Date of Award
Program or Major
Doctor of Philosophy
Steven B Levery
Fungal lectins from the mushrooms Polyporus squamosus and Coprinus cinereus have an affinity for beta-galactosides, with extended binding sites for the non-reducing terminal trisaccharide sequence NeuAcalpha2→6Galbeta1→4Glc/GlcNAc, and substituted beta-galactosides resembling mammalian blood group determinants, respectively. In considering the possibility that glycosylinositol phosphorylceramides (GIPCs) could be endogenous ligands of such fungal lectins, structural characterization of glycosphingolipids (GSLs) from the fruiting bodies of these mushrooms was performed. The extraction, isolation and purification of the lipids, and their subsequent characterization by nuclear magnetic resonance (NMR), mass spectrometry (MS) and gas chromatography-mass spectrometry (GC-MS) was performed. One monohexosylceramide (CMH) and three major GIPCs were identified from P. squamosus. The GIPCs were identified as Manalpha1→2Ins-P-Cer (Ps-1), Galbeta1→6Manalpha1→21ns-P-Cer (Ps-2) and Manalpha1→3Fucalpha1→2Galalpha1→6Galbeta1→6Manalpha1→21ns-P-Cer (Ps-5). Of these GSLs, Ps-2 could be a potential ligand for P. squamosus lectin. From C. cinereus, three components were identified as typical fungal Manalpha1→21ns-P-Cer incorporating unusually short chained ceramides not previously reported in other basidiomycetes.
In addition, preliminary studies towards development of high throughput methods for glycosphingolipidome analysis were performed. These studies explored (i) the use of MSn for disassembly of permethylated glycosylinositols (GIs) and (ii) the construction of GSL-based glycan microarrays. MS n analysis of GIs efficiently provided useful information on linkage and sequence. An essential step in microarray development involved the enzymatic release of the hydroxyl fatty acyl of the ceramide moiety of selected fungal GIPCs with sphingolipid N-deacylase (SCDase). Derivatives were generated from the reaction of the resulting free amino group with N-reactive F-tag (N-[4-(1 H,1H,2H,2H perfluorodecyl)benzyloxycarbonyloxyl]succinimide (F17-Cbz-OSu, C22H14F17NO 5), and with a linker assembled from succinimidyl-[(N-maleimidopropionamido)diethyleneglycol] ester (NHS-PEO2-maleimide) and 2-mercaptoethylamine (2-MEA). The derivatives were immobilized via fluorous-flourous affinity, and by amide bond formation on N-hydroxysuccinimide-activated micro-glass slides, respectively. Preliminary evaluation of their use in assaying protein-carbohydrate binding interactions was carried out. The potential utility of this methodology for designing microarrays that incorporate both mammalian and fungal GSLs for serodiagnosis is discussed.
Arigi, Emma Adhiambo, "Development and application of methods for analysis of fungal glycosphingolipid structure and functional interactions" (2007). Doctoral Dissertations. 407.