Abstract
Previous studies in our laboratory have established ceramide kinase (CERK) as a critical mediator of eicosanoid synthesis. To date, CERK has not been well characterized in vitro. In this study, we investigated the substrate specificity of CERK using baculovirus-expressed human CERK (6 x His) and a newly designed assay based on mixed micelles of Triton X-100. The results indicate that the ability of CERK to recognize ceramide as a substrate is stereospecific. A minimum of a 12 carbon acyl chain was required for normal CERK activity, and the 4-5 trans double bond was important for substrate recognition. A significant discrimination by CERK was not observed between ceramides with long saturated and long unsaturated fatty acyl chains. Methylation of the primary hydroxyl group resulted in a loss of activity, confirming that CERK produces ceramide-1-phosphate versus ceramide-3-phosphate. In addition, methylation of the secondary hydroxyl group drastically decreased the phosphorylation by CERK. These results also indicated that the free hydrogen of the secondary amide group is critical for substrate recognition. Lastly, the sphingoid chain was also required for substrate recognition by CERK. Together, these results indicate a very high specificity for substrate recognition by CERK, explaining the use of ceramide and not sphingosine or diacylglycerol as substrates.
Highlights
Previous studies in our laboratory have established ceramide kinase (CERK) as a critical mediator of eicosanoid synthesis
To determine the molecular basis of the interaction between CERK and its substrate, ceramide, our laboratory focused on key features in the ceramide molecule required for substrate recognition and specificity
These include the stereochemistry at the C2, C3, C4, and C5 positions, the requirement of the acyl chain length, the effect of the functional groups in the sphingoid backbone, the requirement of the secondary amide bond, and the requirement of the primary and secondary hydroxyl groups (Fig. 1)
Summary
Previous studies in our laboratory have established ceramide kinase (CERK) as a critical mediator of eicosanoid synthesis. We examined the structural requirements within the ceramide molecule necessary for recognition and substrate utilization by human CERK. This study shows a high degree of specificity for ceramide by CERK and identifies key features for the development of sphingoid-based inhibitors of the enzyme.
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