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.
Ceramides
are important intermediates in sphingolipid biosynthesis
(and degradation) and are normally present in only small amounts in
unstressed cells. However, following the receptor-mediated activation
of neutral sphingomyelinase, sphingomyelin can acutely give rise to
substantial amounts of ceramides, which dramatically alter membrane
properties. In this study, we have examined the role of the 1-OH and
3-OH functional groups of ceramide for its membrane properties. We
have specifically examined how the oxidation of the primary alcohol
to COOH or COOMe in palmitoyl ceramide (PCer) or the removal of either
the primary alcohol or C(3)–OH (deoxy analogs) affected ceramides’
interlipid interactions in fluid phosphatidylcholine bilayers. Measuring
the time-resolved fluorescence emission of trans-parinaric
acid, or its steady-state anisotropy, we have obtained information
about the propensity of the ceramide analogs to form ceramide-rich
domains and the thermostability of the formed domains. We observed
that the oxidation of the primary alcohol to COOH shifted the ceramide’s
gel-phase onset concentration to slightly higher values in 1-palmitoyl-2-oleoyl-sn-3-glycero-3-phosphocholine (POPC) bilayers.
Methylation of the COOH function of the ceramide did not change the
segregation tendency further. The complete removal of the primary
alcohol dramatically reduced the ability of 1-deoxy-PCer to form ceramide-rich
ordered domains. However, the removal 3-OH (in 3-deoxy-PCer) had only
small effects on the lateral segregation of the ceramide analog. The
thermostability of the ceramide-rich domains in the POPC bilayers
decreased in the following order: 1-OH > COOH > COOMe = 3-deoxy >
1-deoxy. We conclude that ceramide needs a hydrogen-bonding-competent
functional group in the C(1) position to be able to form laterally
segregated ceramide-rich domains of high packing density in POPC bilayers.
The presence or absence of 3-OH was not functionally critical for
ceramide’s lateral segregation properties.
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