In C.elegans, there is evidence that an unusual C17-isobranched-chain sphingoid base is present in glycosylceramides and sphingomyelin (Chitwood, et al. 1995). We have recently shown that RNAi or a deletion of a fatty acid elongation enzyme, ELO-5, results in a reproducible larval arrest. GC analysis of the
elo-5(-) worms indicates a substantial decrease in particular isobranched fatty acids called monomethyl branched-chain fatty acids (mmBCFAs). Growth and development can be restored to these
elo-5(-) worms by addition of mmBCFA to the worms food, indicating that mmBCFAs are essential for C.elegans growth and development (Kniazeva, et al. 2004). In other experiments, we knocked down worm orthologues of the enzyme responsible for the first step in sphingolipid biosynthesis, serine palmitoyl transferase. In mammals, there are two subunits of the serine palmitoyl transferase enzyme (SPT-1 and SPT-2) that form a heterodimer to function (Hanada, K. 2003). We have discovered that there are three subunits in C. elegans. C23H3.4 is most similar to the mammalian SPT-1, and when knocked-down, proves to be essential for worm growth and development. The other two enzymes, F43H9.2 and T22G5.5, are similar to the mammalian SPT-2, and when knocked down individually, seem to be dispensable for worm growth and development. Interestingly, when both of them are knocked-down at the same time via RNAi, the phenotype is virtually identical to knocking down C23H3.4 in phenotypes and FA profile. To explore the relationship between the SPT-like subunits and mmBCFAs, we treated
elo-5(-) worms with RNAi of either of the two SPT-2-like genes. We found that the worms on the T22G5.5 RNAi plates (and to a lesser extent F43H9.2 RNAi plates) displayed an enhancement of the
elo-5(-) phenotype. This result is consistent with the hypothesis that these SPT-2-like enzymes use mmBCFA as a substrate, although it is still possible that the SPT-2-like enzymes act parallel to mmBCFA-mediated functions. We are further investigating these findings by directly testing the fatty acid content of C.elegans sphingolipids in various genetic backgrounds (mutations, RNAi, inhibitors) and +/- FAs, by applying HPLC/MS. Different classes of sphingolipids have specific functions based on their structure ranging from signaling to maintenance of membrane integrity. We hope to further understand the function of mmBCFAs and their role in growth and development by elucidating what sphingolipid molecules they may be incorporated into. Additionally, we are conducting EMS mutagenesis screens for worms that can suppress the necessity for mmBCFAs. Pilot screens suggest it is possible to get this type of mutant and we are mapping and analyzing this mutation.