title: Identification of a sphingolipid-specific phospholipase D activity associated with the generation of phytoceramide-1-phosphate in cabbage leaves.
abstract: 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.
authors.affiliation: Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan. tanaka.tamotsu@tokushima-u.ac.jp
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Tanaka
authors.forename: Takashi
authors.initials: T
authors.lastname: Kida
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Imai
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Morishige
authors.forename: Ryouhei
authors.initials: R
authors.lastname: Yamashita
authors.forename: Hisatsugu
authors.initials: H
authors.lastname: Matsuoka
authors.forename: Sachika
authors.initials: S
authors.lastname: Uozumi
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Satouchi
authors.forename: Minoru
authors.initials: M
authors.lastname: Nagano
authors.forename: Akira
authors.initials: A
authors.lastname: Tokumura
mesh_terms: D029681:Arabidopsis Proteins / Q000302:isolation & purification / Q000378:metabolism; D001937:Brassica / Q000201:enzymology* / Q000378:metabolism; D002518:Ceramides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D008401:Gas Chromatography-Mass Spectrometry; D006028:Glycosphingolipids / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D010739:Phospholipase D / Q000302:isolation & purification / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology* / Q000378:metabolism; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013107:Sphingolipids / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D002518:Ceramides; D006028:Glycosphingolipids; D008565:Membrane Proteins; D010940:Plant Proteins; D013107:Sphingolipids; C065576:ceramide 1-phosphate; C025524:inositolphosphoceramides; D010739:Phospholipase D
keywords: brassicaceous plants; glycosylinositol phosphoceramide; phospholipase D; phytoceramide-1-phosphate; sphingolipid