Preparation and Membrane Properties of Oxidized Ceramide Derivatives

Abstract

Ceramide is a bioactive lipid with important roles in several biological processes including cell proliferation and apoptosis. Although 3-ketoceramides that contain a keto group in place of the 3-OH group of ceramide occur naturally, ceramide derivatives oxidized at the primary 1-OH group have not been identified to date. To evaluate how the oxidative state of the 1-OH group affects the physical properties of membranes, we prepared novel ceramide derivatives in which the 1-OH group was oxidized to a carboxylic acid (PCerCOOH) or methylester (PCerCOOMe) and examined the rigidity of their monolayers and the formation of gel domains in palmitoyloleoylphosphatidylcholine (POPC) or sphingomyelin (SM) bilayers. As a result, PCerCOOH and PCerCOOMe exhibited membrane properties similar to those of native ceramide, although the deprotonated form of PCerCOOH, PCerCOO, exhibited markedly lower rigidity and higher miscibility with POPC and SM. This was attributed to the electrostatic repulsion of the negative charge, which hampered the formation of the ceramide-enriched gel domain. The similarities in the properties of PCerCOOMe and ceramide revealed the potential to introduce various functional groups onto PCerCOOH via ester or amide linkages; therefore, these derivatives will also provide a new strategy for developing molecular probes, such as fluorescent ceramides, and inhibitors of ceramide-related enzymes.

Preparation of Nitrogen Analogues of Ceramide and Studies of Their Aggregation in Sphingomyelin Bilayers

“…Surface pressure π of the Cer monolayers increased as the monolayers were laterally compressed and then reached a plateau at their collapse pressures (π > 50 mN/m). The π– A isotherm of the Cer monolayer is similar to that in the previous reports. , Then, we determined the molecular occupied areas of Cers at 20 mN/m ( A 20 ) from the π– A isotherms and found that the A 20 values of CerNHAc and CerN 3 are larger than that of Cer (Figure b). This result is reasonable because CerNHAc and CerN 3 have larger headgroups than Cer.…”

“…As discussed above, the weakened domain formability of CerNH 2 can be accounted for by the formation of CerNH 3 + in water. In addition, we previously reported that an anionic Cer-analogue CerCOO – has lower domain formability than the neutral Cer-analogues in POPC and SM bilayers . Taken together, we can conclude that the electrostatic repulsion between Cers reduces their domain formation, irrespective of the positive or negative charge.…”

“…The π−A isotherm of the Cer monolayer is similar to that in the previous reports. 20,26 Then, we determined the molecular occupied areas of Cers at 20 mN/m (A 20 ) from the π−A isotherms and found that the A 20 values of CerNHAc and CerN 3 are larger than that of Cer (Figure 5b). This result is reasonable because CerNHAc and CerN 3 have larger headgroups than Cer.…”

“…While 3-ketoceramide, which has a ketone moiety instead of the 3-OH group, occurs naturally, the derivatives in which the C1 position is oxidized to a formyl or a carboxy group has not been identified. Hence, we previously prepared oxidized ceramide analogues such as CerCOOH and CerCOOMe, in which the C1 position is oxidized to COOH and COOMe, respectively, and examined their domain formability in phospholipid bilayers such as palmitoyl-sphingomyelin (SM) and palmitoyloleoylphosphatidylcholine (POPC). , As a result, it was found that these ceramide analogues can form a ceramide-rich phase as in the case with normal ceramide. On the other hand, the deprotonated form of CerCOOH, CerCOO – , showed lower domain formability than the other ceramide analogues, indicating that the electrostatic repulsion between the negative charges of CerCOO – molecules suppresses the domain formation.…”

Preparation and Membrane Distribution of Fluorescent Derivatives of Ceramide

“…Cer lacks the headgroup typical of phospholipids or glycosphingolipids and generally possesses two hydroxy groups at the C1 and C3 positions. Recently, in order to study how the oxidative state of the primary alcohol group of Cer affects membrane physical properties, we prepared Cer derivatives in which the primary alcohol group was oxidized to a carboxylic acid and methylester (PCerCOOH and PCerCOOMe, Figure ) and examined how the oxidative state of the primary alcohol of Cer influences membrane physical properties . As a result, both derivatives exhibited membrane properties similar to those of native Cer.…”