Ceramide Synthase Structure-Function Relationships Determine by a Novel Ceramide Synthase Assay

In mammals, ceramides are synthesized by a family of six ceramide synthases (CerS), transmembrane proteins located in the ER, with each using fatty acyl-CoAs of defined chain length for ceramide synthesis. Little is known about the molecular features of the CerS that determine acyl CoA selectivity. We undertook a systematic analysis to explore CerS structure-function relationships, by constructing chimeric proteins combining sequences from CerS2, which uses C22-C24-CoA for ceramide synthesis, and CerS5, which uses C16-CoA. CerS2 and 5 are 41% identical and 63% similar. A chimeric CerS5/2 protein containing the first 158 residues and the last 83 residues of CerS2 displayed specificity towards C16-CoA, and a chimeric CerS2/5 protein containing the first 150 residues and the last 79 residues of CerS5 displayed specificity towards C22-CoA, demonstrating that a minimal region of 150 residues is sufficient for retaining CerS specificity. Further to these findings, we are investigating additional regions that appear to be important for activity and specificity. This search has led to the discovery of a minimum region of 11 amino acid residues involved in CerS specificity. Moreover, we now routinely assay CerS activity using a fluorescently (NBD) labeled sphinganine rather than the commonly-used radioactive substrate. We have developed a new method in which the fluorescent ceramide products are separated by solid phase extraction (SPE) C18 chromatography using a 96 well plate. We optimized the assay for use of minimal amounts of protein in a minimal volume, while alleviating the use of TLC. We suggest that this assay will permit the rapid assay of CerS activity in large numbers of samples, and will prove particularly useful for laboratories that do not perform this assay routinely.