. Cholesterol-rich fluid membranes solubilize ceramide domains: implications for the structure and dynamics of mammalian intracellular and plasma membranes. J Biol Chem 2009, 284, 22978-22987.

33. Megha, and London, E. Ceramide selectively displaces cholesterol from ordered lipid domains (rafts): implications for lipid raft structure and function. J. Biol. Chem. 2004, 279, 9997-10004.


Langmuir

Page 18 of 23

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

34. Sot, J.; Ibarguren, M.; Busto, J. V.; Montes, L. R.; Goni, F. M. Alonso, A. Cholesterol displacement by ceramide in sphingomyelin-containing liquid-ordered domains, and generation of gel regions in giant lipidic vesicles. FEBS Lett 2008, 582, 3230-3236.

35. Kullberg, A.; Ekholm, O. O. Slotte, J. P. Miscibility of Sphingomyelins and Phosphatidylcholines in Unsaturated Phosphatidylcholine Bilayers. Biophys J 2015, 109, 1907-1916.

36. Brzustowicz, M. R.; Cherezov, V.; Caffrey, M.; Stillwell, W. Wassall, S. R. Molecular organization of cholesterol in polyunsaturated membranes: microdomain formation. Biophys. J. 2002, 82, 285-298.

37. Shaikh, S. R.; Locascio, D. S.; Soni, S. P.; Wassall, S. R. Stillwell, W. Oleic- and docosahexaenoic acidcontaining phosphatidylethanolamines differentially phase separate from sphingomyelin. Biochim Biophys Acta 2009, 1788, 2421-2426.

38. Soni, S. P.; Locascio, D. S.; Liu, Y.; Williams, J. A.; Bittman, R.; Stillwell, W. Wassall, S. R. Docosahexaenoic acid enhances segregation of lipids between : 2H-NMR study. Biophys J. 2008, 95, 203-214.

39. Williams, J. A.; Batten, S. E.; Harris, M.; Rockett, B. D.; Shaikh, S. R.; Stillwell, W. Wassall, S. R. Docosahexaenoic and eicosapentaenoic acids segregate differently between raft and nonraft domains. Biophys J 2012, 103, 228-237.

40. Castro, B. M.; de Almeida, R. F.; Silva, L. C.; Fedorov, A. Prieto, M. Formation of ceramide/sphingomyelin gel domains in the presence of an unsaturated phospholipid. A quantitative multiprobe approach. Biophys J. 2007, 93, 1639-1650.

41. Kuklev, D. V., and Smith, W. L. Synthesis of four isomers of parinaric acid. Chem Phys Lipids 2004, 131, 215-222.

42. Lakowicz, J. R. 1999. Principles of Fluorescence Spectroscopy. Kluwer Academic / Plenum Publishers, New York.


Page 19 of 23

Langmuir

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

43. Nyholm, T. K.; Lindroos, D.; Westerlund, B. Slotte, J. P. Construction of a DOPC/PSM/Cholesterol Phase Diagram Based on the Fluorescence Properties of trans-Parinaric Acid. Langmuir 2011, 27, 8339-8350.

44. Sklar, L. A.; Hudson, B. S. Simoni, R. D. Conjugated polyene fatty acids as fluorescent probes: synthetic phospholipid membrane studies. Biochemistry 1977, 16, 819-828.

45. Shah, J.; Atienza, J. M.; Duclos, R. I., Jr.; Rawlings, A. V.; Dong, Z. Shipley, G. G. Structural and thermotropic properties of synthetic C16:0 (palmitoyl) ceramide: effect of hydration. J Lipid Res. 1995, 36, 1936-1944.

46. Fidorra, M.; Heimburg, T. Bagatolli, L. A. Direct visualization of the lateral structure of porcine brain cerebrosides/POPC mixtures in presence and absence of cholesterol. Biophys J 2009, 97, 142-154.

47. Yasuda, T.; Al Sazzad, M. A.; Jantti, N. Z.; Pentikainen, O. T. Slotte, J. P. The Influence of Hydrogen Bonding on Sphingomyelin/Colipid Interactions in Bilayer Membranes. Biophys J 2016, 110, 431440.

48. Li, L.; Tang, X.; Taylor, K. G.; DuPre, D. B. Yappert, M. C. Conformational characterization of ceramides by nuclear magnetic resonance spectroscopy. Biophys J 2002, 82, 2067-80.

49. Bonosi, F.; Gabrieklli, G.; Margheri, E. Martini, G. Effect of the addition of ceramide to dioleoylphosphatidylcholine vesicles: an ESR and SEM study. Langmuir 1990, 6, 1769-1773.

50. Huang, J., and Feigenson, G. W. A microscopic interaction model of maximum solubility of cholesterol in lipid bilayers. Biophys. J. 1999, 76, 2142-2157.

51. Huang, J.; Buboltz, J. T. Feigenson, G. W. Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers. Biochim Biophys Acta 1999, 1417, 89-100.

52. Heberle, F. A.; Doktorova, M.; Goh, S. L.; Standaert, R. F.; Katsaras, J. Feigenson, G. W. Hybrid and nonhybrid lipids exert common effects on membrane raft size and morphology. J Am Chem Soc 2013, 135, 14932-14935. ACS Paragon Plus Environment

Langmuir

Page 20 of 23

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

53. Litman, B. J.; Lewis, E. N. Levin, I. W. Packing characteristics of highly unsaturated bilayer lipids: Raman spectroscopic studies of multilamellar phosphatidylcholine dispersions. Biochemistry 1991, 30, 313-319.

54. Löfgren, H., and Pascher, I. Molecular arrangements of sphingolipids. The monolayer behaviour of ceramides. Chem Phys Lipids 1977, 20, 273-84.

55. Brockman, H. L.; Momsen, M. M.; Brown, R. E.; He, L.; Chun, J.; Byun, H. S. Bittman, R. The 4,5double bond of ceramide regulates its dipole potential, elastic properties, and packing behavior. Biophys. J. 2004, 87, 1722-1731.

56. Ali, M. R.; Cheng, K. H. Huang, J. Ceramide drives cholesterol out of the ordered lipid bilayer phase into the crystal phase in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol/ceramide ternary mixtures. Biochemistry 2006, 45, 12629-12638.

57. Curatolo, W.; Sears, B. Neuringer, L. J. A calorimetry and deuterium NMR study of mixed model membranes of 1-palmitoyl-2-oleylphosphatidylcholine and saturated phosphatidylcholines. Biochim Biophys Acta 1985, 817, 261-270.

58. Schmidt, M. L.; Ziani, L.; Boudreau, M. Davis, J. H. Phase equilibria in DOPC/DPPC: Conversion from gel to subgel in two component mixtures. J Chem Phys 2009, 131, 175103.

59. Shaikh, S. R.; Cherezov, V.; Caffrey, M.; Soni, S. P.; LoCascio, D.; Stillwell, W. Wassall, S. R. Molecular organization of cholesterol in unsaturated phosphatidylethanolamines: X-ray diffraction and solid state 2H NMR reveal differences with phosphatidylcholines. J. Am. Chem. Soc. 2006, 128, 53755383.

60. Wassall, S. R.; Brzustowicz, M. R.; Shaikh, S. R.; Cherezov, V.; Caffrey, M. Stillwell, W. Order from disorder, corralling cholesterol with chaotic lipids. The role of polyunsaturated lipids in membrane raft formation. Chem. Phys. Lipids 2004, 132, 79-88.


Page 21 of 23

Langmuir

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

61. Hanada, K.; Kumagai, K.; Tomishige, N. Yamaji, T. CERT-mediated trafficking of ceramide. Biochim. Biophys. Acta 2009, 1791, 684-691.


Langmuir

Page 22 of 23

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

Abstract Graphic:


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Langmuir

50 POPC/PCer DOPC/PCer

40 Average tPA lifetime, ns

Page 23 of 23

30

20

10

0 0


5

10

15

20

25


30

Effect of Phosphatidylcholine Unsaturation on the Lateral Segregation

Recommend Documents