On the importance of the C(1)-OH and C(3)-OH functional groups of the long-chain base of ceramide for interlipid interaction and lateral segregation into ceramide-rich domains.

Ceramides are important intermediates in sphingolipid biosynthesis (and degradation) and are normally present in only small amounts in unstressed cells. However, following 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 ceramide's 1-OH and 3-OH functional groups for its membrane properties. We have specifically examined how oxidation of the 1-OH to COOH or COOMe in palmitoyl ceramide (PCer) or removal of either 1-OH or 3-OH (deoxy analogs) affected ceramide's 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 replacing the 1-OH with a COOH-group 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. Complete removal of the 1-OH dramatically reduced the ability of 1-deoxy-PCer to form ceramide-rich ordered domains. However, removal of the 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 POPC bilayers decreased in the following order: 1-OH > 1-COOH > 1-COOMe > 3-deoxy > 1-deoxy. We conclude that ceramide needs a hydrogen bonding competent functional group in the C1 position in order to be able to form laterally segregated ceramide-rich domains of high packing density in POPC bilayers. The presence or absence of the 3-OH was not functionally critical.