Sphingolipid profiling reveals differential functions of sphingolipid biosynthesis isozymes of C. elegans

Multiple isozymes are encoded in the C. elegans genome for the various sphingolipid biosynthesis reactions, but the contributions of individual isozymes are characterized only in part. We developed a simple but effective reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method that enables simultaneous identification and quantification of ceramides (Cer), glucosylceramides (GlcCer), and sphingomyelins (SM), three important classes of sphingolipids from the same MS run. Validating this sphingolipid profiling method, we show that nearly all 47 quantifiable sphingolipid species found in young adult worms were reduced upon RNA interference (RNAi) of sptl-1 or elo-5, which are required for synthesis of the id17:1 sphingoid base. We also confirm that HYL-1 and HYL-2, but not LAGR-1, constitute the major ceramide synthase activity with different preference for fatty acid substrates, and that CGT-3, but not CGT-1 and CGT-2, plays a major role in producing glucosylceramides. Deletion of sms-5 hardly affects SM levels. RNAi of sms-1, -2, and -3 all lower the abundance of sphingomyelins with an odd number of carbon atoms (mostly C21 and C23, with or without hydroxylation) in the N-acyl chain, and only sms-1 RNAi does not elevate sphingomyelins containing even-numbered N-acyl chains. This suggests that sphingolipids containing even-numbered N-acyl chains could be regulated separately, sometimes in opposite directions, with those containing odd-numbered N-acyls, presumably monomethyl branched chain fatty acyls. We also find that ceramide levels are kept in balance with those of glucosylceramides and sphingomyelins.