title: Mass spectrometric analysis of neutral and anionic N-glycans from a Dictyostelium discoideum model for human congenital disorder of glycosylation CDG IL.
abstract: The HL241 mutant strain of the cellular slime mold Dictyostelium discoideum is a potential model for human congenital disorder of glycosylation type IL (ALG9-CDG) and has been previously predicted to possess a lower degree of modification of its N-glycans with anionic moieties than the parental wild-type. In this study, we first showed that this strain has a premature stop codon in its alg9 mannosyltransferase gene compatible with the occurrence of truncated N-glycans. These were subject to an optimized analytical workflow, considering that the mass spectrometry of acidic glycans often presents challenges due to neutral loss and suppression effects. Therefore, the protein-bound N-glycans were first fractionated, after serial enzymatic release, by solid phase extraction. Then primarily single glycan species were isolated by mixed hydrophilic-interaction/anion-exchange or reversed-phase HPLC and analyzed using chemical and enzymatic treatments and MS/MS. We show that protein-linked N-glycans of the mutant are of reduced size as compared to those of wild-type AX3, but still contain core α1,3-fucose, intersecting N-acetylglucosamine, bisecting N-acetylglucosamine, methylphosphate, phosphate, and sulfate residues. We observe that a single N-glycan can carry up to four of these six possible modifications. Due to the improved analytical procedures, we reveal fuller details regarding the N-glycomic potential of this fascinating model organism.
authors: Department für Chemie, Universität für Bodenkultur, A-1190 Wien, Austria.
authors: Alba
authors: A
authors: Hykollari
authors: Crina I A
authors: CI
authors: Balog
authors: Dubravko
authors: D
authors: Rendić
authors: Thomas
authors: T
authors: Braulke
authors: Iain B H
authors: IB
authors: Wilson
authors: Katharina
authors: K
authors: Paschinger
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D018981:Congenital Disorders of Glycosylation / Q000378:metabolism*; D017931:DNA Primers; D004023:Dictyostelium / Q000737:chemistry*; D006801:Humans; D008954:Models, Biological; D011134:Polysaccharides / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D017931:DNA Primers; D011134:Polysaccharides