Characterization of glycoinositolphosphoryl ceramide structure mutant strains of Cryptococcus neoformans.
In fungi, glycoinositolphosphoryl ceramide (GIPC) biosynthetic pathway produces essential molecules for growth, viability, and virulence. In previous studies, we demonstrated that the opportunistic fungus Cryptococcus neoformans synthesizes a complex family of xylose-(Xyl) branched GIPCs, all of which have not been previously reported in fungi. As an effort to understand the biosynthesis of these sphingolipids, we have now characterized the structures of GIPCs from C. neoformans wild-type (KN99alpha) and mutant strains that lack UDP-Xyl, by disruption of either UDP-glucose dehydrogenase (NE321) or UDP-glucuronic acid decarboxylase (NE178). The structures of GIPCs were determined by a combination of nuclear magnetic resonance (NMR) spectroscopy, tandem mass spectrometry (MS), and gas chromatography-MS. The main and largest GIPC from wild-type strain was identified as an alpha-Manp(1 --> 6)alpha-Manp(1 --> 3)alpha-Manp[beta-Xylp(1 --> 2)]alpha-Manp(1 --> 4)beta-Galp(1 --> 6)alpha-Manp(1 --> 2) Ins-1-P-Ceramide, whereas the most abundant GIPC from both mutant strains was found to be an alpha-Manp(1 --> 3)alpha-Manp(1 --> 4)beta-Galp(1 --> 6)alpha-Manp(1 --> 2)Ins-1-P-Ceramide. The ceramide moieties of C. neoformans wild-type and mutant strains were composed of a C(18) phytosphingosine, which was N-acylated with 2-hydroxy tetra-, or hexacosanoic acid, and 2,3-dihydroxy-tetracosanoic acid. Our structural analysis results indicate that the C. neoformans mutant strains are unable to complete the assembly of the GIPC-oligosaccharide moiety due the absence of Xyl side chain.
Gutierrez
Ana L S
AL
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Cidade Universitária, 21944979, Rio de Janeiro, Brasil.
Farage
Layla
L
Melo
Manuel N
MN
Mohana-Borges
Ronaldo S
RS
Guerardel
Yann
Y
Coddeville
Bernadete
B
Wieruszeski
Jean-Michel
JM
Mendonça-Previato
Lucia
L
Previato
Jose O
JO
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002518:Ceramides / Q000737:chemistry* / Q000235:genetics*; D002855:Chromatography, Thin Layer; D003455:Cryptococcus neoformans / Q000737:chemistry* / Q000235:genetics*; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011135:Polysaccharides, Bacterial / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D002518:Ceramides; D011135:Polysaccharides, Bacterial