Sphingolipids desaturated at the ⌬4-position are important signaling molecules in many eukaryotic organisms, including mammals. In a bioinformatics approach, we now identified a new family of protein sequences from animals, plants, and fungi and characterized these sequences biochemically by expression in Saccharomyces cerevisiae. This resulted in the identification of the enzyme sphingolipid ⌬4-desaturase (dihydroceramide desaturase) from Homo sapiens, Mus musculus, Drosophila melanogaster, and Candida albicans, in addition to a bifunctional sphingolipid ⌬4-desaturase/C-4-hydroxylase from M. musculus. Among the sequences investigated are the Homo sapiens membrane lipid desaturase, the M. musculus degenerative spermatocyte, and the Drosophila melanogaster degenerative spermatocyte proteins. During spermatogenesis, but not oogenesis of des mutant flies, both cell cycle and spermatid differentiation are specifically blocked at the entry into the first meiotic division, leading to male sterility. This mutant phenotype can be restored to wild-type by complementation with a functional copy of the des gene (Endo, K., Akiyama, T., Kobayashi S., and Okada, M. (1996) Mol. Gen. Genet. 253, 157-165). These results suggest that ⌬4-desaturated sphingolipids provide an early signal necessary to trigger the entry into both meiotic and spermatid differentiation pathways during Drosophila spermatogenesis.

No abstract

No abstract

In algae, the biosynthesis of docosahexaenoic acid (22:6 3; DHA) proceeds via the elongation of eicosapentaenoic acid (20:5 3; EPA) to 22:5 3, which is required as a substrate for the final ⌬ 4 desaturation. To isolate the elongase specific for this step, we searched expressed sequence tag and genomic databases from the algae Ostreococcus tauri and Thalassiosira pseudonana , from the fish Oncorhynchus mykiss , from the frog Xenopus laevis , and from the sea squirt Ciona intestinalis using as a query the elongase sequence PpPSE1 from the moss Physcomitrella patens . The open reading frames of the identified elongase candidates were expressed in yeast for functional characterization. By this, we identified two types of elongases from O. tauri and T. pseudonana : one specific for the elongation of ( ⌬ 6-)C18-PUFAs and one specific for ( ⌬ 5-)C20-PUFAs, showing highest activity with EPA. The clones isolated from O. mykiss , X. laevis , and C. intestinalis accepted both C18-and C20-PUFAs. By coexpression of the ⌬ 6-and ⌬ 5-elongases from T. pseudonana and O. tauri , respectively, with the ⌬ 5-and ⌬ 4-desaturases from two other algae we successfully implemented DHA synthesis in stearidonic acid-fed yeast. This may be considered an encouraging first step in future efforts to implement this biosynthetic sequence into transgenic oilseed crops. -Meyer, A., H. Kirsch, F. Domergue, A. Abbadi, P. Sperling, J. Bauer, P. Cirpus, T. K. Zank, H. Moreau, T. J. Roscoe, U. Zähringer, and E. Heinz. Novel fatty acid elongases and their use for the reconstitution of docosahexaenoic acid biosynthesis.

The role of D4-unsaturated sphingolipid long-chain bases such as sphingosine was investigated in Arabidopsis (Arabidopsis thaliana). Identification and functional characterization of the sole Arabidopsis ortholog of the sphingolipid D4-desaturase was achieved by heterologous expression in Pichia pastoris. A P. pastoris mutant disrupted in the endogenous sphingolipid D4-desaturase gene was unable to synthesize glucosylceramides. Synthesis of glucosylceramides was restored by the expression of Arabidopsis gene At4g04930, and these sphingolipids were shown to contain D4-unsaturated long-chain bases, confirming that this open reading frame encodes the sphingolipid D4-desaturase. At4g04930 has a very restricted expression pattern, transcripts only being detected in pollen and floral tissues. Arabidopsis insertion mutants disrupted in the sphingolipid D4-desaturase At4g04930 were isolated and found to be phenotypically normal. Sphingolipidomic profiling of a T-DNA insertion mutant indicated the absence of D4-unsaturated sphingolipids in floral tissue, also resulting in the reduced accumulation of glucosylceramides. No difference in the response to drought or water loss was observed between wild-type plants and insertion mutants disrupted in the sphingolipid D4-desaturase At4g04930, nor was any difference observed in stomatal closure after treatment with abscisic acid. No differences in pollen viability between wild-type plants and insertion mutants were detected. Based on these observations, it seems unlikely that D4-unsaturated sphingolipids and their metabolites such as sphingosine-1-phosphate play a significant role in Arabidopsis growth and development. However, D4-unsaturated ceramides may play a previously unrecognized role in the channeling of substrates for the synthesis of glucosylceramides.