Identification of a sphingolipid-specific phospholipase D activity associated with the generation of phytoceramide-1-phosphate in cabbage leaves.

The structure and biosynthetic route for an unidentified lipid (lipid X) detected by TLC of cabbage (Brassica oleracea) lipids was determined. Lipid X is a phospholipid that is resistant to mild alkali and detectable by MALDI-TOF MS as an adduct with Phos-tag, a phosphate-capture zinc complex. Various α-hydroxy fatty acids (16:0, 22:0, 24:0 and 24:1) were detected by GC-MS of fatty acid methyl esters prepared from lipid X. The deacyl derivative of lipid X was determined to be 4-hydroxysphingenine (dehydrophytosphingosine)-1-phosphate by MALDI-TOF MS with Phos-tag. From these results, lipid X was determined to be phytoceramide-1-phosphate (PC1P) with an α-hydroxy fatty acid. When cabbage homogenates were incubated, PC1P was formed, with a concomitant decrease in the amount of glycosylinositol phosphoceramide (GIPC). The formation of PC1P from GIPC was confirmed by treatment of purified cabbage GIPC with a membrane fraction of cabbage homogenates. Using a partially purified enzyme fraction, we found that the enzyme hydrolyzes GIPC specifically, but not glycerophospholipids and sphingomyelin. Arabidopsis thaliana also had this enzyme activity. From these results, we conclude that a previously uncharacterized phospholipase D activity that specifically hydrolyzes GIPC produces PC1P in brassicaceous plants.