Ron Lou Doong

Comparative analysis of ceramide structural modification found in fungal cerebrosides by electrospray tandem mass spectrometry with low energy collision-induced dissociation of Li+ adduct ions.

Fungal cerebrosides (monohexosylceramides, or CMHs) exhibit a number of ceramide structural modifications not found in mammalian glycosphingolipids, which present additional challenges for their complete characterization. The use of Li+ cationization, in conjunction with electrospray ionization mass spectrometry and low energy collision-induced dissociation tandem mass spectrometry (ESI-MS/CID-MS), was found to be particularly effective for detailed structural analysis of complex fungal CMHs, especially minor components present in mixtures at extremely low abundance. A substantial increase in both sensitivity and fragmentation was observed on collision-induced dissociation of [M + Li]+ versus [M + Na]+ of the same CMH components analyzed under similar conditions. The effects of particular modifications on fragmentation were first systematically evaluated by analysis of a wide variety of standard CMHs expressing progressively more functionalized ceramides. These included bovine brain galactocerebrosides with non-hydroxy and 2-hydroxy fatty N-acylation; a plant glucocerebroside having (E/Z)-delta8 in addition to (E)-delta4 unsaturation of the sphingoid base; and a pair of fungal cerebrosides known to be further modified by a branching 9-methyl group on the sphingoid moiety, and to have a 2-hydroxy fatty N-acyl moiety either fully saturated or (E)-delta3 unsaturated. The method was then applied to characterization of both major and minor components in CMH fractions from a non-pathogenic mycelial fungus, Aspergillus niger; and from pathogenic strains of Candida albicans (yeast form); three Cryptococcus spp. (all yeast forms); and Paracoccidioides brasiliensis (both yeast and mycelium forms). The major components of all species examined differed primarily (and widely) in the level of 2-hydroxy fatty N-acyl delta3 unsaturation, but among the minor components a significant degree of additional structural diversity was observed, based on differences in sphingoid or N-acyl chain length, as well as on the presence or absence of the sphingoid delta8 unsaturation or 9-methyl group. Some variants were isobaric, and were not uniformly present in all species, affirming the need for MS/CID-MS analysis for full characterization of all components in a fungal CMH fraction. The diversity in ceramide distribution observed may reflect significant species-specific differences among fungi with respect to cerebroside biosynthesis and function.

Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc:ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth

The opportunistic mycopathogen Aspergillus fumigatus expresses both glucosylceramide and galactosylceramide (GlcCer and GalCer), but their functional significance in Aspergillus species is unknown. We here identified and characterized a GlcCer from Aspergillus nidulans, a non‐pathogenic model fungus. Involvement of GlcCer in fungal development was tested on both species using a family of compounds known to inhibit GlcCer synthase in mammals. Two analogs, D‐threo‐1‐phenyl‐2‐palmitoyl‐3‐pyrrolidinopropanol (P4) and D‐threo‐3′,4′‐ethylenedioxy‐P4, strongly inhibited germination and hyphal growth. Neutral lipids from A. fumigatus cultured in the presence of these inhibitors displayed a significantly reduced GlcCer/GalCer ratio. These results suggest that synthesis of GlcCer is essential for normal development of A. fumigatus and A. nidulans.

Pectin biosynthesis: a solubilized α1,4-galacturonosyltransferase from tobacco catalyzes the transfer of galacturonic acid from UDP-galacturonic acid onto the non-reducing end of homogalacturonan

Abstract. A solubilized α1,4-galacturonosyltransferase (GalAT) from tobacco transfers galacturonic acid (GalA) residues from UDP-GalA onto oligogalacturonide (OGA) exogenous acceptors with degrees of polymerization greater than nine (R.L. Doong and D. Mohnen 1998, Plant J 13: 363–374). The solubilized GalAT has been identified as putative polygalacturonate 4-α-galacturonosyltransferase (PGA-GalAT, EC 2.4.1.43) based on its α1,4-galacturonosyltransferase activity and similar Km for UDP-GalA, pH optimum and Vmax to those of membrane-bound PGA-GalAT (R.L. Doong et al., 1995, Plant Physiol 109: 141–152). The direction of elongation of homogalacturonan catalyzed by solubilized GalAT from microsomes of tobacco (Nicotiana tabacum L. cv. Samsun) cell suspensions has now been determined. Three different types of exogenous acceptor were used to study the direction of synthesis of homogalacturonan: unmodified OGAs, OGAs derivatized by biotinylation at the reducing end, and OGAs containing a 4,5-unsaturated GalA at the non-reducing end. The unmodified OGAs and the OGAs modified at the reducing end functioned equally well as acceptors in the galacturonosyltransferase reaction. In contrast, OGAs with the 4,5-unsaturated residue at the non-reducing end were not acceptors for homogalacturonan biosynthesis. These results show that homogalacturonan biosynthesis by solubilized GalAT occurs via the addition of GalA to the non-reducing end of the polymer chain.

Cell free synthesis of the pectic polysaccharide homogalacturonan

Abstract Polygalacturonate 4-α-galacturonosyltransferase (PGA-GalAT) (EC 2.4.1.43), the enzyme that synthesizes the pectin polysaccharide homogalacturonan (HGA), has been identified in microsomal membranes isolated from tobacco (Nicotiana tabacum L. cv Samsun) cell suspension cultures. The radiolabeled nucleotide sugar substrate, UDP-[14C]galacturonic acid (UDP-GalA), is synthesized by epimerization of UDP-[14C]glucuronic acid using crude particulate preparations from radish roots. Incubation of tobacco membranes with UDP-GalA results in a time-dependent incorporation of [14C]-galacturonic acid into a chloroform-methanol-precipitable and 65% ethanol-insoluble product. The synthesis of HGA in microsomal membranes occurs at a pH of 7.8, a temperature of 25 to 30°C, an apparent Km for UDP-GalA of ∼8.9 μM and a Vmax of ∼150 pmol min−1 mg−1 protein. Treatment of the product with base to hydrolyze ester linkages followed by digestion of the base-treated product with a homogeneous endopolygalacturonase results in cleavage of 34% to 89% of [14C]-labeled product into components that co-chromatograph with mono-, di-, and tri-galacturonic acid, indicating that a large portion of product contains contiguous 1,4-linked α-D-galactosyluronic acid residues. The product synthesized in cell free tobacco membranes has a molecular mass of ∼105,000 daltons based on dextran standards. Approximately 45% to 67% of the galacturonic residues in the synthesized HGA appear to be esterified since base-treatment prior to EPGase-fragmentation is required in order to obtain a maximal yield of mono-, di-, and tri-galacturonic acid. Comparison of the sensitivity of base-treated and pectin methylesterase-treated products to fragmentation by EPGase indicates that at least 40% of the base sensitive linkages are methyl esters. Conditions have been established that allow for up to 90% of the activity in detergent-dispersed membranes to be recovered as soluble enzyme.

Corrigendum to: Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc: ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth (FEBS 26342)

aDepartment of Chemistry, University of New Hampshire, Durham, NH 03824, USA bDepartment of Botany, University of Georgia, Athens, GA 30602-7229, USA cDepartment of Biochemistry, Universidade Federal de Sa‹o Paulo/Escola Paulista de Medicina, Rua Botucatu 862, 04023-900 Sa‹o Paulo SP, Brazil dDivision of Nephrology, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109, USA eThe Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA

Erratum: Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc: Ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth (FEBS 26342): (FEBS Letters (2002) 525 (59-64) PII S001457930030673)

aDepartment of Chemistry, University of New Hampshire, Durham, NH 03824, USA bDepartment of Botany, University of Georgia, Athens, GA 30602-7229, USA cDepartment of Biochemistry, Universidade Federal de Sa‹o Paulo/Escola Paulista de Medicina, Rua Botucatu 862, 04023-900 Sa‹o Paulo SP, Brazil dDivision of Nephrology, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109, USA eThe Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA